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Sample records for rotavirus vp1 protein

  1. Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

    SciTech Connect

    Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra; Tao, Yizhi Jane; Vasquez-Del Carpio, Rodrigo; Nibert, Max L.; Patton, John T.; Harrison, Stephen C.

    2009-04-08

    Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

  2. Hydroxyproline in the major capsid protein VP1 of polyomavirus

    SciTech Connect

    Ludlow, J.W.; Consigli, R.A.

    1989-06-01

    Amino acid analysis of (/sup 3/H)proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of /sup 3/H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from (/sup 35/S)methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.

  3. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein.

    PubMed

    Xu, Juan; Zhang, Chunhua

    2016-05-01

    Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies. PMID:27533931

  4. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

    PubMed Central

    Xu, Juan; Zhang, Chunhua

    2016-01-01

    Abstract Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies.

  5. Polymorphism in the assembly of polyomavirus capsid protein VP1.

    PubMed Central

    Salunke, D M; Caspar, D L; Garcea, R L

    1989-01-01

    Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:2557933

  6. A functional nuclear localization sequence in the VP1 capsid protein of coxsackievirus B3

    SciTech Connect

    Wang, Tianying; Yu, Bohai; Lin, Lexun; Zhai, Xia; Han, Yelu; Qin, Ying; Guo, Zhiwei; Wu, Shuo; Zhong, Xiaoyan; Wang, Yan; Tong, Lei; Zhang, Fengmin; Si, Xiaoning; Zhao, Wenran; Zhong, Zhaohua

    2012-11-25

    The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, the VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.

  7. Characterization of the enterovirus 71 VP1 protein as a vaccine candidate.

    PubMed

    Zhou, Shi-Li; Ying, Xiao-Ling; Han, Xue; Sun, Xian-Xun; Jin, Qi; Yang, Fan

    2015-02-01

    Enterovirus 71 (EV71) is an important agent responsible for hand-foot-and-mouth disease (HFMD), which can cause severe neurological complications and death in children. However, there is no specific treatment for EV71 infection, and a safe and effective vaccine is needed urgently. In this study, an effective and economical method for the production of EV71-VP1 protein was developed, and the VP1 protein was evaluated in humoral and cellular immune responses as an EV71 vaccine. The results revealed that the VP1 protein induced high titers of cross-neutralizing antibodies for different EV71 subtypes, and elicited significant splenocyte proliferation. The high levels of IFN-r and IL-10 showed the VP1 protein induced a mixed Th1 and Th2 immune response. Vaccinated female mice could confer protection in their neonatal offspring. Compared with the inactivated EV71, the VP1 protein elicited similar humoral and cellular responses, but the engineered protein is safer, less expensive and can be produced more efficiently. Therefore, EV71-VP1 protein can induce effective immunologic protection against EV71 and is an ideal candidate against EV71 infection. PMID:25043151

  8. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

  9. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

    PubMed Central

    Haynes, J I; Consigli, R A

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein. Images PMID:1318417

  10. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    NASA Technical Reports Server (NTRS)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  11. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly.

    PubMed Central

    Rodgers, R E; Chang, D; Cai, X; Consigli, R A

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents. Images PMID:8151798

  12. Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

    PubMed Central

    Lien, Yi-Yang; Huang, Chi-Hung; Sun, Fang-Chun; Sheu, Shyang-Chwen; Lu, Tsung-Chi; Lee, Meng-Shiunn; Hsueh, Shu-Chin; Chen, Hsi-Jien

    2012-01-01

    Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future. PMID:22437539

  13. Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

    PubMed

    Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma

    2016-07-01

    A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis. PMID:27038828

  14. Human parvovirus B19 VP1u Protein as inflammatory mediators induces liver injury in naïve mice.

    PubMed

    Hsu, Tsai-Ching; Chiu, Chun-Ching; Chang, Shun-Chih; Chan, Hsu-Chin; Shi, Ya-Fang; Chen, Tzy-Yen; Tzang, Bor-Show

    2016-01-01

    Human parvovirus B19 (B19V) is a human pathogen known to be associated with many non-erythroid diseases, including hepatitis. Although B19V VP1-unique region (B19-VP1u) has crucial roles in the pathogenesis of B19V infection, the influence of B19-VP1u proteins on hepatic injury is still obscure. This study investigated the effect and possible inflammatory signaling of B19-VP1u in livers from BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. The in vivo effects of B19-VP1u were analyzed by using live animal imaging system (IVIS), Haematoxylin-Eosin staining, gel zymography, and immunoblotting after inoculation. Markedly hepatocyte disarray and lymphocyte infiltration, enhanced matrix metalloproteinase (MMP)-9 activity and increased phosphorylation of p38, ERK, IKK-α, IκB and NF-κB (p-p65) proteins were observed in livers from BALB/c mice receiving COS-7 cells expressing B19-VP1u as well as the significantly increased CRP, IL-1β and IL-6. Notably, IFN-γ and phosphorylated STAT1, but not STAT3, were also significantly increased in the livers of BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. These findings revealed the effects of B19-VP1u on liver injury and suggested that B19-VP1u may have a role as mediators of inflammation in B19V infection. PMID:26632342

  15. Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles

    PubMed Central

    Yuan, Shilin; Li, Guiming; Wang, Ying; Gao, Qianqian; Wang, Yizhuo; Cui, Rui

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra- and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles. IMPORTANCE Infection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are

  16. Subcellular localisation of Theiler's murine encephalomyelitis virus (TMEV) capsid subunit VP1 vis-á-vis host protein Hsp90.

    PubMed

    Ross, Caroline; Upfold, Nicole; Luke, Garry A; Bishop, Özlem Tastan; Knox, Caroline

    2016-08-15

    The VP1 subunit of the picornavirus capsid is the major antigenic determinant and mediates host cell attachment and virus entry. To investigate the localisation of Theiler's murine encephalomyelitis virus (TMEV) VP1 during infection, a bioinformatics approach was used to predict a surface-exposed, linear epitope region of the protein for subsequent expression and purification. This region, comprising the N-terminal 112 amino acids of the protein, was then used for rabbit immunisation, and the resultant polyclonal antibodies were able to recognise full length VP1 in infected cell lysates by Western blot. Following optimisation, the antibodies were used to investigate the localisation of VP1 in relation to Hsp90 in infected cells by indirect immunofluorescence and confocal microscopy. At 5h post infection, VP1 was distributed diffusely in the cytoplasm with strong perinuclear staining but was absent from the nucleus of all cells analysed. Dual-label immunofluorescence using anti-TMEV VP1 and anti-Hsp90 antibodies indicated that the distribution of both proteins colocalised in the cytoplasm and perinuclear region of infected cells. This is the first report describing the localisation of TMEV VP1 in infected cells, and the antibodies produced provide a valuable tool for investigating the poorly understood mechanisms underlying the early steps of picornavirus assembly. PMID:27269472

  17. Interaction between Simian Virus 40 Major Capsid Protein VP1 and Cell Surface Ganglioside GM1 Triggers Vacuole Formation

    PubMed Central

    Luo, Yong; Motamedi, Nasim; Magaldi, Thomas G.; Gee, Gretchen V.; Atwood, Walter J.

    2016-01-01

    ABSTRACT Simian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40. PMID:27006465

  18. Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity ▿

    PubMed Central

    Yang, Su-Lin; Chou, Ying-Ting; Wu, Cheng-Nan; Ho, Mei-Shang

    2011-01-01

    Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose. PMID:21900167

  19. Bioinformatic analysis of non-VP1 capsid protein of coxsackievirus A6.

    PubMed

    Liu, Hong-Bo; Yang, Guang-Fei; Liang, Si-Jia; Lin, Jun

    2016-08-01

    This study bioinformatically analyzed the non-VP1 capsid proteins (VP2-VP4) of Coxasckievirus A6 (CVA6), with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools SubLoc, TargetP and the others from ExPASy Bioinformatics Resource Portal, and SWISS-MODEL (an online protein structure modeling server), were utilized to analyze the amino acid (AA) sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices (AI) of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents. PMID:27465341

  20. Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

    SciTech Connect

    Bucks, Michelle A.; O'Regan, Kevin J.; Murphy, Michael A.; Wills, John W.; Courtney, Richard J. . E-mail: rcourtney@psu.edu

    2007-05-10

    The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.

  1. Molecular characterization of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of bovine group B rotaviruses: identification of a novel VP4 genotype.

    PubMed

    Ghosh, Souvik; Kobayashi, N; Nagashima, S; Chawla-Sarkar, M; Krishnan, T; Ganesh, B; Naik, T N

    2010-02-01

    Studies on bovine group B rotaviruses (GBRs) are limited. To date, only the VP6 gene of a single bovine GBR strain and the VP7 and NSP5 genes of a few bovine GBR strains have been sequenced and analyzed. In the present study, using a single-primer amplification method, we have determined the full-length nucleotide sequences of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of three bovine GBR strains from eastern India. In all six of these genes, the bovine GBR strains shared high genetic relatedness among themselves but exhibited high genetic diversity with cognate genes of human, murine and ovine GBRs. Interestingly, as with group A rotaviruses, the bovine GBR VP1, VP2, VP6 and NSP2 genes appeared to be more conserved than the VP4 and NSP1 genes among strains of different species. The present study provides important insights into the genetic makeup and diversity of bovine GBRs, and also identifies a novel GBR VP4 genotype. PMID:19936611

  2. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Guo, Yuanli; Guo, Rui; Zhou, Quan; Sun, Changgui; Zhang, Xinmei; Liu, Yuanjun; Liu, Quanzhong

    2016-01-01

    Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis. PMID:27089359

  3. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway.

    PubMed

    Guo, Yuanli; Guo, Rui; Zhou, Quan; Sun, Changgui; Zhang, Xinmei; Liu, Yuanjun; Liu, Quanzhong

    2016-04-01

    Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis. PMID:27089359

  4. The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

  5. High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    PubMed Central

    2011-01-01

    Background Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests. PMID:21781331

  6. Distinguishing the genotype 1 genes and proteins of human Wa-like rotaviruses vs. porcine rotaviruses.

    PubMed

    Silva, Fernanda D F; Gregori, F; McDonald, Sarah M

    2016-09-01

    Group A rotaviruses (RVAs) are 11-segmented, double-stranded RNA viruses and important causes of gastroenteritis in the young of many animal species. Previous studies have suggested that human Wa-like RVAs share a close evolutionary relationship with porcine RVAs. Specifically, the VP1-VP3 and NSP2-5/6 genes of these viruses are usually classified as genotype 1 with >81% nucleotide sequence identity. Yet, it remains unknown whether the genotype 1 genes and proteins of human Wa-like strains are distinguishable from those of porcine strains. To investigate this, we performed comprehensive bioinformatic analyses using all known genotype 1 gene sequences. The RVAs analyzed represent wildtype strains isolated from humans or pigs at various geographical locations during the years of 2004-2013, including 11 newly-sequenced porcine RVAs from Brazil. We also analyzed archival strains that were isolated during the years of 1977-1992 as well as atypical strains involved in inter-species transmission between humans and pigs. We found that, in general, the genotype 1 genes of typical modern human Wa-like RVAs clustered together in phylogenetic trees and were separate from those of typical modern porcine RVAs. The only exception was for the NSP5/6 gene, which showed no host-specific phylogenetic clustering. Using amino acid sequence alignments, we identified 34 positions that differentiated the VP1-VP3, NSP2, and NSP3 genotype 1 proteins of typical modern human Wa-like RVAs versus typical modern porcine RVAs and documented how these positions vary in the archival/unusual isolates. No host-specific amino acid positions were identified for NSP4, NSP5, or NSP6. Altogether, the results of this study support the notion that human Wa-like RVAs and porcine RVAs are evolutionarily related, but indicate that some of their genotype 1 genes and proteins have diverged over time possibly as a reflection of sequestered replication and protein co-adaptation in their respective hosts. PMID

  7. Detection of HIV-1 Tat and JCV capsid protein, VP1, in AIDS brain with progressive multifocal leukoencephalopathy.

    PubMed

    Del Valle, L; Croul, S; Morgello, S; Amini, S; Rappaport, J; Khalili, K

    2000-06-01

    HIV-1 infection can lead to severe central nervous system (CNS) clinical syndromes in more than 50% of HIV-1 positive individuals. Progressive multifocal leukoencephalopathy (PML) is the frequent opportunistic infection of the CNS which is seen in as high as 5% of AIDS patients. Results from previous cell culture studies showed that the HIV-1 regulatory protein, Tat can potentiate transcription of the human neurotropic virus, JCV, the causative agent for PML in cells derived from the human CNS. In this communication we examine the presence of the HIV-1 regulatory protein, Tat, as well as the HIV-1 and JCV structural proteins, p24 and VP1, respectively in AIDS/PML clinical samples. We demonstrate high level expression of the JCV capsid protein, VP1, in oligodendrocytes and to some degree in astrocytes of AIDS with PML. In HIV-1+ samples expression of HIV-1 core protein, p24 was detected in perivascular monocytic cells and to a lesser extent in astrocytes and endothelial cells. A lack of p24 expression in oligodendrocytes suggested no infection of these cells with HIV-1. Interestingly, HIV-1 Tat was detected in various infected cells as well as in uninfected oligodendrocytes from HIV-1+ tissue, supporting the earlier in vitro findings that secreted Tat from the infected cells can be localized in the neighboring uninfected cells. The presence of Tat in oligodendrocytes was particularly interesting as this protein can up-modulate JCV gene transcription and several key cell cycle regulatory proteins including cyclin E, Cdk2, and pRb. The data presented here provide in vivo evidence for a role of HIV-1 Tat in the pathogenesis of AIDS/PML by acting as a positive regulatory protein that affects the expression of JCV and other cell regulatory proteins in the CNS. PMID:10878711

  8. Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

    PubMed

    Miño, Samuel; Barrandeguy, María; Parreño, Viviana; Parra, Gabriel I

    2016-04-01

    Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments. PMID:26758293

  9. Evolutionary Analysis of Structural Protein Gene VP1 of Foot-and-Mouth Disease Virus Serotype Asia 1

    PubMed Central

    Zhang, Qingxun; Liu, Xinsheng; Fang, Yuzhen; Pan, Li; Lv, Jianliang; Zhang, Zhongwang; Zhou, Peng; Ding, Yaozhong; Chen, Haotai; Shao, Junjun; Zhao, Furong; Lin, Tong; Chang, Huiyun; Zhang, Jie; Wang, Yonglu; Zhang, Yongguang

    2015-01-01

    Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I–VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10−3 substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown. PMID:25793223

  10. Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1

    PubMed Central

    Tan, Chee Wah; Chan, Yoke Fun; Sim, Kooi Mow; Tan, Eng Lee; Poh, Chit Laa

    2012-01-01

    Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC50 values ranging from 6–9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71. PMID:22563456

  11. MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1

    PubMed Central

    Nandi, Satabdi; Chanda, Shampa; Bagchi, Parikshit; Nayak, Mukti Kant; Bhowmick, Rahul; Chawla-Sarkar, Mamta

    2014-01-01

    Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. In developing countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Among various pattern recognition receptors (PAMPs) of the host, the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein (MAVS), which regulates cellular interferon response. With an aim to identify the role of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independent manner. Rotavirus non-structural protein 1 (NSP1) which is a known IFN antagonist, interacted with MAVS and degraded it in a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of our knowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. In addition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signaling cascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit by various back up strategies. PMID:24643253

  12. Expression of a recombinant chimeric protein of hepatitis A virus VP1-Fc using a replicating vector based on Beet curly top virus in tobacco leaves and its immunogenicity in mice.

    PubMed

    Chung, Ho Yong; Lee, Hyun Ho; Kim, Kyung Il; Chung, Ha Young; Hwang-Bo, Jeon; Park, Jong Hwa; Sunter, Garry; Kim, Jong Bum; Shon, Dong Hwa; Kim, Wonyong; Chung, In Sik

    2011-08-01

    We describe the expression and immunogenicity of a recombinant chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus VP1 and an Fc antibody fragment using a replicating vector based on Beet curly top virus (BCTV) in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Recombinant HAV VP1-Fc was expressed with a molecular mass of approximately 68 kDa. Recombinant HAV VP1-Fc, purified using Protein A Sepharose affinity chromatography, elicited production of specific IgG antibodies in the serum after intraperitoneal immunization. Following vaccination with recombinant HAV VP1-Fc protein, expressions of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Recombinant VP1-Fc from infiltrated tobacco plants can be used as an effective experimental immunogen for research into vaccine development. PMID:21442402

  13. Rotavirus.

    PubMed

    Esona, Mathew D; Gautam, Rashi

    2015-06-01

    Group A rotavirus (RVA) is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live, attenuated rotavirus vaccines, Rotarix® and RotaTeq®, has dramatically reduced RVA-associated AGE and mortality. High-throughput, sensitive and specific techniques are required to rapidly diagnose and characterize rotavirus strains in stool samples for proper patient treatment and to monitor circulating vaccine and wild-type rotavirus strains. New molecular assays are rapidly developed that are more sensitive and specific than the conventional assays for detection, genotyping and full genome characterization of circulating rotavirus wild-type and vaccine (Rotarix® and RotaTeq®) strains causing AGE. PMID:26004648

  14. Different Antibody Response against the Coxsackievirus A16 VP1 Capsid Protein: Specific or Non-Specific.

    PubMed

    Ding, Yingying; Wang, Zhihong; Zhang, Xi; Teng, Zheng; Gao, Caixia; Qian, Baohua; Wang, Lili; Feng, Jiaojiao; Wang, Jinhong; Zhao, Chunyan; Guo, Cunjiu; Pan, Wei

    2016-01-01

    Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease worldwide. The non-neutralizing antibody response that targets CA16 VP1 remains poorly elucidated. In the present study, antibody responses against CA16 VP1 in Shanghai blood donors and Shanxi individuals were analyzed by ELISA and inhibitory ELISA using five CA16 VP1 antigens: VP11-297, VP141-297, VP11-60, VP145-58 and VP161-297. The correlation coefficients for most of the reactions against each of the five antigens and the inhibition of the anti-CA16 VP1 antibody response produced by the various antigens were higher in Shanghai blood donors compared to those in Shanxi individuals. VP11-297 and VP141-297 strongly inhibited the anti-CA16 VP1 response in serum samples from both populations, while VP145-58 and VP161-297 intermediately and weakly inhibited the anti-CA16 VP1 response, respectively, in only Shanghai group. A specific type of inhibition (anti-CA16 VP1 was completely inhibited by both VP11-60 and VP141-297) characterized by high neutralizing antibody titers was identified and accounted for 71.4% of the strongly reactive samples from the Shanghai group. These results indicate that the Shanghai blood donors exhibited a consistent and specific antibody response, while the Shanxi individuals showed an inconsistent and non-specific antibody response. These findings may improve the understanding of host humoral immunity against CA16 and help to identify an effective approach for seroepidemiological surveillance and specific diagnosis of CA16 infection based on normal and competitive ELISA. PMID:27622652

  15. Codon Optimization, Expression in Escherichia coli, and Immunogenicity of Recombinant Chinese Sacbrood Virus (CSBV) Structural Proteins VP1, VP2, and VP3

    PubMed Central

    Zhang, Haochun; Jiang, Lili; Wang, Qiang; Zhong, Yi; Fan, Zhaobin; Ma, Mingxiao

    2015-01-01

    Chinese sacbrood virus (CSBV) is a small RNA virus family belonging to the genus Iflavirus that causes larval death, and even the collapse of entire bee colonies. The virus particle is spherical, non-enveloped, and its viral capsid is composed of four proteins, although the functions of the structural proteins are unclear. In this study, we used codon recoding to express the recombinant proteins VP1, VP2, and VP3 in Escherichia coli. SDS-PAGE analysis and Western blotting revealed that the target genes were expressed at high levels. Mice were then immunized with the purified, recombinant proteins, and antibody levels and lymphocyte proliferation were analyzed by ELISA and the MTT assay, respectively. The results show that the recombinant proteins induced high antibody levels and promoted lymphocyte proliferation. Polyclonal antibodies directed against these proteins will aid future studies of the molecular pathogenesis of CSBV. PMID:26067659

  16. Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87

    PubMed Central

    Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

    2011-01-01

    The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3Dpol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3Dpol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp26→Glu substitution in a beta sheet located within a small groove of the 3Dpol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment. PMID:22122902

  17. Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1.

    PubMed

    Premanand, Balraj; Kiener, Tanja K; Meng, Tao; Tan, Yun Rui; Jia, Qiang; Chow, Vincent T K; Kwang, Jimmy

    2012-09-01

    EV71 is a major causative agent of hand, foot and mouth disease (HFMD) and is responsible for large outbreaks in various Asian Pacific countries. In the present study, we generated the recombinant baculovirus (Bac-VP1) encoding VP1 in a novel expression cassette. The transmembrane domain of hemagglutinin of the H3N2 influenza virus was included in the cassette as a minimal membrane anchor for VP1. The protective immunity of Bac-VP1 was investigated in a mouse model. The results showed that mice vaccinated with live Bac-VP1 had strong VP1 specific antibody responses. In an in vitro neutralization assay Bac-VP1 sera exhibited cross-neutralization against homologous and heterologous EV71 strains with a maximum titer of 1:512. Passive immunization studies confirmed that these sera were able to provide 100% protection against 5 MLD(50) of mouse adapted EV71 (B4 strain). This study revealed that baculovirus displaying VP1 with a HA transmembrane domain efficiently induced cross-neutralizing antibody responses in mice. PMID:22691220

  18. Reassortant group A rotavirus from straw-colored fruit bat (Eidolon helvum).

    PubMed

    Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Conrardy, Christina; Tong, Suxiang; Kuzmin, Ivan V; Agwanda, Bernard; Breiman, Robert F; Banyai, Krisztian; Niezgoda, Michael; Rupprecht, Charles E; Gentsch, Jon R; Bowen, Michael D

    2010-12-01

    Bats are known reservoirs of viral zoonoses. We report genetic characterization of a bat rotavirus (Bat/KE4852/07) detected in the feces of a straw-colored fruit bat (Eidolon helvum). Six bat rotavirus genes (viral protein [VP] 2, VP6, VP7, nonstructural protein [NSP] 2, NSP3, and NSP5) shared ancestry with other mammalian rotaviruses but were distantly related. The VP4 gene was nearly identical to that of human P[6] rotavirus strains, and the NSP4 gene was closely related to those of previously described mammalian rotaviruses, including human strains. Analysis of partial sequence of the VP1 gene indicated that it was distinct from cognate genes of other rotaviruses. No sequences were obtained for the VP3 and NSP1 genes of the bat rotavirus. This rotavirus was designated G25-P[6]-I15-R8(provisional)-C8-Mx-Ax-N8-T11-E2-H10. Results suggest that several reassortment events have occurred between human, animal, and bat rotaviruses. Several additional rotavirus strains were detected in bats. PMID:21122212

  19. Rotavirus RNA Polymerases Resolve into Two Phylogenetically Distinct Classes that Differ in Their Mechanism of Template Recognition

    PubMed Central

    Ogden, Kristen M.; Johne, Reimar; Patton, John T.

    2012-01-01

    Rotaviruses (RVs) are segmented double-stranded RNA viruses that cause gastroenteritis in mammals and birds. Within the RV genus, eight species (RVA-RVH) have been proposed. Here, we report the first RVF and RVG sequences for the viral RNA polymerase (VP1)-encoding segments and compare them to those of other RV species. Phylogenetic analyses indicate that the VP1 RNA segments and proteins resolve into two major clades, with RVA, RVC, RVD and RVF in clade A, and RVB, RVG and RVH in clade B. Plus-strand RNA of clade A viruses, and not clade B viruses, contain a 3′-proximal UGUG cassette that serves as the VP1 recognition signal. VP1 structures for a representative of each RV species were predicted using homology modeling. Structural elements involved in interactions with the UGUG cassette were conserved among VP1 of clade A, suggesting a conserved mechanism of viral RNA recognition for these viruses. PMID:22687427

  20. Analysis of Amino Acid Variation in the P2 Domain of the GII-4 Norovirus VP1 Protein Reveals Putative Variant-Specific Epitopes

    PubMed Central

    Allen, David J.; Gray, Jim J.; Gallimore, Chris I.; Xerry, Jacqueline; Iturriza-Gómara, Miren

    2008-01-01

    Background Human noroviruses are a highly diverse group of viruses classified into three of the five currently recognised Norovirus genogroups, and contain numerous genotypes or genetic clusters. Noroviruses are the major aetiological agent of endemic gastroenteritis in all age groups, as well as the cause of periodic epidemic gastroenteritis. The noroviruses most commonly associated with outbreaks of gastroenteritis are genogroup II genotype 4 (GII-4) strains. The relationship between genotypes of noroviruses with their phenotypes and antigenic profile remains poorly understood through an inability to culture these viruses and the lack of a suitable animal model. Methodology/Principal Findings Here we describe a study of the diversity of amino acid sequences of the highly variable P2 region in the major capsid protein, VP1, of the GII-4 human noroviruses strains using sequence analysis and homology modelling techniques. Conclusions/Significance Our data identifies two sites in this region, which show significant amino acid substitutions associated with the appearance of variant strains responsible for epidemics with major public health impact. Homology modelling studies revealed the exposed nature of these sites on the capsid surface, providing supportive structural data that these two sites are likely to be associated with putative variant-specific epitopes. Furthermore, the patterns in the evolution of these viruses at these sites suggests that noroviruses follow a neutral network pattern of evolution. PMID:18213393

  1. Ovine rotaviruses

    PubMed Central

    Gazal, S.; Mir, I.A.; Iqbal, A.; Taku, A.K.; Kumar, B.; Bhat, M.A.

    2011-01-01

    Rotavirus has been recognized as a predominant cause of acute diarrhea in young animals and humans. Rotavirus has segmented genome composed of 11 segments of double stranded RNA. The virus has a triple layered protein shell consisting of a core, an inner capsid and an outer capsid. The inner capsid protein is responsible for group specificity and based on it rotaviruses are classified into seven groups. Ovine rotavirus strains have only been identified into two serogroups (A and B). The two outer capsid proteins (VP7 and VP4) are responsible for G and P typing of rotavirus, respectively. Although rotavirus has been frequently reported in many animal species, data regarding ovine rotavirus strains is very scanty and limited. Only a few ovine rotaviruses have been isolated and characterized so far. Recently, the G and P types circulating in ovines have been identified. The ovine rotavirus strain NT isolated from a diarrheic lamb in China is being considered as a promising vaccine candidate for human infants. PMID:26623281

  2. Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection

    PubMed Central

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A.; Meng, Jihong

    2015-01-01

    Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11–21 aa contain EV-71-specific antigenic sites, whereas positions 1–5 and 51–100 contain epitopes shared with human coxsackievirus A16 (CV-A16) and human echovirus 6 (E-6). The N-terminal truncated protein of VP1, VP16–43, exhibited good stability and was recognized by anti-EV-71 specific rabbit sera. Alignment analysis showed that VP16–43 is highly conserved among EV-71 strains from different genotypes but was heterologous among other enteroviruses. When the GST-VP16–43 fusion protein was incorporated as antibody-capture agent in a WB assay and an ELISA for detecting anti-EV-71 IgM in human sera, sensitivities of 91.7% and 77.8% were achieved, respectively, with 100% specificity for both. The characterized EV-71 VP1 protein truncated to positions 6–43 aa has potential as an antigen for detection of anti-EV-71 IgM for early diagnosis of EV-71 infection in a WB format. PMID:24952304

  3. Antigenic relationships among human rotaviruses as determined by outer capsid protein VP4.

    PubMed

    Gorziglia, M; Larralde, G; Kapikian, A Z; Chanock, R M

    1990-09-01

    cDNA clones representing the VP4 gene of symptomatic human rotavirus strain KU (VP7 serotype 1) or DS-1 (VP7 serotype 2) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2) were constructed and inserted into a baculovirus expression vector under the control of the polyhedrin promoter. The resulting recombinants expressed the appropriate authentic VP4 rotavirus outer capsid protein. Guinea pigs immunized with these VP4 proteins developed antibodies that neutralized infectivity of the rotavirus from which the immunizing VP4 was derived. These antisera were then used in neutralization tests to define the extent and distribution of VP4 antigenic polymorphism among human rotaviruses. Three distinct serotypes and one subtype of the VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. For the most part, VP4 serotype segregated independently of VP7 serotype. Ten strains of human rotavirus that were associated with symptomatic infection and that exhibited VP7 serotype 1, 3, 4, or 9 specificity, each possessed a VP4 of the same serotype and subtype, designated VP4 serotype 1A. Both symptomatic human rotavirus strains with VP7 serotype 2 specificity were related by neutralization to the VP4 serotype 1A strains and were classified as a subtype of VP4 serotype 1--i.e., serotype 1B--since viruses of serotype 1A appeared to be prime strains. Four human rotavirus strains that were recovered from healthy infants in newborn nurseries in which virus transmission persisted over a long interval, belonged to VP7 serotype 1, 2, 3, or 4, but each strain possessed the same VP4 antigenic specificity that was designated VP4 serotype 2. Finally, a single strain of symptomatic human rotavirus of VP7 serotype 1 specificity possessed a unique VP4 that was provisionally classified as VP4 serotype 3 but this remains to be confirmed because neutralization tests were performed in only one direction. Among

  4. Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

    PubMed Central

    Couderc, T; Hogle, J; Le Blay, H; Horaud, F; Blondel, B

    1993-01-01

    Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation

  5. Electron microscopic analysis of rotavirus assembly-replication intermediates

    SciTech Connect

    Boudreaux, Crystal E.; Kelly, Deborah F.; McDonald, Sarah M.

    2015-03-15

    Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy.

  6. Effect of animal plasma proteins on intestinal damage and recovery of neonatal pigs infected with rotavirus.

    PubMed

    Corl, Benjamin A; Harrell, Robert J; Moon, Hong Kil; Phillips, Oulayvahn; Weaver, Eric M; Campbell, Joy M; Arthington, John D; Odle, Jack

    2007-12-01

    Rotaviruses infect and elicit diarrhea in neonates of most mammalian species and cause 800,000 infant deaths a year. We used neonatal piglets to study the effects of dietary animal plasma proteins on intestinal health following rotavirus infection. Plasma protein contains a diverse mixture of functional components with biological activity and improves the health of animals challenged with other diarrhea-causing pathogens. In a 2 x 2 factorial design, we compared plasma protein- and soy protein-based diets in rotavirus-infected and noninfected piglets to determine if plasma protein reduced acute rotavirus intestinal damage or improved recovery. All infected animals shed rotavirus particles in their feces. Infected, plasma protein-fed piglets maintained growth rates similar to noninfected piglets in the first 3 days of infection; however, soy protein-fed piglets experienced reduced gains. Furthermore, infected, plasma protein-fed piglets showed no clinical signs of diarrhea. Infection reduced intestinal villus height and the villus height/crypt depth ratio by Day 3 of infection; however, reductions were not attenuated with dietary plasma protein. Infected, plasma protein-fed pigs maintained greater intestinal mucosa protein and estimated total lactase activity than infected, soy protein-fed piglets. Plasma proteins contain growth factors that may aid in rate of recovery as well as virus-binding proteins that may reduce infection pressure in the intestine. These data, combined with findings from other studies using plasma proteins in animal models of diarrhea, indicate the potential for using plasma proteins to improve the health of diarrheic neonates. PMID:17475463

  7. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens.

    PubMed

    Xu, Juan; Wang, Shixia; Gan, Weihua; Zhang, Wenhong; Ju, Liwen; Huang, Zuhu; Lu, Shan

    2012-04-20

    Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines. PMID:22450314

  8. GITRL as a genetic adjuvant enhances enterovirus 71 VP1 DNA vaccine immunogenicity.

    PubMed

    Yuan, Jing; Tang, Xinyi; Yin, Kai; Tian, Jie; Rui, Ke; Ma, Jie; Mao, Chaoming; Chen, Jianguo; Lu, Liwei; Xu, Huaxi; Wang, Shengjun

    2015-05-01

    VP1 protein is the immunodominant capsid protein of enterovirus 71 (EV71) which is responsible for large outbreaks of hand, foot and mouth disease. It has been reported that glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and its ligand (GITRL) are involved in modulating both innate and adaptive immune responses. In this study, a DNA vaccine vector encoding EV71 VP1 gene and mGITRL gene (pIRES/VP1/mGITRL) was constructed. And female Balb/c mice were immunized intramuscularly with the DNA vaccine. Compared with the groups immunized with pIRES, pIRES/VP1, pIRES/mGITRL and PBS, the inoculation of pIRES/VP1/mGITRL induced a higher levels of EV71 VP1-specific antibody and specific antibody-forming cells. However, significantly higher levels of CD4(+)Th1, Th2 and CD8(+)IFN-γ(+)T cells were found in the pIRES/VP1/mGITRL group compared with control groups. Our results demonstrate that a novel DNA vaccine, expressing VP1 and mGITRL, could effectively elicit both humoral and cell-mediated immune responses against EV71 VP1 in mice. Thus, the mGITRL may be used as molecular adjuvant for EV71 DNA vaccine. PMID:25772201

  9. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

    SciTech Connect

    Xu, Juan; Wang, Shixia; Gan, Weihua; Zhang, Wenhong; Ju, Liwen; Huang, Zuhu; Lu, Shan

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

  10. Oral immunization with recombinant enterovirus 71 VP1 formulated with chitosan protects mice against lethal challenge

    PubMed Central

    2014-01-01

    Background Enterovirus 71 (EV71) is the etiologic agent of hand-foot-and-mouth disease (HFMD) in the Asia-Pacific region, Many strategies have been applied to develop EV71 vaccines but no vaccines are currently available. Mucosal immunization of the VP1, a major immunogenic capsid protein of EV71, may be an alternative way to prevent EV71 infection. Results In this study, mucosal immunogenicity and protect function of recombinant VP1 protein (rVP1) in formulation with chitosan were tested and assessed in female ICR mouse model. The results showed that the oral immunization with rVP1 induced VP1-specific IgA antibodies in intestine, feces, vagina, and the respiratory tract and serum-specific IgG and neutralization antibodies in vaccinated mice. Splenocytes from rVP1-immunized mice induced high levels of Th1 (cytokine IFN-γ), Th2 (cytokine IL-4) and Th3 (cytokine TGF-β) type immune responses after stimulation. Moreover, rVP1-immunized mother mice conferred protection (survival rate up to 30%) on neonatal mice against a lethal challenge of 103 plaque-forming units (PFU) EV71. Conclusions These data indicated that oral immunization with rVP1 in formulation with chitosan was effective in inducing broad-spectrum immune responses and might be a promising subunit vaccine candidate for preventing EV71 infection. PMID:24885121

  11. Replication of the Rotavirus Genome Requires an Active Ubiquitin-Proteasome System▿

    PubMed Central

    López, Tomás; Silva-Ayala, Daniela; López, Susana; Arias, Carlos F.

    2011-01-01

    Here we show that the ubiquitin-proteasome system is required for the efficient replication of rotavirus RRV in MA104 cells. The proteasome inhibitor MG132 decreased the yield of infectious virus under conditions where it severely reduces the synthesis of not only viral but also cellular proteins. Addition of nonessential amino acids to the cell medium restored both viral protein synthesis and cellular protein synthesis, but the production of progeny viruses was still inhibited. In medium supplemented with nonessential amino acids, we showed that MG132 does not affect rotavirus entry but inhibits the replication of the viral genome. It was also shown that it prevents the efficient incorporation into viroplasms of viral polymerase VP1 and the capsid proteins VP2 and VP6, which could explain the inhibitory effect of MG132 on genome replication and infectious virus yield. We also showed that ubiquitination is relevant for rotavirus replication since the yield of rotavirus progeny in cells carrying a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme was reduced at the restrictive temperature. In addition, overexpression of ubiquitin in MG132-treated MA104 cells partially reversed the effect of the inhibitor on virus yield. Altogether, these data suggest that the ubiquitin-proteasome (UP) system has a very complex interaction with the rotavirus life cycle, with both the ubiquitination and proteolytic activities of the system being relevant for virus replication. PMID:21900156

  12. Evidence that Equine Rhinitis A Virus VP1 Is a Target of Neutralizing Antibodies and Participates Directly in Receptor Binding

    PubMed Central

    Warner, Simone; Hartley, Carol A.; Stevenson, Rachel A.; Ficorilli, Nino; Varrasso, Annalisa; Studdert, Michael J.; Crabb, Brendan S.

    2001-01-01

    Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathione S-transferase (GST) fusion protein (GST-VP1). GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV. An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro. Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated GST-VP1 in receptor binding. Importantly, anti-GST-VP1 antibodies inhibited the binding of ERAV virions to Vero cells, suggesting that these antibodies exert their neutralizing effect by blocking viral attachment. Thus ERAV VP1, like its counterpart in FMDV, appears to be both a target of protective antibodies and involved directly in receptor binding. This study reveals the potential of recombinant VP1 molecules to serve as vaccines and diagnostic reagents for the control of ERAV infections. PMID:11533189

  13. Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV) for downstream use in virus characterization.

    PubMed

    Church, Molly E; Dela Cruz, Florante N; Kim, Kevin; Persiani, Michele; Woods, Leslie W; Pesavento, Patricia A; Woolard, Kevin D

    2016-06-01

    Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA), and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV) [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus. PMID:26955649

  14. Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV) for downstream use in virus characterization

    PubMed Central

    Church, Molly E.; Dela Cruz, Florante N.; Kim, Kevin; Persiani, Michele; Woods, Leslie W.; Pesavento, Patricia A.; Woolard, Kevin D.

    2016-01-01

    Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA), and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV) [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus. PMID:26955649

  15. RELATIVE CONCENTRATIONS OF SERUM NEUTRALIZING ANTIBODY TO VP3 AND VP7 PROTEINS IN ADULTS INFECTED WITH A HUMAN ROTAVIRUS (JOURNAL VERSION)

    EPA Science Inventory

    Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and...

  16. Pros and cons of VP1-specific maternal IgG for the protection of Enterovirus 71 infection.

    PubMed

    Kim, Young-In; Song, Jae-Hyoung; Kwon, Bo-Eun; Kim, Ha-Neul; Seo, Min-Duk; Park, KwiSung; Lee, SangWon; Yeo, Sang-Gu; Kweon, Mi-Na; Ko, Hyun-Jeong; Chang, Sun-Young

    2015-11-27

    Enterovirus 71 (EV71) causes hand, foot, and mouth diseases and can result in severe neurological disorders when it infects the central nervous system. Thus, there is a need for the development of effective vaccines against EV71 infection. Here we report that viral capsid protein 1 (VP1), one of the main capsid proteins of EV71, efficiently elicited VP1-specific immunoglobulin G (IgG) in the serum of mice immunized with recombinant VP1. The VP1-specific IgG produced in female mice was efficiently transferred to their offspring, conferring protection against EV71 infection immediately after birth. VP1-specific antibody can neutralize EV71 infection and protect host cells. VP1-specific maternal IgG in offspring was maintained for over 6 months. However, the pre-existence of VP1-specific maternal IgG interfered with the production of VP1-specific IgG antibody secreting cells by active immunization in offspring. Therefore, although our results showed the potential for VP1-specific maternal IgG protection against EV71 in neonatal mice, other strategies must be developed to overcome the hindrance of maternal IgG in active immunization. In this study, we developed an effective and feasible animal model to evaluate the protective efficacy of humoral immunity against EV71 infection using a maternal immunity concept. PMID:26529069

  17. The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells

    PubMed Central

    Díaz-Salinas, Marco A.; Romero, Pedro; Espinosa, Rafaela; Hoshino, Yasutaka; López, Susana

    2013-01-01

    Rotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface protein VP7 and the spike protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway. PMID:23175367

  18. Facilitation of Rice Stripe Virus Accumulation in the Insect Vector by Himetobi P Virus VP1

    PubMed Central

    Li, Shuo; Ge, Shangshu; Wang, Xi; Sun, Lijuan; Liu, Zewen; Zhou, Yijun

    2015-01-01

    The small brown planthopper (SBPH) is the main vector for rice stripe virus (RSV), which causes serious rice stripe disease in East Asia. To characterize the virus-vector interactions, the SBPH cDNA library was screened with RSV ribonucleoprotein (RNP) as bait using a GAL4-based yeast two-hybrid system. The interaction between RSV-RNP and the Himetobi P virus (HiPV, an insect picorna-like virus) VP1 protein was identified. The relationships between HiPV and RSV in SBPH were further investigated, and the results showed that the titer of RSV was commonly higher in single insect that exhibited more VP1 expression. After the VP1 gene was repressed by RNA silencing, the accumulation of RSV decreased significantly in the insect, whereas the virus acquisition ability of SBPH was unaffected, which suggests that HiPV VP1 potentially facilitates the accumulation of RSV in SBPH. PMID:25807055

  19. Rotavirus Vaccine

    MedlinePlus

    Why get vaccinated?Rotavirus is a virus that causes diarrhea, mostly in babies and young children. The diarrhea can be severe, and lead ... and fever are also common in babies with rotavirus.Before rotavirus vaccine, rotavirus disease was a common ...

  20. Monoclonal antibodies directed against VP7 protein of human group B rotavirus.

    PubMed

    Deng, Xiaojie; Xiong, Guomei; Cong, Wenjuan; Liu, Zhonglai; Qi, Chao; Yang, Jihong

    2014-02-01

    The aim of this study was to prepare and identify a monoclonal antibody that binds the viral proteins 7 (VP7 protein) of human group B rotavirus (GBRV) and to describe its immunologic characterization. Human group B rotavirus vp7 gene was successfully ligated into pGEX-KG vector and transformed into Escherichia coli TOP10 cells. The glutathione S-transferases (GST)-fusion protein GST-VP7 was induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and immediately purified to immunize BALB/c mice. Splenocytes were then prepared from the immunized mouse and fused with SP2/0 myeloma cell line. In the end we obtained one positive hybridoma cell line stably secreting monoclonal antibody against GST-VP7 protein by indirect enzyme-linked immunosorbent assay (ELISA) and limiting dilution. The production of the monoclonal antibody against GBRV will benefit the further study of GBRV's structures and functions and also lay a solid foundation for the research of disease prevention, clinical diagnosis, and treatment. PMID:24555935

  1. Bovine Rotavirus Nonstructural Protein 4 Produced by Lactococcus lactis Is Antigenic and Immunogenic

    PubMed Central

    Enouf, Vincent; Langella, Philippe; Commissaire, Jacqueline; Cohen, Jean; Corthier, Gérard

    2001-01-01

    Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens. PMID:11282586

  2. Display of VP1 on the Surface of Baculovirus and Its Immunogenicity against Heterologous Human Enterovirus 71 Strains in Mice

    PubMed Central

    Kiener, Tanja K.; Chow, Vincent T. K.; Kwang, Jimmy

    2011-01-01

    Background Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization. Methodology/Principal Finding In the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1) of EV71-Fuyang (subgenogroup C4) was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV) immediate early ie1 promoter. Baculovirus expressed VP1 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that VP1 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4) in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains. Conclusion Our results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns. PMID:21747954

  3. Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

  4. Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

    SciTech Connect

    Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.; Greenberg, Harry B.; Harrison, Stephen C.; Dormitzer, Philip R.

    2009-06-17

    Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

  5. Rotavirus Infections

    MedlinePlus

    Rotavirus is a virus that causes gastroenteritis. Symptoms include severe diarrhea, vomiting, fever, and dehydration. Almost all ... the U.S. are likely to be infected with rotavirus before their 5th birthday. Infections happen most often ...

  6. Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex

    PubMed Central

    Gratia, Matthieu; Sarot, Emeline; Vende, Patrice; Charpilienne, Annie; Baron, Carolina Hilma; Duarte, Mariela

    2015-01-01

    ABSTRACT Through its interaction with the 5′ translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5′-capped and 3′-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3′ GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3′ end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCE To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation

  7. The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells.

    PubMed

    Martínez-Álvarez, Laura; Piña-Vázquez, Carolina; Zarco, Wilbert; Padilla-Noriega, Luis

    2013-06-01

    A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover. PMID:23827992

  8. The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

    PubMed Central

    Martínez-Álvarez, Laura; Piña-Vázquez, Carolina; Zarco, Wilbert; Padilla-Noriega, Luis

    2013-01-01

    A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover. PMID:23827992

  9. Phylogenetic and structural analysis of merkel cell polyomavirus VP1 in Brazilian samples.

    PubMed

    Baez, Camila F; Diaz, Nuria C; Venceslau, Marianna T; Luz, Flávio B; Guimarães, Maria Angelica A M; Zalis, Mariano G; Varella, Rafael B

    2016-08-01

    Our understanding of the phylogenetic and structural characteristics of the Merkel Cell Polyomavirus (MCPyV) is increasing but still scarce, especially in samples originating from South America. In order to investigate the properties of MCPyV circulating in the continent in more detail, MCPyV Viral Protein 1 (VP1) sequences from five basal cell carcinoma (BCC) and four saliva samples from Brazilian individuals were evaluated from the phylogenetic and structural standpoint, along with all complete MCPyV VP1 sequences available at Genbank database so far. The VP1 phylogenetic analysis confirmed the previously reported pattern of geographic distribution of MCPyV genotypes and the complexity of the South-American clade. The nine Brazilian samples were equally distributed in the South-American (3 saliva samples); North American/European (2 BCC and 1 saliva sample); and in the African clades (3 BCC). The classification of mutations according to the functional regions of VP1 protein revealed a differentiated pattern for South-American sequences, with higher number of mutations on the neutralizing epitope loops and lower on the region of C-terminus, responsible for capsid formation, when compared to other continents. In conclusion, the phylogenetic analysis showed that the distribution of Brazilian VP1 sequences agrees with the ethnic composition of the country, indicating that VP1 can be successfully used for MCPyV phylogenetic studies. Finally, the structural analysis suggests that some mutations could have impact on the protein folding, membrane binding or antibody escape, and therefore they should be further studied. PMID:27173789

  10. The rotavirus surface protein VP8 modulates the gate and fence function of tight junctions in epithelial cells.

    PubMed

    Nava, Porfirio; López, Susana; Arias, Carlos F; Islas, Socorro; González-Mariscal, Lorenza

    2004-11-01

    Rotaviruses constitute a major cause of diarrhea in young mammals. Rotaviruses utilize different integrins as cell receptors, therefore upon their arrival to the intestinal lumen their integrin receptors will be hidden below the tight junction (TJ), on the basolateral membrane. Here we have studied whether the rotavirus outer capsid proteins are capable of opening the paracellular space sealed by the TJ. From the outermost layer of proteins of the rotavirus, 60 spikes formed of protein VP4 are projected. VP4 is essential for virus-cell interactions and is cleaved by trypsin into peptides VP5 and VP8. Here we found that when these peptides are added to confluent epithelial monolayers (Madin-Darby canine kidney cells), VP8 is capable of diminishing in a dose dependent and reversible manner the transepithelial electrical resistance. VP5 exerted no effect. VP8 can also inhibit the development of newly formed TJs in a Ca-switch assay. Treatment with VP8 augments the paracellular passage of non-ionic tracers, allows the diffusion of a fluorescent lipid probe and the apical surface protein GP135, from the luminal to the lateral membrane, and triggers the movement of the basolateral proteins Na+-K+-ATPase, alphanubeta3 integrin and beta1 integrin subunit, to the apical surface. VP8 generates a freeze-fracture pattern of TJs characterized by the appearance of loose end filaments, that correlates with an altered distribution of several TJ proteins. VP8 given orally to diabetic rats allows the enteral administration of insulin, thus indicating that it can be employed to modulate epithelial permeability. PMID:15494377

  11. Rotavirus Infection Induces the Unfolded Protein Response of the Cell and Controls It through the Nonstructural Protein NSP3▿

    PubMed Central

    Trujillo-Alonso, Vicenta; Maruri-Avidal, Liliana; Arias, Carlos F.; López, Susana

    2011-01-01

    The unfolded protein response (UPR) is a cellular mechanism that is triggered in order to cope with the stress caused by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). This response is initiated by the endoribonuclease inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER kinase, which increase the expression of the genes involved in the folding and degradation processes and decrease the protein input into the ER by inhibiting translation. It has been shown that viruses both induce and manipulate the UPR in order to protect the host cells from an ER stress-mediated death, thus permitting the translation of viral proteins and the efficient replication of the virus. To understand the cellular events that occur during the rotavirus replication cycle, we examined the activation of the three UPR arms following infection, using luciferase reporters driven by promoters of the ER stress-responsive genes and real-time reverse transcription-PCR to determine the levels of the stress-induced mRNAs. Our findings indicated that during rotavirus infection two of the three arms of the UPR (IRE1 and ATF6) become activated; however, these pathways are interrupted at the translational level by the general inhibition of protein synthesis caused by NSP3. This response seems to be triggered by more than one viral protein synthesized during the replication of the virus, but not by the viral double-stranded RNA (dsRNA), since cells transfected with psoralen-inactivated virions, or with naked viral dsRNA, did not induce UPR. PMID:21937647

  12. Functional Analysis of Nuclear Localization Signals in VP1-2 Homologues from All Herpesvirus Subfamilies

    PubMed Central

    Hennig, T.; Abaitua, F.

    2014-01-01

    ABSTRACT The herpes simplex virus (HSV) tegument protein VP1-2 contains an N-terminal nuclear localization signal (NLS) that is critical for capsid routing to the nuclear pore. Here we analyzed positionally conserved determinants in VP1-2 homologues from each of the alpha, beta, and gamma classes of human herpesviruses. The overall architectures of the VP1-2s were similar, with a conserved N-terminal ubiquitin-specific protease domain separated from an internal region by a linker that was quite poorly conserved in length and sequence. Within this linker region all herpesviruses contained a conserved, highly basic motif which nevertheless exhibited distinct class-specific features. The motif in HSV functioned as a monopartite NLS, while in varicella-zoster virus (VZV) activity required an adjacent basic section defining the motif as a bipartite NLS. Neither the beta- nor gammaherpesvirus VP1-2 motifs were identified by prediction algorithms, but they nevertheless functioned as efficient NLS motifs both in heterologous transfer assays and in HSV VP1-2. Furthermore, though with different efficiencies and with the exception of human herpesvirus 8 (HHV-8), these chimeric variants rescued the replication defect of an HSV mutant lacking its NLS motif. We demonstrate that the lysine at position 428 of HSV is critical for replication, with a single alanine substitution being sufficient to abrogate NLS function and virus growth. We conclude that the basic motifs of each of the VP1-2 proteins are likely to confer a similar function in capsid entry in the homologous setting and that while there is flexibility in the exact type of motif employed, specific individual residues are critical for function. IMPORTANCE To successfully infect cells, all herpesviruses, along with many other viruses, e.g., HIV, hepatitis B virus, and influenza virus, must navigate through the cytoplasmic environment and dock with nuclear pores for transport of their genomes into the nucleus. However, we

  13. Rotavirus spike protein VP5* binds alpha2beta1 integrin on the cell surface and competes with virus for cell binding and infectivity.

    PubMed

    Graham, Kate L; Takada, Yoshikazu; Coulson, Barbara S

    2006-05-01

    Rotaviruses recognize several cell-surface molecules, including the alpha2beta1 integrin, and the processes of rotavirus cell attachment and entry appear to be multifactorial. The VP5* subunit of the rotavirus spike protein VP4 contains the alpha2beta1 ligand sequence Asp-Gly-Glu at residues 308-310. Binding to alpha2beta1 and infectivity of monkey rotavirus strain RRV and human rotavirus strain Wa, but not porcine rotavirus strain CRW-8, are inhibited by peptides containing Asp-Gly-Glu. Asp308 and Gly309 are necessary for the binding of RRV VP5* (aa 248-474) to expressed I domain of the alpha2 integrin subunit. Here, the ability of RRV VP5* to bind cells and affect rotavirus-integrin interactions was determined. Interestingly, VP5* bound to cells at 4 and 37 degrees C, both via alpha2beta1 and independently of this integrin. Prior VP5* binding at 37 degrees C eliminated RRV binding to cellular alpha2beta1 and reduced RRV and Wa infectivity in MA104 cells by 38-46 %. VP5* binding did not affect the infectivity of CRW-8. VP5* binding at 4 degrees C did not affect permissive-cell infection by RRV, indicating an energy requirement for VP5* competition with virus for infectivity. Mutagenesis of VP5* Asp308 and Gly309 eliminated VP5* binding to alpha2beta1 and the VP5* inhibition of rotavirus cell binding and infection, but not alpha2beta1-independent cell binding by VP5*. These studies show for the first time that expressed VP5* binds cell-surface alpha2beta1 using Asp308 and Gly309 and inhibits the infection of homologous and heterologous rotaviruses that use alpha2beta1 as a receptor. PMID:16603530

  14. Antibodies induced with recombinant VP1 from human rhinovirus exhibit cross-neutralisation.

    PubMed

    Edlmayr, J; Niespodziana, K; Popow-Kraupp, T; Krzyzanek, V; Focke-Tejkl, M; Blaas, D; Grote, M; Valenta, R

    2011-01-01

    Human rhinoviruses (HRVs) are the major cause of the common cold and account for 30-50% of all acute respiratory illnesses. Although HRV infections are usually harmless and invade only the upper respiratory tract, several studies demonstrate that HRV is involved in the exacerbation of asthma. VP1 is one of the surface-exposed proteins of the viral capsid that is important for the binding of rhinoviruses to the corresponding receptors on human cells. Here we investigated its potential usefulness for vaccination against the common cold. We expressed VP1 proteins from two distantly related HRV strains, HRV89 and HRV14, in Escherichia coli. Mice and rabbits were immunised with the purified recombinant proteins. The induced antibodies reacted with natural VP1 and with whole virus particles as shown by immunoblotting and immunogold electron microscopy. They exhibited strong cross-neutralising activity for different HRV strains. Therefore, recombinant VP1 may be considered a candidate HRV vaccine to prevent HRV-induced asthma exacerbations. PMID:20530036

  15. Diversity of Interferon Antagonist Activities Mediated by NSP1 Proteins of Different Rotavirus Strains▿

    PubMed Central

    Arnold, Michelle M.; Patton, John T.

    2011-01-01

    Studies involving limited numbers of rotavirus (RV) strains have shown that the viral gene 5 product, NSP1, can antagonize beta interferon (IFN-β) expression by inducing the degradation of IFN-regulatory factors (IRFs) (IRF3, IRF5, and IRF7) or a component of the E3 ubiquitin ligase complex responsible for activating NF-κB (β-transducin repeat-containing protein [β-TrCP]). To gain a broader perspective of NSP1 activities, we examined various RV strains for the ability to inhibit IFN-β expression in human cells. We found that all strains encoding wild-type NSP1 impeded IFN-β expression but not always through IRF3 degradation. To identify other degradation targets involved in suppressing IFN-β expression, we used transient expression vectors to test the abilities of a diverse collection of NSP1 proteins to target IRF3, IRF5, IRF7, and β-TrCP for degradation. The results indicated that human RVs rely predominantly on the NSP1-induced degradation of IRF5 and IRF7 to suppress IFN signaling, whereas NSP1 proteins of animal RVs tended to target IRF3, IRF5, and IRF7, allowing the animal viruses a broader attack on the IFN-β signaling pathway. The results also suggested that the NSP1-induced degradation of β-TrCP is an uncommon mechanism of subverting IFN-β signaling but is one that can be shared with NSP1 proteins that induce IRF degradation. Our analysis reveals that the activities of NSP1 proteins are diverse, with no obvious correlations between degradations of pairs of target proteins. Thus, RVs have evolved functionally distinct approaches for subverting the host antiviral response, a property consistent with the immense sequence variation noted for NSP1 proteins. PMID:21177809

  16. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

    PubMed Central

    Bugli, Francesca; Caprettini, Valeria; Cacaci, Margherita; Martini, Cecilia; Paroni Sterbini, Francesco; Torelli, Riccardo; Della Longa, Stefano; Papi, Massimiliano; Palmieri, Valentina; Giardina, Bruno; Posteraro, Brunella; Sanguinetti, Maurizio; Arcovito, Alessandro

    2014-01-01

    In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes. PMID:24936129

  17. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein.

    PubMed

    Bugli, Francesca; Caprettini, Valeria; Cacaci, Margherita; Martini, Cecilia; Paroni Sterbini, Francesco; Torelli, Riccardo; Della Longa, Stefano; Papi, Massimiliano; Palmieri, Valentina; Giardina, Bruno; Posteraro, Brunella; Sanguinetti, Maurizio; Arcovito, Alessandro

    2014-01-01

    In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes. PMID:24936129

  18. Comparison of the rotavirus nonstructural protein NSP1 (NS53) from different species by sequence analysis and northern blot hybridization.

    PubMed

    Dunn, S J; Cross, T L; Greenberg, H B

    1994-08-15

    The nucleotide sequence of gene 5 encoding the rotavirus nonstructural protein NSP1 (NS53) of 6 strains (EW, EHP, RRV, I321, OSU, and Gottfried) was determined and compared to 6 previously reported strains (SA11, UK, RF, Hu803, DS-1, and Wa). The 12 rotavirus strains were derived from a total of five separate species (murine, bovine, simian, porcine, and human). Gene sizes ranged from 1564 to 1611 nucleotides in length and the deduced protein sequences were found to be 486 to 495 amino acids in length. Comparisons of NSP1 amino acid sequences showed identities ranging from 36 to 92%. This diversity was most evident between strains from different species. Phylogenetic analysis revealed a clustering of NSP1 sequences according to species origin with the exception that the human and porcine strains were included in a single grouping. Northern blot hybridizations using additional rotavirus strains from the five species confirmed the grouping found by sequence analysis. The species specificity of NSP1 is consistent with the hypothesis that NSP1 plays a role in host range restriction. PMID:8030275

  19. Rotavirus Symptoms

    MedlinePlus

    ... Rotavirus Vaccine Program American Academy of Pediatrics Symptoms Language: English Español (Spanish) Recommend on Facebook Tweet Share Compartir ... PATH's Rotavirus Vaccine Program American Academy of Pediatrics Language: English Español (Spanish) File Formats Help: How do I ...

  20. Rotavirus Treatment

    MedlinePlus

    ... Rotavirus Vaccine Program American Academy of Pediatrics Treatment Language: English Español (Spanish) Recommend on Facebook Tweet Share Compartir ... PATH's Rotavirus Vaccine Program American Academy of Pediatrics Language: English Español (Spanish) File Formats Help: How do I ...

  1. The TATA-less promoter of VP1, a plant gene controlling seed germination.

    PubMed

    Carrari, F; Frankel, N; Lijavetzky, D; Benech-Arnold, R; Sánchez, R; Iusem, N D

    2001-01-01

    Vp1 is a seed-specific gene involved in the control of dormancy and germination. We here present the complete sequence of the sorghum vp1 promoter/enhancer region highlighting its main features, especially the lack of canonical TATA and CAAT boxes and the presence of elements responsive to abscisic acid and light. The region closest to the start of transcription is highly homologous to the partial proximal sequence reported for the maize vp1 promoter. This region is interrupted by a 57-nt stretch containing 14 CT microsatellite repeats. We observed a poor overall homology to the promoter from abi3 gene, the Arabidopsis counterpart bearing a similar coding sequence. However, there exists a high degree of homology (89%) between a TATA-rich 103-bp stretch of the sorghum vp1 promoter located about 700 nt upstream of the startpoint and miniature inverted transposable elements (MITEs) interspersed within the sorghum seed-specific kafirin cluster. This sorghum MITE-like element displays considerable homology (68%) to the TATA-less promoter from the sorghum NADP-malate dehydrogenase gene and lesser similarity to the Tourist, Pilgrim and Batuta MITEs previously identified within the promoter from the maize Abp1 (auxin-binding protein) gene. PMID:11761708

  2. The Receptor-Binding Domain in the VP1u Region of Parvovirus B19

    PubMed Central

    Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos

    2016-01-01

    Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5–80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system. PMID:26927158

  3. The Receptor-Binding Domain in the VP1u Region of Parvovirus B19.

    PubMed

    Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos

    2016-03-01

    Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5-80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system. PMID:26927158

  4. Molecular Evolution of the Human Enteroviruses: Correlation of Serotype with VP1 Sequence and Application to Picornavirus Classification

    PubMed Central

    Oberste, M. Steven; Maher, Kaija; Kilpatrick, David R.; Pallansch, Mark A.

    1999-01-01

    Sixty-six human enterovirus serotypes have been identified by serum neutralization, but the molecular determinants of the serotypes are unknown. Since the picornavirus VP1 protein contains a number of neutralization domains, we hypothesized that the VP1 sequence should correspond with neutralization (serotype) and, hence, with phylogenetic lineage. To test this hypothesis and to analyze the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 sequences of the prototype strains of 47 human enterovirus serotypes and 10 antigenic variants. Our sequences, together with those available from GenBank, comprise a database of complete VP1 sequences for all 66 human enterovirus serotypes plus additional strains of seven serotypes. Phylogenetic trees constructed from complete VP1 sequences produced the same four major clusters as published trees based on partial VP2 sequences; in contrast to the VP2 trees, however, in the VP1 trees strains of the same serotype were always monophyletic. In pairwise comparisons of complete VP1 sequences, enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15 viruses should be classified as strains of the same serotype, as should coxsackie A13 and A18 viruses. Pairwise identity scores also distinguished between enteroviruses of different clusters and enteroviruses from picornaviruses of different genera. The data suggest that VP1 sequence comparisons may be valuable in enterovirus typing and in picornavirus taxonomy by assisting in the genus assignment of unclassified picornaviruses. PMID:9971773

  5. The VP1 S154D mutation of type Asia1 foot-and-mouth disease virus enhances viral replication and pathogenicity.

    PubMed

    Lian, Kaiqi; Yang, Fan; Zhu, Zixiang; Cao, Weijun; Jin, Ye; Liu, Huanan; Li, Dan; Zhang, Keshan; Guo, Jianhong; Liu, Xiangtao; Zheng, Haixue

    2016-04-01

    One of the proteins encoded by the foot-and-mouth disease virus (FMDV), the VP1 protein, a capsid protein, plays an important role in integrin receptor attachment and humoral immunity-mediated host responses. The integrin receptor recognition motif and an important antigenic epitope exist within the G-H loop, which is comprised of amino acids 134-160 of the VP1 protein. FMDV strain, Asia1/HN/CHA/06, isolated from a pig, was passaged four times in suckling mice and sequenced. Sequencing analyses showed that there was a mutation of the integrin receptor recognition motif Arg-Gly-Asp/Arg-Asp-Asp (RGD/RDD, VP1 143-145) and a VP1 154 serine/Asp (VP1 S154D) mutation in the G-H loop of the VP1 protein. The influence of the RGD/RDD mutation on Asia1 FMDV disease phenotype has been previously studied. In this study, to determine the influence of the VP1 S154D mutation on FMDV Asia1 replication and pathogenicity, two recombinant FMDVs with different residues only at the VP1 154 site were rescued by reverse genetics techniques and their infectious potential in host cells and pathogenicity in pigs were compared. Our data indicates that the VP1 S154D mutation increases the replication level of FMDV Asia1/HN/CHA/06 in BHK-21, IB-RS-2, and PK-15 cells and enhances pathogenicity in pigs. Through the transient transfection-infection assay to compare integrin receptor usage of two recombinant viruses, the result shows that the VP1 S154D mutation markedly increases the ability of type Asia1 FMDV to use the integrin receptors αυβ6 and αυβ8 from pig. This study identifies a key research target for illuminating the role of residues located at G-H loop in FMDV pathogenicity. PMID:26792712

  6. Identification of Cellular Calcium Binding Protein Calmodulin as a Regulator of Rotavirus A Infection during Comparative Proteomic Study

    PubMed Central

    Chattopadhyay, Shiladitya; Basak, Trayambak; Nayak, Mukti Kant; Bhardwaj, Gourav; Mukherjee, Anupam; Bhowmick, Rahul; Sengupta, Shantanu; Chakrabarti, Oishee; Chatterjee, Nabendu S.; Chawla-Sarkar, Mamta

    2013-01-01

    Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca2+. Ca2+-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca2+ chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca2+/CaM during viral pathogenesis. PMID:23437200

  7. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    SciTech Connect

    Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

  8. Rhinovirus-induced VP1-specific Antibodies are Group-specific and Associated With Severity of Respiratory Symptoms

    PubMed Central

    Niespodziana, Katarzyna; Cabauatan, Clarissa R.; Jackson, David J.; Gallerano, Daniela; Trujillo-Torralbo, Belen; del Rosario, Ajerico; Mallia, Patrick; Valenta, Rudolf; Johnston, Sebastian L.

    2014-01-01

    Background Rhinoviruses (RVs) are a major cause of common colds and induce exacerbations of asthma and chronic inflammatory lung diseases. Methods We expressed and purified recombinant RV coat proteins VP1-4, non-structural proteins as well as N-terminal fragments of VP1 from four RV strains (RV14, 16, 89, C) covering the three known RV groups (RV-A, RV-B and RV-C) and measured specific IgG-subclass-, IgA- and IgM-responses by ELISA in subjects with different severities of asthma or without asthma before and after experimental infection with RV16. Findings Before infection subjects showed IgG1 > IgA > IgM > IgG3 cross-reactivity with N-terminal fragments from the representative VP1 proteins of the three RV groups. Antibody levels were higher in the asthmatic group as compared to the non-asthmatic subjects. Six weeks after infection with RV16, IgG1 antibodies showed a group-specific increase towards the N-terminal VP1 fragment, but not towards other capsid and non-structural proteins, which was highest in subjects with severe upper and lower respiratory symptoms. Interpretation Our results demonstrate that increases of antibodies towards the VP1 N-terminus are group-specific and associated with severity of respiratory symptoms and suggest that it may be possible to develop serological tests for identifying causative RV groups. PMID:26137535

  9. Co-Expression of Anti-Rotavirus Proteins (Llama VHH Antibody Fragments) in Lactobacillus: Development and Functionality of Vectors Containing Two Expression Cassettes in Tandem

    PubMed Central

    Günaydın, Gökçe; Álvarez, Beatriz; Lin, Yin; Hammarström, Lennart; Marcotte, Harold

    2014-01-01

    Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3) as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11), by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules. PMID:24781086

  10. Rotavirus vaccines.

    PubMed

    Barnes, G

    1998-01-01

    Encouraging results have been reported from several large trials of tetravalent rhesus rotavirus vaccine, with efficacy of 70-80% against severe disease. A recent Venezuelan study showed similar results to trials in USA and Europe. The vaccine may soon be licensed in USA. It provides the exciting prospect of a strategy to prevent one of the world's major child killers. Other candidate vaccines are under development including human-bovine reassortants, neonatal strains, non-replicating rotaviruses, vector vaccines and other genetically engineered products. Second and third generation rotavirus vaccines are on the horizon. The need for a rotavirus vaccine is well accepted by paediatricians, but public health authorities need to be lobbied. Other issues which need to be addressed include relative importance of non-group A rotaviruses, possible administration with OPV, the influence of breast feeding, and most importantly, cost. It is essential that rotavirus vaccine is somehow made available to all of the world's children, not just those in developed countries. PMID:9553287

  11. Complete Genome Sequence of a Genotype G23P[37] Pheasant Rotavirus Strain Identified in Hungary

    PubMed Central

    Gál, János; Marton, Szilvia; Ihász, Katalin; Papp, Hajnalka; Jakab, Ferenc; Malik, Yashpal S.; Bányai, Krisztián

    2016-01-01

    We investigated the genomic properties of a rotavirus A strain isolated from diarrheic pheasant poults in Hungary in 2015. Sequence analyses revealed a shared genomic constellation (G23-P[37]-I4-R4-C4-M4-A16-N10-T4-E4-H4) and close relationship (range of nucleotide sequence similarity: VP2, 88%; VP1 and NSP4, 98%) with another pheasant rotavirus strain isolated previously in Germany. PMID:27034484

  12. Complete Genome Sequence of a Genotype G23P[37] Pheasant Rotavirus Strain Identified in Hungary.

    PubMed

    Gál, János; Marton, Szilvia; Ihász, Katalin; Papp, Hajnalka; Jakab, Ferenc; Malik, Yashpal S; Bányai, Krisztián; Farkas, Szilvia L

    2016-01-01

    We investigated the genomic properties of a rotavirus A strain isolated from diarrheic pheasant poults in Hungary in 2015. Sequence analyses revealed a shared genomic constellation (G23-P[37]-I4-R4-C4-M4-A16-N10-T4-E4-H4) and close relationship (range of nucleotide sequence similarity: VP2, 88%; VP1 and NSP4, 98%) with another pheasant rotavirus strain isolated previously in Germany. PMID:27034484

  13. Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

    PubMed Central

    Estes, M K; Mason, B B; Crawford, S; Cohen, J

    1984-01-01

    The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic. Images PMID:6322125

  14. Full genome characterization of the first G3P[24] rotavirus strain detected in humans provides evidence of interspecies reassortment and mutational saturation in the VP7 gene.

    PubMed

    Mijatovic-Rustempasic, Slavica; Roy, Sunando; Teel, Elizabeth N; Weinberg, Geoffrey A; Payne, Daniel C; Parashar, Umesh D; Bowen, Michael D

    2016-02-01

    During the 2008-2009 rotavirus season of the Centers for Disease Control and Prevention New Vaccine Surveillance Network, one case of paediatric acute gastroenteritis associated with a rotavirus G14P[24] strain was identified. This was the first detection of the genotype G14 and P[24] in humans, and the first detection of the G14P[24] combination. To gain an insight into the origins and the evolution of this strain, we determined the complete ORF sequences of all 11 genes. A majority of the genes identified were similar to the simian strain TUCH, except for the VP1 and VP7 genes that clustered only distantly with the bovine and equine strains, respectively. In addition, this strain carried AU-1-like NSP2 and NSP4 genes. Using codon-partitioning and protein-based phylogenetic approaches, we determined that the VP7 genotype of strain 2009727118 was actually G3; therefore, the proposed full genomic classification of the 2009727118 strain is G3-P[24]-I9-R2-C3-M3-A9-N3-T3-E3-H6. These findings indicate the possibility that the 2009727118 strain originated by interspecies transmission and multiple reassortment events involving human, bovine and equine rotaviruses, resulting in the introduction of some genes into the genome of simian rotaviruses. Additionally, we found evidence of mutational saturation in the third codon position of the VP7 ORF which presented an issue with homoplasy in phylogenetic analyses. PMID:26590163

  15. Full genome characterization of the first G3P[24] rotavirus strain detected in humans provides evidence of interspecies reassortment and mutational saturation in the VP7 gene

    PubMed Central

    Mijatovic-Rustempasic, Slavica; Roy, Sunando; Teel, Elizabeth N.; Weinberg, Geoffrey A.; Payne, Daniel C.; Parashar, Umesh D.; Bowen, Michael D.

    2016-01-01

    During the 2008–2009 rotavirus season of the Centers for Disease Control and Prevention New Vaccine Surveillance Network, one case of paediatric acute gastroenteritis associated with a rotavirus G14P[24] strain was identified. This was the first detection of the genotype G14 and P[24] in humans, and the first detection of the G14P[24] combination. To gain an insight into the origins and the evolution of this strain, we determined the complete ORF sequences of all 11 genes. A majority of the genes identified were similar to the simian strain TUCH, except for the VP1 and VP7 genes that clustered only distantly with the bovine and equine strains, respectively. In addition, this strain carried AU-1-like NSP2 and NSP4 genes. Using codon-partitioning and protein-based phylogenetic approaches, we determined that the VP7 genotype of strain 2009727118 was actually G3; therefore, the proposed full genomic classification of the 2009727118 strain is G3-P[24]-I9-R2-C3-M3-A9-N3-T3-E3-H6. These findings indicate the possibility that the 2009727118 strain originated by interspecies transmission and multiple reassortment events involving human, bovine and equine rotaviruses, resulting in the introduction of some genes into the genome of simian rotaviruses. Additionally, we found evidence of mutational saturation in the third codon position of the VP7 ORF which presented an issue with homoplasy in phylogenetic analyses. PMID:26590163

  16. Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation

    PubMed Central

    Sen, Adrish; Rott, Lusijah; Phan, Nguyen; Mukherjee, Gourab

    2014-01-01

    Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the

  17. Rotavirus NSP1 protein inhibits interferon-mediated STAT1 activation.

    PubMed

    Sen, Adrish; Rott, Lusijah; Phan, Nguyen; Mukherjee, Gourab; Greenberg, Harry B

    2014-01-01

    Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the

  18. Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3)*

    PubMed Central

    Dutta, Dipanjan; Chattopadhyay, Shiladitya; Bagchi, Parikshit; Halder, Umesh Chandra; Nandi, Satabdi; Mukherjee, Anupam; Kobayashi, Nobumichi; Taniguchi, Koki; Chawla-Sarkar, Mamta

    2011-01-01

    Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225–258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225–258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225–258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225–258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells. PMID:21489987

  19. Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery.

    PubMed

    Zhao, Qinghuan; Chen, Weihong; Chen, Yuanding; Zhang, Liming; Zhang, Jinping; Zhang, Zhijun

    2011-03-16

    Proteins of viral capsid may self-assemble into virus-like particles (VLPs) that can find many biomedical applications such as platform for drug delivery. In this paper, we describe preparation of VLPs by self-assembly of VP6, a rotavirus capsid protein that was chemically conjugated with doxorubicin (DOX), an anticancer drug. VP6 was first highly expressed in E. Coli, followed by purification and renaturation. DOX was then covalently attached to VP6 to form DOX-VP6 (DVP6) conjugates, which were subsequently self-assembled into VLPs under appropriate condition. Next, lactobionic acid (LA) was chemically linked to the surface of the VLPs. We demonstrated that the aforementioned nanosystem shows specific targeting to hepatoma cell line HepG2. The chemically functionalized VLPs, a kind of biological nanoparticles with excellent biocompatibility and biodegradability, can be prepared in large scale from E. Coli through our method, which may find practical applications in biomedicine. PMID:21338097

  20. Uncatalyzed assembly of spherical particles from SV40 VP1 pentamers and linear dsDNA incorporates both low and high cooperativity elements

    SciTech Connect

    Mukherjee, Santanu; Kler, Stanislav; Oppenheim, Ariella; Zlotnick, Adam

    2010-02-05

    The capsid of SV40 virion is comprised of 72 pentamers of the major capsid protein, VP1. We examined the synergism between pentamer-pentamer interaction and pentamer-DNA interaction using a minimal system of purified VP1 and a linear dsDNA 600-mer, comparing electrophoresis with electron microscopy and size exclusion chromatography. At low VP1/DNA ratios, large tubes were observed that apparently did not survive native agarose gel electrophoresis. As the VP1 concentration increased, electrophoretic migration was slower and tubes were replaced by 200 A diameter particles and excess free pentamer. At high VP1/DNA ratios, a progressively larger fraction of particles was similar to 450 A diameter virions. VP1 association with DNA is very strong compared to the concentrations in these experiments yet, paradoxically, stable complexes appear only at high ratios of VP1 to DNA. These data suggest a DNA saturation-dependent nucleation event based on non-specific pentamer-DNA interaction that controls assembly and the ultimate capsid geometry.

  1. Rotavirus antigen test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003349.htm Rotavirus antigen test To use the sharing features on this page, please enable JavaScript. The rotavirus antigen test detects rotavirus in the feces. This ...

  2. Genetic Variation of Capsid Protein VP7 in Genotype G4 Human Rotavirus Strains: Simultaneous Emergence and Spread of Different Lineages in Argentina

    PubMed Central

    Bok, Karin; Matson, David O.; Gomez, Jorge A.

    2002-01-01

    Rotavirus is the most-common cause of severe diarrhea in young children. Complete rotavirus characterization includes determination of the antigenic type of the two outer capsid proteins, VP7 and VP4, designated G and P types, respectively. During a nationwide rotavirus surveillance study, genotype G4 frequency increased during the second year. To evaluate further the mechanism of emergence and the relationship among G4 strains, the genetic diversity of VP7 capsid protein in these samples was studied in detail. Overall nucleotide sequence divergence ranged from less than 0.1 to 19.5%, a higher divergence than that observed for other rotavirus G types (0.1 to 9%). Sequences were classified into two major lineages (designated I and II) based on their nucleotide distances. The most heterogeneous lineage was further subdivided into four sublineages (designated Ia to Id). Most Argentine sequences were of sublineages Ib and Ic, which were confirmed to be independent sequence clusters by parsimony analysis. This study describes different lineages and sublineages within G4 strains and shows that Argentine strains are distantly related to reference strain ST3. The appearance of at least two G4 genotype (sub)lineages during 1998 demonstrates that the increased frequency of these strains was due to the synchronized emergence of different groups of strains. PMID:12037057

  3. Inflammatory and oxidative stress in rotavirus infection.

    PubMed

    Guerrero, Carlos A; Acosta, Orlando

    2016-05-12

    Rotaviruses are the single leading cause of life-threatening diarrhea affecting children under 5 years of age. Rotavirus entry into the host cell seems to occur by sequential interactions between virion proteins and various cell surface molecules. The entry mechanisms seem to involve the contribution of cellular molecules having binding, chaperoning and oxido-reducing activities. It appears to be that the receptor usage and tropism of rotaviruses is determined by the species, cell line and rotavirus strain. Rotaviruses have evolved functions which can antagonize the host innate immune response, whereas are able to induce endoplasmic reticulum (ER) stress, oxidative stress and inflammatory signaling. A networking between ER stress, inflammation and oxidative stress is suggested, in which release of calcium from the ER increases the generation of mitochondrial reactive oxygen species (ROS) leading to toxic accumulation of ROS within ER and mitochondria. Sustained ER stress potentially stimulates inflammatory response through unfolded protein response pathways. However, the detailed characterization of the molecular mechanisms underpinning these rotavirus-induced stressful conditions is still lacking. The signaling events triggered by host recognition of virus-associated molecular patterns offers an opportunity for the development of novel therapeutic strategies aimed at interfering with rotavirus infection. The use of N-acetylcysteine, non-steroidal anti-inflammatory drugs and PPARγ agonists to inhibit rotavirus infection opens a new way for treating the rotavirus-induced diarrhea and complementing vaccines. PMID:27175349

  4. Inflammatory and oxidative stress in rotavirus infection

    PubMed Central

    Guerrero, Carlos A; Acosta, Orlando

    2016-01-01

    Rotaviruses are the single leading cause of life-threatening diarrhea affecting children under 5 years of age. Rotavirus entry into the host cell seems to occur by sequential interactions between virion proteins and various cell surface molecules. The entry mechanisms seem to involve the contribution of cellular molecules having binding, chaperoning and oxido-reducing activities. It appears to be that the receptor usage and tropism of rotaviruses is determined by the species, cell line and rotavirus strain. Rotaviruses have evolved functions which can antagonize the host innate immune response, whereas are able to induce endoplasmic reticulum (ER) stress, oxidative stress and inflammatory signaling. A networking between ER stress, inflammation and oxidative stress is suggested, in which release of calcium from the ER increases the generation of mitochondrial reactive oxygen species (ROS) leading to toxic accumulation of ROS within ER and mitochondria. Sustained ER stress potentially stimulates inflammatory response through unfolded protein response pathways. However, the detailed characterization of the molecular mechanisms underpinning these rotavirus-induced stressful conditions is still lacking. The signaling events triggered by host recognition of virus-associated molecular patterns offers an opportunity for the development of novel therapeutic strategies aimed at interfering with rotavirus infection. The use of N-acetylcysteine, non-steroidal anti-inflammatory drugs and PPARγ agonists to inhibit rotavirus infection opens a new way for treating the rotavirus-induced diarrhea and complementing vaccines. PMID:27175349

  5. Two proline residues are essential in the calcium-binding activity of rotavirus VP7 outer capsid protein.

    PubMed Central

    Gajardo, R; Vende, P; Poncet, D; Cohen, J

    1997-01-01

    Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7. PMID:9032355

  6. Typing of human enteroviruses by partial sequencing of VP1.

    PubMed

    Oberste, M S; Maher, K; Kilpatrick, D R; Flemister, M R; Brown, B A; Pallansch, M A

    1999-05-01

    Human enteroviruses (family Picornaviridae) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3' half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an "unknown" may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related. PMID:10203472

  7. Typing of Human Enteroviruses by Partial Sequencing of VP1

    PubMed Central

    Oberste, M. Steven; Maher, Kaija; Kilpatrick, David R.; Flemister, Mary R.; Brown, Betty A.; Pallansch, Mark A.

    1999-01-01

    Human enteroviruses (family Picornaviridae) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3′ half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an “unknown” may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related. PMID:10203472

  8. Rotavirus Infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The avian rotaviruses are members of the Reoviridae family, which is characterized by virions that contain 10-12 linear double-stranded RNA (dsRNA) segments. The Reoviridae consists of 15 genera which can be placed into two recognized subfamilies based upon the presence or absence of structural “tur...

  9. Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

    PubMed Central

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J.; Ströh, Luisa; Nitsch, Roger M.; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2016-01-01

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show “recognition holes”; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  10. Broadly neutralizing human monoclonal JC polyomavirus VP1-specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy.

    PubMed

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J; Ströh, Luisa; Nitsch, Roger M; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2015-09-23

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show "recognition holes"; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  11. Identification of the two rotavirus genes determining neutralization specificities

    SciTech Connect

    Offit, P.A.; Blavat, G.

    1986-01-01

    Bovine rotavirus NCDV and simian rotavirus SA-11 represent two distinct rotavirus serotypes. A genetic approach was used to determine which viral gene segments segregated with serotype-specific viral neutralization. There were 16 reassortant rotarviruses derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant rotavirus double-stranded RNA segments was determined by gene segment mobility in polyacrylamide gels and by hybridization with radioactively labeled parental viral transcripts. The authors found that two rotavirus gene segments found previously to code for outer capsid proteins vp3 and vp7 cosegreated with virus neutralization specificities.

  12. Binding specificity of P[8] VP8* proteins of rotavirus vaccine strains with histo-blood group antigens.

    PubMed

    Sun, Xiaoman; Guo, Nijun; Li, Dandi; Jin, Miao; Zhou, Yongkang; Xie, Guangcheng; Pang, Lili; Zhang, Qing; Cao, Youde; Duan, Zhao-Jun

    2016-08-01

    RotaTeq(®) and Rotarix™ are two common human rotavirus (RV) vaccines currently on the market worldwide. Recent studies indicate histo-blood group antigens (HBGAs) may be attachment factors for RVs. The P[8] VP8* proteins of RotaTeq and Rotarix were expressed and purified, and their binding specificities were evaluated. Saliva-based binding assays showed that the VP8* proteins bound to the saliva samples of secretors irrespective of ABO blood types. However, in the oligosaccharide binding assay, the VP8* proteins displayed no specific binding to the HBGAs tested, including Lewis b and H1. The structure of RotaTeq P[8] VP8* was solved at 1.9Å. Structural comparisons revealed that the putative receptor binding site was different to that of other genotypes and displayed a novel potential binding region. These findings indicate RotaTeq and Rotarix may have better efficiency in areas with a high percentage of secretors. PMID:27209447

  13. Rotavirus gene structure and function.

    PubMed Central

    Estes, M K; Cohen, J

    1989-01-01

    Knowledge of the structure and function of the genes and proteins of the rotaviruses has expanded rapidly. Information obtained in the last 5 years has revealed unexpected and unique molecular properties of rotavirus proteins of general interest to virologists, biochemists, and cell biologists. Rotaviruses share some features of replication with reoviruses, yet antigenic and molecular properties of the outer capsid proteins, VP4 (a protein whose cleavage is required for infectivity, possibly by mediating fusion with the cell membrane) and VP7 (a glycoprotein), show more similarities with those of other viruses such as the orthomyxoviruses, paramyxoviruses, and alphaviruses. Rotavirus morphogenesis is a unique process, during which immature subviral particles bud through the membrane of the endoplasmic reticulum (ER). During this process, transiently enveloped particles form, the outer capsid proteins are assembled onto particles, and mature particles accumulate in the lumen of the ER. Two ER-specific viral glycoproteins are involved in virus maturation, and these glycoproteins have been shown to be useful models for studying protein targeting and retention in the ER and for studying mechanisms of virus budding. New ideas and approaches to understanding how each gene functions to replicate and assemble the segmented viral genome have emerged from knowledge of the primary structure of rotavirus genes and their proteins and from knowledge of the properties of domains on individual proteins. Localization of type-specific and cross-reactive neutralizing epitopes on the outer capsid proteins is becoming increasingly useful in dissecting the protective immune response, including evaluation of vaccine trials, with the practical possibility of enhancing the production of new, more effective vaccines. Finally, future analyses with recently characterized immunologic and gene probes and new animal models can be expected to provide a basic understanding of what regulates the

  14. Analysis of Human Rotaviruses from a Single Location Over an 18-Year Time Span Suggests that Protein Coadaption Influences Gene Constellations

    PubMed Central

    Zhang, Shu; McDonald, Paul W.; Thompson, Travis A.; Dennis, Allison F.; Akopov, Asmik; Kirkness, Ewen F.; Patton, John T.

    2014-01-01

    ABSTRACT Rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses that cause severe gastroenteritis in children. In addition to an error-prone genome replication mechanism, RVs can increase their genetic diversity by reassorting genes during host coinfection. Such exchanges allow RVs to acquire advantageous genes and adapt in the face of selective pressures. However, reassortment may also impose fitness costs if it unlinks genes/proteins that have accumulated compensatory, coadaptive mutations and that operate best when kept together. To better understand human RV evolutionary dynamics, we analyzed the genome sequences of 135 strains (genotype G1/G3/G4-P[8]-I1-C1-R1-A1-N1-T1-E1-H1) that were collected at a single location in Washington, DC, during the years 1974 to 1991. Intragenotypic phylogenetic trees were constructed for each viral gene using the nucleotide sequences, thereby defining novel allele level gene constellations (GCs) and illuminating putative reassortment events. The results showed that RVs with distinct GCs cocirculated during the vast majority of the collection years and that some of these GCs persisted in the community unchanged by reassortment. To investigate the influence of protein coadaptation on GC maintenance, we performed a mutual information-based analysis of the concatenated amino acid sequences and identified an extensive covariance network. Unexpectedly, amino acid covariation was highest between VP4 and VP2, which are structural components of the RV virion that are not thought to directly interact. These results suggest that GCs may be influenced by the selective constraints placed on functionally coadapted, albeit noninteracting, viral proteins. This work raises important questions about mutation-reassortment interplay and its impact on human RV evolution. IMPORTANCE Rotaviruses are devastating human pathogens that cause severe diarrhea and kill >450,000 children each year. The virus can evolve by accumulating mutations and by

  15. Polyomavirus replication in mice: influences of VP1 type and route of inoculation.

    PubMed Central

    Dubensky, T W; Freund, R; Dawe, C J; Benjamin, T L

    1991-01-01

    Patterns of polyomavirus replication and spread have been studied following inoculation of virus into newborn mice. Levels of virus replication in different tissues were followed in situ by using whole mouse section blots and immunoperoxidase staining for the major capsid protein VP1, as well as by tissue extraction and direct quantitation of viral DNA and infectious virus. Patterns of replication and spread were compared between the "high tumor" strain (inducing a high incidence of tumors) PTA and and the "low tumor" strain (inducing a low incidence of tumors) RA, following different routes of inoculation. The ability to induce a high tumor profile correlated with the ability to establish disseminated productive infection, with the kidney as a major site of amplification. Furthermore, results with PTA-RA recombinant viruses and site-directed mutants showed that the VP1 specificity of PTA, demonstrated earlier to be a critical determinant for induction of a high tumor profile (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991), is also critical for amplification in the kidney and for establishment of disseminated infections. Images PMID:1845895

  16. Natural history of human rotavirus infection.

    PubMed

    Bishop, R F

    1996-01-01

    Rotavirus infections occur repeatedly in humans from birth to old age. Most are asymptomatic or are associated with mild enteric symptoms. Infection in young children can be accompanied by severe life-threatening diarrhea, most commonly after primary infection. Annual childhood morbidity rates for severe diarrhea are similar worldwide. Mortality rates are low in developed countries but approach 1,000,000 annually in young children in developing countries. Rotaviruses can be classified into Groups A-E according to antigenic groups on VP6, the major capsid antigen. Only Group A,B and C rotaviruses have been shown to infect humans, and most human rotavirus disease is caused by Group A viruses. These are further classified into G and P types based on identification of antigens on the outer capsid proteins VP7 and VP4 respectively. Most severe infections in young children are caused by serotypes G1-4, and during the last two decades, G1 infections appear to have predominated worldwide. In general the more densely populated countries show the most complex patterns of occurrence of serotypes. Clinical rotavirus disease can be accompanied by shedding of > 10(12) rotavirus particles/gm feces. The virus is highly infectious and appears to retain infectivity over many months. In temperate climates, disease is most common during the colder months, when it is likely that rapid spread within families and communities occurs. Nosocomial infections are frequent, and rotaviruses can become endemic within obstetric hospital nurseries for the newborn. Few (if any) human rotavirus infections appear to be zoonoses, even though Group A rotaviruses are widespread in the young of all mammalian species. However infection of humans with reassortant rotavirus strains derived from human-animal sources can occur. The extent to which this contributes to new epidemic strains within particular countries (or worldwide) remains to be determined. PMID:9015109

  17. Complete Genome Characterization of Recent and Ancient Belgian Pig Group A Rotaviruses and Assessment of Their Evolutionary Relationship with Human Rotaviruses

    PubMed Central

    Heylen, Elisabeth; Zeller, Mark; Roukaerts, Inge D. M.; Desmarets, Lowiese M. B.; Van Ranst, Marc; Nauwynck, Hans J.; Matthijnssens, Jelle

    2014-01-01

    ABSTRACT Group A rotaviruses (RVAs) are an important cause of diarrhea in young pigs and children. An evolutionary relationship has been suggested to exist between pig and human RVAs. This hypothesis was further investigated by phylogenetic analysis of the complete genomes of six recent (G2P[27], G3P[6], G4P[7], G5P[7], G9P[13], and G9P[23]) and one historic (G1P[7]) Belgian pig RVA strains and of all completely characterized pig RVAs from around the globe. In contrast to the large diversity of genotypes found for the outer capsid proteins VP4 and VP7, a relatively conserved genotype constellation (I5-R1-C1-M1-A8-N1-T7-E1-H1) was found for the other 9 genes in most pig RVA strains. VP1, VP2, VP3, NSP2, NSP4, and NSP5 genes of porcine RVAs belonged to genotype 1, which is shared with human Wa-like RVAs. However, for most of these gene segments, pig strains clustered distantly from human Wa-like RVAs, indicating that viruses from both species have entered different evolutionary paths. However, VP1, VP2, and NSP3 genes of some archival human strains were moderately related to pig strains. Phylogenetic analysis of the VP6, NSP1, and NSP3 genes, as well as amino acid analysis of the antigenic regions of VP7, further confirmed this evolutionary segregation. The present results also indicate that the species barrier is less strict for pig P[6] strains but that chances for successful spread of these strains in the human population are hampered by the better adaptation of pig RVAs to pig enterocytes. However, future surveillance of pig and human RVA strains is warranted. IMPORTANCE Rotaviruses are an important cause of diarrhea in many species, including pigs and humans. Our understanding of the evolutionary relationship between rotaviruses from both species is limited by the lack of genomic data on pig strains. In this study, recent and ancient Belgian pig rotavirus isolates were sequenced, and their evolutionary relationship with human Wa-like strains was investigated

  18. Mimicking Retention and Transport of Rotavirus and Adenovirus in Sand Media Using DNA-labeled, Protein-coated Silica Nanoparticles

    NASA Astrophysics Data System (ADS)

    Pang, Liping; Farkas, Kata; Bennett, Grant; Varsani, Arvind; Easingwood, Richard; Tilley, Richard; Nowostawska, Urszula; Lin, Susan

    2014-05-01

    Rotavirus (RoV) and adenovirus (AdV) are important viral pathogens for the risk analysis of drinking water. Despite this, little is known about their retention and transport behaviors in porous media (e.g. sand filtered used for water treatment and groundwater aquifers due to a lack of representative surrogates. In this study, we developed RoV and AdV surrogates by covalently coating 70-nm sized silica nanoparticles with specific proteins and a DNA marker for sensitive detection. Filtration experiments using beach sand columns demonstrated the similarity of the surrogates' concentrations, attachment, and filtration efficiencies to the target viruses. The surrogates showed the same magnitude of concentration reduction as the viruses. Conversely, MS2 phage (a traditional virus model) over predicted concentrations of AdV and RoV by 1- and 2-orders of magnitude, respectively. The surrogates remained stable in size, surface charge and DNA concentration for at least one year. They can be easily and rapidly detected at concentrations down to one particle per PCR reaction and are readily detectable in natural waters and even in effluent. With up-scaling validation in pilot trials, the surrogates can be a useful cost-effective new tool for studying virus retention and transport in porous media, e.g. for assessing filter efficiency in water and wastewater treatment, tracking virus migration in groundwater after effluent land disposal, and establishing safe setback distances for groundwater protection.

  19. Mimicking filtration and transport of rotavirus and adenovirus in sand media using DNA-labeled, protein-coated silica nanoparticles.

    PubMed

    Pang, Liping; Farkas, Kata; Bennett, Grant; Varsani, Arvind; Easingwood, Richard; Tilley, Richard; Nowostawska, Urszula; Lin, Susan

    2014-10-01

    Rotavirus (RoV) and adenovirus (AdV) are important viral pathogens for the risk analysis of drinking water. Despite this, little is known about their retention and transport behaviors in porous media due to a lack of representative surrogates. We developed RoV and AdV surrogates by covalently coupling 70-nm sized silica nanoparticles with specific proteins and a DNA marker for sensitive detection. Filtration experiments using beach sand columns demonstrated the similarity of the surrogates' concentrations, filtration efficiencies and attachment kinetics to those of the target viruses. The surrogates showed the same magnitude of concentration reduction as the viruses. Conversely, MS2 phage (a traditional virus model) over-predicted concentrations of AdV and RoV by 1- and 2-orders of magnitude respectively. The surrogates remained stable in size, surface charge and DNA concentration for at least one year. They can be easily and rapidly detected down to a single particle. Preliminary tests suggest that they were readily detectable in a number of environmental waters and treated effluent. With up-scaling validation in pilot trials, the surrogates developed here could be a cost-effective new tool for studying virus retention and transport in porous media. Examples include assessing filter efficacy in water and wastewater treatment, tracking virus migration in groundwater after effluent land disposal, and establishing safe setback distances for groundwater protection. PMID:24954130

  20. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

    PubMed Central

    Sircar, Subhankar; Saurabh, Sharad; Gulati, Baldev R.; Singh, Neeraj; Singh, Arvind Kumar; Joshi, Vinay G.; Banyai, Krisztian; Dhama, Kuldeep

    2016-01-01

    We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings. PMID:27391106

  1. Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

    PubMed

    Yuki, Yoshikazu; Kurokawa, Shiho; Kozuka-Hata, Hiroko; Tokuhara, Daisuke; Mejima, Mio; Kuroda, Masaharu; Oyama, Masaaki; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Kiyono, Hiroshi

    2016-04-01

    To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application. PMID:26851506

  2. Mutations in the GM1 Binding Site of Simian Virus 40 VP1 Alter Receptor Usage and Cell Tropism

    PubMed Central

    Magaldi, Thomas G.; Buch, Michael H. C.; Murata, Haruhiko; Erickson, Kimberly D.; Neu, Ursula; Garcea, Robert L.; Peden, Keith; Stehle, Thilo

    2012-01-01

    Polyomaviruses are nonenveloped viruses with capsids composed primarily of 72 pentamers of the viral VP1 protein, which forms the outer shell of the capsid and binds to cell surface oligosaccharide receptors. Highly conserved VP1 proteins from closely related polyomaviruses recognize different oligosaccharides. To determine whether amino acid changes restricted to the oligosaccharide binding site are sufficient to determine receptor specificity and how changes in receptor usage affect tropism, we studied the primate polyomavirus simian virus 40 (SV40), which uses the ganglioside GM1 as a receptor that mediates cell binding and entry. Here, we used two sequential genetic screens to isolate and characterize viable SV40 mutants with mutations in the VP1 GM1 binding site. Two of these mutants were completely resistant to GM1 neutralization, were no longer stimulated by incorporation of GM1 into cell membranes, and were unable to bind to GM1 on the cell surface. In addition, these mutant viruses displayed an infection defect in monkey cells with high levels of cell surface GM1. Interestingly, one mutant infected cells with low cell surface GM1 more efficiently than wild-type virus, apparently by utilizing a different ganglioside receptor. Our results indicate that a small number of mutations in the GM1 binding site are sufficient to alter ganglioside usage and change tropism, and they suggest that VP1 divergence is driven primarily by a requirement to accommodate specific receptors. In addition, our results suggest that GM1 binding is required for vacuole formation in permissive monkey CV-1 cells. Further study of these mutants will provide new insight into polyomavirus entry, pathogenesis, and evolution. PMID:22514351

  3. Molecular and phylogenetic analysis of the porcine kobuvirus VP1 region using infected pigs from Sichuan Province, China

    PubMed Central

    2013-01-01

    Background Porcine kobuvirus (PKoV) is a member of the Kobuvirus genus within the Picornaviridae family. PKoV is distributed worldwide with high prevalence in clinically healthy pigs and those with diarrhea. Methods Fecal and intestinal samples (n = 163) from pig farms in Sichuan Province, China were obtained to determine the presence of PKoV using reverse transcription polymerase chain reaction assays. Specific primers were used for the amplification of the gene encoding the PKoV VP1 protein sequence. Sequence and phylogenetic analyses were conducted to clarify evolutionary relationships with other PKoV strains. Results Approximately 53% (87/163) of pigs tested positive for PKoV. PKoV was widespread in asymptomatic pigs and those with diarrhea. A high prevalence of PKoV was observed in pigs younger than 4 weeks and in pigs with diarrhea. Phylogenetic analysis of 36 PKoV VP1 protein sequences showed that Sichuan PKoV strains formed four distinct clusters. Two pigs with diarrhea were found to be co-infected with multiple PKoV strains. Sequence and phylogenetic analyses revealed diversity within the same host and between different hosts. Significant recombination breakpoints were observed between the CHN/SC/31-A1 and CHN/SC/31-A3 strains in the VP1 region, which were isolated from the same sample. Conclusion PKoV was endemic in Sichuan Province regardless of whether pigs were healthy or suffering from diarrhea. Based on our statistical analyses, we suggest that PKoV was the likely causative agent of high-mortality diarrhea in China from 2010. For the first time, we provide evidence for the co-existence of multiple PKoV strains in one pig, and possible recombination events in the VP1 region. Our findings provide further insights into the molecular properties of PKoV, along with its epidemiology. PMID:24025093

  4. Overlap of Viviparous1 (VP1) and abscisic acid response elements in the Em promoter: G-box elements are sufficient but not necessary for VP1 transactivation.

    PubMed Central

    Vasil, V; Marcotte, W R; Rosenkrans, L; Cocciolone, S M; Vasil, I K; Quatrano, R S; McCarty, D R

    1995-01-01

    The relationship between promoter sequences that mediate Viviparous1 (VP1) transactivation and regulation by abscisic acid (ABA) in the wheat Em promoter was investigated using deletion analysis and directed mutagenesis. The Em1a G-box is strongly coupled to VP1 transactivation as well as to ABA regulation; however, the Em promoter includes additional components that can support VP1 transactivation without ABA responsiveness or synergism. Oligonucleotide tetramers of several G-box sequences, including Em1a, Em1b, and the dyad G-box element from the UV light-regulated parsley chalcone synthase gene, were sufficient to confer VP1 transactivation and the synergistic interaction with ABA to the -45 cauliflower mosaic virus 35S core promoter. These data suggest that VP1 can activate transcription through at least two classes of cis-acting sequences, including the G-box elements and the Sph regulatory motif found in the C1 promoter. The contrasting roles of these motifs in the Em and C1 promoters suggest a basis for the differential regulation of the corresponding genes by VP1. PMID:8589631

  5. A Medicago truncatula H+-pyrophosphatase gene, MtVP1, improves sucrose accumulation and anthocyanin biosynthesis in potato (Solanum tuberosum L.).

    PubMed

    Wang, J W; Wang, H Q; Xiang, W W; Chai, T Y

    2014-01-01

    We recently cloned MtVP1, a type I vacuolar-type H(+)-translocating inorganic pyrophosphatase from Medicago truncatula. In the present study, we investigated the cellular location and the function of this H(+)-PPase in Arabidopsis and potato (Solanum tuberosum L.). An MtVP1::enhanced green fluorescent protein fusion was constructed, which localized to the plasma membrane of onion epidermal cells. Transgenic Arabidopsis thaliana overexpressing MtVP1 had more robust root systems and redder shoots than wild-type (WT) plants under conditions of cold stress. Furthermore, overexpression of MtVP1 in potato accelerated the formation and growth of vegetative organs. The tuber buds and stem base of transgenic potatoes became redder than those of WT plants, but flowering was delayed by approximately half a month. Interestingly, anthocyanin biosynthesis was promoted in transgenic Arabidopsis seedlings and potato tuber buds. The sucrose concentration of transgenic potato tubers and tuber buds was enhanced compared with that of WT plants. Furthermore, sucrose concentration in tubers was higher than that in tuber buds. Although there was no direct evidence to support Fuglsang's hypothetical model regarding the effects of H(+)-PPase on sucrose phloem loading, we speculated that sucrose concentration was increased in tuber buds owing to the increased concentration in tubers. Therefore, overexpressed MtVP1 enhanced sucrose accumulation of source organs, which might enhance sucrose transport to sink organs, thus affecting anthocyanin biosynthesis. PMID:24854441

  6. Substantial Receptor-induced Structural Rearrangement of Rotavirus VP8*: Potential Implications for Cross-Species Infection.

    PubMed

    Yu, Xing; Mishra, Rahul; Holloway, Gavan; von Itzstein, Mark; Coulson, Barbara S; Blanchard, Helen

    2015-10-12

    Rotavirus-cell binding is the essential first step in rotavirus infection. This binding is a major determinant of rotavirus tropism, as host cell invasion is necessary to initiate infection. Initial rotavirus-cell interactions are mediated by carbohydrate-recognizing domain VP8* of the rotavirus capsid spike protein VP4. Here, we report the first observation of significant structural rearrangement of VP8* from human and animal rotavirus strains upon glycan receptor binding. The structural adaptability of rotavirus VP8* delivers important insights into how human and animal rotaviruses utilize the wider range of cellular glycans identified as VP8* binding partners. Furthermore, our studies on rotaviruses with atypical genetic makeup provide information expected to be critical for understanding the mechanisms of animal rotavirus gene emergence in humans and support implementation of epidemiologic surveillance of animal reservoirs as well as future vaccination schemes. PMID:26250751

  7. The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model

    PubMed Central

    Kataoka, Chikako; Suzuki, Tadaki; Kotani, Osamu; Iwata-Yoshikawa, Naoko; Nagata, Noriyo; Ami, Yasushi; Wakita, Takaji; Nishimura, Yorihiro; Shimizu, Hiroyuki

    2015-01-01

    Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral

  8. Rotavirus vaccine: a review.

    PubMed

    Kumar, Goel Manish; Arun, Kumar; Bilas, Jain Ram; Ruchi, Jain; Pardeep, Khanna; Pradeep, Siwach

    2012-12-01

    Worldwide, large proportion i.e., 37% of deaths due to diarrhea in young children is attributed to rotavirus. A monovalent P1A[8] G1 vaccine and a pentavalent bovine-human reassortant vaccine human rotavirus vaccine had shown good clinical efficacy without any increase in intussusception among vaccine recipients. WHO recommends that the first dose of rotavirus vaccine should be administered to infants up to age of 6-15 weeks irrespective of the prior history of rotavirus infection and the maximum age for administering the last dose of the vaccine should be 32 weeks. Booster doses are not recommended. The current update reviews the issues related to rotavirus vaccines and their usages like milestones in the development of rotavirus vaccines, concerns regarding their efficacy and cost-effectiveness, immunity after natural infection, potential for changes in virus strains, current recommendations, post marketing surveillance, and future challenges and scope for further research regarding rotavirus vaccines. PMID:25145068

  9. H1PVAT is a novel and potent early-stage inhibitor of poliovirus replication that targets VP1.

    PubMed

    Tijsma, Aloys; Thibaut, Hendrik Jan; Spieser, Stéphane A H; De Palma, Armando; Koukni, Mohamed; Rhoden, Eric; Oberste, Steve; Pürstinger, Gerhard; Volny-Luraghi, Antonia; Martin, Javier; Marchand, Arnaud; Chaltin, Patrick; Neyts, Johan; Leyssen, Pieter

    2014-10-01

    A novel small molecule, H1PVAT, was identified as a potent and selective inhibitor of the in vitro replication of all three poliovirus serotypes, whereas no activity was observed against other enteroviruses. Time-of-drug-addition studies revealed that the compound interfered with an early stage of virus replication. Four independently-selected H1PVAT-resistant virus variants uniformly carried the single amino acid substitution I194F in the VP1 capsid protein. Poliovirus type 1 strain Sabin, reverse-engineered to contain this substitution, proved to be completely insensitive to the antiviral effect of H1PVAT and was cross-resistant to the capsid-binding inhibitors V-073 and pirodavir. The VP1 I194F mutant had a smaller plaque phenotype than wild-type virus, and the amino acid substitution rendered the virus more susceptible to heat inactivation. Both for the wild-type and VP1 I194F mutant virus, the presence of H1PVAT increased the temperature at which the virus was inactivated, providing evidence that the compound interacts with the viral capsid, and that capsid stabilization and antiviral activity are not necessarily correlated. Molecular modeling suggested that H1PVAT binds with high affinity in the pocket underneath the floor of the canyon that is involved in receptor binding. Introduction of the I194F substitution in the model of VP1 induced a slight concerted rearrangement of the core β-barrel in this pocket, which disfavors binding of the compound. Taken together, the compound scaffold, to which H1PVAT belongs, may represent another promising class of poliovirus capsid-binding inhibitors next to V-073 and pirodavir. Potent antivirals against poliovirus will be essential in the poliovirus eradication end-game. PMID:25043639

  10. Rotavirus-associated diarrhoea in foals in Greece.

    PubMed

    Ntafis, V; Fragkiadaki, Eir; Xylouri, E; Omirou, A; Lavazza, A; Martella, V

    2010-08-26

    Severe outbreaks of diarrhoeic syndrome occurred in young foals at the same stud farm during two consecutive breeding periods namely spring 2006 and 2007. Rotavirus-like particles were detected by electron microscopy in the faeces of the affected foals and group A rotavirus infection was confirmed by Reverse-Transcription (RT)-PCR with selected sets of rotavirus-specific primers. Sequence analysis of the genes encoding the outer capsid rotavirus proteins VP7 and VP4 enabled classification of the viruses as G3AP[12] and revealed that the viruses were highly similar to recently reported equine rotavirus strains circulating in Europe. All Greek equine rotavirus isolates were genetically identical, suggesting persistence of the same viral strain in the stud farm, over the two consecutive foaling periods. PMID:20197218

  11. Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1 G-H loop as a new tool for studying FMDV pathogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, we generated a recombinant foot-and-mouth disease virus (FMDV) particle derived from A24 Cruzeiro with a FLAG tag (DYKDDDDK) substitution in the hypervariable antigenic site of the G-H loop of the VP1 capsid protein in an effort to expand the immunogenicity of the virus particle and t...

  12. Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus-like particles produced in Escherichia coli.

    PubMed

    Saccardo, Paolo; Rodríguez-Carmona, Escarlata; Villaverde, Antonio; Ferrer-Miralles, Neus

    2014-01-01

    Protein nanoparticles such as virus-like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self-assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK(-) E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. PMID:24574306

  13. A Developmental Switch of Gene Expression in the Barley Seed Mediated by HvVP1 (Viviparous-1) and HvGAMYB Interactions.

    PubMed

    Abraham, Zamira; Iglesias-Fernández, Raquel; Martínez, Manuel; Rubio-Somoza, Ignacio; Díaz, Isabel; Carbonero, Pilar; Vicente-Carbajosa, Jesús

    2016-04-01

    The accumulation of storage compounds in the starchy endosperm of developing cereal seeds is highly regulated at the transcriptional level. These compounds, mainly starch and proteins, are hydrolyzed upon germination to allow seedling growth. The transcription factor HvGAMYB is a master activator both in the maturation phase of seed development and upon germination, acting in combination with other transcription factors. However, the precise mechanism controlling the switch from maturation to germination programs remains unclear. We report here the identification and molecular characterization of Hordeum vulgare VIVIPAROUS1 (HvVP1), orthologous to ABA-INSENSITIVE3 from Arabidopsis thaliana HvVP1 transcripts accumulate in the endosperm and the embryo of developing seeds at early stages and in the embryo and aleurone of germinating seeds up to 24 h of imbibition. In transient expression assays, HvVP1 controls the activation of Hor2 and Amy6.4 promoters exerted by HvGAMYB. HvVP1 interacts with HvGAMYB in Saccharomyces cerevisiae and in the plant nuclei, hindering its interaction with other transcription factors involved in seed gene expression programs, like BPBF. Similarly, this interaction leads to a decrease in the DNA binding of HvGAMYB and the Barley Prolamine-Box binding Factor (BPBF) to their target sequences. Our results indicate that the HvVP1 expression pattern controls the full Hor2 expression activated by GAMYB and BPBF in the developing endosperm and the Amy6.4 activation in postgerminative reserve mobilization mediated by GAMYB. All these data demonstrate the participation of HvVP1 in antagonistic gene expression programs and support its central role as a gene expression switch during seed maturation and germination. PMID:26858366

  14. The Critical Role Of VP1 In Forming The Necessary Cavities For Receptor-mediated Entry Of FMDV To The Host Cell.

    PubMed

    Ashkani, Jahanshah; Rees, D J G

    2016-01-01

    The antigenic inconsistency of the foot-and-mouth disease virus (FMDV) is very broad, such that a vaccine made from one isolate will not offer protection against infection with other isolates from the same serotype. Viral particles (VPs) or surface exposed capsid proteins, VP1-VP3, of FMDV determine both the antigenicity of the virus and its receptor-mediated entry into the host cell. Therefore, modifications of these structural proteins may alter the properties of the virus. Here we show putative cavities on the FMDV-SAT1 (FMDV Southern African Territories1) capsid as possible binding sites for the receptor-mediated viral entry into the host cell. We identified three possible cavities on the FMDV capsid surface, from which the largest one (C2) is shaped in the contact regions of VP1-VP3. Our results demonstrate the significance of VP1, in the formation of FMDV-SAT1 surface cavities, which is the main component in all the identified cavities. Our findings can have profound implications in the protein engineering of FMDV in the contact region of VP1-VP3 found to be embedded in several cavities. Such information is of great significance in the context of vaccine design, as it provides the ground for future improvement of synthetic vaccines to control FMD caused by FMDV-SAT1 serotypes. PMID:27249937

  15. The Critical Role Of VP1 In Forming The Necessary Cavities For Receptor-mediated Entry Of FMDV To The Host Cell

    PubMed Central

    Ashkani, Jahanshah; Rees, D. J. G.

    2016-01-01

    The antigenic inconsistency of the foot-and-mouth disease virus (FMDV) is very broad, such that a vaccine made from one isolate will not offer protection against infection with other isolates from the same serotype. Viral particles (VPs) or surface exposed capsid proteins, VP1–VP3, of FMDV determine both the antigenicity of the virus and its receptor-mediated entry into the host cell. Therefore, modifications of these structural proteins may alter the properties of the virus. Here we show putative cavities on the FMDV-SAT1 (FMDV Southern African Territories1) capsid as possible binding sites for the receptor-mediated viral entry into the host cell. We identified three possible cavities on the FMDV capsid surface, from which the largest one (C2) is shaped in the contact regions of VP1–VP3. Our results demonstrate the significance of VP1, in the formation of FMDV-SAT1 surface cavities, which is the main component in all the identified cavities. Our findings can have profound implications in the protein engineering of FMDV in the contact region of VP1–VP3 found to be embedded in several cavities. Such information is of great significance in the context of vaccine design, as it provides the ground for future improvement of synthetic vaccines to control FMD caused by FMDV-SAT1 serotypes. PMID:27249937

  16. Some serum acute phase proteins and immunoglobulins concentrations in calves with rotavirus, coronavirus, E. coli F5 and Eimeria species

    PubMed Central

    Balikci, E; Al, M

    2014-01-01

    The purpose of this study was to evaluate the changes in the serum concentrations of haptoglobin (Hp), serum amyloid A (SAA) and IgG, IgA in calves with diarrhea caused by rotavirus, coronavirus, Escherichia coli F5 and Eimeria species. The experiment was carried out on 40 diarrhoeic and 10 non-diarrhoeic calves (group C). A total of 13 calves were infected with rotavirus or coronavirus (group V), 12 calves with E. coli F5 (group B) and 15 calves with Eimeria species (group P). SAA and Hp levels of calves in groups V, B and P were statistically higher than group C (P<0.05). SAA and Hp levels of the group B and group P were significantly higher than the group V (P<0.05). SAA and Hp levels in group B were not significantly higher than the group P. The levels of IgG and IgA were found to be lower in groups B and V compared to other groups. There was a negative correlation between immunoglobulins and the levels of serum Hp and SAA in groups B and V (r=-0.315 and r=-0.369, respectively, P<0.05). Serum SAA, Hp, IgA and IgG levels could be useful for the diagnosis and differential diagnosis of diarrhea caused by rotavirus, coronavirus, E. coli F5 and Eimeria species. PMID:27175138

  17. [Rotavirus and other viruses of diarrhea].

    PubMed

    Bajolet, O; Chippaux-Hyppolite, C

    1998-01-01

    Rotaviruses represent 80% of recognized viral etiologies and 140 million cases of diarrhea per year. They strike young children with similar frequency throughout the world, but the mortality rate is high in developing countries only, with some 870,000 deaths per year (WHO, 1997). Rotaviruses belong to the family of Reoviridae; they are segmented bicatenary RNA viruses, which explains their genetic variability, the presence of mixed infections, the establishment for some time already of a molecular epidemiology by electrophore types. The viruses are "naked" and thus resistant to the outside environment; their massive elimination, 10(8) to 10(10)viral particles per gram of faeces, begins with the first day of diarrhea. They are found in used water and can also be concentrated by shellfish; the environment thus constitutes a notable reservoir for the virus. Oral-faecal transmission is facilitated by deficient sanitary conditions. The 11 fragments of the genome each codify for 1 viral protein; 2 surface proteins, VP4 and VP7, bring about the formation of neutralizing antibodies, which are important for the protection and determination of different serotypes. A non structural protein--NSP4--would seem to intervene in the cytopathogenic effect and may act as a veritable viral enterotoxine. Numerous animal species are infected by rotaviruses which are district from the human ones. The pathology as it affects animals is of economic importance and of interest as an experimental and vaccinal model. Between human and animal rotaviruses there can be genetic rematchings and the VP6 protein is an antigen common to the group. The description of the other viruses responsible for diarrhea has benefited from widespread use of electronic microscopes from the very first years of study of rotaviruses. These other viruses belong to 6 different types: adenovirus, calcivirus, astrovirus, Norwalk agent and related viruses, coronavirus, enterovirus. They therefore have a structural and

  18. The outer capsid protein VP4 of equine rotavirus strain H-2 represents a unique VP4 type by amino acid sequence analysis.

    PubMed

    Hardy, M E; Gorziglia, M; Woode, G N

    1993-03-01

    The nucleotide and deduced amino acid sequence of G serotype 3 equine rotavirus strain H-2 was determined. A predicted 776-amino-acid H-2 VP4 shows less than or equal to 85.3% identity to other rotavirus VP4 types sequenced to date and thus represents a new P serotype. A PCR-generated probe derived from a cDNA clone of H-2 gene 4 hybridized to gene 4 of several tissue-culture-adapted equine rotavirus isolates, demonstrating that the gene 4 allele present in the H-2 strain is present in the equine rotavirus population. PMID:8382410

  19. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    SciTech Connect

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. )

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  20. The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication

    PubMed Central

    Zhang, Jie; Guo, Hong; Chen, Qingxiu; Zhang, Fuxian; Fang, Qin

    2016-01-01

    Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1–471) of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection. PMID:26871941

  1. Molecular and antigenic characterization of porcine rotavirus YM, a possible new rotavirus serotype.

    PubMed Central

    Ruiz, A M; López, I V; López, S; Espejo, R T; Arias, C F

    1988-01-01

    In 1983, we isolated a porcine rotavirus (strain YM) that was prevalent in several regions of Mexico, as judged by the frequency of its characteristic electropherotype. By a focus reduction neutralization test, rotavirus YM was clearly distinguished from prototype rotavirus strains belonging to serotypes 1 (Wa), 2 (S2), 3 (SA11), 4 (ST3), 5 (OSU), and 6 (NCDV). Minor, one-way cross-neutralization (1 to 5%) was observed when antisera to the various rotavirus strains were incubated with rotavirus YM. In addition, the YM virus was not neutralized by neutralizing monoclonal antibodies with specificity to serotypes 1, 2, 3, and 5. The subgroup of the virus was determined to be I by enzyme-linked immunosorbent assay. To characterize the serotype-specific glycoprotein of the virus at the molecular level, we cloned and sequenced the gene coding for VP7. Comparison of the deduced amino acid sequence with reported homologous sequences from human and animal rotavirus strains belonging to six different serotypes further supported the distinct immunological identity of the YM VP7 protein. Images PMID:2845146

  2. Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein.

    PubMed

    Kearney, Karen; Chen, Dayue; Taraporewala, Zenobia F; Vende, Patrice; Hoshino, Yasutaka; Tortorici, Maria Alejandra; Barro, Mario; Patton, John T

    2004-10-13

    Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3'-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0-5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3'CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines. PMID:15372078

  3. PCR-based approach to distinguish group A human rotavirus genotype 1 vs. genotype 2 genes.

    PubMed

    McKell, Allison O; Nichols, Joshua C; McDonald, Sarah M

    2013-12-01

    Group A rotaviruses (RVs) are eleven-segmented, double-stranded RNA viruses and important causes of severe diarrhea in children. A full-genome classification system is readily used to describe the genetic makeup of individual RV strains. In this system, each viral gene is assigned a specific genotype based upon its nucleotide sequence and established percent identity cut-off values. However, a faster and more cost-effective approach to determine RV gene genotypes is to utilize specific oligonucleotide primer sets in RT-PCR/PCR. Such primer sets and PCR-based genotyping methods have already been developed for the VP7-, VP6-, VP4- and NSP4-coding gene segments. In this study, primers were developed for the remaining seven RV gene segments, which encode proteins VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5/6. Specifically, primers were designed to distinguish the two most common human RV genotypes (1 vs. 2) for these genes and were validated on several cell culture-adapted human and animal RV strains, as well as on human RVs from clinical fecal specimens. As such, primer sets now exist for all eleven genes of common human RVs, allowing for the identification of reassortant strains with mixed constellations of both genotype 1 and 2 genes using a rapid and economical RT-PCR/PCR method. PMID:24012969

  4. Interaction of rotavirus particles with liposomes.

    PubMed

    Nandi, P; Charpilienne, A; Cohen, J

    1992-06-01

    We have studied the interactions of purified viral particles with liposomes as a model to understand the mechanism of entry of rotavirus into the cell. Liposomes, made from pure as well as mixed lipids, that contained encapsulated self-quenching concentrations of the fluorophore carboxyfluorescein (CF) were used. Rotavirus-liposome interactions were studied from the fluorescence dequenching of CF resulting from its release to the bulk solution. Purified infectious double-shelled virus particles induced a concentration- and temperature-dependent release of CF. The rate and extent of CF release was maximum between pH 7.3 and 7.6. The removal of outer structural proteins VP4 and VP7 from virus, which results in the formation of single-shelled particles, prevented virus interaction with liposomes. Rotavirus particles with uncleaved VP4 did not interact with liposomes, but treatment in situ of these particles with trypsin restored the interaction with the liposomes and resulted in CF dequenching. Our data support the view that rotavirus enters the cell through direct penetration of the plasma membrane. In contrast, adenovirus, the only other nonenveloped virus studied by this method, shows the optimum rate of marker release from liposomes at around pH 6 (R. Blumenthal, P. S. Seth, M. C. Willingham, and I. Pastan, Biochemistry 25:2231-2237, 1986). The interaction between rotavirus and liposomes is sensitive to specific divalent metal ions, unlike the adenovirus-liposome interaction, which is independent of them. PMID:1316453

  5. Neutralizing linear epitopes of B19 parvovirus cluster in the VP1 unique and VP1-VP2 junction regions.

    PubMed Central

    Saikawa, T; Anderson, S; Momoeda, M; Kajigaya, S; Young, N S

    1993-01-01

    Presentation of linear epitopes of the B19 parvovirus capsid proteins as peptides might be a useful vaccine strategy. We produced overlapping fusion proteins to span the viral capsid sequence, inoculated rabbits, and determined whether the resulting antisera contained antibodies that neutralized the ability of the virus to infect human erythroid progenitor cells. Antibodies that bound to virus in an enzyme-linked immunosorbent assay were present in antisera raised against 10 of 11 peptides; strongest activity was found for antisera against the carboxyl-terminal half of the major capsid protein. However, strong neutralizing activity was elicited in animals immunized with peptides from the amino-terminal portion of the unique region of the minor capsid protein and peptides containing the sequence of the junction region between the minor and major capsid proteins. The development of neutralizing activity in animals was elicited most rapidly with the fusion peptide from the first quarter of the unique region. A 20-amino-acid region of the unique region of the minor capsid protein was shown to contain a neutralizing epitope. Multiple antigenic peptides, based on the sequence of the unique region and produced by covalent linkage through a polylysine backbone, elicited strong neutralizing antibody responses. Synthetic peptides and fusion proteins containing small regions of the unique portion of the minor capsid protein might be useful as immunogens in a human vaccine against B19 parvovirus. Images PMID:7684458

  6. Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain).

    PubMed

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Meng, Tao; Chow, Vincent Tk; Chua, Kaw Bing

    2016-01-01

    Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1(K98E,E145A,L169F) with three substitutions in the VP1 protein-K98E, E145A and L169F-productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection. PMID:27329847

  7. Rotavirus (For Parents)

    MedlinePlus

    ... spring months. It is particularly a problem in childcare centers and children's hospitals because rotavirus infection is ... who care for kids, including health care and childcare workers, also can spread the virus, especially if ...

  8. Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)

    PubMed Central

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Meng, Tao; Chow, Vincent TK; Chua, Kaw Bing

    2016-01-01

    Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1K98E,E145A,L169F with three substitutions in the VP1 protein—K98E, E145A and L169F—productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection. PMID:27329847

  9. Frequently Asked Questions about Rotavirus

    MedlinePlus

    ... from being exposed to rotavirus. Better hygiene and sanitation have not been very good at reducing rotavirus disease. Because the virus is so widespread, even the cleanest environments can be infected. Children who have previously had ...

  10. Recombinant Outer Capsid Glycoprotein (VP7) of Rotavirus Expressed in Insect Cells Induces Neutralizing Antibodies in Rabbits

    PubMed Central

    Khodabandehloo, M; Shahrabadi, M Shamsi; Keyvani, H; Bambai, B; Sadigh, ZA

    2012-01-01

    Background: Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection. Many efforts have been done to produce recombinant VP7 that maintain native characteristics. We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Methods: Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Results: Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants. Injection of recombinant VP7 in rabbits elicited the production of serum antibodies, which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture. Conclusion: Recombinant outer capsid glycoprotein (VP7) of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine. PMID:23113180

  11. Mapping the hemagglutination domain of rotaviruses.

    PubMed Central

    Fuentes-Pananá, E M; López, S; Gorziglia, M; Arias, C F

    1995-01-01

    Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein. PMID:7884915

  12. Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein.

    PubMed

    Arnold, Michelle M; Brownback, Catie Small; Taraporewala, Zenobia F; Patton, John T

    2012-07-01

    The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3'-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus. Herein, we report the isolation and characterization of SA11-4Fg7re, an SA11-4F RV derivative that contains a large sequence duplication initiating within the genome segment (gene 7) encoding NSP3. Our analysis showed that mutant NSP3 (NSP3m) encoded by SA11-4Fg7re is almost twice the size of the wild-type protein and retains the capacity to dimerize. However, in comparison to wild-type NSP3, NSP3m has a decreased capacity to interact with eIF4G and to suppress the translation of polyadenylated mRNAs. In addition, NSP3m fails to induce the nuclear accumulation of PABP in infected cells. Despite the defective activities of NSP3m, the levels of viral protein and progeny virus produced in SA11-4Fg7re- and SA11-4F-infected cells were indistinguishable. Collectively, these data are consistent with a role for NSP3 in suppressing host protein synthesis through antagonism of PABP activity, but also suggest that NSP3 functions may have little or no impact on the efficiency of virus replication in widely used RV-permissive cell lines. PMID:22442114

  13. Whole genomic characterization of Korean porcine G8P[7] reassortant rotaviruses.

    PubMed

    Park, Jun-Gyu; Park, Sang-Ik; Woo, Nam-Il; Kim, Deok-Song; Seo, Ja-Young; Alfajaro, Mia Madel; Kim, Ji-Yun; Soliman, Mahmoud; Baek, Yeong-Bin; Cho, Eun-Hyo; Kwon, Joseph; Choi, Jong-Soon; Kang, Mun-Il; Matthijnssens, Jelle; Cho, Kyoung-Oh

    2016-10-01

    This study analyzed eleven genomic segments of three Korean porcine G8P[7] group A rotavirus (RVA) strains. Phylogenetically, these strains contained two bovine-like and nine porcine-like genomic segments. Eight genes (VP1, VP2, VP6 and NSP1-NSP5) of strains 156-1 and 42-1 and seven genes (VP1, VP2, VP6 and NSP2-NSP5) of strain C-1 clustered closely with porcine and porcine-like animal strains and distantly from typical human Wa-like strains. The VP3-M2 genotype of these strains clustered closely with bovine-like strains, but distantly with typical human DS-1-like strains. These data indicate that multiple reassortments involving porcine and bovine RVA strains in Korea must have occurred. PMID:27393603

  14. Evidence of multiple reassortment events of feline-to-human rotaviruses based on a rare human G3P[9] rotavirus isolated from a patient with acute gastroenteritis.

    PubMed

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Kim, Wonyong

    2016-06-01

    A rare human/feline-like rotavirus G3P[9] strain, CAU14-1-262, from a 2-year-old girl with severe gastroenteritis was isolated and sequenced. The 11 gene segments of the CAU14-1-262 strain possessed a novel genotype constellation, G3-P[9]-I3-R3-C3-M3-A3-N3-T1-E3-H6, which was identified for the first time. Phylogenetic analysis of this strain identified the following genome origins: VP7, VP4, VP6, VP1-VP3, NSP1, NSP2, and NSP4 genes possessed an AU-1-like genotype 3 constellation with high sequence identity to those of the feline and human/feline-like rotaviruses; NSP5 possessed a H6 lineage, with highest sequence identity to the human/feline-like E2541 strain; and the NSP3 gene possessed a Wa-like genotype 1 constellation with high sequence identity to those of the of human rotaviruses. These results provided evidence of multiple reassortment events in G3P[9] rotavirus CAU14-1-262 and possibility of feline-to-human interspecies transmission. PMID:27260811

  15. Molecular Characterization of Equine Rotavirus in Ireland▿

    PubMed Central

    Collins, P. J.; Cullinane, A.; Martella, V.; O'Shea, H.

    2008-01-01

    Group A rotaviruses are important causative agents of severe, acute dehydrating diarrhea in foals. A total of 86 rotavirus-positive fecal samples, collected from diarrheic foals from 11 counties in three of the four provinces of Ireland, were obtained from the Irish Equine Centre in Kildare during a 7-year (1999 to 2005) passive surveillance study and were characterized molecularly to establish the VP7 (G type) and VP4 (P type) antigenic specificities. Fifty-eight samples (67.5%) were found to contain G3 viruses, while in 26 samples (30.2%) the rotaviruses were typed as G14 and in 2 samples (2.3%) there was a mixed infection, G3 plus G14. All samples except for two, which were untypeable, were characterized as P[12]. Fifty-eight percent of the samples were obtained from County Kildare, the center of the Irish horse industry, where an apparent shift from G3P[12] to G14P[12] was observed in 2003. By sequence analysis of the VP7 protein, the G3 Irish strains were shown to resemble viruses of the G3A subtype (H2-like) (97.1 to 100% amino acid [aa] identity), while the G14 Irish strains displayed 93.9 to 97.1% aa identity to other G14 viruses. In the VP8* fragment of the VP4 protein, the P[12] Irish viruses displayed high conservation (92.3 to 100% aa) with other equine P[12] viruses. Worldwide, G3P[12] and G14P[12] are the most prevalent equine rotavirus strains, and G3P[12] vaccines have been developed for prevention of rotavirus-associated diarrhea in foals. Investigations of the VP7/VP4 diversity of the circulating equine viruses and the dynamics of strain replacement are important for better assessing the efficacies of the vaccines. PMID:18716232

  16. Rotavirus infection activates the UPR but modulates its activity

    PubMed Central

    2011-01-01

    Background Rotaviruses are known to modulate the innate antiviral defense response driven by IFN. The purpose of this study was to identify changes in the cellular proteome in response to rotavirus infection in the context of the IFN response. We also sought to identify proteins outside the IFN induction and signaling pathway that were modulated by rotavirus infection. Methods 2D-DIGE and image analysis were used to identify cellular proteins that changed in levels of expression in response to rotavirus infection, IFN treatment, or IFN treatment prior to infection. Immunofluorescence microscopy was used to determine the subcellular localization of proteins associated with the unfolded protein response (UPR). Results The data show changes in the levels of multiple proteins associated with cellular stress in infected cells, including levels of ER chaperones GRP78 and GRP94. Further investigations showed that GRP78, GRP94 and other proteins with roles in the ER-initiated UPR including PERK, CHOP and GADD34, were localized to viroplasms in infected cells. Conclusions Together the results suggest rotavirus infection activates the UPR, but modulates its effects by sequestering sensor, transcription factor, and effector proteins in viroplasms. The data consequently also suggest that viroplasms may directly or indirectly play a fundamental role in regulating signaling pathways associated with cellular defense responses. PMID:21774819

  17. Crystallographic and Glycan Microarray Analysis of Human Polyomavirus 9 VP1 Identifies N-Glycolyl Neuraminic Acid as a Receptor Candidate

    PubMed Central

    Khan, Zaigham Mahmood; Liu, Yan; Neu, Ursula; Gilbert, Michel; Ehlers, Bernhard

    2014-01-01

    ABSTRACT Human polyomavirus 9 (HPyV9) is a closely related homologue of simian B-lymphotropic polyomavirus (LPyV). In order to define the architecture and receptor binding properties of HPyV9, we solved high-resolution crystal structures of its major capsid protein, VP1, in complex with three putative oligosaccharide receptors identified by glycan microarray screening. Comparison of the properties of HPyV9 VP1 with the known structure and glycan-binding properties of LPyV VP1 revealed that both viruses engage short sialylated oligosaccharides, but small yet important differences in specificity were detected. Surprisingly, HPyV9 VP1 preferentially binds sialyllactosamine compounds terminating in 5-N-glycolyl neuraminic acid (Neu5Gc) over those terminating in 5-N-acetyl neuraminic acid (Neu5Ac), whereas LPyV does not exhibit such a preference. The structural analysis demonstrated that HPyV9 makes specific contacts, via hydrogen bonds, with the extra hydroxyl group present in Neu5Gc. An equivalent hydrogen bond cannot be formed by LPyV VP1. IMPORTANCE The most common sialic acid in humans is 5-N-acetyl neuraminic acid (Neu5Ac), but various modifications give rise to more than 50 different sialic acid variants that decorate the cell surface. Unlike most mammals, humans cannot synthesize the sialic acid variant 5-N-glycolyl neuraminic acid (Neu5Gc) due to a gene defect. Humans can, however, still acquire this compound from dietary sources. The role of Neu5Gc in receptor engagement and in defining viral tropism is only beginning to emerge, and structural analyses defining the differences in specificity for Neu5Ac and Neu5Gc are still rare. Using glycan microarray screening and high-resolution protein crystallography, we have examined the receptor specificity of a recently discovered human polyomavirus, HPyV9, and compared it to that of the closely related simian polyomavirus LPyV. Our study highlights critical differences in the specificities of both viruses

  18. Rotavirus capsid VP6 protein acts as an adjuvant in vivo for norovirus virus-like particles in a combination vaccine

    PubMed Central

    Blazevic, Vesna; Malm, Maria; Arinobu, Daisuke; Lappalainen, Suvi; Vesikari, Timo

    2016-01-01

    ABSTRACT Rotavirus (RV) and norovirus (NoV) are the 2 leading causes of acute viral gastroenteritis worldwide. We have developed a non-live NoV and RV vaccine candidate consisting of NoV virus-like particles (VLPs) and recombinant polymeric RV VP6 protein produced in baculovirus-insect cell expression system. Both components have been shown to induce strong potentially protective immune responses. As VP6 nanotubes are highly immunogenic, we investigated here a possible adjuvant effect of these structures on NoV-specific immune responses in vivo. BALB/c mice were immunized intramuscularly with a suboptimal dose (0.3 μg) of GII.4 or GI.3 VLPs either alone or in a combination with 10 μg dose of VP6 and induction of NoV-specific antibodies in sera of experimental animals were measured. Blocking assay using human saliva or synthetic histo-blood group antigens was employed to test NoV blocking antibodies. Suboptimal doses of the VLPs alone did not induce substantial anti-NoV antibodies. When co-administered with the VP6, considerable titers of not only type-specific but also cross-reactive IgG antibodies against NoV VLP genotypes not included in the vaccine composition were induced. Most importantly, NoV-specific blocking antibodies, a surrogate for neutralizing antibodies, were generated. Our results show that RV VP6 protein has an in vivo adjuvant effect on NoV-specific antibody responses and support the use of VP6 protein as a part of the NoV-RV combination vaccine, especially when addition of external adjuvants is not desirable. PMID:26467630

  19. Rotavirus capsid VP6 protein acts as an adjuvant in vivo for norovirus virus-like particles in a combination vaccine.

    PubMed

    Blazevic, Vesna; Malm, Maria; Arinobu, Daisuke; Lappalainen, Suvi; Vesikari, Timo

    2016-03-01

    Rotavirus (RV) and norovirus (NoV) are the 2 leading causes of acute viral gastroenteritis worldwide. We have developed a non-live NoV and RV vaccine candidate consisting of NoV virus-like particles (VLPs) and recombinant polymeric RV VP6 protein produced in baculovirus-insect cell expression system. Both components have been shown to induce strong potentially protective immune responses. As VP6 nanotubes are highly immunogenic, we investigated here a possible adjuvant effect of these structures on NoV-specific immune responses in vivo. BALB/c mice were immunized intramuscularly with a suboptimal dose (0.3 μg) of GII.4 or GI.3 VLPs either alone or in a combination with 10 μg dose of VP6 and induction of NoV-specific antibodies in sera of experimental animals were measured. Blocking assay using human saliva or synthetic histo-blood group antigens was employed to test NoV blocking antibodies. Suboptimal doses of the VLPs alone did not induce substantial anti-NoV antibodies. When co-administered with the VP6, considerable titers of not only type-specific but also cross-reactive IgG antibodies against NoV VLP genotypes not included in the vaccine composition were induced. Most importantly, NoV-specific blocking antibodies, a surrogate for neutralizing antibodies, were generated. Our results show that RV VP6 protein has an in vivo adjuvant effect on NoV-specific antibody responses and support the use of VP6 protein as a part of the NoV-RV combination vaccine, especially when addition of external adjuvants is not desirable. PMID:26467630

  20. Rotaviruses from Canadian farm samples.

    PubMed

    Lamhoujeb, Safaa; Cook, Angela; Pollari, Frank; Bidawid, Sabah; Farber, Jeff; Mattison, Kirsten

    2010-07-01

    Animal rotavirus (RoV) strains detected in Canadian swine and dairy cattle farms were characterized by sequence analysis of viral protein 4 (VP4), VP6, VP7 and non-structural protein 4 segments from 15 RoV strains. Some porcine strains were found to contain a mixture of segments typical of human and animal viruses. One strain represented a novel VP6 genotype "I14", G2-P[27]-I14. Other strains detected in porcine samples represented multiple different segment types. These results illustrate the active evolution of animal RoV strains and underline the need for surveillance of both animal and human strains in public health-monitoring programs. PMID:20517624

  1. Systemic features of rotavirus infection.

    PubMed

    Rivero-Calle, Irene; Gómez-Rial, José; Martinón-Torres, Federico

    2016-07-01

    A growing body of evidence warrants a revision of the received/conventional wisdom of rotavirus infection as synonymous with acute gastroenteritis. Rotavirus vaccines have boosted our interest and knowledge of this virus, but also importantly, they may have changed the landscape of the disease. Extraintestinal spread of rotavirus is well documented, and the clinical spectrum of the disease is widening. Furthermore, the positive impact of current rotavirus vaccines in reducing seizure hospitalization rates should prompt a reassessment of the actual burden of extraintestinal manifestations of rotavirus diseases. This article discusses current knowledge of the systemic extraintestinal manifestations of rotavirus infection and their underlying mechanisms, and aims to pave the way for future clinical, public health and research questions. PMID:27181101

  2. Disease Caused by Rotavirus Infection

    PubMed Central

    Lin, Che-Liang; Chen, Shou-Chien; Liu, Shyun-Yeu; Chen, Kow-Tong

    2014-01-01

    Although rotavirus vaccines are available, rotaviruses remain the major cause of childhood diarrheal disease worldwide. The Rotarix (GlaxoSmithKline Biologicals Rixensart, Belgium) and RotaTeq (Merck and Co., Inc. Whitehouse Station, New Jersey, USA) vaccines are effective for reducing the morbidity and mortality of rotavirus infection. This article aims to assess the epidemiology of rotaviral gastroenteritis and the efficacy and effectiveness of licensed rotavirus vaccines. This review concludes by presenting challenges in the field that require further exploration by and perspectives from basic and translational research in the future. PMID:25553142

  3. Predicted Structure and Domain Organization of Rotavirus Capping Enzyme and Innate Immune Antagonist VP3

    PubMed Central

    Snyder, Matthew J.; Dennis, Allison F.; Patton, John T.

    2014-01-01

    ABSTRACT Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5′-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2′-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2′-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2′-5′ oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3. IMPORTANCE Rotaviruses are an

  4. Distinct function of COAR and B3 domains of maize VP1 in induction of ectopic gene expression and plant developmental phenotypes in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize VP1 enhancement of ABA sensitivity in roots is B3 independent. ABA and VP1 mediated suppression of auxin induced lateral root development is B3 independent. Arabidopsis transgenic system to delineate B3 dependent and COAR domain dependent regulatory functions of VP1. Analyses of ectopic ABA re...

  5. Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains.

    PubMed

    Komoto, Satoshi; Tacharoenmuang, Ratana; Guntapong, Ratigorn; Ide, Tomihiko; Tsuji, Takao; Yoshikawa, Tetsushi; Tharmaphornpilas, Piyanit; Sangkitporn, Somchai; Taniguchi, Koki

    2016-01-01

    The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating

  6. Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

    PubMed Central

    Komoto, Satoshi; Tacharoenmuang, Ratana; Guntapong, Ratigorn; Ide, Tomihiko; Tsuji, Takao; Yoshikawa, Tetsushi; Tharmaphornpilas, Piyanit; Sangkitporn, Somchai; Taniguchi, Koki

    2016-01-01

    The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating

  7. Trivalent Combination Vaccine Induces Broad Heterologous Immune Responses to Norovirus and Rotavirus in Mice

    PubMed Central

    Tamminen, Kirsi; Lappalainen, Suvi; Huhti, Leena; Vesikari, Timo; Blazevic, Vesna

    2013-01-01

    Rotavirus (RV) and norovirus (NoV) are the two major causes of viral gastroenteritis (GE) in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1) derived virus-like particles (VLPs) of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6), the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined in vitro. The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50%) as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization (GII-4 NO, GII-12 and GI-1 VLPs) and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes. PMID:23922988

  8. Diversity and relationships of cocirculating modern human rotaviruses revealed using large-scale comparative genomics.

    PubMed

    McDonald, Sarah M; McKell, Allison O; Rippinger, Christine M; McAllen, John K; Akopov, Asmik; Kirkness, Ewen F; Payne, Daniel C; Edwards, Kathryn M; Chappell, James D; Patton, John T

    2012-09-01

    Group A rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses and are primary causes of gastroenteritis in young children. Despite their medical relevance, the genetic diversity of modern human RVs is poorly understood, and the impact of vaccine use on circulating strains remains unknown. In this study, we report the complete genome sequence analysis of 58 RVs isolated from children with severe diarrhea and/or vomiting at Vanderbilt University Medical Center (VUMC) in Nashville, TN, during the years spanning community vaccine implementation (2005 to 2009). The RVs analyzed include 36 G1P[8], 18 G3P[8], and 4 G12P[8] Wa-like genogroup 1 strains with VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 genotype constellations of I1-R1-C1-M1-A1-N1-T1-E1-H1. By constructing phylogenetic trees, we identified 2 to 5 subgenotype alleles for each gene. The results show evidence of intragenogroup gene reassortment among the cocirculating strains. However, several isolates from different seasons maintained identical allele constellations, consistent with the notion that certain RV clades persisted in the community. By comparing the genes of VUMC RVs to those of other archival and contemporary RV strains for which sequences are available, we defined phylogenetic lineages and verified that the diversity of the strains analyzed in this study reflects that seen in other regions of the world. Importantly, the VP4 and VP7 proteins encoded by VUMC RVs and other contemporary strains show amino acid changes in or near neutralization domains, which might reflect antigenic drift of the virus. Thus, this large-scale, comparative genomic study of modern human RVs provides significant insight into how this pathogen evolves during its spread in the community. PMID:22696651

  9. Diversity and Relationships of Cocirculating Modern Human Rotaviruses Revealed Using Large-Scale Comparative Genomics

    PubMed Central

    McKell, Allison O.; Rippinger, Christine M.; McAllen, John K.; Akopov, Asmik; Kirkness, Ewen F.; Payne, Daniel C.; Edwards, Kathryn M.; Chappell, James D.; Patton, John T.

    2012-01-01

    Group A rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses and are primary causes of gastroenteritis in young children. Despite their medical relevance, the genetic diversity of modern human RVs is poorly understood, and the impact of vaccine use on circulating strains remains unknown. In this study, we report the complete genome sequence analysis of 58 RVs isolated from children with severe diarrhea and/or vomiting at Vanderbilt University Medical Center (VUMC) in Nashville, TN, during the years spanning community vaccine implementation (2005 to 2009). The RVs analyzed include 36 G1P[8], 18 G3P[8], and 4 G12P[8] Wa-like genogroup 1 strains with VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 genotype constellations of I1-R1-C1-M1-A1-N1-T1-E1-H1. By constructing phylogenetic trees, we identified 2 to 5 subgenotype alleles for each gene. The results show evidence of intragenogroup gene reassortment among the cocirculating strains. However, several isolates from different seasons maintained identical allele constellations, consistent with the notion that certain RV clades persisted in the community. By comparing the genes of VUMC RVs to those of other archival and contemporary RV strains for which sequences are available, we defined phylogenetic lineages and verified that the diversity of the strains analyzed in this study reflects that seen in other regions of the world. Importantly, the VP4 and VP7 proteins encoded by VUMC RVs and other contemporary strains show amino acid changes in or near neutralization domains, which might reflect antigenic drift of the virus. Thus, this large-scale, comparative genomic study of modern human RVs provides significant insight into how this pathogen evolves during its spread in the community. PMID:22696651

  10. A computational study of the interaction of the foot and mouth disease virus VP1 with monoclonal antibodies.

    PubMed

    Marrero, Ruben; Limardo, Ramiro Rodríguez; Carrillo, Elisa; König, Guido A; Turjanski, Adrián G

    2015-10-01

    Foot and mouth disease is caused by a non-enveloped virus (FMDV), which disposes several antigenic sites at the surface of their capsid proteins. The most relevant and immunodominant antigenic site of FMDV (site A or AnSA) includes a key virus-cell interaction element (RGD motif) located in the Viral Protein 1 (VP1), more precisely at the GH loop. AnSA includes a set of overlapped and mainly linear epitopes, which are the main targets of the humoral immune response. Taking advantage over specific structural features of the GH loop, we have evaluated the influence of every amino acid residue at AnSA in the interaction with 2 neutralizing antibodies by molecular modeling techniques. Additionally, we constructed diverse interaction complexes with multiple site A mutants and discussed about the structural influence of amino acidic insertions in such relevant antigenic site of FMDV. Our approach is in agreement with previous ELISA experiments and allows the understanding of how FMDV mutations may alter the interaction with different antibodies, as we can estimate the contribution of each amino acid to the interaction. Overall, our work contributes to the development of specific vaccination strategies for FMD control. PMID:26093030

  11. The Rotavirus Interferon Antagonist NSP1: Many Targets, Many Questions.

    PubMed

    Arnold, Michelle M

    2016-06-01

    Rotavirus is a leading cause of death due to diarrhea among young children across the globe. Despite the limited coding capacity that is characteristic of RNA viruses, rotavirus dedicates substantial resources to avoiding the host innate immune response. Among these strategies is use of the interferon antagonist protein NSP1, which targets cellular proteins required for interferon production to be degraded by the proteasome. Although numerous cellular targets have been described, there remain many questions about the mechanism of NSP1 activity and its role in promoting replication in specific host species. PMID:27009959

  12. Global Seasonality of Rotavirus Disease

    PubMed Central

    Patel, Manish M.; Pitzer, Virginia; Alonso, Wladimir J.; Vera, David; Lopman, Ben; Tate, Jacqueline; Viboud, Cecile; Parashar, Umesh D.

    2012-01-01

    Background A substantial number of surveillance studies have documented rotavirus prevalence among children admitted for dehydrating diarrhea. We sought to establish global seasonal patterns of rotavirus disease before widespread vaccine introduction. Methods We reviewed studies of rotavirus detection in children with diarrhea published since 1995. We assessed potential relationships between seasonal prevalence and locality by plotting the average monthly proportion of diarrhea cases positive for rotavirus according to geography, country development, and latitude. We used linear regression to identify variables that were potentially associated with the seasonal intensity of rotavirus. Results Among a total of 99 studies representing all six geographical regions of the world, patterns of year-round disease were more evident in low- and low-middle income countries compared with upper-middle and high income countries where disease was more likely to be seasonal. The level of country development was a stronger predictor of strength of seasonality (P=0.001) than geographical location or climate. However, the observation of distinctly different seasonal patterns of rotavirus disease in some countries with similar geographical location, climate and level of development indicate that a single unifying explanation for variation in seasonality of rotavirus disease is unlikely. Conclusion While no unifying explanation emerged for varying rotavirus seasonality globally, the country income level was somewhat more predictive of the likelihood of having seasonal disease than other factors. Future evaluation of the effect of rotavirus vaccination on seasonal patterns of disease in different settings may help understand factors that drive the global seasonality of rotavirus disease. PMID:23190782

  13. A Broadly Cross-protective Vaccine Presenting the Neighboring Epitopes within the VP1 GH Loop and VP2 EF Loop of Enterovirus 71.

    PubMed

    Xu, Longfa; He, Delei; Yang, Lisheng; Li, Zhiqun; Ye, Xiangzhong; Yu, Hai; zhao, Huan; Li, Shuxuan; Yuan, Lunzhi; Qian, Hongliu; Que, Yuqiong; Shih, James Wai Kuo; Zhu, Hua; Li, Yimin; Cheng, Tong; Xia, Ningshao

    2015-01-01

    Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops. PMID:26243660

  14. Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition*

    PubMed Central

    Civra, Andrea; Giuffrida, Maria Gabriella; Donalisio, Manuela; Napolitano, Lorenzo; Takada, Yoshikazu; Coulson, Barbara S.; Conti, Amedeo; Lembo, David

    2015-01-01

    Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding. PMID:25814665

  15. Whole genome sequencing reveals genetic heterogeneity of G3P[8] rotaviruses circulating in Italy.

    PubMed

    Medici, Maria Cristina; Tummolo, Fabio; Martella, Vito; Arcangeletti, Maria Cristina; De Conto, Flora; Chezzi, Carlo; Magrì, Alessandro; Fehér, Enikő; Marton, Szilvia; Calderaro, Adriana; Bányai, Krisztián

    2016-06-01

    After a sporadic detection in 1990s, G3P[8] rotaviruses emerged as a predominant genotype during recent years in many areas worldwide, including parts of Italy. The present study describes the molecular epidemiology and evolution of G3P[8] rotaviruses detected in Italian children with gastroenteritis during two survey periods (2004-2005 and 2008-2013). Whole genome of selected G3P[8] strains was determined and antigenic differences between these strains and rotavirus vaccine strains were analyzed. Among 819 (271 in 2004-2005 and 548 in 2008-2013) rotaviruses genotyped during the survey periods, the number of G3P[8] rotavirus markedly varied over the years (0/83 in 2004, 30/188 in 2005 and 0/96 in 2008, 6/88 in 2009, 4/97 in 2010, 0/83 in 2011, 9/82 in 2012, 56/102 cases in 2013). The genotypes of the 11 gene segments of 15 selected strains were assigned to G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1; thus all strains belonged to the Wa genogroup. Phylogenetic analysis of the Italian G3P[8] strains showed a peculiar picture of segregation with a 2012 lineage for VP1-VP3, NSP1, NSP2, NSP4 and NSP5 genes and a 2013 lineage for VP6, NSP1 and NSP3 genes, with a 1.3-20.2% nucleotide difference from the oldest Italian G3P[8] strains. The genetic variability of the Italian G3P[8] observed in comparison with sequences of rotaviruses available in GenBank suggested a process of selection acting on a global scale, rather than the emergence of local strains, as several lineages were already circulating globally. Compared with the vaccine strains, the Italian G3P[8] rotaviruses segregated in different lineages (5-5.3% and 7.2-11.4% nucleotide differences in the VP7 and VP4, respectively) with some mismatches in the putative neutralizing epitopes of VP7 and VP4 antigens. The accumulation of point mutations and amino acid differences between vaccine strains and currently circulating rotaviruses might generate, over the years, vaccine-resistant variants. PMID:26980605

  16. Phylogenetic analysis of probable non-human genes of group A rotaviruses isolated from children with acute gastroenteritis in Belém, Brazil.

    PubMed

    Maestri, Régis Piloni; Kaiano, Jane Haruko Lima; Neri, Darivaldo Luz; Soares, Luana da Silva; Guerra, Sylvia de Fatima Dos Santos; Oliveira, Darleise de Souza; Farias, Yasmin Nascimento; Gabbay, Yvone Benchimol; Leite, José Paulo Gagliardi; Linhares, Alexandre da Costa; Mascarenhas, Joana D'Arc Pereira

    2012-12-01

    Rotaviruses (RVs) are the main cause of acute viral gastroenteritis in both humans and young animals of various species such as calves, horses, pigs, dogs, cats, and birds. The genetic diversity of RVs is related to a variety of evolutionary mechanisms, including point mutation, and genome reassortment. The objective of this study was to characterize molecularly genes that encode structural and nonstructural proteins in unusual RV strains. The clinical specimens selected for this study were obtained from children and newborn with RV gastroenteritis, who participated in research projects on viral gastroenteritis conducted at the Evandro Chagas Institute. Structural (VP1-VP4, VP6, and VP7) and nonstructural (NSP1-NSP6) genes were amplified from stool samples by the polymerase chain reaction and subsequently sequenced. Eight unusual RV strains isolated from children and newborn with gastroenteritis were studied. Reassortment between genes of animal origin were observed in 5/8 (62.5%) strains analyzed. These results demonstrate that, although rare, interspecies (animal-human) transmission of RVs occurs in nature, as observed in the present study in strains NB150, HSP034, HSP180, HST327, and RV10109. This study is the first to be conducted in the Amazon region and supports previous data showing a close relationship between genes of human and animal origin, representing a challenge to the large-scale introduction of RV vaccines in national immunization programs. PMID:23080508

  17. Fabrication of gold nanoparticles in Therminol VP-1 by laser ablation and fragmentation with fs pulses

    NASA Astrophysics Data System (ADS)

    Torres-Mendieta, R.; Mondragón, R.; Juliá, E.; Mendoza-Yero, O.; Cordoncillo, E.; Lancis, J.; Mínguez-Vega, G.

    2014-12-01

    This letter reports on a physical method to produce highly pure, size-controlled and well-dispersed spherical gold nanoparticles (NPs) in Therminol VP-1 by pulsed laser ablation in liquids (PLAL) using a 30 fs Ti:Sapphire laser at a fluence of 1 J cm-2. A second photo-fragmentation of the ablated colloid solution by subsequent treatment with the same laser light yields a mean size and size dispersion of the NPs of 58 ± 31 nm. A study of the nanofluid properties reveals a low agglomeration over time and an enhancement of thermal conductivity of the base fluid by up to 4%. These results improve the characteristics of current nanofluids in thermal oils that may have a potential impact on the improvement of the efficiency of harvesting of solar light.

  18. Mean protein evolutionary distance: a method for comparative protein evolution and its application.

    PubMed

    Wise, Michael J

    2013-01-01

    Proteins are under tight evolutionary constraints, so if a protein changes it can only do so in ways that do not compromise its function. In addition, the proteins in an organism evolve at different rates. Leveraging the history of patristic distance methods, a new method for analysing comparative protein evolution, called Mean Protein Evolutionary Distance (MeaPED), measures differential resistance to evolutionary pressure across viral proteomes and is thereby able to point to the proteins' roles. Different species' proteomes can also be compared because the results, consistent across virus subtypes, concisely reflect the very different lifestyles of the viruses. The MeaPED method is here applied to influenza A virus, hepatitis C virus, human immunodeficiency virus (HIV), dengue virus, rotavirus A, polyomavirus BK and measles, which span the positive and negative single-stranded, doubled-stranded and reverse transcribing RNA viruses, and double-stranded DNA viruses. From this analysis, host interaction proteins including hemagglutinin (influenza), and viroporins agnoprotein (polyomavirus), p7 (hepatitis C) and VPU (HIV) emerge as evolutionary hot-spots. By contrast, RNA-directed RNA polymerase proteins including L (measles), PB1/PB2 (influenza) and VP1 (rotavirus), and internal serine proteases such as NS3 (dengue and hepatitis C virus) emerge as evolutionary cold-spots. The hot spot influenza hemagglutinin protein is contrasted with the related cold spot H protein from measles. It is proposed that evolutionary cold-spot proteins can become significant targets for second-line anti-viral therapeutics, in cases where front-line vaccines are not available or have become ineffective due to mutations in the hot-spot, generally more antigenically exposed proteins. The MeaPED package is available from www.pam1.bcs.uwa.edu.au/~michaelw/ftp/src/meaped.tar.gz. PMID:23613826

  19. Rotavirus Vaccine -- Questions and Answers

    MedlinePlus

    ... to these vaccines. The infant's immune response to influenza vaccine administered at the same time as rotavirus vaccine ... previously that an inactivated vaccine (e.g., inactivated influenza vaccine) may be administered either simultaneously or at any ...

  20. Sudden Death from Systemic Rotavirus Infection and Detection of Nonstructural Rotavirus Proteins▿

    PubMed Central

    Nakano, Ineko; Taniguchi, Koki; Ishibashi-Ueda, Hatsue; Maeno, Yoshimasa; Yamamoto, Naoki; Yui, Akiko; Komoto, Satoshi; Wakata, Yasushi; Matsubara, Tamehito; Ozaki, Nozomu

    2011-01-01

    A 2.5-year-old girl died suddenly during the course of rotavirus gastroenteritis. The autopsy showed encephalopathy with rotavirus systemic infection. Here, we provide evidence of rotavirus replication in multiple organs. Our findings clarify that rotavirus infection in children can extend beyond the intestinal tract through viremia. PMID:21998424

  1. Rotavirus-Like Particles: A Novel Nanocarrier for the Gut

    PubMed Central

    Cortes-Perez, Naima G.; Sapin, Catherine; Jaffrelo, Loïc; Daou, Sabine; Grill, Jean Pierre; Langella, Philippe; Seksik, Philippe; Beaugerie, Laurent; Chwetzoff, Serge; Trugnan, Germain

    2010-01-01

    The delivery of bioactive molecules directly to damaged tissues represents a technological challenge. We propose here a new system based on virus-like particles (VLP) from rotavirus, with a marked tropism for the gut to deliver bio-active molecules to intestinal cells. For this, nonreplicative VLP nanoparticles were constructed using a baculovirus expression system and used to deliver an exogenous biomolecule, the green fluorescent protein (GFP), into either MA104 cells or intestinal cells from healthy and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated mice. Our results show that expression of rotavirus capsid proteins in baculovirus led to the auto assembly of VLP that display similar properties to rotavirus. In vitro experiments showed that VLP were able to enter into MA104 cells and deliver the reporter protein. Intragastric administration of fluorescent VLP in healthy and TNBS-treated mice resulted in the detection of GFP and viral proteins in intestinal samples. Our results demonstrate an efficient entry of non-replicative rotavirus VLP into the epithelial cell line MA104 and provide the first in vivo evidence of the potential of these nanoparticles as a promising safe candidate for drug delivery to intestinal cells. PMID:20414353

  2. Genistein inhibits rotavirus replication and upregulates AQP4 expression in rotavirus-infected Caco-2 cells.

    PubMed

    Huang, Haohai; Liao, Dan; Liang, Liping; Song, Lijun; Zhao, Wenchang

    2015-06-01

    Rotavirus (RV) is the primary cause of severe dehydrating gastroenteritis and acute diarrheal disease in infants and young children. Previous studies have revealed that genistein can inhibit the infectivity of enveloped or nonenveloped viruses. Although the biological properties of genistein are well studied, the mechanisms of action underlying their anti-rotavirus properties have not been fully elucidated. Here, we report that genistein significantly inhibits RV-Wa replication in vitro by repressing viral RNA transcripts, and possibly viral protein synthesis. Interestingly, we also found that aquaporin 4 (AQP4) mRNA and protein expression, which was downregulated in RV-infected Caco-2 cells, can be upregulated by genistein in a time- and dose-dependent manner. Further experiments confirmed that genistein triggers CREB phosphorylation through PKA activation and subsequently promotes AQP4 gene transcription. These findings suggest that the pathophysiological mechanism of RV infection involves decreased expression of AQP4 and that genistein may be a useful candidate for developing a new anti-RV strategy by inhibiting rotavirus replication and upregulating AQP4 expression via the cAMP/PKA/CREB signaling pathway. Further studies on the effect of genistein on RV-induced diarrhea are warranted. PMID:25877820

  3. Pathogenicity of rotavirus in rabbits.

    PubMed Central

    Thouless, M E; DiGiacomo, R F; Deeb, B J; Howard, H

    1988-01-01

    The role of rotavirus in diarrheal disease of rabbits was investigated, and a model for human rotavirus infection was established. Orogastric inoculation of 8- and 12-week-old New Zealand White rabbits with a rabbit strain of rotavirus (L:ALA:84) resulted in fecal shedding of virus for 6 to 8 days from 2 to 5 days after inoculation. Most rabbits exhibited diarrhea, coincident with the onset of viral shedding, which persisted for 2 to 4 days. Diarrhea was characterized by soft or fluid stools and fecal staining of the perineum. Inoculation of 3-week-old rabbits resulted in a briefer period of viral shedding and diarrhea of a milder nature. Histopathologic examination during the period of viral shedding revealed a mild, nonsuppurative enteritis. Inoculated rabbits exhibited antibodies in serum to rotavirus by enzyme-linked immunosorbent assay. Sham-inoculated or uninoculated rabbits maintained in the same cage or the same room with inoculated rabbits acquired rotavirus infection. The mild diarrheal disease which resulted with a rotavirus isolate from severe field cases suggests that cofactors were involved. Images PMID:2838507

  4. Rotavirus Prevalence in the Primary Care Setting in Nicaragua after Universal Infant Rotavirus Immunization

    PubMed Central

    Becker-Dreps, Sylvia; Paniagua, Margarita; Zambrana, Luis Enrique; Bucardo, Filemon; Hudgens, Michael G.; Weber, David J.; Morgan, Douglas R.; Espinoza, Félix

    2011-01-01

    Nicaragua was the first developing nation to implement universal infant rotavirus immunization with the pentavalent rotavirus vaccine (RV5). Initial studies of vaccine effectiveness in Nicaragua and other developing nations have focused on the prevention of hospitalizations and severe rotavirus diarrhea. However, rotavirus diarrhea is more commonly treated in the primary care setting, with only 1–3% of rotavirus cases receiving hospital care. We measured the prevalence of rotavirus infection in primary care clinics in León, Nicaragua, after introduction of the immunization program. In the post-vaccine period, 3.5% (95% confidence interval = 1.9–5.8) of children seeking care for diarrhea tested positive for rotavirus. A high diversity of rotavirus genotypes was encountered among the few positive samples. In conclusion, rotavirus was an uncommon cause of childhood diarrhea in this primary care setting after implementation of a rotavirus immunization program. PMID:22049057

  5. Identification of Novel Ghanaian G8P[6] Human-Bovine Reassortant Rotavirus Strain by Next Generation Sequencing

    PubMed Central

    Dennis, Francis E.; Fujii, Yoshiki; Haga, Kei; Damanka, Susan; Lartey, Belinda; Agbemabiese, Chantal A.; Ohta, Nobuo; Armah, George E.; Katayama, Kazuhiko

    2014-01-01

    Group A rotaviruses (RVAs) are the most important etiological agent of acute gastroenteritis in children <5 years of age worldwide. The monovalent rotavirus vaccine Rotarix was introduced into the national Expanded Programme on Immunization (EPI) in Ghana in May 2012. However, there is a paucity of genetic and phylogenetic data on the complete genomes of human RVAs in circulation pre-vaccine introduction. The common bovine rotavirus VP7 genotype G8 has been sporadically detected in Ghanaian children, usually in combination with the VP4 genotype P[6]. To investigate the genomic constellations and phylogeny of RVA strains in circulation prior to vaccine introduction, the full genomes of two unusual G8P[6] strains, GH018-08 and GH019-08, detected during burden of disease surveillance, were characterized by Illumina MiSeq sequencing. The Ghanaian isolates, GH018-08 and GH019-08, exhibited the unusual, previously unreported genotype constellation G8-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H3. Phylogenetic analyses confirmed that 10 out of the 11 genes of GH018-08 and GH019-08 were identical/nearly identical, with significant variation detected only in their VP1 genes, and clearly established the occurrence of multiple independent interspecies transmission and reassortment events between co-circulating bovine/ovine/caprine rotaviruses and human DS-1-like RVA strains. These findings highlight the contribution of reassortment and interspecies transmission events to the high rotavirus diversity in this region of Africa, and justify the need for simultaneous monitoring of animal and human rotavirus strains. PMID:24971993

  6. Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

    PubMed Central

    Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

    2014-01-01

    As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity. PMID:25268969

  7. Rotavirus Infections - Multiple Languages: MedlinePlus

    MedlinePlus

    ... Are Here: Home → Multiple Languages → All Health Topics → Rotavirus Infections URL of this page: https://medlineplus.gov/ ... V W XYZ List of All Topics All Rotavirus Infections - Multiple Languages To use the sharing features ...

  8. Protect Your Child from Rotavirus Disease

    MedlinePlus

    ... within the first week after the first or second dose of rotavirus vaccine.​ CDC continues to recommend that infants receive rotavirus vaccine. The benefits of the vaccine far outweigh the small risk ...

  9. Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG).

    PubMed

    Matthijnssens, Jelle; Ciarlet, Max; McDonald, Sarah M; Attoui, Houssam; Bányai, Krisztián; Brister, J Rodney; Buesa, Javier; Esona, Mathew D; Estes, Mary K; Gentsch, Jon R; Iturriza-Gómara, Miren; Johne, Reimar; Kirkwood, Carl D; Martella, Vito; Mertens, Peter P C; Nakagomi, Osamu; Parreño, Viviana; Rahman, Mustafizur; Ruggeri, Franco M; Saif, Linda J; Santos, Norma; Steyer, Andrej; Taniguchi, Koki; Patton, John T; Desselberger, Ulrich; Van Ranst, Marc

    2011-08-01

    In April 2008, a nucleotide-sequence-based, complete genome classification system was developed for group A rotaviruses (RVs). This system assigns a specific genotype to each of the 11 genome segments of a particular RV strain according to established nucleotide percent cutoff values. Using this approach, the genome of individual RV strains are given the complete descriptor of Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx. The Rotavirus Classification Working Group (RCWG) was formed by scientists in the field to maintain, evaluate and develop the RV genotype classification system, in particular to aid in the designation of new genotypes. Since its conception, the group has ratified 51 new genotypes: as of April 2011, new genotypes for VP7 (G20-G27), VP4 (P[28]-P[35]), VP6 (I12-I16), VP1 (R5-R9), VP2 (C6-C9), VP3 (M7-M8), NSP1 (A15-A16), NSP2 (N6-N9), NSP3 (T8-T12), NSP4 (E12-E14) and NSP5/6 (H7-H11) have been defined for RV strains recovered from humans, cows, pigs, horses, mice, South American camelids (guanaco), chickens, turkeys, pheasants, bats and a sugar glider. With increasing numbers of complete RV genome sequences becoming available, a standardized RV strain nomenclature system is needed, and the RCWG proposes that individual RV strains are named as follows: RV group/species of origin/country of identification/common name/year of identification/G- and P-type. In collaboration with the National Center for Biotechnology Information (NCBI), the RCWG is also working on developing a RV-specific resource for the deposition of nucleotide sequences. This resource will provide useful information regarding RV strains, including, but not limited to, the individual gene genotypes and epidemiological and clinical information. Together, the proposed nomenclature system and the NCBI RV resource will offer highly useful tools for investigators to search for, retrieve, and analyze the ever-growing volume of RV genomic data. PMID:21597953

  10. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    NASA Technical Reports Server (NTRS)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  11. 18β-glycyrrhetinic acid inhibits rotavirus replication in culture

    PubMed Central

    2012-01-01

    Background Glycyrrhizin (GA) and primary metabolite 18β-glycyrrhetinic acid (GRA) are pharmacologically active components of the medicinal licorice root, and both have been shown to have antiviral and immunomodulatory properties. Although these properties are well established, the mechanisms of action are not completely understood. In this study, GA and GRA were tested for the ability to inhibit rotavirus replication in cell culture, toward a long term goal of discovering natural compounds that may complement existing vaccines. Methods Epithelial cells were treated with GA or GRA various times pre- or post-infection and virus yields were measured by immunofluorescent focus assay. Levels of viral proteins VP2, VP6, and NSP2 in GRA treated cells were measured by immunoblot to determine if there was an effect of GRA treatment on the accumulation of viral protein. Results GRA treatment reduced rotavirus yields by 99% when added to infected cultures post-- virus adsorption, whereas virus yields in GA treated cultures were similar to mock treated controls. Time of addition experiments indicated that GRA-mediated replication inhibition likely occurs at a step or steps subsequent to virus entry. The amounts of VP2, VP6 and NSP2 were substantially reduced when GRA was added to cultures up to two hours post-entry. Conclusions GRA, but not GA, has significant antiviral activity against rotavirus replication in vitro, and studies to determine whether GRA attenuates rotavirus replication in vivo are underway. PMID:22616823

  12. Phylogenetic analysis of VP1 gene sequences of waterfowl parvoviruses from the Mainland of China revealed genetic diversity and recombination.

    PubMed

    Wang, Shao; Cheng, Xiao-Xia; Chen, Shao-Ying; Lin, Feng-Qiang; Chen, Shi-Long; Zhu, Xiao-Li; Wang, Jin-Xiang; Huang, Mei-Qing; Zheng, Min

    2016-03-01

    To determine the origin and evolution of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) in the Mainland of China, phylogenetic and recombination analyses in the present study were performed on 32 complete VP1 gene sequences from China and other countries. Based on the phylogenetic analysis of the VP1 gene, GPV strains studied here from Mainland China (PRC) could be divided into three genotypes, namely PRC-I, PRC-II and PRC-III. Genotype PRC-I is indigenous to Mainland China. Only one GPV strain from Northeast China was of Genotype PRC-II and was thought to be imported from Europe. Genotype PRC-III, which was the most isolated genotype during 1999-2012, is related to GPVs in Taiwan and has been the predominant pathogen responsible for recent Derzy's disease outbreaks in Mainland China. Current vaccine strains used in Mainland China belong to Genotype PRC-I that is evolutionary distant from Genotypes PRC-II and PRC-III. In comparison, MDPV strains herein from Mainland China are clustered in a single group which is closely related to Taiwanese MDPV strains, and the full-length sequences of the VP1 gene of China MDPVs are phylogenetic closely related to the VP1 sequence of a Hungarian MDPV strain. Moreover, We also found that homologous recombination within VP1 gene plays a role in generating genetic diversity in GPV evolution. The GPV GDFSh from Guangdong Province appears to be the evolutionary product of a recombination event between parental GPV strains GD and B, while the major parent B proved to be a reference strain for virulent European GPVs. Our findings provide valuable information on waterfowl parvoviral evolution in Mainland China. PMID:26692144

  13. Candidate new rotavirus species in sheltered dogs, Hungary.

    PubMed

    Mihalov-Kovács, Eszter; Gellért, Ákos; Marton, Szilvia; Farkas, Szilvia L; Fehér, Enikő; Oldal, Miklós; Jakab, Ferenc; Martella, Vito; Bányai, Krisztián

    2015-04-01

    We identified unusual rotavirus strains in fecal specimens from sheltered dogs in Hungary by viral metagenomics. The novel rotavirus species displayed limited genome sequence homology to representatives of the 8 rotavirus species, A-H, and qualifies as a candidate new rotavirus species that we tentatively named Rotavirus I. PMID:25811414

  14. Structural Correlates of Rotavirus Cell Entry

    PubMed Central

    Abdelhakim, Aliaa H.; Salgado, Eric N.; Fu, Xiaofeng; Pasham, Mithun; Nicastro, Daniela; Kirchhausen, Tomas; Harrison, Stephen C.

    2014-01-01

    Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration. PMID:25211455

  15. Ability of TESTPACK ROTAVIRUS enzyme immunoassay to diagnose rotavirus gastroenteritis.

    PubMed Central

    Chernesky, M; Castriciano, S; Mahony, J; Spiewak, M; Schaefer, L

    1988-01-01

    TESTPACK ROTAVIRUS, a simple 10-min enzyme immunoassay, was compared with electron microscopy and Pathfinder enzyme immunoassay on feces from 172 patients of various ages with gastroenteritis. The percent sensitivities and specificities before blocking with antiserum were as follows: TESTPACK, 100% sensitivity and 99% specificity; Pathfinder, 95% sensitivity and 98% specificity. After blocking, the sensitivity and specificity, respectively, were 100% and 100% for TESTPACK and 95% and 99% for Pathfinder. TESTPACK ROTAVIRUS was more sensitive, but not significantly, than Pathfinder (P greater than 0.1) and the direct electron microscopy technique (P greater than 0.1). PMID:3069866

  16. PPARγ Agonists as an Anti-Inflammatory Treatment Inhibiting Rotavirus Infection of Small Intestinal Villi

    PubMed Central

    Gómez, Dory; Muñoz, Natalia; Guerrero, Rafael; Acosta, Orlando; Guerrero, Carlos A.

    2016-01-01

    Rotavirus infection has been reported to induce an inflammatory response in the host cell accompanied by the increased expression or activation of some cellular molecules including ROS, NF-κB, and COX-2. PPARγ stimulation and N-acetylcysteine (NAC) treatment have been found to interfere with viral infections including rotavirus infection. Small intestinal villi isolated from in vivo infected mice with rotavirus ECwt were analyzed for the percentage of ECwt-infected cells, the presence of rotavirus antigens, and infectious virion yield following treatment with pioglitazone. Isolated villi were also infected in vitro and treated with PPARγ agonists (PGZ, TZD, RGZ, DHA, and ALA), all-trans retinoic acid (ATRA), and NAC. After treatments, the expression of cellular proteins including PPARγ, NF-κB, PDI, Hsc70, and COX-2 was analyzed using immunochemistry, ELISA, immunofluorescence, and Western blotting. The results showed that rotavirus infection led to an increased accumulation of the cellular proteins studied and ROS. The virus infection-induced accumulation of the cellular proteins studied and ROS was reduced upon pioglitazone treatment, causing also a concomitant reduction of the infectious virion yield. We hypothesized that rotavirus infection is benefiting from the induction of a host cell proinflammatory response and that the interference of the inflammatory pathways involved leads to decreased infection. PMID:27382365

  17. Recurrent rotavirus diarrhoea outbreaks in a stud farm, in Italy.

    PubMed

    Monini, M; Biasin, A; Valentini, S; Cattoli, G; Ruggeri, F M

    2011-04-21

    A total of 47 stool samples were collected at the same stud farm from young foals with rotavirus diarrhoea and from their stud mares. Illness involved foals during three consecutive winter seasons. Infection in the farm appeared firstly in January-February 2008. After vanishing in the warm seasons, cases reappeared in March 2009 and 2010. Determination of the rotavirus G- and P-types was carried out using nested RT-PCR in samples collected in 2009 and 2010. A total of 19 of 47 samples resulted positive for rotavirus. The G type was determined in 19/47 samples, whereas the P genotype was determined in 17/47 samples. All equine strains presented a G14 VP7 in combination with a P[12] VP4, suggesting persistence of the same viral strain in the stud farm, during at least two consecutive winter periods. Sequence analysis of the genes encoding the outer capsid rotavirus proteins VP7 and VP4 revealed that the virus had a close relationship between strains recently isolated in the rest of Europe. PMID:21129862

  18. Distribution of serotypes of human rotavirus in different populations.

    PubMed Central

    Woods, P A; Gentsch, J; Gouvea, V; Mata, L; Santosham, M; Bai, Z S; Urasawa, S; Glass, R I

    1992-01-01

    Serotyping is a useful tool to study the epidemiologic characteristics of rotaviruses in large populations and to assess the need for a vaccine to protect against all strains. By using an enzyme immunoassay with serotype-specific monoclonal antibodies to the four most common rotavirus serotypes, we analyzed 1,183 rotavirus-positive specimens from 16 stool collections in eight countries on four continents that were obtained from 1978 to 1989. Of the 926 strains (78%) that could be serotyped, 48% were serotype 1, 8% were serotype 2, 15% were serotype 3, and 7% were serotype 4. Twenty-two percent had insufficient numbers of double-shelled virus particles to react with the monoclonal antibody of the VP4 rotavirus protein and therefore could not be serotyped. Our results indicate that vaccines being developed must provide the greatest coverage against serotype 1 and that the serotype distribution cannot be predicted currently by the geographic area or prevalence in the preceding year. PMID:1315333

  19. In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments

    PubMed Central

    Aladin, Farah; Einerhand, Alexandra W. C.; Bouma, Janneke; Bezemer, Sandra; Hermans, Pim; Wolvers, Danielle; Bellamy, Kate; Frenken, Leon G. J.; Gray, Jim; Iturriza-Gómara, Miren

    2012-01-01

    Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains. PMID:22403728

  20. Rotavirus antagonizes cellular antiviral responses by inhibiting the nuclear accumulation of STAT1, STAT2, and NF-kappaB.

    PubMed

    Holloway, Gavan; Truong, Thanhmai T; Coulson, Barbara S

    2009-05-01

    A vital arm of the innate immune response to viral infection is the induction and subsequent antiviral effects of interferon (IFN). Rotavirus reduces type I IFN induction in infected cells by the degradation of IFN regulatory factors. Here, we show that the monkey rotavirus RRV and human rotavirus Wa also block gene expression induced by type I and II IFNs through a mechanism allowing signal transducer and activator of transcription 1 (STAT1) and STAT2 activation but preventing their nuclear accumulation. In infected cells, this may allow rotavirus to block the antiviral actions of IFN produced early in infection or by activated immune cells. As the intracellular expression of rotavirus nonstructural proteins NSP1, NSP3, and NSP4 individually did not inhibit IFN-stimulated gene expression, their involvement in this process is unlikely. RRV and Wa rotaviruses also prevented the tumor necrosis factor alpha-stimulated nuclear accumulation of NF-kappaB and NF-kappaB-driven gene expression. In addition, NF-kappaB was activated by rotavirus infection, confirming earlier findings by others. As NF-kappaB is important for the induction of IFN and other cytokines during viral infection, this suggests that rotavirus prevents cellular transcription as a means to evade host responses. To our knowledge, this is the first report of the use of this strategy by a double-stranded RNA virus. PMID:19244315

  1. Mice develop effective but delayed protective immune responses when immunized as neonates either intranasally with nonliving VP6/LT(R192G) or orally with live rhesus rotavirus vaccine candidates.

    PubMed

    VanCott, John L; Prada, Anne E; McNeal, Monica M; Stone, Susan C; Basu, Mitali; Huffer, Bert; Smiley, Kristi L; Shao, Mingyuan; Bean, Judy A; Clements, John D; Choi, Anthony H-C; Ward, Richard L

    2006-05-01

    Rotavirus vaccines are delivered early in life, when the immune system is immature. To determine the effects of immaturity on responses to candidate vaccines, neonatal (7 days old) and adult mice were immunized with single doses of either Escherichia coli-expressed rotavirus VP6 protein and the adjuvant LT(R192G) or live rhesus rotavirus (RRV), and protection against fecal rotavirus shedding following challenge with the murine rotavirus strain EDIM was determined. Neonatal mice immunized intranasally with VP6/LT(R192G) were unprotected at 10 days postimmunization (dpi) and had no detectable rotavirus B-cell (antibody) or CD4(+) CD8(+) T-cell (rotavirus-inducible, Th1 [gamma interferon and interleukin-2 {IL-2}]-, Th2 [IL-5 and IL-4]-, or ThIL-17 [IL-17]-producing spleen cells) responses. However, by 28 and 42 dpi, these mice were significantly (P >or= 0.003) protected and contained memory rotavirus-specific T cells but produced no rotavirus antibody. In contrast, adult mice were nearly fully protected by 10 dpi and contained both rotavirus immunoglobulin G and memory T cells. Neonates immunized orally with RRV were also less protected (P=0.01) than adult mice by 10 dpi and produced correspondingly less rotavirus antibody. Both groups contained few rotavirus-specific memory T cells. Protection levels by 28 dpi for neonates or adults were equal, as were rotavirus antibody levels. This report introduces a neonatal mouse model for active protection studies with rotavirus vaccines. It indicates that, with time, neonatal mice develop full protection after intranasal immunization with VP6/LT(R192G) or oral immunization with a live heterologous rotavirus and supports reports that protection depends on CD4(+) T cells or antibody, respectively. PMID:16641286

  2. Rotavirus virus-like particles as surrogates in environmental persistence and inactivation studies.

    PubMed

    Caballero, Santiago; Abad, F Xavier; Loisy, Fabienne; Le Guyader, Françoise S; Cohen, Jean; Pintó, Rosa M; Bosch, Albert

    2004-07-01

    Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20 degrees C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20 degrees C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced. PMID:15240262

  3. Analysis of host range restriction determinants in the rabbit model: comparison of homologous and heterologous rotavirus infections.

    PubMed

    Ciarlet, M; Estes, M K; Barone, C; Ramig, R F; Conner, M E

    1998-03-01

    The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV x D (G1), RRV x DS-1 (G2), and RRV x ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10(3) 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction

  4. A reverse evidence of rotavirus vaccines impact.

    PubMed

    Martinón-Torres, Federico; Aramburo, Angela; Martinón-Torres, Nazareth; Cebey, Miriam; Seoane-Pillado, María Teresa; Redondo-Collazo, Lorenzo; Martinón-Sánchez, Jose Maria

    2013-06-01

    In 2010, and due to a quality problem identified in the vaccine manufacture, the rotavirus (RV) vaccination was withheld in Spain during 5 months. Our study aimed to evaluate the impact that this sudden cease had on rotavirus acute gastroenteritis (RAGE) hospitalizations. An increase in RAGE hospitalization was observed in parallel to the drop in vaccine coverage. Here, we report the first reverse evidence of rotavirus vaccine impact. PMID:23836258

  5. A reverse evidence of rotavirus vaccines impact

    PubMed Central

    Martinón-Torres, Federico; Aramburo, Angela; Martinón-Torres, Nazareth; Cebey, Miriam; Seoane-Pillado, María Teresa; Redondo-Collazo, Lorenzo; Martinón-Sánchez, Jose Maria

    2013-01-01

    In 2010, and due to a quality problem identified in the vaccine manufacture, the rotavirus (RV) vaccination was withheld in Spain during 5 months. Our study aimed to evaluate the impact that this sudden cease had on rotavirus acute gastroenteritis (RAGE) hospitalizations. An increase in RAGE hospitalization was observed in parallel to the drop in vaccine coverage. Here, we report the first reverse evidence of rotavirus vaccine impact. PMID:23836258

  6. New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

    PubMed Central

    Rodríguez, Javier M.; Chichón, Francisco J.; Martín-Forero, Esther; González-Camacho, Fernando; Carrascosa, José L.; Castón, José R.; Luque, Daniel

    2014-01-01

    The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry. PMID:24873828

  7. Construction and Characterization of Human Rotavirus Recombinant VP8* Subunit Parenteral Vaccine Candidates

    PubMed Central

    Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W.; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka

    2012-01-01

    Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in E. coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., (P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines. PMID:22885016

  8. Detection of Novel Rotavirus Strain by Vaccine Postlicensure Surveillance

    PubMed Central

    Teel, Elizabeth N.; Mijatovic-Rustempasic, Slavica; Payne, Daniel C.; Roy, Sunando; Foytich, Kimberly; Parashar, Umesh D.; Gentsch, Jon R.; Bowen, Michael D.

    2013-01-01

    Surveillance for rotavirus-associated diarrhea after implementation of rotavirus vaccination can assess vaccine effectiveness and identify disease-associated genotypes. During active vaccine postlicensure surveillance in the United States, we found a novel rotavirus genotype, G14P[24], in a stool sample from a child who had diarrhea. Unusual rotavirus strains may become more prevalent after vaccine implementation. PMID:23876297

  9. Comparative study on the mechanisms of rotavirus inactivation by sodium dodecyl sulfate and ethylenediaminetetraacetate

    SciTech Connect

    Ward, R.L.; Ashley, C.S.

    1980-06-01

    This report describes a comparative study on the effects of the anionic detergent sodium dodecyl sulfate and the chelating agent ethylenediaminetetraacetate on purified rotavirus SA-11 particles. Both chemicals readily inactivated rotavirus at quite low concentrations and under very mild conditions. In addition, both agents modified the viral capsid and prevented the adsorption of inactivated virions to cells. Capsid damage by ethylenediaminetetraacetate caused a shift in the densities of rotavirions from about l.35 to about 1.37 g/ml and a reduction in their sedimentation coefficients. Sodium dodcyl sulfate, on the other hand, did not detectably alter either of these physical properties of rotavirions. Both agents caused some alteration of the isoelectric points of the virions. Finally, analysis of rotavirus proteins showed that ethylenediaminetetraacetate caused the loss of two protein peaks from the electrophoretic pattern of virions but sodium dodecyl sulfate caused the loss of only one of these same protein peaks.

  10. Serological detection and analysis of anti-VP1 responses against various enteroviruses (EV) (EV-A, EV-B and EV-C) in Chinese individuals.

    PubMed

    Gao, Caixia; Ding, Yingying; Zhou, Peng; Feng, Jiaojiao; Qian, Baohua; Lin, Ziyu; Wang, Lili; Wang, Jinhong; Zhao, Chunyan; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Rui, Bing; Pan, Wei

    2016-01-01

    The overall serological prevalence of EV infections based on ELISA remains unknown. In the present study, the antibody responses against VP1 of the EV-A species (enterovirus 71 (EV71), Coxsackievirus A16 (CA16), Coxsackievirus A5 (CA5) and Coxsackievirus A6 (CA6)), of the EV-B species (Coxsackievirus B3 (CB3)), and of the EV-C species (Poliovirus 1 (PV1)) were detected and analyzed by a NEIBM (novel evolved immunoglobulin-binding molecule)-based ELISA in Shanghai blood donors. The serological prevalence of anti-CB3 VP1 antibodies was demonstrated to show the highest level, with anti-PV1 VP1 antibodies at the second highest level, and anti-CA5, CA6, CA16 and EV71 VP1 antibodies at a comparatively low level. All reactions were significantly correlated at different levels, which were approximately proportional to their sequence similarities. Antibody responses against EV71 VP1 showed obvious differences with responses against other EV-A viruses. Obvious differences in antibody responses between August 2013 and May 2014 were revealed. These findings are the first to describe the detailed information of the serological prevalence of human antibody responses against the VP1 of EV-A, B and C viruses, and could be helpful for understanding of the ubiquity of EV infections and for identifying an effective approach for seroepidemiological surveillance based on ELISA. PMID:26917423

  11. Protection against Foot-and-Mouth Disease Virus in Guinea Pigs via Oral Administration of Recombinant Lactobacillus plantarum Expressing VP1

    PubMed Central

    Wang, Miao; Pan, Li; Zhou, Peng; Lv, Jianliang; Zhang, Zhongwang; Wang, Yonglu; Zhang, Yongguang

    2015-01-01

    Mucosal vaccination is an effective strategy for generating antigen-specific immune responses against mucosal infections of foot-and-mouth disease virus (FMDV). In this study, Lactobacillus plantarum strains NC8 and WCFS1 were used as oral delivery vehicles containing a pSIP411-VP1 recombinant plasmid to initiate mucosal and systemic immune responses in guinea pigs. Guinea pigs were orally vaccinated (three doses) with NC8-pSIP411, NC8-pSIP411-VP1, WCFS1-pSIP411, WCFS1-pSIP411-VP1 or milk. Animals immunized with NC8-pSIP411-VP1 and WCFS1-pSIP411-VP1 developed high levels of antigen-specific serum IgG, IgA, IgM, mucosal secretory IgA (sIgA) and neutralizing antibodies, and revealed stronger cell-mediated immune responses and enhanced protection against FMDV challenge compared with control groups. The recombinant pSIP411-VP1 effectively improved immunoprotection against FMDV in guinea pigs. PMID:26629822

  12. Marker vaccine potential of a foot-and-mouth disease virus with a partial VP1 G-H loop deletion.

    PubMed

    Fowler, V L; Knowles, N J; Paton, D J; Barnett, P V

    2010-04-26

    Previous work in cattle and pigs demonstrated that protection against foot-and-mouth disease (FMD) could be achieved following vaccination with chimeric foot-and-mouth disease virus (FMDV) vaccines, in which the VP1 G-H loop had been substituted with that from another serotype. This indicated that the VP1 G-H loop may not be essential for the protection of natural hosts against FMDV. If this could be substantiated there would be potential to develop FMD marker vaccines, characterised by the absence of this region. Here, we investigate the serological responses to vaccination with a virus with a partial VP1 G-H loop deletion in order to determine the likelihood of achieving protection and the potential of this virus as a marker vaccine. Inactivated, oil adjuvanted, vaccines, consisting of chemically inactivated virus with or without a partially deleted VP1 G-H loop, were used to immunise cattle. Serum was collected on days 0, 7, 14 and 21 and antibody titres calculated using the virus neutralisation test (VNT) to estimate the likelihood of protection. We predict a good likelihood that cattle vaccinated with a vaccine characterised by a partial VP1 G-H loop would be protected against challenge with the same virus containing the VP1 G-H loop. We also present evidence on the potential of such a construct to act as a marker vaccine, when used in conjunction with a novel serological test. PMID:20199761

  13. Serological detection and analysis of anti-VP1 responses against various enteroviruses (EV) (EV-A, EV-B and EV-C) in Chinese individuals

    PubMed Central

    Gao, Caixia; Ding, Yingying; Zhou, Peng; Feng, Jiaojiao; Qian, Baohua; Lin, Ziyu; Wang, Lili; Wang, Jinhong; Zhao, Chunyan; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Rui, Bing; Pan, Wei

    2016-01-01

    The overall serological prevalence of EV infections based on ELISA remains unknown. In the present study, the antibody responses against VP1 of the EV-A species (enterovirus 71 (EV71), Coxsackievirus A16 (CA16), Coxsackievirus A5 (CA5) and Coxsackievirus A6 (CA6)), of the EV-B species (Coxsackievirus B3 (CB3)), and of the EV-C species (Poliovirus 1 (PV1)) were detected and analyzed by a NEIBM (novel evolved immunoglobulin-binding molecule)-based ELISA in Shanghai blood donors. The serological prevalence of anti-CB3 VP1 antibodies was demonstrated to show the highest level, with anti-PV1 VP1 antibodies at the second highest level, and anti-CA5, CA6, CA16 and EV71 VP1 antibodies at a comparatively low level. All reactions were significantly correlated at different levels, which were approximately proportional to their sequence similarities. Antibody responses against EV71 VP1 showed obvious differences with responses against other EV-A viruses. Obvious differences in antibody responses between August 2013 and May 2014 were revealed. These findings are the first to describe the detailed information of the serological prevalence of human antibody responses against the VP1 of EV-A, B and C viruses, and could be helpful for understanding of the ubiquity of EV infections and for identifying an effective approach for seroepidemiological surveillance based on ELISA. PMID:26917423

  14. Rotavirus vaccines: successes and challenges.

    PubMed

    Glass, Roger I; Parashar, Umesh; Patel, Manish; Gentsch, Jon; Jiang, Baoming

    2014-01-01

    Since 2006, the availability of two new rotavirus vaccines has raised enthusiasm to consider the eventual control and elimination of severe rotavirus diarrhea through the global use of vaccines. Rotavirus remains the most severe cause of acute diarrhea in children worldwide responsible for several hundred thousands of deaths in low income countries and up to half of hospital admissions for diarrhea around the world. The new vaccines have been recommended by WHO for all infants and in more than 47 countries, their introduction into routine childhood immunization programs has led to a remarkable decline in hospital admissions and even deaths within 3 years of introduction. Challenges remain with issues of vaccine finance globally and the problem that these live oral vaccines perform less well in low income settings where they are needed most. Ongoing research that will accompany vaccine introduction might help address these issues of efficacy and new vaccines and novel financing schemes may both help make these vaccines universally available and affordable in the decade. PMID:24156947

  15. Identification of the interaction of VP1 with GM130 which may implicate in the pathogenesis of CVB3-induced acute pancreatitis

    PubMed Central

    Li, Xiuzhen; Xia, Yanhua; Huang, Shengping; Liu, Fadi; Ying, Ying; Xu, Qiufang; Liu, Xin; Jin, Guili; Papasian, Christopher J.; Chen, Jack; Fu, Mingui; Huang, Xiaotian

    2015-01-01

    Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans. Although the susceptibility of CVB3-induced acute pancreatitis is age-dependent, the underlying mechanisms remain unclear. Here we identified the host factor Golgi matrix protein 130 (GM130) as a novel target of CVB3 during CVB3-induced acute pancreatitis. The viral protein VP1 interacted with GM130, disrupted GM130-GRASP65 complexes, and caused GM130 degradation, which may lead to disruption of the Golgi ribbon and development of acute pancreatitis in mice. Interestingly, the expression level of GM130 in mouse pancreas was age-dependent, which was nicely correlated with the age-associated susceptibility of CVB3-induced acute pancreatitis. Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells. Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis. PMID:26314804

  16. Establishment of indirect immunofluorescence assay for rotavirus.

    PubMed

    Tao, J; Zhang, J; Liu, X; Jin, H; Jiang, C; Yin, Y

    2016-03-01

    Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a significant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most effective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofluorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. The activating concentration of trypsin was 1 µg/ml for healthy monolayers of MA104 cells at 100% confluence. After incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fluorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID50-ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certification of rotavirus vaccine production. PMID:26982471

  17. Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

    PubMed

    Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

    2016-08-28

    The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR. PMID:27197668

  18. Longitudinal survey of rotavirus infection in calves.

    PubMed

    McNulty, M S; Logan, E F

    1983-10-01

    A longitudinal survey of rotavirus infection in heifer calves was carried out on a closed Friesian dairy herd over two successive calving seasons. Rotavirus was detected by electron microscopy in the faeces of 45 of 57 (79 per cent) calves examined. On average the virus was first detected at 6.1 days of age. Clinically the disease associated with rotavirus infection was of mild to moderate severity. Only one infected calf required intravenous fluid therapy. Diarrhoea or excretion of abnormal faeces was associated with rotavirus infection in 58 per cent of infected calves, while in the remaining 42 per cent infection was subclinical. The cycle of rotavirus infection was broken by thorough cleansing and disinfection of the calf house. PMID:6316619

  19. Burden of Norovirus and Rotavirus in Children After Rotavirus Vaccine Introduction, Cochabamba, Bolivia.

    PubMed

    McAtee, Casey L; Webman, Rachel; Gilman, Robert H; Mejia, Carolina; Bern, Caryn; Apaza, Sonia; Espetia, Susan; Pajuelo, Mónica; Saito, Mayuko; Challappa, Roxanna; Soria, Richard; Ribera, Jose P; Lozano, Daniel; Torrico, Faustino

    2016-01-01

    The effectiveness of rotavirus vaccine in the field may set the stage for a changing landscape of diarrheal illness affecting children worldwide. Norovirus and rotavirus are the two major viral enteropathogens of childhood. This study describes the prevalence of norovirus and rotavirus 2 years after widespread rotavirus vaccination in Cochabamba, Bolivia. Stool samples from hospitalized children with acute gastroenteritis (AGE) and outpatients aged 5-24 months without AGE were recruited from an urban hospital serving Bolivia's third largest city. Both viruses were genotyped, and norovirus GII.4 was further sequenced. Norovirus was found much more frequently than rotavirus. Norovirus was detected in 69/201 (34.3%) of specimens from children with AGE and 13/71 (18.3%) of those without diarrhea. Rotavirus was detected in 38/201 (18.9%) of diarrheal specimens and 3/71 (4.2%) of non-diarrheal specimens. Norovirus GII was identified in 97.8% of norovirus-positive samples; GII.4 was the most common genotype (71.4% of typed specimens). Rotavirus G3P[8] was the most prevalent rotavirus genotype (44.0% of typed specimens) and G2P[4] was second most prevalent (16.0% of typed specimens). This community is likely part of a trend toward norovirus predominance over rotavirus in children after widespread vaccination against rotavirus. PMID:26598569

  20. Roles of VP4 and NSP1 in determining the distinctive replication capacities of simian rotavirus RRV and bovine rotavirus UK in the mouse biliary tract.

    PubMed

    Feng, Ningguo; Sen, Adrish; Wolf, Marie; Vo, Phuoc; Hoshino, Yasutaka; Greenberg, Harry B

    2011-03-01

    Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1. Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4. Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/β and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1. PMID:21191030

  1. Innate immune responses to rotavirus infection in macrophages depend on MAVS but involve neither the NLRP3 inflammasome nor JNK and p38 signaling pathways.

    PubMed

    Di Fiore, Izabel J M; Holloway, Gavan; Coulson, Barbara S

    2015-10-01

    Rotavirus infection is a major cause of life-threatening infantile gastroenteritis. The innate immune system provides an immediate mechanism of suppressing viral replication and is necessary for an effective adaptive immune response. Innate immunity involves host recognition of viral infection and establishment of a powerful antiviral state through the expression of pro-inflammatory cytokines such as type-1 interferon (IFN). Macrophages, the front-line cells of innate immunity, produce IFN and other cytokines in response to viral infection. However, the role of macrophages during rotavirus infection is not well defined. We demonstrate here that RRV rotavirus triggers the production of proinflammatory cytokines from mouse bone marrow-derived macrophages. IFN and antiviral cytokine production was abolished in rotavirus-infected MAVS (-/-) macrophages. This indicates that rotavirus triggers innate immunity in macrophages through RIG-I and/or MDA5 viral recognition, and MAVS signaling is essential for cytokine responses in macrophages. Rotavirus induced IFN expression in both wild type and MDA5 (-/-) macrophages, showing that MDA5 is not essential for IFN secretion following infection, and RIG-I and MDA5 may act redundantly in promoting rotavirus recognition. Interestingly, rotavirus neither stimulated mitogen-activated protein kinases p38 and JNK nor activated the NLRP3 inflammasome, demonstrating that these components might not be involved in innate responses to rotavirus infection in macrophages. Our results indicate that rotavirus elicits intracellular signaling in macrophages, resulting in the induction of IFN and antiviral cytokines, and advance our understanding of the involvement of these cells in innate responses against rotavirus. PMID:26079065

  2. The Evolution of Vp1 Gene in Enterovirus C Species Sub-Group That Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

    PubMed Central

    Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

    2014-01-01

    Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific ‘signature’ amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific ‘signature’ amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared. PMID:24695547

  3. Therapeutics Insight with Inclusive Immunopharmacology Explication of Human Rotavirus A for the Treatment of Diarrhea

    PubMed Central

    Hossain, Mohammad Uzzal; Hashem, Abu; Keya, Chaman Ara; Salimullah, Md.

    2016-01-01

    Rotavirus is the most common cause of severe infant and childhood diarrhea worldwide, and the morbidity and mortality rate is going to be outnumbered in developing countries like Bangladesh. To mitigate this substantial burden of disease, new therapeutics such as vaccine and drug are swiftly required against rotavirus. The present therapeutics insight study was performed with comprehensive immunoinformatics and pharmacoinformatics approach. T and B-cell epitopes were assessed in the conserved region of outer capsid protein VP4 among the highly reviewed strains from different countries including Bangladesh. The results suggest that epitope SU1 (TLKNLNDNY) could be an ideal candidate among the predicted five epitopes for both T and B-cell epitopes for the development of universal vaccine against rotavirus. This research also suggests five novel drug compounds from medicinal plant Rhizophora mucronata Lamk. for better therapeutics strategies against rotavirus diarrhea based on 3D structure building, pharmacophore, ADMET, and QSAR properties. The exact mode of action between drug compounds and target protein VP4 were revealed by molecular docking analysis. Drug likeness and oral bioavailability further confirmed the effectiveness of the proposed drugs against rotavirus diarrhea. This study might be implemented for experimental validation to facilitate the novel vaccine and drug design. PMID:27445802

  4. Overexpression of a Populus trichocarpa H+-pyrophosphatase gene PtVP1.1 confers salt tolerance on transgenic poplar.

    PubMed

    Yang, Y; Tang, R J; Li, B; Wang, H H; Jin, Y L; Jiang, C M; Bao, Y; Su, H Y; Zhao, N; Ma, X J; Yang, L; Chen, S L; Cheng, X H; Zhang, H X

    2015-06-01

    The Arabidopsis vacuolar H(+)-pyrophosphatase (AVP1) has been well studied and subsequently employed to improve salt and/or drought resistance in herbaceous plants. However, the exact function of H(+)-pyrophosphatase in woody plants still remains unknown. In this work, we cloned a homolog of type I H(+)-pyrophosphatase gene, designated as PtVP1.1, from Populus trichocarpa, and investigated its function in both Arabidopsis and poplar. The deduced translation product PtVP1.1 shares 89.74% identity with AVP1. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR analyses revealed a ubiquitous expression pattern of PtVP1.1 in various tissues, including roots, stems, leaves and shoot tips. Heterologous expression of PtVP1.1 rescued the retarded-root-growth phenotype of avp1, an Arabidopsis knock out mutant of AVP1, on low carbohydrate medium. Overexpression of PtVP1.1 in poplar (P. davidiana × P. bolleana) led to more vigorous growth of transgenic plants in the presence of 150 mM NaCl. Microsomal membrane vesicles derived from PtVP1.1 transgenic plants exhibited higher H(+)-pyrophosphatase hydrolytic activity than those from wild type (WT). Further studies indicated that the improved salt tolerance was associated with a decreased Na(+) and increased K(+) accumulation in the leaves of transgenic plants. Na(+) efflux and H(+) influx in the roots of transgenic plants were also significantly higher than those in the WT plants. All these results suggest that PtVP1.1 is a functional counterpart of AVP1 and can be genetically engineered for salt tolerance improvement in trees. PMID:25877769

  5. VP1 crystal structure-guided exploration and optimization of 4,5-dimethoxybenzene-based inhibitors of rhinovirus 14 infection.

    PubMed

    Da Costa, Laurène; Roche, Manon; Scheers, Els; Coluccia, Antonio; Neyts, Johan; Terme, Thierry; Leyssen, Pieter; Silvestri, Romano; Vanelle, Patrice

    2016-06-10

    Human rhinoviruses (HRV) are the predominant cause of common colds and flu-like illnesses, but are also responsible for virus-induced exacerbations of asthma and chronic obstructive pulmonary disease. However, to date, no drug has been approved yet for clinical use. In this study, we present the results of the structure-based lead optimization of a class of new small-molecule inhibitors that we previously reported to bind into the pocket beneath the canyon of the VP1 protein. A small series of analogues that we designed based on the available structure and interaction data were synthesized and evaluated for their potency to inhibit the replication of HRV serotype 14. 2-(4,5-Dimethoxy-2-nitrophenyl)-1-(4-(pyridin-4-yl)phenyl)ethanol (3v) was found to be a potent inhibitor exhibiting micromolar activity (EC50 = 3.4 ± 1.0 μM) with a toxicity for HeLa cells that was significantly lower than that of our previous hit (LPCRW_0005, CC50 = 104.0 ± 22.2 μM; 3v, CC50 > 263 μM). PMID:27049678

  6. Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

    PubMed

    Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong

    2016-02-01

    Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection. PMID:26790940

  7. Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli.

    PubMed

    Favacho, Alexsandra R M; Kurtenbach, Eleonora; Sardi, Silvia I; Gouvea, Vera S

    2006-04-01

    Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys. PMID:16275130

  8. Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1.

    PubMed

    Liu, Lin; Ma, Yongping; Zhou, Huicong; Wu, Mingjun

    2016-01-01

    The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system. PMID:27618897

  9. Rabbit model of rotavirus infection.

    PubMed Central

    Conner, M E; Estes, M K; Graham, D Y

    1988-01-01

    A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines. Images PMID:2833612

  10. Simian rhesus rotavirus is a unique heterologous (non-lapine) rotavirus strain capable of productive replication and horizontal transmission in rabbits.

    PubMed

    Ciarlet, M; Estes, M K; Conner, M E

    2000-05-01

    Simian rhesus rotavirus (RRV) is the only identified heterologous (non-lapine) rotavirus strain capable of productive replication at a high inoculum dose of virus (>10(8) p.f.u.) in rabbits. To evaluate whether lower doses of RRV would productively infect rabbits and to obtain an estimate of the 50% infectious dose, rotavirus antibody-free rabbits were inoculated orally with RRV at inoculum doses of 10(3), 10(5) or 10(7) p.f.u. Based on faecal virus antigen or infectious virus shedding, RRV replication was observed with inoculum doses of 10(7) and 10(5) p.f.u., but not 10(3) p.f.u. Horizontal transmission of RRV to one of three mock-inoculated rabbits occurred 4-5 days after onset of virus antigen shedding in RRV-infected rabbits. Rabbits infected at 10(7) and 10(5), but not 10(3), p.f.u. of RRV developed rotavirus-specific immune responses and were completely (100%) protected from lapine ALA rotavirus challenge. These data confirm that RRV can replicate productively and spread horizontally in rabbits. In attempts to elucidate the genetic basis of the unusual replication efficacy of RRV in rabbits, the sequence of the gene encoding the lapine non-structural protein NSP1 was determined. Sequence analysis of the NSP1 of three lapine rotaviruses revealed a high degree of amino acid identity (85-88%) with RRV. Since RRV and lapine strains also share similar VP7s (96-97%) and VP4s (69-70%), RRV might replicate efficiently in rabbits because of the high relatedness of these three gene products, each implicated in host range restriction. PMID:10769066

  11. A serotype 10 human rotavirus.

    PubMed

    Beards, G; Xu, L; Ballard, A; Desselberger, U; McCrae, M A

    1992-06-01

    Rotaviruses with genome rearrangements isolated from a chronically infected immunodeficient child (F. Hundley, M. McIntyre, B. Clark, G. Beards, D. Wood, I. Chrystie, and U. Desselberger, J. Virol 61:3365-3372, 1987) are the first recognized human isolates of serotype 10. This was shown by both a direct enzyme-linked immunosorbent assay and virus neutralization assays using serotype specific monoclonal antibodies. The serotype was confirmed by sequence analysis of the gene encoding VP7, which revealed a 96% amino acid homology to the bovine serotype 10 isolate B223. PMID:1320627

  12. [Universal vaccination for Rotavirus infection control].

    PubMed

    Mita, Valentin; Capanna, Alessandra; Gervasi, Giuseppe; Zaratti, Laura; Franco, Elisabetta

    2015-01-01

    Rotaviruses are the most common etiological cause for pediatric acute gastroenteritis, particularly in children under 5 years of age or immunocompromised. Since 2008, vaccination program has determined a decrease in Rotavirus-related hospitalization, outpatient's visits, emergency department visits and mortality. These indicators of illness for Rotaviruses diseases remain high in those countries where there is no access to rehydrating therapies. In Italy vaccine coverage is very low, even if the burden of RV disease is well known, and at present vaccination is offered free of charge in a single region. PMID:26519750

  13. Whole-genomic analysis of a human G1P[9] rotavirus strain reveals intergenogroup-reassortment events.

    PubMed

    Ghosh, Souvik; Shintani, Tsuzumi; Urushibara, Noriko; Taniguchi, Koki; Kobayashi, Nobumichi

    2012-08-01

    Group A rotavirus (RVA) strain K8 (RVA/Human-tc/JPN/K8/1977/G1P[9]) was found to have Wa-like VP7 and NSP1 genes and AU-1-like VP4 and NSP5 genes. To determine the exact origin and overall genetic makeup of this unusual RVA strain, the remaining genes (VP1-VP3, VP6 and NSP2-NSP4) of K8 were analysed in this study. Strain K8 exhibited a G1-P[9]-I1-R3-C3-M3-A1-N1-T3-E3-H3 genotype constellation, not reported previously. The VP6 and NSP2 genes of strain K8 were related closely to those of common human Wa-like G1P[8] and/or G3P[8] strains, whilst its VP1-VP3, NSP3 and NSP4 genes were related more closely to those of AU-1-like RVAs and/or AU-1-like genes of multi-reassortant strains than to those of other RVAs. Therefore, strain K8 might have originated from intergenogroup-reassortment events involving acquisition of four Wa-like genes, possibly from G1P[8] RVAs, by an AU-1-like P[9] strain. Whole-genomic analysis of strain K8 has provided important insights into the complex genetic diversity of RVAs. PMID:22592265

  14. Rotavirus vaccine - what you need to know

    MedlinePlus

    ... caused by other germs. Another virus called porcine circovirus (or parts of it) can be found in ... get rotavirus vaccine. Babies who have had a type of bowel blockage called "intussusception" should not get ...

  15. Rotavirus immune responses and correlates of protection

    PubMed Central

    Angel, Juana; Franco, Manuel A.; Greenberg, Harry B.

    2012-01-01

    Selected topics in the field of rotavirus immunity are reviewed focusing on recent developments that may improve efficacy and safety of current and future vaccines. Rotaviruses have developed multiple mechanisms to evade interferon-mediated innate immunity. Compared to more developed regions of the world, protection induced by natural infection and vaccination is reduced in developing countries where, among other factors, high viral challenge loads are common and where infants are infected at an early age. Studies in developing countries indicate that rotavirus-specific serum IgA levels are not an optimal correlate of protection following vaccination, and better correlates need to be identified. Protection against rotavirus following vaccination is substantially heterotypic; nonetheless, a role for homotypic immunity in selection of circulating post vaccination strains needs further study. PMID:22677178

  16. Are hospitalizations for rotavirus gastroenteritis associated with meteorologic factors?

    PubMed

    Hervás, D; Hervás-Masip, J; Rosell, A; Mena, A; Pérez, J L; Hervás, J A

    2014-09-01

    Local climatic factors might explain seasonal patterns of rotavirus infections, but few models have been proposed to determine the effects of weather conditions on rotavirus activity. Here, we study the association of meteorologic factors with rotavirus activity, as determined by the number of children hospitalized for rotavirus gastroenteritis on the Mediterranean island of Mallorca (Spain). We conducted a retrospective review of the medical records of children aged 0-5 years admitted for rotavirus gastroenteritis between January 2000 and December 2010. The number of rotavirus hospitalizations was correlated to temperature, humidity, rainfall, atmospheric pressure, water vapor pressure, wind speed, and solar radiation using regression and time-series techniques. A total of 311 patients were hospitalized for rotavirus gastroenteritis in the 11-year study period, with a seasonal pattern from December to June, and a peak incidence in February. After multiple regressions, weekly rotavirus activity could be explained in 82 % of cases (p < 0.001) with a one-week lag meteorologic model. Rotavirus activity was negatively associated to temperature and positively associated to atmospheric pressure, solar radiation, and wind speed. Temperature and solar radiation were the factors that contributed most to the model, with a peak rotavirus activity at 9 °C and 800 10KJ/m(2), respectively. In conclusion, hospitalization for rotavirus was strongly associated with mean temperature, but an association of rotavirus activity with solar radiation, atmospheric pressure, and wind speed was also demonstrated. This model predicted more than 80 % of rotavirus hospitalizations. PMID:24760250

  17. Impact of rotavirus vaccine on premature infants.

    PubMed

    Roué, Jean-Michel; Nowak, Emmanuel; Le Gal, Grégoire; Lemaitre, Thomas; Oger, Emmanuel; Poulhazan, Elise; Giroux, Jean-Dominique; Garenne, Armelle; Gagneur, Arnaud

    2014-10-01

    Infants born preterm are at a higher risk of complications and hospitalization in cases of rotavirus diarrhea than children born at term. We evaluated the impact of a rotavirus vaccination campaign (May 2007 to May 2010) on hospitalizations for rotavirus gastroenteritis in a population of children under 3 years old born prematurely (before 37 weeks of gestation) in the Brest University Hospital birth zone. Active surveillance from 2002 to 2006 and a prospective collection of hospitalizations for rotavirus diarrhea were initiated in the pediatric units of Brest University Hospital until May 2010. Numbers of hospitalizations for rotavirus diarrhea among the population of children born prematurely, before and after the start of the vaccination program, were compared using a Poisson regression model controlling for epidemic-to-epidemic variation. A total of 217 premature infants were vaccinated from 2007 to 2010. Vaccine coverage for a complete course of three doses was 41.9%. The vaccine safety in premature infants was similar to that in term infants. The vaccination program led to a division by a factor of 2.6 (95% confidence interval [CI], 1.3 to 5.2) in the number of hospitalizations for rotavirus diarrhea during the first two epidemic seasons following vaccine introduction and by a factor of 11 (95% CI, 3.5 to 34.8) during the third season. We observed significant effectiveness of the pentavalent rotavirus vaccine on the number of hospitalizations in a population of prematurely born infants younger than 3 years of age. A multicenter national study would provide better assessment of this impact. (This study [Impact of Systematic Infants Vaccination Against Rotavirus on Gastroenteritis Hospitalization: a Prospective Study in Brest District, France (IVANHOE)] has been registered at ClinicalTrials.gov under registration no. NCT00740935.). PMID:25080553

  18. The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to Type II Precursor Glycans

    PubMed Central

    Ramani, Sasirekha; Cortes-Penfield, Nicolas W.; Hu, Liya; Crawford, Sue E.; Czako, Rita; Smith, David F.; Kang, Gagandeep; Ramig, Robert F.; Le Pendu, Jacques; Prasad, B. V. Venkataram

    2013-01-01

    Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P[11] showed specific binding to glycans with the Galβ1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-restricted infectivity of neonatal viruses. PMID:23616650

  19. Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

    PubMed

    Foo, D G W; Ang, R X; Alonso, S; Chow, V T K; Quak, S H; Poh, C L

    2008-03-01

    A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6. PMID:18076666

  20. Fusion of the mouse IgG1 Fc domain to the VHH fragment (ARP1) enhances protection in a mouse model of rotavirus

    PubMed Central

    Günaydın, Gökçe; Yu, Shengze; Gräslund, Torbjörn; Hammarström, Lennart; Marcotte, Harold

    2016-01-01

    A variable fragment of a heavy chain antibody (VHH) directed against rotavirus, also referred to as anti-rotavirus protein 1 (ARP1), was shown to confer protection against rotavirus induced diarrhea in infant mouse model of rotavirus induced diarrhea. In this study, we have fused the mouse IgG1 Fc to ARP1 to improve the protective capacity of ARP1 by inducing an Fc-mediated effector function. We have shown that the Fc-ARP1 fusion protein confers significantly increased protection against rotavirus in a neonatal mouse model of rotavirus-induced diarrhea by reducing the prevalence, duration and severity of diarrhea and the viral load in the small intestines, suggesting that the Fc part of immunoglobulins may be engaged in Fc-mediated neutralization of rotavirus. Engineered conventional-like antibodies, by fusion of the Fc part of immunoglobulins to antigen-specific heavy-chain only VHH fragments, might be applied to novel antibody-based therapeutic approaches to enhance elimination of pathogens by activation of distinct effector signaling pathways. PMID:27439689

  1. Fusion of the mouse IgG1 Fc domain to the VHH fragment (ARP1) enhances protection in a mouse model of rotavirus.

    PubMed

    Günaydın, Gökçe; Yu, Shengze; Gräslund, Torbjörn; Hammarström, Lennart; Marcotte, Harold

    2016-01-01

    A variable fragment of a heavy chain antibody (VHH) directed against rotavirus, also referred to as anti-rotavirus protein 1 (ARP1), was shown to confer protection against rotavirus induced diarrhea in infant mouse model of rotavirus induced diarrhea. In this study, we have fused the mouse IgG1 Fc to ARP1 to improve the protective capacity of ARP1 by inducing an Fc-mediated effector function. We have shown that the Fc-ARP1 fusion protein confers significantly increased protection against rotavirus in a neonatal mouse model of rotavirus-induced diarrhea by reducing the prevalence, duration and severity of diarrhea and the viral load in the small intestines, suggesting that the Fc part of immunoglobulins may be engaged in Fc-mediated neutralization of rotavirus. Engineered conventional-like antibodies, by fusion of the Fc part of immunoglobulins to antigen-specific heavy-chain only VHH fragments, might be applied to novel antibody-based therapeutic approaches to enhance elimination of pathogens by activation of distinct effector signaling pathways. PMID:27439689

  2. Measuring indirect effects of rotavirus vaccine in low income countries.

    PubMed

    Bennett, Aisleen; Bar-Zeev, Naor; Cunliffe, Nigel A

    2016-08-17

    Widespread introduction of rotavirus vaccines has led to major reductions in the burden of rotavirus gastroenteritis worldwide. Vaccine effectiveness is diminished, however, in low income countries, that harbour the greatest burden of rotavirus attributed morbidity and mortality. Indirect effects of rotavirus vaccine (herd immunity and herd protection) could increase population level impact and improve vaccine cost effectiveness in such settings. While rotavirus vaccine indirect effects have been demonstrated in high and middle income countries, there are very little data from low income countries where force of infection, population structures and vaccine schedules differ. Targeted efforts to evaluate indirect effects of rotavirus vaccine in low income countries are required to understand the total impact of rotavirus vaccine on the global burden of rotavirus disease. PMID:27443593

  3. Epidemiology of rotavirus diarrhoea in Africa: a review to assess the need for rotavirus immunization.

    PubMed Central

    Cunliffe, N. A.; Kilgore, P. E.; Bresee, J. S.; Steele, A. D.; Luo, N.; Hart, C. A.; Glass, R. I.

    1998-01-01

    Rapid progress towards the development of rotavirus vaccines has prompted a reassessment of the disease burden of rotavirus diarrhoea in developing countries and the possible impact of these vaccines in reducing diarrhoeal morbidity and mortality among infants and young children. We examined the epidemiology and disease burden of rotavirus diarrhoea among hospitalized and clinic patients in African countries through a review of 43 published studies of the etiology of diarrhoea. The studies were carried out from 1975 through 1992, and only those in which a sample of more than 100 patients with diarrhoea were specifically screened for rotavirus by using an established diagnostic test were included. Rotavirus was detected in a median of 24% of children hospitalized for diarrhoea and in 23% who were treated as outpatients; 38% of the hospitalized patients with rotavirus were < 6 months and 81% were < 1 year of age. Rotavirus was detected year-round in nearly every country and generally exhibited distinct seasonal peaks during the dry months. In 5 countries where rotavirus strains had been G-typed, 74% of strains were of one of the four common serotypes (G1 to G4), G1 was the predominant serotype, and 26% were non-typeable. This cumulative experience from 15 African countries suggests that rotavirus is the most important cause of severe diarrhoea in African children and that most strains in circulation today belong to common G types that are included in reassortant vaccines. Wherever large numbers of cases of rotavirus diarrhoea occur early in infancy, immunization at birth may protect the children before their first symptomatic infection. PMID:9868844

  4. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  5. Association of Rotavirus Gastroenteritis with Histo-blood Group Antigens.

    PubMed

    Mohanty, E; Dwibedi, B; Kar, S K; Pandey, R M

    2016-07-01

    Association of rotavirus gastroenteritis with histo-blood group antigens in children younger than 5 years admitted with diarrhea (n=389) was studied. Distribution of blood groups in rotavirus positive (n=96) and rotavirus negative (n=51) diarrhea gastroenteritis cases did not show any susceptibility to any blood group; blood group O seemed to be protective. PMID:27508550

  6. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.).

    PubMed

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants. PMID:26284097

  7. Rotavirus-associated hospitalization and emergency department costs and rotavirus vaccine program impact☆

    PubMed Central

    Kilgore, April; Donauer, Stephanie; Edwards, Kathryn M.; Weinberg, Geoffrey A.; Payne, Daniel C.; Szilagyi, Peter G.; Rice, Marilyn; Cassedy, Amy; Ortega-Sanchez, Ismael R.; Parashar, Umesh D.; Staat, Mary Allen

    2015-01-01

    Objectives To determine the medical costs of laboratory-confirmed rotavirus hospitalizations and emergency department (ED) visits and estimate the economic impact of the rotavirus vaccine program. Patients and methods During 4 rotavirus seasons (2006–2009), children <3 years of age hospitalized or seen in the ED with laboratory-confirmed rotavirus were identified through active population-based rotavirus surveillance in three US counties. Medical costs were obtained from hospital and physician billing data, and factors associated with increased costs were examined. Annual national costs were estimated using rotavirus hospitalization and ED visit rates and medical costs for rotavirus hospitalizations and ED visits from our surveillance program for pre- (2006–2007) and post-vaccine (2008–2009) time periods. Results Pre-vaccine, for hospitalizations, the median medical cost per child was $3581, the rotavirus hospitalization rate was 22.1/10,000, with an estimated annual national cost of $91 million. Post-vaccine, the median medical cost was $4304, the hospitalization rate was 6.3/10,000 and the estimated annual national cost was $31 million. Increased costs were associated with study site, age <3 months, underlying medical conditions and an atypical acute gastroenteritis presentation. For ED visits, the pre-vaccine median medical cost per child was $574, the ED visit rate was 291/10,000 resulting in an estimated annual national cost of $192 million. Post-vaccine, the median medical cost was $794, the ED visit rate was 71/10,000 with an estimated annual national cost of $65 million. Conclusions After implementation of rotavirus immunization, the total annual medical costs decreased from $283 million to $96 million, an annual reduction of $187 million PMID:23845802

  8. Isolation and molecular characterisation of equine rotaviruses from Germany.

    PubMed

    Elschner, Mandy; Schrader, Christina; Hotzel, Helmut; Prudlo, Jutta; Sachse, Konrad; Eichhorn, Werner; Herbst, Werner; Otto, Peter

    2005-01-31

    A total of 26 rotavirus positive faecal samples of diarrhoeal foals, and 8 equine rotavirus isolates were examined. Viral RNA patterns were generated, G typing was performed by PCR, and a P[12]-specific DNA probe was developed for P typing. Furthermore, five equine rotavirus isolates were sequenced in the genomic regions coding for VP7 and part of VP4. Rotaviruses of genotype G3 P[12] were found in 22 faecal samples and G14 P[12] type could be found in 4 faecal samples. These findings confirm that in Germany G3 P[12] is the predominating type of equine rotaviruses. PMID:15627523

  9. Full genomic analysis of rabbit rotavirus G3P[14] strain N5 in China: identification of a novel VP6 genotype.

    PubMed

    Guo, Dongchun; Liu, Jiasen; Lu, Yan; Sun, Yan; Yuan, Dongwei; Jiang, Qian; Lin, Huan; Li, Changwen; Si, Changde; Qu, Liandong

    2012-10-01

    Group A rotaviruses (RVAs) are major pathogens associated with acute gastroenteritis in young children and in a wide variety of domestic animals. The full-length genome of a rabbit RVA strain, RVA/Rabbit-tc/CHN/N5/1992/G3P[14], showed a G3-P[14]-I17-R3-C3-M3-A9-N1-T1-E3-H2 genomic configuration. A novel VP6 genotype, I17, was confirmed by the Rotavirus Classification Working Group. Phylogenetic analyses revealed that strain N5 possessed VP1-3, VP7, NSP1-2 and NSP4 genes closely related to those of the simian strain TUCH, NSP3 and NSP5 genes closely related to the human strains Wa and 69M, and a VP4 gene closely related to the rabbit strain 30/96 and sheep strain OVR762. The RRV and TUCH shared their ancestry with canine/feline RVAs and showed a close relationship to the human T152/feline-like RVAs. Comparison with the genotypes of the simian strains TUCH and RRV, canine strains A79-10, CU-1, K9, feline strains Cat2 and Cat97, and human strains T152 and 69M showed that RVA/Rabbit-tc/CHN/N5/1992/G3P[14] was possibly of feline/canine origin, or was a multiple reassortment involving canine, feline and human rotaviruses. The sequencing and phylogenetic analyses of rotavirus genomes is critical to the elucidation of the patterns of virus evolution. PMID:22750552

  10. Rescue of noncultivatable human rotavirus by gene reassortment during mixed infection with ts mutants of a cultivatable bovine rotavirus.

    PubMed Central

    Greenberg, H B; Kalica, A R; Wyatt, R G; Jones, R W; Kapikian, A Z; Chanock, R M

    1981-01-01

    Fastidious human rotaviruses that did not undergo productive infection in tissue culture were rescued by genetic reassortment during mixed infection with a temperature-sensitive (ts) mutant of a cultivatable bovine rotavirus. In this manner, the genes of the fastidious rotavirus that restricted growth in vitro were replaced by the corresponding genes from a tissue culture-adapted rotavirus. We recovered genetically reassorted viruses that grew to high titer and were neutralized specifically by hyperimmune guinea pig type 1 or type 2 human rotavirus antiserum. Preliminary RNA analysis of these clones disclosed that they were indeed viruses with reassorted genes. Images PMID:6264442

  11. Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja Saponaria Molina

    PubMed Central

    Tam, Ka Ian; Roner, Michael R.

    2011-01-01

    Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide with approximately 300,000 pre-adolescent deaths each year. Quillaja saponins are a natural aqueous extract obtained from the Chilean soapbark tree. The extract is approved for use in humans by the FDA for use in beverages as a food addictive. We have demonstrated that Quillaja extracts have strong antiviral activities in vitro against six different viruses. In this study, we evaluated the in vivo antiviral activity of these extracts against rhesus rotavirus (RRV) using a mouse model. We established that at a dosage of 0.015 mg/mouse of saponin extract, RRV induced diarrhea can be significantly reduced from 79% to 11% when mice are exposed to 500 plaque-forming-units (PFU) for each of five consecutive days. Additionally, while a reduction of RRV induced diarrhea depended both on the concentration of virus introduced and on the amount of Quillaja extract given to each mouse, the severity and interval of diarrhea under a variety of conditions tested, in all the treated mice were greatly reduced when compared to those that did not receive the Quillaja extracts. Mechanistically, there is strong evidence that the Quillaja extracts are able to “block” rotavirus infection by inhibiting virus-host attachment through disruption of cellular membrane proteins and/or virus receptors. We believe that Quillaja extracts have promise as antivirals to reduce rotavirus infection and the severity of the disease in humans. PMID:21549151

  12. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

    SciTech Connect

    Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B. Palo Alto Veterans Administration Medical Center, CA ); Rubin, D.H. )

    1988-04-01

    Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

  13. African horse sickness virus serotype 4 antigens, VP1-1, VP2-2, VP4, VP7 and NS3, induce cytotoxic T cell responses in vitro.

    PubMed

    Faber, F E; van Kleef, M; Tshilwane, S I; Pretorius, A

    2016-07-15

    It was shown in a previous study that proliferating CD8+ T cells could be detected in immune horse peripheral blood mononuclear cells (PBMC) when stimulated with African horse sickness virus serotype 4 (AHSV4). In this study the cytotoxicity of CD8+ T cells were tested by using the fluorescent antigen-transfected target cells-cytotoxic T lymphocytes (FATT-CTL) assay, for both the virus and its individual proteins expressed in Escherichia coli. This CTL assay measures the killing of viral protein expressing cells. AHSV proteins were successfully expressed in E. coli using the pET102/D-TOPO expression vector and the effector cells were stimulated with these recombinant proteins or with live viable virulent AHSV4. The AHSV genes were amplified and cloned into the pIRES-hrGFP II (pGFPempty) vector and these plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used to nucleofect PBMC, the target cells. The elimination of antigen-GFP expressing cells by CTL was quantified by flowcytometry. VP1-1, VP2-2, VP4, VP7 and NS3, antigen-specific CD8+ T cells resulted in cell lysis suggesting that CTL may play a role in the immune response induced against the AHSV4 vaccine strain. PMID:27063332

  14. Effectiveness of rotavirus vaccines in preventing cases and hospitalizations due to rotavirus gastroenteritis in Navarre, Spain.

    PubMed

    Castilla, Jesús; Beristain, Xabier; Martínez-Artola, Víctor; Navascués, Ana; García Cenoz, Manuel; Alvarez, Nerea; Polo, Isabel; Mazón, Ana; Gil-Setas, Alberto; Barricarte, Aurelio

    2012-01-11

    Two rotavirus vaccines have been available since 2006. This study evaluates the effectiveness of these vaccines using a test-negative case-control design in Navarre, Spain. We included children 3-59 months of age who sought medical care for gastroenteritis and for whom stool samples were taken between January 2008 and June 2011. About 9% had received the pentavalent vaccine (RotaTeq) and another 8% received the monovalent vaccine (Rotarix). Cases were the 756 children with confirmed rotavirus and controls were the 6036 children who tested negative for rotavirus. Thirty-five percent of cases and 9% of controls had required hospitalization (p<0.0001). The adjusted effectiveness of complete vaccination was 78% (95% CI: 68-85%) in preventing rotavirus gastroenteritis and 83% (95% CI: 65-93%) in preventing hospitalization for rotavirus gastroenteritis. No differences between the two vaccines were detected (p=0.4523). Both vaccines were highly effective in preventing cases and hospital admissions in children due to rotavirus gastroenteritis. PMID:22122860

  15. Characterization of VP1 gene of coxsackievirus A16 prevalent among hand foot mouth disease suffered children in Taizhou, P. R. China, between 2010 and 2013.

    PubMed

    Ma, Zhilong; Zha, Jie

    2016-02-01

    A total of 453 strains of Coxsackievirus A16 (CV-A16) were screened out of 1,509 hand-foot-mouth disease (HFMD) samples collected in Taizhou during the period from 2010 to 2013. And between first quarter of 2011 and first quarter of 2013, an outbreak of CV-A16 was found among the HFMD sufferers in Taizhou. Phylogenic analysis of VP1 sequences indicated a major CV-A16 sub-group in Taizhou, whose change pattern of effective population sizes was found to be similar to the pattern of the actual percentage changes of CV-A16 during the outbreak over the same period. More importantly, the sub-group all displayed a Leu (L) to Met (M) mutation at site-23 of capsid VP1 which might be correlated with the outbreak of CV-A16 in Taizhou. PMID:26174468

  16. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.)

    PubMed Central

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet’s salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na+/H+ antiporter and H+-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na+ and K+ in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na+-toxicity for plants. PMID:26284097

  17. Patterns of polymorphism and divergence in the VP1 gene of enterovirus 71 circulating in the Asia-Pacific region between 1994 and 2013.

    PubMed

    Wu, Jun-Song; Zhao, Na; Pan, Hao; Wang, Cheng-Min; Wu, Bin; Zhang, Hong-Mei; He, Hong-Xuan; Liu, Dan; Amer, Said; Liu, She-Lan

    2013-11-01

    Enterovirus 71 has been implicated in several outbreaks of hand, foot and mouth disease in the Asia-Pacific region. The present study aimed to achieve comprehensive evolutionary dynamic aspects of EV71 during 1994-2013, based on phylogenetic analyses of the VP1 sequences. The results indicated that 4 genotypes, namely C4, C1, C2 and B4 are the predominant strains, especially in Southeast Asian countries. No common ancestor was shared in different countries. Fourteen sites of substitutions were detected in the VP1 gene sequences; including the most common sites related to neutralization at position V249I [47.1% (189/401)] and A289T [42.6% (171/401)]. However, the sites Q22H and Q22R associated with increased virulence were recognized only in 13.7% (55/401) and 18% (72/401), respectively. None of the above mutations seemed to become fixed because the ratio of Ka/Ks was greater than 1.0. Mutations K43E, A58T, S184T, and T240S could possibly change the spatial structure. Two mutations, G145E and T240S, could obviously affect the hydrophobicity of VP1 and thus alter the EV71 immunoreactivity. In conclusion, the VP1 gene of EV71 strains circulating in the Asia-Pacific region during 1994-2013, showed polymorphisms and divergence with very slow evolution rate, which may be one of the reasons for periodic outbreaks in this area. PMID:23933074

  18. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-01-01

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71. PMID:27188732

  19. Interactions between Multiple Genetic Determinants in the 5′ UTR and VP1 Capsid Control Pathogenesis of Chronic Post-Viral Myopathy caused by Coxsackievirus B1

    PubMed Central

    Sandager, Maribeth M.; Nugent, Jaime L.; Schulz, Wade L.; Messner, Ronald P.; Tam, Patricia E.

    2008-01-01

    Mice infected with coxsackievirus B1 Tucson (CVB1T) develop chronic, post-viral myopathy (PVM) with clinical manifestations of hind limb muscle weakness and myositis. The objective of the current study was to establish the genetic basis of myopathogenicity in CVB1T. Using a reverse genetics approach, full attenuation of PVM could only be achieved by simultaneously mutating four sites located at C706U in the 5′ untranslated region (5′ UTR) and at Y87F, V136A, and T276A in the VP1 capsid. Engineering these four myopathic determinants into an amyopathic CVB1T variant restored the ability to cause PVM. Moreover, these same four determinants controlled PVM expression in a second strain of mice, indicating that the underlying mechanism is operational in mice of different genetic backgrounds. Modeling studies predict that C706U alters both local and long-range pairing in the 5′ UTR, and that VP1 determinants are located on the capsid surface. However, these differences did not affect viral titers, temperature stability, pH stability, or the antibody response to virus. These studies demonstrate that PVM develops from a complex interplay between viral determinants in the 5′ UTR and VP1 capsid and have uncovered intriguing similarities between genetic determinants that cause PVM and those involved in pathogenesis of other enteroviruses. PMID:18029287

  20. A lattice-based model of rotavirus epidemics

    NASA Astrophysics Data System (ADS)

    Lara-Sagahón, A.; Govezensky, T.; Méndez-Sánchez, R. A.; José, M. V.

    2006-01-01

    The cyclic recurrence of childhood rotavirus epidemics in unvaccinated populations provides one of the best documented phenomena in population dynamics and can become a paradigm for epidemic studies. Herein we analyse the monthly incidence of rotavirus infection from the city of Melbourne, Australia during 1976-2003. We show that there is an inverse nonlinear relationship of the cumulative distribution of the number of cases per month in a log-log plot. It is also shown that the rate of transmission of rotavirus infection follows a symmetric distribution centered on zero. A wavelet phase analysis of rotavirus epidemics is also carried out. We test the hypothesis that rotavirus dynamics could be a realization of a forest-fire model with sparks and with immune trees. Some statistical properties of this model turn out to be similar to the above results of actual rotavirus data.

  1. Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM

    PubMed Central

    Chen, James Z.; Settembre, Ethan C.; Aoki, Scott T.; Zhang, Xing; Bellamy, A. Richard; Dormitzer, Philip R.; Harrison, Stephen C.; Grigorieff, Nikolaus

    2009-01-01

    Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 Å) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal “arms” of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca2+-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding. PMID:19487668

  2. Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

    PubMed Central

    Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

    2014-01-01

    ABSTRACT Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3′ consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCE Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its

  3. The effectiveness of rotavirus vaccine in preventing acute gastroenteritis during rotavirus seasons among Polish children

    PubMed Central

    Kieltyka, Agnieszka; Majewska, Renata; Augustyniak, Malgorzata

    2016-01-01

    Introduction Rotavirus is the main etiological cause of intestinal infections in children. Voluntary rotavirus vaccines were included in the Polish vaccination schedule in 2007. The aim of this study was to assess the effectiveness of a completed rotavirus vaccination course in preventing acute gastroenteritis in Polish infants during their first five years of life. Material and methods This was a retrospective cohort study conducted in Lesser Poland (Malopolska Province). The sample population included a group of 303 children who received the completed rotavirus vaccination course and 303 children not vaccinated against rotavirus. The date of the child's acute gastroenteritis diagnosis and his or her vaccination history were extracted from the physicians’ records. Each kind of diagnosed acute gastroenteritis during winter-spring rotavirus seasons was treated as the endpoint. The relative risk of having gastrointestinal infection was assessed using the hazard ratio from the Cox proportional hazards regression model. Results In the examined group, 96 (15.8%) children had winter-spring gastrointestinal infections. In the non-vaccinated children, the cumulative incidence of these infections in the first 5 years of life was 20.8%, whereas in the children vaccinated with Rotarix it was only 10.9%. Those who were vaccinated with Rotarix had a 44% reduction in the risk of a winter-spring acute gastroenteritis infection compared to those not vaccinated with Rotarix (p = 0.005). Birth weight less than 2500 g increased the risk of the infection twofold and also reached statistical significance (p = 0.044). Conclusions The results showed that Rotarix is effective in preventing acute gastroenteritis in Polish children during rotavirus seasons. PMID:27279856

  4. Three infants with rotavirus gastroenteritis complicated by severe gastrointestinal bleeding.

    PubMed

    Kawamura, Yoshiki; Miura, Hiroki; Mori, Yuji; Sugata, Ken; Nakajima, Yoichi; Yamamoto, Yasuto; Morooka, Masashi; Tsuge, Ikuya; Yoshikawa, Akiko; Taniguchi, Koki; Yoshikawa, Tetsushi

    2016-01-01

    Rotavirus gastroenteritis causes substantial morbidity and mortality worldwide in children. We report three infants with rotavirus gastroenteritis complicated by various severity of gastrointestinal bleeding. Two patients (cases 1 and 2) recovered completely without any specific treatments. One patient (case 3) died despite extensive treatments including a red blood cell transfusion and endoscopic hemostatic therapy. Rotavirus genotypes G1P[8] and G9P[8] were detected in cases 2 and 3, respectively. Rotavirus antigenemia levels were not high at the onset of melena, suggesting that systemic rotaviral infection does not play an important role in causing melena. PMID:26100228

  5. Asymptomatic rotavirus infections in England: prevalence, characteristics, and risk factors.

    PubMed

    Phillips, Gemma; Lopman, Ben; Rodrigues, Laura C; Tam, Clarence C

    2010-05-01

    Rotavirus is a major cause of infectious intestinal disease in young children; a substantial prevalence of asymptomatic infection has been reported across all age groups. In this study, the authors determined characteristics of asymptomatic rotavirus infection and potential risk factors for infection. Healthy persons were recruited at random from the general population of England during the Study of Infectious Intestinal Disease in England (1993-1996). Rotavirus infection was identified using reverse-transcription polymerase chain reaction. Multivariable logistic regression was used to compare exposures reported by participants with rotavirus infection with those of participants who tested negative. Multiple imputation was used to account for missing responses in the data set. The age-adjusted prevalence of asymptomatic rotavirus infection was 11%; prevalence was highest in children under age 18 years. Attendance at day care was a risk factor for asymptomatic rotavirus infection in children under age 5 years; living in a household with a baby that was still in diapers was a risk factor in older adults. The results suggest that asymptomatic rotavirus infection is transmitted through the same route as rotavirus infectious intestinal disease: person-to-person contact. More work is needed to understand the role of asymptomatic infections in transmission leading to rotavirus disease. PMID:20392863

  6. 75 FR 48706 - Proposed Vaccine Information Materials for Rotavirus Vaccine

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-11

    ... these materials is included in a December 17, 1999 Federal Register notice (64 FR 70914). Proposed..., rotavirus, hepatitis A, meningococcal, human papillomavirus (HPV), and trivalent influenza...

  7. Formation of empty B19 parvovirus capsids by the truncated minor capsid protein.

    PubMed Central

    Wong, S; Momoeda, M; Field, A; Kajigaya, S; Young, N S

    1994-01-01

    We previously reported that empty capsids of B19 parvovirus were formed by the major capsid protein (VP2) alone expressed in a baculovirus system, but the minor capsid protein (VP1), longer by 227 amino acids, alone did not form empty capsids. We report here further investigations of the constraints on capsid formation by truncated versions of VP1. Studies were performed with recombinant baculoviruses expressed in Sf9 cells. Severely shortened VP1, extended beyond the VP2 core sequence by about 70 amino acids of the unique region, formed capsids normal in appearance; longer versions of VP1 also formed capsids but did so progressively less efficiently and produced capsids of more markedly dysmorphic appearance as the VP1-unique region was lengthened. Images PMID:8207846

  8. Impact of rotavirus disease in Spain: an estimate of hospital admissions due to rotavirus.

    PubMed

    Visser, L E; Cano Portero, R; Gay, N J; Martínez Navarro, J F

    1999-01-01

    The epidemiology of rotavirus infection in Spain was investigated using information from existing surveillance systems. Reports from laboratories and data on hospital admissions were used to estimate the number of hospital admissions due to rotavirus infection. Between January 1989 and December 1995 there were 8265 reports of rotavirus identification in the Sistema de Información Microbiológica, a voluntary national laboratory surveillance system. Where age was reported, 89.2% were in children under 5 y old. This represents 21.8% of all pathogens identified in faecal specimens in children of this age group. Between January and December 1994 there were 5639 admissions coded as "infectious intestinal disease" and 8225 as "non-infective gastroenteritis" in children under 5 y of age in the Conjunto Mínimo Básico de Datos, an obligatory hospital admissions surveillance system which covers 83% of public hospitals. Using regression analysis of admission data and laboratory reports it was estimated that rotavirus infection accounted for 3519 (25.3%) of the hospital admissions. We estimate that 4239 admissions attributable to rotavirus occurred in children under 5 y of age in Spain in 1994, a rate of 2.5/1000 population in this age group. These estimates are lower than those published for other countries, where the burden of disease is considered high enough to warrant a national vaccination programme. A cost-effectiveness study with Spanish information is essential before embarking on a national vaccination programme. PMID:10088916

  9. Stress Response and Translation Control in Rotavirus Infection.

    PubMed

    López, Susana; Oceguera, Alfonso; Sandoval-Jaime, Carlos

    2016-01-01

    The general stress and innate immune responses are closely linked and overlap at many levels. The outcomes of these responses serve to reprogram host expression patterns to prevent viral invasions. In turn, viruses counter attack these cell responses to ensure their replication. The mechanisms by which viruses attempt to control host cell responses are as varied as the number of different virus families. One of the most recurrent strategies used by viruses to control the antiviral response of the cell is to hijack the translation machinery of the host, such that viral proteins are preferentially synthesized, while the expression of the stress and antiviral responses of the cell are blocked at the translation level. Here, we will review how rotaviruses, an important agent of acute severe gastroenteritis in children, overcome the stress responses of the cell to establish a productive infectious cycle. PMID:27338442

  10. Stress Response and Translation Control in Rotavirus Infection

    PubMed Central

    López, Susana; Oceguera, Alfonso; Sandoval-Jaime, Carlos

    2016-01-01

    The general stress and innate immune responses are closely linked and overlap at many levels. The outcomes of these responses serve to reprogram host expression patterns to prevent viral invasions. In turn, viruses counter attack these cell responses to ensure their replication. The mechanisms by which viruses attempt to control host cell responses are as varied as the number of different virus families. One of the most recurrent strategies used by viruses to control the antiviral response of the cell is to hijack the translation machinery of the host, such that viral proteins are preferentially synthesized, while the expression of the stress and antiviral responses of the cell are blocked at the translation level. Here, we will review how rotaviruses, an important agent of acute severe gastroenteritis in children, overcome the stress responses of the cell to establish a productive infectious cycle. PMID:27338442

  11. [Genomic Characterization of an Unusual Human G3P[3] Rotavirus with Multiple Cross-species Reassortment].

    PubMed

    Dong, Huijin; Qian, Yuan; Nong, Yi; Zhang, You; Mo, Zhaojun; Li, Rongcheng

    2016-03-01

    One unusual human G3P[3] group A rotavirus (RVA) strain M2-102 was identified in stool sample collected from a child with diarrhea in Guangxi Province, China in 2014. It is well known that G3P[3] is a genotype commonly identified in feline and canine RVAs. However, the preliminary phylogenetic analyses of the VP7 and VP4 genes of strain M2-102 indicated that these two genes were closely related to bat RVA strain MYAS33 and simian strain RRV, respectively, whereas both clustered distantly to feline/canine-like RVA strains. In this study, full genome sequencing and molecular analyses were conducted to obtain the true origin of strain M2-102. It was revealed that strain RVA/Human-wt/CHN/M2-102/2014/G3P[3] exhibited a G3-P[3]-I3-R3-C3-M3-A9-N3-T3-E3-H6 genotype constellation for VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes. Phylogenetic analyses revealed that 5 genes (VP7, VP1, VP2, NSP2 and NSP3) from strain M2-102 were closely related to those of bat strain MYAS33 from Yunnan Province which was thought a true bat RVA strain rather than a virus transmitted between species, while another 5 genes (VP4, VP3, NSP1, NSP4 and NSP5) clustered closely with those of simian strain RRV, yet the VP6 gene was closely related to that of human G3P[9] strain AU-1 and AU-1-like RVAs. The epidemiological data indicated that the child infected with M2-102 came from a countryside village, located in Dong Autonomous County of Sanjiang (subtropical hilly wooded area), Liuzhou city in Guangxi Province which might provide natural environment for reassortment events occurring among animal and human RVAs. Therefore, the data suggest that human strain M2-102 might originate from multiple reassortment events among bat, simian and human AU-1-like RVAs, yet it is not clear whether the genomic backbone based on bat MYAS33 (5 genes) and simian RRV (5 genes) like rotaviruses had been obtained through reassortment before being transmitted to the human. This is the first report on whole

  12. In Silico Study of Rotavirus VP7 Surface Accessible Conserved Regions for Antiviral Drug/Vaccine Design

    PubMed Central

    Ghosh, Ambarnil; Chattopadhyay, Shiladitya; Chawla-Sarkar, Mamta; Nandy, Papiya; Nandy, Ashesh

    2012-01-01

    Background Rotaviral diarrhoea kills about half a million children annually in developing countries and accounts for one third of diarrhea related hospitalizations. Drugs and vaccines against the rotavirus are handicapped, as in all viral diseases, by the rapid mutational changes that take place in the DNA and protein sequences rendering most of these ineffective. As of now only two vaccines are licensed and approved by the WHO (World Health Organization), but display reduced efficiencies in the underdeveloped countries where the disease is more prevalent. We approached this issue by trying to identify regions of surface exposed conserved segments on the surface glycoproteins of the virion, which may then be targeted by specific peptide vaccines. We had developed a bioinformatics protocol for these kinds of problems with reference to the influenza neuraminidase protein, which we have refined and expanded to analyze the rotavirus issue. Results Our analysis of 433 VP7 (Viral Protein 7 from rotavirus) surface protein sequences across 17 subtypes encompassing mammalian hosts using a 20D Graphical Representation and Numerical Characterization method, identified four possible highly conserved peptide segments. Solvent accessibility prediction servers were used to identify that these are predominantly surface situated. These regions analyzed through selected epitope prediction servers for their epitopic properties towards possible T-cell and B-cell activation showed good results as epitopic candidates (only dry lab confirmation). Conclusions The main reasons for the development of alternative vaccine strategies for the rotavirus are the failure of current vaccines and high production costs that inhibit their application in developing countries. We expect that it would be possible to use the protein surface exposed regions identified in our study as targets for peptide vaccines and drug designs for stable immunity against divergent strains of the rotavirus. Though this

  13. Rotavirus and Serotonin Cross-Talk in Diarrhoea

    PubMed Central

    Nordgren, Johan; Karlsson, Thommie; Sharma, Sumit; Magnusson, Karl-Eric; Svensson, Lennart

    2016-01-01

    Rotavirus (RV) has been shown to infect and stimulate secretion of serotonin from human enterochromaffin (EC) cells and to infect EC cells in the small intestine of mice. It remains to identify which intracellularly expressed viral protein(s) is responsible for this novel property and to further establish the clinical role of serotonin in RV infection. First, we found that siRNA specifically silencing NSP4 (siRNANSP4) significantly attenuated secretion of serotonin from Rhesus rotavirus (RRV) infected EC tumor cells compared to siRNAVP4, siRNAVP6 and siRNAVP7. Second, intracellular calcium mobilization and diarrhoeal capacity from virulent and avirulent porcine viruses correlated with the capacity to release serotonin from EC tumor cells. Third, following administration of serotonin, all (10/10) infants, but no (0/8) adult mice, responded with diarrhoea. Finally, blocking of serotonin receptors using Ondansetron significantly attenuated murine RV (strain EDIM) diarrhoea in infant mice (2.9 vs 4.5 days). Ondansetron-treated mice (n = 11) had significantly (p < 0.05) less diarrhoea, lower diarrhoea severity score and lower total diarrhoea output as compared to mock-treated mice (n = 9). Similarly, Ondansetron-treated mice had better weight gain than mock-treated animals (p < 0.05). A most surprising finding was that the serotonin receptor antagonist significantly (p < 0.05) also attenuated total viral shedding. In summary, we show that intracellularly expressed NSP4 stimulates release of serotonin from human EC tumor cells and that serotonin participates in RV diarrhoea, which can be attenuated by Ondansetron. PMID:27459372

  14. Rotavirus and Serotonin Cross-Talk in Diarrhoea.

    PubMed

    Bialowas, Sonja; Hagbom, Marie; Nordgren, Johan; Karlsson, Thommie; Sharma, Sumit; Magnusson, Karl-Eric; Svensson, Lennart

    2016-01-01

    Rotavirus (RV) has been shown to infect and stimulate secretion of serotonin from human enterochromaffin (EC) cells and to infect EC cells in the small intestine of mice. It remains to identify which intracellularly expressed viral protein(s) is responsible for this novel property and to further establish the clinical role of serotonin in RV infection. First, we found that siRNA specifically silencing NSP4 (siRNANSP4) significantly attenuated secretion of serotonin from Rhesus rotavirus (RRV) infected EC tumor cells compared to siRNAVP4, siRNAVP6 and siRNAVP7. Second, intracellular calcium mobilization and diarrhoeal capacity from virulent and avirulent porcine viruses correlated with the capacity to release serotonin from EC tumor cells. Third, following administration of serotonin, all (10/10) infants, but no (0/8) adult mice, responded with diarrhoea. Finally, blocking of serotonin receptors using Ondansetron significantly attenuated murine RV (strain EDIM) diarrhoea in infant mice (2.9 vs 4.5 days). Ondansetron-treated mice (n = 11) had significantly (p < 0.05) less diarrhoea, lower diarrhoea severity score and lower total diarrhoea output as compared to mock-treated mice (n = 9). Similarly, Ondansetron-treated mice had better weight gain than mock-treated animals (p < 0.05). A most surprising finding was that the serotonin receptor antagonist significantly (p < 0.05) also attenuated total viral shedding. In summary, we show that intracellularly expressed NSP4 stimulates release of serotonin from human EC tumor cells and that serotonin participates in RV diarrhoea, which can be attenuated by Ondansetron. PMID:27459372

  15. Rotavirus in India: Forty Years of Research.

    PubMed

    Kang, Gagandeep

    2016-07-01

    Rotavirus was first identified as a human pathogen just over 40 years ago, and work on this pathogen in India started shortly thereafter. Subsequent studies have confirmed its pre-eminent role in gastroenteritis in children in India. Standardized surveillance has enabled the documentation of the high burden of disease, and has demonstrated that there is considerable geographic and temporal variation in strain circulation. Internationally licensed vaccines, vaccine candidates based on indigenous strains and out-licensed strains have been tested for safety, immunogenicity and efficacy; three vaccines are now licensed in India and are used in the private sector. Public sector vaccination has begun, and it will be path-breaking for Indian vaccinologists to measure impact of vaccine introduction in terms of safety and effectiveness. So far, India has kept pace with international epidemiologic and vaccine research on rotavirus, and these efforts should continue. PMID:27508532

  16. Rotavirus P[8] Infections in Persons with Secretor and Nonsecretor Phenotypes, Tunisia.

    PubMed

    Ayouni, Siwar; Sdiri-Loulizi, Khira; de Rougemont, Alexis; Estienney, Marie; Ambert-Balay, Katia; Aho, Serge; Hamami, Sabeur; Aouni, Mahjoub; Neji-Guediche, Mohamed; Pothier, Pierre; Belliot, Gaël

    2015-11-01

    To determine whether rotavirus infections are linked to secretor status, we studied samples from children in Tunisia with gastroenteritis. We phenotyped saliva for human blood group antigens and tested feces for rotavirus. Rotavirus was detected in 32/114 patients. Secretor genotyping showed that P[8] rotavirus infected secretors and nonsecretors, and infection correlated with presence of Lewis antigen. PMID:26488868

  17. Serotype-specific glycoprotein of simian 11 rotavirus: coding assignment and gene sequence.

    PubMed Central

    Both, G W; Mattick, J S; Bellamy, A R

    1983-01-01

    Cloned DNA copies of the double-stranded RNA genomic segments of simian 11 rotavirus have been used to determine the coding assignment for VP7, the type-specific antigen of this virus. Translation of hybrid-selected mRNAs in an in vitro system supplemented with canine pancreatic microsomes permitted VP7 to be assigned to segment 9 and the two nonstructural viral proteins NCVP4 and NCVP3, to segments 7 and 8, respectively. Hybridization of cloned DNA probes for segments 7-9 with the corresponding segments of human rotavirus Wa confirmed these assignments. The complete nucleotide sequence of gene 9 has been determined. The deduced amino acid sequence reveals VP7 to be 326 amino acids in length with two NH2-terminal hydrophobic regions and a single glycosylation site at residues 69-71. Images PMID:6304692

  18. The Battle between Rotavirus and Its Host for Control of the Interferon Signaling Pathway

    PubMed Central

    Arnold, Michelle M.; Sen, Adrish; Greenberg, Harry B.; Patton, John T.

    2013-01-01

    Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ∼450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses. PMID:23359266

  19. Prevention of rotavirus gastroenteritis in infants and children: rotavirus vaccine safety, efficacy, and potential impact of vaccines

    PubMed Central

    Chandran, Aruna; Fitzwater, Sean; Zhen, Anjie; Santosham, Mathuram

    2010-01-01

    Rotavirus infection is the most common cause of severe gastroenteritis globally, with greater than 86% of deaths occurring in low-income and middle-income countries. There are two rotavirus vaccines currently licensed in the United States and prequalified by the World Health Organization. RV1 is a monovalent attenuated human rotavirus strain, given orally in two doses. RV5 is a pentavalent human-bovine reassortant rotavirus vaccine, given orally in three doses. A third rotavirus vaccine, LLV, is a lamb rotavirus strain given orally as a single dose, which is currently available only in China. RV1 and RV5 have been shown to be highly efficacious in developed countries, and initial results from trials in Africa and Asia are promising as well. At least three other vaccines are in development, which are being developed by manufacturers of developing countries. Further studies are needed to clarify issues including administration of oral rotavirus vaccines with breastfeeding and other oral vaccines, and alterations in dosing schedule. Using new data on global diarrheal burden, rotavirus is estimated to cause 390,000 deaths in children younger than 5 years. Should rotavirus vaccines be introduced in the routine immunization programs of all countries, a potential of 170,000 deaths could be prevented annually. The largest impact on mortality would be seen in low-income and middle-income countries, despite poor immunization coverage and lower efficacy. Therefore, international efforts are needed to ensure that rotavirus vaccines reach the populations with highest burden of rotavirus disease. PMID:20714358

  20. SIMPLIFIED RADIOIMMUNOASSAY FOR DETECTION OF HUMAN ROTAVIRUS IN STOOLS

    EPA Science Inventory

    A simplified radioimmunoassay (RIA) technique was developed to facilitate the diagnosis of human rotavirus in stools of infants with diarrhea. This microtiter solid-phase RIA utilizes as a critical reagent hyperimmune serum against a tissue culture-grown simian rotavirus that is ...

  1. Immunochromatography-based Diagnosis of Rotavirus Infection in Acute Diarrhea.

    PubMed

    Vashishtha, Vipin M; Thacker, Sandeep; Namjoshi, Gajanan Sudhir

    2016-07-01

    Documentation of rotavirus diarrhea in a rural, resource-poor setting is a difficult task. We analyzed stool samples of 103 children admitted for acute diarrhea in a pediatric hospital in Bijnor, UP, India, using a simple bedside immunochromatography kit. Rotavirus infection was detected in 47 out of total of 103 children (45.6%). PMID:27508549

  2. Serological response to rotavirus infection in newborn infants.

    PubMed

    Flores, J; White, L; Blanco, M; Perez-Schael, I

    1994-01-01

    We report the identification of rotavirus in stools of newborn infants at the "Hospital Materno Infantil de Caricuao" (HMIC) as well as the infants' serological responses to various rotavirus strains. The serological responses of another group of rotavirus-positive neonates studied previously at the "Maternidad Concepcion Palacios" (MCP) hospital was also evaluated. Fifty-four of 266 (20%) newborns examined at HMIC shed rotavirus. The infection rate was higher among infants admitted to the nursery (75%) than in those "rooming in" with their mothers (7%) (P < .01). Eleven of the 54 neonates (20%) had diarrhea; seven of them experienced mild, short-lived episodes, whereas five had frequent diarrhea bouts or diarrhea lasting for over 3 days; the remaining 43 infants were asymptomatic. Twenty-seven of 28 rotavirus specimens tested at HMIC had VP7 serotype 4 specificity and one belonged to VP7 serotype 1; VP4 typing performed on 24 of the viruses by RNA hybridization showed these viruses to be similar to the M37 strain, a rotavirus previously associated with asymptomatic infections in newborns at MCP. IgA seroresponses were detected in eight of 11 infants born at HMIC (73%), but most failed to developed neutralization responses to homologous or heterologous strains. Newborn infants who had shed the M37 rotavirus strain at MCP reacted similarly: 16 of 24 (67%) developed a rotavirus IgA rise, but only 29% developed a neutralization response. PMID:8308526

  3. Multispecies reassortant bovine rotavirus strain carries a novel simian G3-like VP7 genotype.

    PubMed

    Malik, Yashpal Singh; Kumar, Naveen; Sharma, Kuldeep; Saurabh, Sharad; Dhama, Kuldeep; Prasad, Minakshi; Ghosh, Souvik; Bányai, Krisztián; Kobayashi, Nobumichi; Singh, Raj Kumar

    2016-07-01

    Rotavirus-A (RVAs), are the major cause of severe gastroenteritis in the young of mammals and birds. RVA strains possessing G6, G8, and G10 genotypes in combination with P[1] or P[11] have been commonly detected in cattle. During a routine surveillance for enteric viruses in a bovine population on North-Western temperate Himalayan region of India, an uncommon bovine RVA strain, designated as RVA/Cow-wt/IND/M1/09/2009 was detected in a diarrhoeic crossbred calf. The examination of nearly complete genome sequence of this RVA strain revealed an unusual G-P combination (G3P[11]) on a typical bovine RVA genotype backbone (I2-R2-C2-M2-A11-N2-T6-E2-H3). The VP7 gene of M1/09 isolate displayed a maximum nucleotide sequence identity of 73.8% with simian strain (RVA/Simian-tc/USA/RRV/1975/G3P[3]). The VP4 and NSP5 genes clustered with an Indian pig strain, RVA/Pig-wt/IND/AM-P66/2012/G10P[11] (99.6%), and a caprine strain, RVA/Goat-tc/BGD/GO34/1999/G6P[1] (98.9%) from Bangladesh, respectively, whilst the, VP6, NSP1, NSP3 and NSP4 genes were identical or nearly identical to Indian bovine strains (RVA/Cow-wt/IND/B-72/2008/G10P[X], RVA/Cow-wt/IND/B85/2010/GXP[X], and RVA/Cow-wt/IND/C91/2011/G6P[X]). The remaining four genes (VP1, VP2, VP3 and NSP2) were more closely related to RVA/Human-wt/ITA/PAI11/1996/G2P[4] (93.5%), RVA/Sheep-wt/CHN/LLR/1985/G10P[15] (88.8%), RVA/Human-tc/SWE/1076/1983/G2P2A[6] (93.2%) and RVA/Human-wt/AUS/CK20003/2000/G2P[4] (91.2%), respectively. Altogether, these findings are suggestive of multiple independent interspecies transmission and reassortment events between co-circulating bovine, porcine, ovine and human rotaviruses. The complete genome sequence information is necessary to establish the evolutionary relationship, interspecies transmission and ecological features of animal RVAs from different geographical regions. PMID:27033751

  4. Diarrheal Diseases Hospitalization in Yemen before and after Rotavirus Vaccination.

    PubMed

    Amood Al-Kamarany, Mohammed; Al-Areqi, Lina; Mujally, Abulatif; Alkarshy, Fawzya; Nasser, Arwa; Jumaan, Aisha O

    2016-01-01

    The study aims to assess the impact of rotavirus vaccine introduction on diarrheal diseases hospitalization and to identify the rotavirus genotypes most prevalent before and after vaccine introduction among children ≤ 5 years of age. Rotarix™ ® rotavirus vaccine is currently licensed for infants in Yemen and was introduced in 2012. The vaccination course consists of two doses. The first dose is administrated at 6 weeks of age and the second dose is completed by 10 weeks. Based on a longitudinal observational study, we assessed the impact of vaccination on rotavirus hospitalization before and after vaccination among children ≤ 5 years of age at the Yemeni-Swedish Hospital (YSH) in Taiz, Yemen. Prevaccination covered January 2009-July 2012 during which 2335 fecal samples were collected from children ≤ 5 years old. Postvaccination covered January 2013-December 2014 during which 1114 fecal samples were collected. Rotavirus was detected by Enzyme Linkage Immunosorbent Assay (ELISA). The incidence of rotavirus hospitalization decreased from 43.79% in 2009 to 10.54% in 2014. Hospitalization due to rotavirus diarrhea was reduced by 75.93%. Vaccine coverage increased from 23% in 2012 to 72% in 2014. Also, the results showed that the most predominant genotypes in prevaccination period were G2P[4] (55.0%), followed by G1P[8] (15.0%), while in postvaccination period G1P[8] (31%) was the predominant genotype, followed by G9P[8] (27.5%). In conclusion, rotavirus vaccination in Yemen resulted in sharp reduction in diarrheal hospitalization. A successful rotavirus vaccination program in Yemen will rely upon efficient vaccine delivery systems and sustained vaccine efficacy against diverse and evolving rotavirus strains. PMID:27437161

  5. Diarrheal Diseases Hospitalization in Yemen before and after Rotavirus Vaccination

    PubMed Central

    Al-Areqi, Lina; Mujally, Abulatif; Alkarshy, Fawzya; Nasser, Arwa; Jumaan, Aisha O.

    2016-01-01

    The study aims to assess the impact of rotavirus vaccine introduction on diarrheal diseases hospitalization and to identify the rotavirus genotypes most prevalent before and after vaccine introduction among children ≤ 5 years of age. Rotarix™ ® rotavirus vaccine is currently licensed for infants in Yemen and was introduced in 2012. The vaccination course consists of two doses. The first dose is administrated at 6 weeks of age and the second dose is completed by 10 weeks. Based on a longitudinal observational study, we assessed the impact of vaccination on rotavirus hospitalization before and after vaccination among children ≤ 5 years of age at the Yemeni-Swedish Hospital (YSH) in Taiz, Yemen. Prevaccination covered January 2009–July 2012 during which 2335 fecal samples were collected from children ≤ 5 years old. Postvaccination covered January 2013–December 2014 during which 1114 fecal samples were collected. Rotavirus was detected by Enzyme Linkage Immunosorbent Assay (ELISA). The incidence of rotavirus hospitalization decreased from 43.79% in 2009 to 10.54% in 2014. Hospitalization due to rotavirus diarrhea was reduced by 75.93%. Vaccine coverage increased from 23% in 2012 to 72% in 2014. Also, the results showed that the most predominant genotypes in prevaccination period were G2P[4] (55.0%), followed by G1P[8] (15.0%), while in postvaccination period G1P[8] (31%) was the predominant genotype, followed by G9P[8] (27.5%). In conclusion, rotavirus vaccination in Yemen resulted in sharp reduction in diarrheal hospitalization. A successful rotavirus vaccination program in Yemen will rely upon efficient vaccine delivery systems and sustained vaccine efficacy against diverse and evolving rotavirus strains. PMID:27437161

  6. Variations in DREB1A and VP1.1 Genes Show Association with Salt Tolerance Traits in Wild Tomato (Solanum pimpinellifolium)

    PubMed Central

    Rao, Eguru Sreenivasa; Kadirvel, Palchamy; Symonds, Rachael C.; Geethanjali, Subramaniam; Thontadarya, Ramadihalli N.; Ebert, Andreas W.

    2015-01-01

    Association analysis was conducted in a core collection of 94 genotypes of Solanum pimpinellifolium to identify variations linked to salt tolerance traits (physiological and yield traits under salt stress) in four candidate genes viz., DREB1A, VP1.1, NHX1, and TIP. The candidate gene analysis covered a concatenated length of 4594 bp per individual and identified five SNP/Indels in DREB1A and VP1.1 genes explaining 17.0% to 25.8% phenotypic variation for various salt tolerance traits. Out of these five alleles, one at 297 bp in DREB1A had in-frame deletion of 6 bp (CTGCAT) or 12 bp (CTGCATCTGCAT), resulting in two alleles, viz., SpDREB1A_297_6 and SpDREB1A_297_12. These alleles individually or as haplotypes accounted for maximum phenotypic variance of about 25% for various salt tolerance traits. Design of markers for selection of the favorable alleles/haplotypes will hasten marker-assisted introgression of salt tolerance from S. pimpinellifolium into cultivated tomato. PMID:26161546

  7. Variations in DREB1A and VP1.1 Genes Show Association with Salt Tolerance Traits in Wild Tomato (Solanum pimpinellifolium).

    PubMed

    Rao, Eguru Sreenivasa; Kadirvel, Palchamy; Symonds, Rachael C; Geethanjali, Subramaniam; Thontadarya, Ramadihalli N; Ebert, Andreas W

    2015-01-01

    Association analysis was conducted in a core collection of 94 genotypes of Solanum pimpinellifolium to identify variations linked to salt tolerance traits (physiological and yield traits under salt stress) in four candidate genes viz., DREB1A, VP1.1, NHX1, and TIP. The candidate gene analysis covered a concatenated length of 4594 bp per individual and identified five SNP/Indels in DREB1A and VP1.1 genes explaining 17.0% to 25.8% phenotypic variation for various salt tolerance traits. Out of these five alleles, one at 297 bp in DREB1A had in-frame deletion of 6 bp (CTGCAT) or 12 bp (CTGCATCTGCAT), resulting in two alleles, viz., SpDREB1A_297_6 and SpDREB1A_297_12. These alleles individually or as haplotypes accounted for maximum phenotypic variance of about 25% for various salt tolerance traits. Design of markers for selection of the favorable alleles/haplotypes will hasten marker-assisted introgression of salt tolerance from S. pimpinellifolium into cultivated tomato. PMID:26161546

  8. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes.

    PubMed

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-08-01

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

  9. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

    PubMed Central

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-01-01

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

  10. [Seasonality of rotavirus infection in Venezuela: relationship between monthly rotavirus incidence and rainfall rates].

    PubMed

    González Chávez, Rosabel

    2015-09-01

    In general, it has been reported that rotavirus infection was detected year round in tropical countries. However, studies in Venezuela and Brazil suggest a seasonal behavior of the infection. On the other hand, some studies link infection with climatic variables such as rainfall. This study analyzes the pattern of behavior of the rotavirus infection in Carabobo-Venezuela (2001-2005), associates the seasonality of the infection with rainfall, and according to the seasonal pattern, estimates the age of greatest risk for infection. The analysis of the rotavirus temporal series and accumulated precipitation was performed with the software SPSS. The infection showed two periods: high incidence (November-April) and low incidence (May-October). Accumulated precipitation presents an opposite behavior. The highest frequency of events (73.8% 573/779) for those born in the period with a low incidence of the virus was recorded at an earlier age (mean age 6.5 +/- 2.0 months) when compared with those born in the station of high incidence (63.5% 568/870, mean age 11.7 +/- 2.2 months). Seasonality of the infection and the inverse relationship between virus incidence and rainfall was demonstrated. In addition, it was found that the period of birth determines the age and risk of infection. This information generated during the preaccine period will be helpful to measure the impact of the vaccine against the rotavirus. PMID:26710540

  11. In vitro detection of porcine rotavirus-like virus (group B rotavirus) and its antibody.

    PubMed Central

    Theil, K W; Saif, L J

    1985-01-01

    Four isolates of porcine rotavirus-like virus (PRVLV) infected MA104 cells and induced syncytium formation after low-speed centrifugation of the inoculum onto the monolayer. Ten of 44 (23%) Ohio swine sera had PRVLV antibodies when tested by indirect immunofluorescence, using PRVLV-infected MA104 cell monolayers as antigen. Images PMID:2987303

  12. Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8

    SciTech Connect

    Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Szyczew, Alex J.; Kiefel, Milton J.; Itzstein, Mark von; Blanchard, Helen

    2005-06-01

    The sialic acid-binding domain (VP8*) component of the porcine CRW-8 rotavirus spike protein has been overexpressed in E. coli, purified and co-crystallized with an N-acetylneuraminic acid derivative. X-ray diffraction data have been collected to 2.3 Å, which has enabled determination of the structure by molecular replacement. Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by the virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 Å resolution, enabling the determination of the VP8* structure by molecular replacement.

  13. Analysis of codon usage pattern evolution in avian rotaviruses and their preferred host.

    PubMed

    Kattoor, Jobin Jose; Malik, Yashpal Singh; Sasidharan, Aravind; Rajan, Vishnuraj Mangalathu; Dhama, Kuldeep; Ghosh, Souvik; Bányai, Krisztián; Kobayashi, Nobumichi; Singh, Raj Kumar

    2015-08-01

    Rotavirus infection is a worldwide problem, with occurrence of highly divergent viruses classified in 8 species (A-H). We report here the evolution assessment of codon usage patterns in virus-host system in avian rotavirus (AvRV) of species RVA, RVD, RVF and RVG (preferentially affecting birds). The nucleotide contents, codon usage bias (CUB), relative synonymous codon usage (RSCU), and effective number of codons (ENCs) values were investigated targeting overexpressing major inner capsid viral protein (VP6) of these AvRV species. The results confirm that the evolutionary characteristics influences the rotavirus (RV) genetic diversity and impact of host's natural selection on the AvRVs codons. Synonymous codon usage patterns were evaluated following multivariate statistical procedures on all available AvRV coding gene sequences. RSCU trees accommodated all AvRV species and preferred host sequences in one topology confirming greater imminence of AvRVs with the host chicken cell genes. Similarly, the codon adaptation index (CAI) results also displayed a higher adaptation of AvRVs to its chicken host. The codon preference analysis of RVs revealed that VP6 gene express more proficiently in the yeast system, whereas, codon optimization might be required for the effectual expression in Escherichia coli and Homo sapiens. The findings provide basic evidence on the dynamics of AvRV evolution and its host adaptation, which could be exploited for additional research on avian species in future. PMID:26086995

  14. Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene.

    PubMed

    Bezerra, Delana Andreza Melo; da Silva, René Ribeiro; Kaiano, Jane Haruko Lima; Silvestre, Rodrigo Vellasco Duarte; de Souza Oliveira, Darleise; Linhares, Alexandre C; Gabbay, Yvone Benchimol; Mascarenhas, Joana D'Arc Pereira

    2012-11-01

    Group D rotaviruses (RVs-D) have been documented in birds and, while they may be common in these animals, few molecular studies are available for this specific group. In this study, specific primers for the gene that encodes for the RVs-D VP6 protein were designed and used in a reverse transcription polymerase chain reaction (RT-PCR). Thirty pools of samples were tested by polyacrylamide gel electrophoresis (PAGE) yielding a 30% (9/30) positivity. These pools were subjected subsequently to RT-PCR, with a 53% (16/30) positivity rate. The sensitivity of the PCR assay was demonstrated up to a dilution of 5 × 10(-4)ng/μL (0.5 pg/μL) of the cloned VP6 gene. The four samples were sequenced and showed 90.8-91.1% similarity with regards to the RVs-D VP6 gene. To assess for specificity our RT-PCR was applied to nine samples known to contain enteric viral agents other than group D rotaviruses including picobirnavirus, rotavirus group A, and reovirus with negative results. Overall, the data confirm the specificity of the primers used for detecting the RVs-D by RT-PCR, suggesting that this assay can be used for diagnostic purposes. PMID:22820073

  15. Distribution of VP4 gene alleles in human rotaviruses by using probes to the hyperdivergent region of the VP4 gene.

    PubMed Central

    Steele, A D; Garcia, D; Sears, J; Gerna, G; Nakagomi, O; Flores, J

    1993-01-01

    The rotavirus VP4 protein elicits the production of neutralizing antibodies and is known to play a role in inducing resistance to disease. At least five human rotavirus VP4 gene alleles have been described on the basis of antigenic polymorphism and/or nucleotide sequence differences. In the present study, we developed cDNA probes directed at the hyperdivergent region of the VP4 gene of the five described human rotavirus VP4 alleles (Wa, DS1, M37, AU228, and 69M) and used them in hybridization assays with human rotavirus strains from Latin America and Europe to determine the distribution of the VP4 gene alleles in nature. The Wa-like allele was detected most frequently, occurring in 57% of the 402 rotavirus strains tested, and the DS1-like allele was the next most common, occurring in 14% of the strains tested. The M37- and AU228-like alleles were detected in only 4 and 3% of the rotavirus strains tested, respectively, whereas the 69M-like VP4 gene allele was not detected. Several rotavirus strains from Europe did not react with any of the VP4 gene probes, although they did hybridize to a probe generated from a representative strain from the group. These data indicate the global distribution of various VP4 gene alleles and raise the possibility that other, unrecognized human VP4 alleles exist in nature because almost one-fourth of the strains could not be classified into any of the established VP4 groups. Images PMID:8394374

  16. Human rotavirus strains bearing VP4 gene P[6] allele recovered from asymptomatic or symptomatic infections share similar, if not identical, VP4 neutralization specificities.

    PubMed

    Hoshino, Yasutaka; Jones, Ronald W; Ross, Jerri; Santos, Norma; Kapikian, Albert Z

    2003-11-10

    A rotavirus VP4 gene P[6] allele has been documented in a number of countries to be characteristically associated with an endemic predominantly asymptomatic infection in neonates in maternity hospital nurseries. The mechanisms underlying the endemicity and asymptomatic nature of such neonatal infections remain unknown. Rotavirus strains sharing this same P genotype, however, have more recently been recovered from an increasing number of symptomatic diarrheal episodes in infants and young children in various parts of the world. Previously, we have shown that an asymptomatic P[6] rotavirus neonatal infection is not associated with a unique VP7 (G) serotype but may occur in conjunction with various G types. Although amino acid sequence comparisons of the VP4 gene between selected "asymptomatic" and "symptomatic" P[6] rotavirus strains have been reported and yielded information concerning their VP4 genotypes, serotypic comparisons of the outer capsid spike protein VP4 of such viruses have not been studied systematically by two-way cross-neutralizations. We determined the VP4 neutralization specificities of four asymptomatic and four symptomatic P[6] strains: two each of asymptomatic and symptomatic strains by two-way tests, and two each of additional asymptomatic and symptomatic strains by one-way tests. Both asymptomatic and symptomatic P[6] strains were shown to bear similar, if not identical, VP4 neutralization specificities. Thus, P[6] rotavirus strains causing asymptomatic or symptomatic infections did not appear to belong to unique P (VP4) serotypes. In addition, a close VP4 serotypic relationship between human P[6] rotavirus strains and the porcine P[6] rotavirus Gottfried strain was confirmed. PMID:14599785

  17. Whole Genomic Analysis of an Unusual Human G6P[14] Rotavirus Strain Isolated from a Child with Diarrhea in Thailand: Evidence for Bovine-To-Human Interspecies Transmission and Reassortment Events

    PubMed Central

    Tacharoenmuang, Ratana; Komoto, Satoshi; Guntapong, Ratigorn; Ide, Tomihiko; Haga, Kei; Katayama, Kazuhiko; Kato, Takema; Ouchi, Yuya; Kurahashi, Hiroki; Tsuji, Takao; Sangkitporn, Somchai; Taniguchi, Koki

    2015-01-01

    An unusual rotavirus strain, SKT-27, with the G6P[14] genotypes (RVA/Human-wt/THA/SKT-27/2012/G6P[14]), was identified in a stool specimen from a hospitalized child aged eight months with severe diarrhea. In this study, we sequenced and characterized the complete genome of strain SKT-27. On whole genomic analysis, strain SKT-27 was found to have a unique genotype constellation: G6-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The non-G/P genotype constellation of this strain (I2-R2-C2-M2-A3-N2-T6-E2-H3) is commonly shared with rotavirus strains from artiodactyls such as cattle. Phylogenetic analysis indicated that nine of the 11 genes of strain SKT-27 (VP7, VP4, VP6, VP2-3, NSP1, NSP3-5) appeared to be of artiodactyl (likely bovine) origin, while the remaining VP1 and NSP2 genes were assumed to be of human origin. Thus, strain SKT-27 was found to have a bovine rotavirus genetic backbone, and thus is likely to be of bovine origin. Furthermore, strain SKT-27 appeared to be derived through interspecies transmission and reassortment events involving bovine and human rotavirus strains. Of note is that the VP7 gene of strain SKT-27 was located in G6 lineage-5 together with those of bovine rotavirus strains, away from the clusters comprising other G6P[14] strains in G6 lineages-2/6, suggesting the occurrence of independent bovine-to-human interspecies transmission events. To our knowledge, this is the first report on full genome-based characterization of human G6P[14] strains that have emerged in Southeast Asia. Our observations will provide important insights into the origin of G6P[14] strains, and into dynamic interactions between human and bovine rotavirus strains. PMID:26421718

  18. Avian rotavirus enteritis - an updated review.

    PubMed

    Dhama, Kuldeep; Saminathan, Mani; Karthik, Kumaragurubaran; Tiwari, Ruchi; Shabbir, Muhammad Zubair; Kumar, Naveen; Malik, Yashpal Singh; Singh, Raj Kumar

    2015-01-01

    Rotaviruses (RVs) are among the leading causes of enteritis and diarrhea in a number of mammalian and avian species, and impose colossal loss to livestock and poultry industry globally. Subsequent to detection of rotavirus in mammalian hosts in 1973, avian rotavirus (AvRV) was first reported in turkey poults in USA during 1977 and since then RVs of group A (RVA), D (RVD), F (RVF) and G (RVG) have been identified around the globe. Besides RVA, other AvRV groups (RVD, RVF and RVG) may also contribute to disease. However, their significance has yet to be unraveled. Under field conditions, co-infection of AvRVs occurs with other infectious agents such as astroviruses, enteroviruses, reoviruses, paramyxovirus, adenovirus, Salmonella, Escherichia coli, cryptosporidium and Eimeria species prospering severity of disease outcome. Birds surviving to RV disease predominantly succumb to secondary bacterial infections, mostly E. coli and Salmonella spp. Recent developments in molecular tools including state-of-the-art diagnostics and vaccine development have led to advances in our understanding towards AvRVs. Development of new generation vaccines using immunogenic antigens of AvRV has to be explored and given due importance. Till now, no effective vaccines are available. Although specific as well as sensitive approaches are available to identify and characterize AvRVs, there is still need to have point-of-care detection assays to review disease burden, contemplate new directions for adopting vaccination and follow improvements in public health measures. This review discusses AvRVs, their epidemiology, pathology and pathogenesis, immunity, recent trends in diagnostics, vaccines, therapeutics as well as appropriate prevention and control strategies. PMID:25917772

  19. The Prevalence of Rotavirus and Adenovirus in the Childhood Gastroenteritis

    PubMed Central

    Ozsari, Tamer; Bora, Gulhan; Kaya, Bulent; Yakut, Kahraman

    2016-01-01

    Background Acute gastroenteritis stemming from viral causes is very common during the childhood period. Rotavirus and enteric adenovirus are the most common factors of acute gastroenteritis encountered in infants and children. However, the epidemiology of rotavirus and enteric adenovirus gastroenteritis in the east Anatolia region is not well-known. Objectives We aimed to evaluate the relationship between the distribution of antigen positivity in rotavirus and enteric adenovirus antigen tests required cases and demographic data retrospectively in pediatric patients admitted to our hospital. Patients and Methods The records of stool sample analyses for 1154 patients admitted to our hospital from June 2011 to December 2011 with complaints of diarrhea were retrospectively examined. The presence of rotavirus and enteric adenovirus antigens in stool specimens was investigated by means of an immunochromatographic test. Results Viral antigens were detected in 327 (28.3%) stool specimens out of 1154. Among the positive results, the frequency was 73.7% for rotavirus and 26.2% for adenovirus. While the detected rotavirus antigen rate was high for all age groups, it was highest for children under the age of 2, with a rate of 57.1%. Moreover, the rotavirus infections were observed at a rate of 44.3% in winter and of 24.6% in autumn. Conclusions The most important factor in childhood acute gastroenteritis in east Anatolia is the rotavirus. Rotavirus and adenovirus antigens should be routinely investigated as a factor in fresh stool samples for the accurate diagnosis and treatment of gastroenteritis in children in the winter and autumn months.

  20. Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

    PubMed Central

    Ku, Zhiqiang; Ye, Xiaohua; Shi, Jinping; Wang, Xiaoli

    2015-01-01

    ABSTRACT Antibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre- and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs. IMPORTANCE Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during

  1. Inactivation of human and simian rotaviruses by ozone

    SciTech Connect

    Vaughn, J.M.; Chen, Y.S.; Lindburg, K.; Morales, D.

    1987-09-01

    The inactivation of simian rotavirus Sa-11 and human rotavirus type 2 (Wa) by ozone was compared at 4/sup 0/C by using single-particle virus stocks. Although the human strain was clearly more sensitive, both virus types were rapidly inactivated by ozone concentrations of 0.25 mg/liter or greater at all pH levels tested. Comparison of the virucidal activity of ozone with that of chlorine in identical experiments indicated little significant difference in rotavirus-inactivating efficiencies when the disinfectants were used at concentrations of 0.25 mg/liter or greater.

  2. Inhibition of Rotavirus Infectivity by a Neoglycolipid Receptor Mimetic

    PubMed Central

    Bergner, Daniel W.; Kuhlenschmidt, Theresa B.; Hanafin, William P.; Firkins, Lawrence D.; Kuhlenschmidt, Mark S.

    2011-01-01

    Group A rotaviruses are a major cause of diarrhea in the young of many mammalian species. In rotavirus infected piglets mortality can be as high as 60%. Previous research in this laboratory has identified a porcine intestinal GM3 ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. In addition, we previously demonstrated exogenously added GM3 can competitively inhibit porcine rotavirus binding and infectivity of host cells in vitro. Sialyllactose, the carbohydrate moiety of GM3, is approximately 3 orders of magnitude less effective than GM3 at inhibiting rotavirus binding to cells. Furthermore, production of therapeutic quantities of GM3 ganglioside for use as an oral carbomimetic in swine is cost prohibitive. In an effort to circumvent these problems, a sialyllactose-containing neoglycolipid was synthesized and evaluated for its ability to inhibit rotavirus binding and infectivity of host cells. Sialyllactose was coupled to dipalmitoylphosphatidylethanolamine (PE) by reductive amination and the product (SLPE) purified by HPLC. Characterization of the product showed a single primulin (lipid) and resorcinol (sialic acid) positive band by thin layer chromatography and quantification of phosphate and sialic acid yielded a 1:1 molar ratio. Mass spectroscopy confirmed a molecular weight coinciding with SLPE. Concentration-dependent binding of rotavirus to SLPE was demonstrated using a thin-layer overlay assay. Using concentrations comparable to GM3, SLPE was also shown to inhibit rotavirus binding to host cells by 80%. Furthermore, SLPE was shown to decrease rotavirus infection of host cells by over 90%. Finally, preliminary results of in vivo animal challenge studies using newborn piglets in their natural environment, demonstrated SLPE afforded complete protection from rotavirus disease. The efficacy of SLPE in inhibiting rotavirus binding and infection in vitro and in vivo, coupled with its relatively low-cost, large

  3. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

    PubMed

    Silva, Haroldo Cid da; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-08-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  4. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

    PubMed Central

    da Silva, Haroldo Cid; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-01-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  5. Prevalence of Rotavirus Genotypes in Children Younger than 5 Years of Age before the Introduction of a Universal Rotavirus Vaccination Program: Report of Rotavirus Surveillance in Turkey

    PubMed Central

    Durmaz, Riza; Kalaycioglu, Atila Taner; Acar, Sumeyra; Bakkaloglu, Zekiye; Karagoz, Alper; Korukluoglu, Gulay; Ertek, Mustafa; Torunoglu, Mehmet Ali

    2014-01-01

    Background Group A rotaviruses are the most common causative agent of acute gastroenteritis among children less than 5 years of age throughout the world. This sentinel surveillance study was aimed to obtain baseline data on the rotavirus G and P genotypes across Turkey before the introduction of a universal rotavirus vaccination program. Methods Rotavirus antigen-positive samples were collected from 2102 children less than 5 years of age who attended hospitals participating in the Turkish Rotavirus Surveillance Network. Rotavirus antigen was detected in the laboratories of participating hospitals by commercial serological tests such as latex agglutination, immunochromatographic test or enzyme immunoassay. Rotavirus G and P genotypes were determined by reverse transcription polymerase chain reaction (RT-PCR) using consensus primers detecting the VP7 and VP4 genes, followed by semi-nested type-specific multiplex PCR. Results RT-PCR found rotavirus RNA in 1644 (78.2%) of the samples tested. The highest rate of rotavirus positivity (38.7%) was observed among children in the 13 to 24 month age group, followed by children in the age group of 25 to 36 months (28.3%). A total of eight different G types, six different P types, and 42 different G–P combinations were obtained. Four common G types (G1, G2, G3, and G9) and two common P types (P[8] and P[4]) accounted for 95.1% and 98.8% of the strains, respectively. G9P[8] was the most common G/P combination found in 40.5% of the strains followed by G1P[8] (21.6%), G2P[8] (9.3%), G2P[4] (6.5%), G3P[8] (3.5%), and finally, G4P[8] (3.4%). These six common genotypes included 83.7% of the strains tested in this study. The rate of uncommon genotypes was 14%. Conclusion The majority of the strains analyzed belonged to the G1–G4 and G9 genotypes, suggesting high coverage of current rotavirus vaccines. This study also demonstrates a dramatic increase in G9 genotype across the country. PMID:25437502

  6. Reconciliation of rotavirus temperature-sensitive mutant collections and assignment of reassortment groups D, J, and K to genome segments.

    PubMed

    Criglar, Jeanette; Greenberg, Harry B; Estes, Mary K; Ramig, Robert F

    2011-05-01

    Four rotavirus SA11 temperature-sensitive (ts) mutants and seven rotavirus RRV ts mutants, isolated at the National Institutes of Health (NIH) and not genetically characterized, were assigned to reassortment groups by pairwise crosses with the SA11 mutant group prototypes isolated and characterized at Baylor College of Medicine (BCM). Among the NIH mutants, three of the RRV mutants and all four SA11 mutants contained mutations in single reassortment groups, and four RRV mutants contained mutations in multiple groups. One NIH mutant [RRVtsK(2)] identified the previously undefined 11th reassortment group (K) expected for rotavirus. Three NIH single mutant RRV viruses, RRVtsD(7), RRVtsJ(5), and RRVtsK(2), were in reassortment groups not previously mapped to genome segments. These mutants were mapped using classical genetic methods, including backcrosses to demonstrate reversion or suppression in reassortants with incongruent genotype and temperature phenotype. Once located to specific genome segments by genetic means, the mutations responsible for the ts phenotype were identified by sequencing. The reassortment group K mutant RRVtsK(2) maps to genome segment 9 and has a Thr280Ileu mutation in the capsid surface glycoprotein VP7. The group D mutant RRVtsD(7) maps to segment 5 and has a Leu140Val mutation in the nonstructural interferon (IFN) antagonist protein NSP1. The group J mutant RRVtsJ(5) maps to segment 11 and has an Ala182Gly mutation affecting only the NSP5 open reading frame. Rotavirus ts mutation groups are now mapped to 9 of the 11 rotavirus genome segments. Possible segment locations of the two remaining unmapped ts mutant groups are discussed. PMID:21367894

  7. Genetic characterization of VP1 gene of seven Sacbrood virus isolated from three provinces in northern China during the years 2008-2012.

    PubMed

    Mingxiao, Ma; Yanna, Yin; Xiaoli, Xu; Lin, Zhang; Yongfei, Li; Zhidong, Luan

    2013-09-01

    The genetic diversity of seven northern China isolated Sacbrood virus strains (SBV) has been analyzed, and hypervariable regions of the VP1 gene of 7 SBV were sequenced and characterized, in order to obtain epidemiological and immunological information, and to suggest typing criteria for SBV. Sequence analysis of hypervariable regions of the VP1 gene in the genome of these isolates revealed a sequence homology of 91.0-99.3% among all seven local SBV isolates from Apis cerana from China, with a similarity of 93.3-100.0% in deduced amino acid sequences. These local isolates shared 87.4-92.8% sequence homology with six SBV reference strains in GenBank (including two SBV reference strains from Apis cerana from China), which represents a 91.8-97.6% similarity in deduced amino acid sequences. Genetic analysis also showed that five SBV strains from Apis cerana from China had a 13-amino-acid deletion at amino acid positions 287-299, and two SBV strains infecting the Korean honeybee had a 17-amino-acid deletion at amino acid positions 284-300 in comparison with other SBV. Phylogenetic analysis revealed two major groups (AC genotype SBV infecting Apis cerana and AM genotype SBV infecting Apis mellifera). The AC genotype could be further divided into subgroups. Based on the results of phylogenetic analysis, a similarity scan of SBV nucleotide sequences was carried out by using Simplot software and results in similar results. Our results suggest possible typing criteria for SBV based on the phylogenetic tree and sequence homology, and also that the virus has host specificity and regional variations. PMID:23722004

  8. Large-scale whole genome sequencing identifies country-wide spread of an emerging G9P[8] rotavirus strain in Hungary, 2012.

    PubMed

    Dóró, Renáta; Mihalov-Kovács, Eszter; Marton, Szilvia; László, Brigitta; Deák, Judit; Jakab, Ferenc; Juhász, Ágnes; Kisfali, Péter; Martella, Vito; Melegh, Béla; Molnár, Péter; Sántha, Ildikó; Schneider, Ferenc; Bányai, Krisztián

    2014-12-01

    With the availability of rotavirus vaccines routine strain surveillance has been launched or continued in many countries worldwide. In this study relevant information is provided from Hungary in order to extend knowledge about circulating rotavirus strains. Direct sequencing of the RT-PCR products obtained by VP7 and VP4 genes specific primer sets was utilized as routine laboratory method. In addition we explored the advantage of random primed RT-PCR and semiconductor sequencing of the whole genome of selected strains. During the study year, 2012, we identified an increase in the prevalence of G9P[8] strains across the country. This genotype combination predominated in seven out of nine study sites (detection rates, 45-83%). In addition to G9P[8]s, epidemiologically major strains included genotypes G1P[8] (34.2%), G2P[4] (13.5%), and G4P[8] (7.4%), whereas unusual and rare strains were G3P[8] (1%), G2P[8] (0.5%), G1P[4] (0.2%), G3P[4] (0.2%), and G3P[9] (0.2%). Whole genome analysis of 125 Hungarian human rotaviruses identified nine major genotype constellations and uncovered both intra- and intergenogroup reassortment events in circulating strains. Intergenogroup reassortment resulted in several unusual genotype constellations, including mono-reassortant G1P[8] and G9P[8] strains whose genotype 1 (Wa-like) backbone gene constellations contained DS1-like NSP2 and VP3 genes, respectively, as well as, a putative bovine-feline G3P[9] reassortant strain. The conserved genomic constellations of epidemiologically major genotypes suggested the clonal spread of the re-emerging G9P[8] genotype and several co-circulating strains (e.g., G1P[8] and G2P[4]) in many study sites during 2012. Of interest, medically important G2P[4] strains carried bovine-like VP1 and VP6 genes in their genotype constellation. No evidence for vaccine associated selection, or, interaction between wild-type and vaccine strains was obtained. In conclusion, this study reports the reemergence of G9P[8

  9. Whole genomic analyses of asymptomatic human G1P[6], G2P[6] and G3P[6] rotavirus strains reveal intergenogroup reassortment events and genome segments of artiodactyl origin.

    PubMed

    Ghosh, Souvik; Urushibara, Noriko; Chawla-Sarkar, Mamta; Krishnan, Triveni; Kobayashi, Nobumichi

    2013-06-01

    Although P[6] group A rotaviruses (RVA) cause diarrhoea in humans, they have been also associated with endemics of predominantly asymptomatic neonatal infections. Interestingly, strains representing the endemic and asymptomatic P[6] RVAs were found to possess one of the four common human VP7 serotypes (G1-G4), and exhibited little antigenic/genetic differences with the VP4 proteins/VP4 encoding genome segments of P[6] RVAs recovered from diarrhoeic children, raising interest on their complete genetic constellations. In the present study, we report the overall genetic makeup and possible origin of three such asymptomatic human P[6] RVA strains, RVA/Human-tc/VEN/M37/1982/G1P2A[6], RVA/Human-tc/SWE/1076/1983/G2P2A[6] and RVA/Human-tc/AUS/McN13/1980/G3P2A[6]. G1P[6] strain M37 exhibited an unusual genotype constellation (G1-P[6]-R1-C1-M1-A1-N1-T2-E1-H1), not reported previously, and was found to originate from possible intergenogroup reassortment events involving acquisition of a DS-1-like NSP3 encoding genome segment by a human Wa-like RVA strain. On the other hand, G2P[6] strain 1076 exhibited a DS-1-like genotype constellation, and was found to possess several genome segments (those encoding VP1, VP3, VP6 and NSP4) of possible artiodactyl (ruminants) origin on a human RVA genetic backbone. The whole genome of G3P[6] strain McN13 was closely related to that of asymptomatic human Wa-like G3P[6] strain RV3, and both strains shared unique amino acid changes, which might have contributed to their attenuation. Taken together, the present study provided insights into the origin and complex genetic diversity of P[6] RVAs possessing the common human VP7 genotypes. This is the first report on the whole genomic analysis of a G1P[6] RVA strain. PMID:23347969

  10. Rotavirus Diversity and Evolution in the Post-Vaccine World

    PubMed Central

    Patton, John T.

    2013-01-01

    Rotaviruses (RVs) are a large genetically diverse population of segmented double-stranded (ds) RNA viruses that are important causes of gastroenteritis in many animal species. The human RVs are responsible for the deaths of nearly 450,000 infants and young children each year, most occurring in developing countries. Recent large-scale sequencing efforts have revealed that the genomes of human RVs typically consist of phylogenetically linked constellations of eleven dsRNA segments. The presence of such preferred constellations indicate that the human RV genes have co-evolved to produce protein sets that work optimally together to support virus replication. Two of the viral genes encode virion outer capsid proteins (VP7 and VP4) whose antigenic properties define the G/P type of the virus. From year-to-year and place-to-place, the G/P type of human RVs associated with disease can fluctuate dramatically, phenomena that can be associated with the presence and behavior of genetically distinct RV clades. The recent introduction of two live attenuated RV vaccines (RotaReq™ and Rotarix™) into the childhood vaccination programs of various countries has been highly effective in reducing the incidence of RV diarrheal disease. Whether the widespread use of these vaccines will introduce selective pressures on human RVs, triggering genetic and antigenic changes that undermine the effectiveness of vaccinations programs, is uncertain and will require continued surveillance of human RVs. PMID:22284787

  11. Production of rotavirus core-like particles in Sf9 cells using recombinase-mediated cassette exchange.

    PubMed

    Fernandes, Fabiana; Dias, Mafalda M; Vidigal, João; Sousa, Marcos F Q; Patrone, Marco; Teixeira, Ana P; Alves, Paula M

    2014-02-10

    A flexible Sf9 insect cell line was recently developed leveraging the recombinase-mediated cassette exchange (RMCE) technology, which competes with the popular baculovirus expression vector system (BEVS) in terms of speed to produce new proteins. Herein, the ability of this cell platform to produce complex proteins, such as rotavirus core-like particles, was evaluated. A gene construct coding for a VP2-GFP fusion protein was targeted to a pre-characterized high recombination efficiency locus flanked by flipase (Flp) recognition target sites and, after three weeks in selection, an isogenic cell population was obtained. Despite the lower cell specific productivities with respect to those obtained by baculovirus infection, the titers of VP2-GFP reached in shake flask batch cultures were comparable as a result of higher cell densities. To further improve the VP2-GFP levels from stable expression, analysis of exhausted medium was undertaken to design feeding strategies enabling higher cell densities as well as increased culture duration. The implementation of the best strategy allowed reaching 20 million cells per ml in bioreactor cultures; the integrity of the rotavirus core-like particles could be confirmed by electron microscopy. Overall, we show that this Sf9-Flp cell platform represents a valuable alternative to the BEVS for producing complex recombinant proteins, such as rotavirus core-like particles. PMID:24333128

  12. Rotavirus Prevents the Expression of Host Responses by Blocking the Nucleocytoplasmic Transport of Polyadenylated mRNAs

    PubMed Central

    Rubio, Rosa M.; Mora, Silvia I.; Romero, Pedro; Arias, Carlos F.

    2013-01-01

    Rotaviruses are the most important agent of severe gastroenteritis in young children. Early in infection, these viruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis while viral proteins are efficiently synthesized. In infected cells, there is an accumulation of the cytoplasmic poly(A)-binding protein in the nucleus, induced by the viral protein NSP3. Here we found that poly(A)-containing mRNAs also accumulate and become hyperadenylated in the nuclei of infected cells. Using reporter genes bearing the untranslated regions (UTRs) of cellular or viral genes, we found that the viral UTRs do not determine the efficiency of translation of mRNAs in rotavirus-infected cells. Furthermore, we showed that while a polyadenylated reporter mRNA directly delivered into the cytoplasm of infected cells was efficiently translated, the same reporter introduced as a plasmid that needs to be transcribed and exported to the cytoplasm was poorly translated. Altogether, these results suggest that nuclear retention of poly(A)-containing mRNAs is one of the main strategies of rotavirus to control cell translation and therefore the host antiviral and stress responses. PMID:23536677

  13. Complement-fixing antibody response to rotavirus infection.

    PubMed Central

    Gust, I D; Pringle, R C; Barnes, G L; Davidson, G P; Bishop, R F

    1977-01-01

    A human rotavirus complement-fixing (CF) antigen, prepared by purification of large volumes of fluid feces collected from children with winter diarrhea, was used to study the development and persistence of antibody in children with diarrhea and the prevalence of rotavirus antibody in Melbourne. In children with diarrhea, antibody rises were detectable within 4 to 6 weeks of the onset of illness, and the titers usually remained elevated for the next 1 to 2 years. CF antibody did not develop in two children with proven rotavirus infection aged less than 6 months, an age at which poor CF responses to other viruses have also been observed. A study of CF antibody levels in the general community showed that in Melbourne, most children have been infected with human rotavirus by the age of 3 years. PMID:403196

  14. Correlates of protection against rotavirus infection and disease.

    PubMed

    Offit, P A

    2001-01-01

    Repeated infections with the 'mucosal' pathogen rotavirus are common in children. Subsequent rotavirus infections usually cause milder symptoms than first-time infections. Therefore, although natural rotavirus infection attenuates the severity of subsequent infections, it does not prevent reinfection or mild disease. On the other hand, natural infection with 'systemic' viruses such as measles, mumps, rubella, or varicella often confers life-long protection against mild disease associated with reinfection. The degree to which differences in the pathogenesis of systemic and mucosal pathogens determines differences in the capacity of natural infection to induce life-long protective immunity will be discussed. This paradigm will be used to explore the immunological effector functions associated with protection against rotavirus challenge. PMID:11444023

  15. Binding Patterns of Rotavirus Genotypes P[4], P[6], and P[8] in China with Histo-Blood Group Antigens.

    PubMed

    Ma, Xin; Li, Dan-di; Sun, Xiao-Man; Guo, Yan-Qing; Xiang, Jing-Yao; Wang, Wei-Huan; Zhang, Li-Xia; Gu, Qing-Jiu; Duan, Zhao-Jun

    2015-01-01

    Rotaviruses (RVs) are an important cause of severe gastroenteritis in children. It has been found that RV may recognize the histo-blood group antigens (HBGAs) as ligands or receptors and bind HBGAs in a type-dependent manner. In this study, we investigated the binding specificity of VP8* proteins from human rotaviruses (RV) that are prevalent in China including genotypes P[4], P[6], and P[8]. Through the saliva- and oligosaccharide-based binding assays, we found that the VP8* proteins of P[4] and P[8] RV showed similar reactivity with the Leb and H type 1 antigens, while P[6] RV weakly bound the Leb antigen. These findings may facilitate further research into RV host specificity and vaccine development. PMID:26274396

  16. Binding Patterns of Rotavirus Genotypes P[4], P[6], and P[8] in China with Histo-Blood Group Antigens

    PubMed Central

    Ma, Xin; Li, Dan-di; Sun, Xiao-man; Guo, Yan-qing; Xiang, Jing-yao; Wang, Wei-huan; Zhang, Li-xia; Gu, Qing-jiu; Duan, Zhao-jun

    2015-01-01

    Rotaviruses (RVs) are an important cause of severe gastroenteritis in children. It has been found that RV may recognize the histo-blood group antigens (HBGAs) as ligands or receptors and bind HBGAs in a type-dependent manner. In this study, we investigated the binding specificity of VP8* proteins from human rotaviruses (RV) that are prevalent in China including genotypes P[4], P[6], and P[8]. Through the saliva- and oligosaccharide-based binding assays, we found that the VP8* proteins of P[4] and P[8] RV showed similar reactivity with the Leb and H type 1 antigens, while P[6] RV weakly bound the Leb antigen. These findings may facilitate further research into RV host specificity and vaccine development. PMID:26274396

  17. Molecular Epidemiology of Rotavirus in Cats in the United Kingdom

    PubMed Central

    Iturriza-Gómara, M.; Dove, W.; Sandrasegaram, M.; Nakagomi, T.; Nakagomi, O.; Cunliffe, N.; Radford, A. D.; Morgan, K. L.

    2014-01-01

    Rotaviruses are leading causes of gastroenteritis in the young of many species. Molecular epidemiological studies in children suggest that interspecies transmission contributes to rotavirus strain diversity in people. However, population-based studies of rotaviruses in animals are few. We investigated the prevalence, risk factors for infection, and genetic diversity of rotavirus A in a cross-sectional survey of cats housed within 25 rescue catteries across the United Kingdom. Morning litter tray fecal samples were collected during the winter and summer in 2012 from all pens containing kittens and a random sample of those housing adult cats. Group A rotavirus RNA was detected by real-time reverse transcription-PCR, and positive samples were G and P genotyped using nested VP4 and VP7 PCR assays. A total of 1,727 fecal samples were collected from 1,105 pens. Overall, the prevalence of rotavirus was 3.0% (95% confidence interval [CI], 1.2 to 4.9%). Thirteen out of 25 (52%; 95% CI, 31.3 to 72.2%) centers housed at least one rotavirus-positive cat. The prevalence of rotavirus was associated with season (odds ratio, 14.8 [95% CI, 1.1 to 200.4]; P = 0.04) but not age or diarrhea. It was higher during the summer (4.7%; 95% CI, 1.2 to 8.3%) than in winter (0.8%; 95% CI, 0.2 to 1.5%). Asymptomatic epidemics of infection were detected in two centers. G genotypes were characterized for 19 (33.3%) of the 57 rotavirus-positive samples and P genotypes for 36 (59.7%). Two rotavirus genotypes were identified, G3P[9] and G6P[9]. This is the first population-based study of rotavirus in cats and the first report of feline G6P[9], which questions the previous belief that G6P[9] in people is of bovine origin. PMID:25411173

  18. Human and bovine serotype G8 rotaviruses may be derived by reassortment.

    PubMed

    Browning, G F; Snodgrass, D R; Nakagomi, O; Kaga, E; Sarasini, A; Gerna, G

    1992-01-01

    The origin of, and relationship between human and bovine serotype G8 rotaviruses were investigated by genomic hybridisation. Radiolabelled mRNAs of human G8 rotaviruses 69M (isolated in Indonesia) and HAL1271 (isolated in Finland), and bovine rotaviruses KK3 (G10) and NCDV (G6), were used as probes. The products of liquid hybridisation between the probes and the genomic RNA of human and bovine rotaviruses, including bovine G8 rotavirus 678 (isolated in Scotland) and two other Finnish human G8 rotaviruses HAL1166 and HAL8590, were examined by separation in polyacrylamide gels. The genomes of Finnish human G8 rotaviruses were similar to those of bovine G6 and G10 rotaviruses. Neither Indonesian human G8 nor bovine G8 viruses had high levels of similarity to each other or to other bovine and human rotaviruses. Thus these three epidemiologically distinct G8 rotaviruses have different origins and may be derived by reassortment with rotaviruses of a third, as yet unknown, host species. The similarity between the Finnish isolates and the bovine isolate NCDV suggests that they have diverged recently and that these human G8 rotaviruses may be derived from a zoonotic infection, or alternatively, from the live rotavirus vaccine of bovine origin which has been used to vaccinate Finnish children. PMID:1322648

  19. Genetic heterogeneity, evolution and recombination in emerging G9 rotaviruses.

    PubMed

    Phan, Tung Gia; Okitsu, Shoko; Maneekarn, Niwat; Ushijima, Hiroshi

    2007-09-01

    G9 rotavirus is recognized as the emerging genotype spreading around the world. The rapidly increasing detection of this virus in association with the genetic heterogeneity raises questions regarding its origin and epidemiological importance. A total of 380 sequences of rotavirus G9 strains including our sequence data from Vietnam and Japan, which were detected from 1983 to 2006 in five different continents, were collected from GenBank to investigate their heterogeneity and evolution. A novel nomenclature for G9 rotaviruses is proposed, in which these viruses are clustered into 6 lineages with 11 sublineages. Multiple amino acid substitutions of VP7 specific for lineages and sublineages were found. Interestingly, six short amino acid motifs correctly defined phylogenetic lineages and sublineages. Another interesting finding was the identification of recombinant G9 rotavirus, bearing different genotype sequence. In view of rotavirus evolution, this report is an additional evidence to support the notion that there might exist a genomic relatedness between human and porcine rotaviruses. PMID:17544926

  20. Estimating rotavirus vaccine effectiveness in Japan using a screening method.

    PubMed

    Araki, Kaoru; Hara, Megumi; Sakanishi, Yuta; Shimanoe, Chisato; Nishida, Yuichiro; Matsuo, Muneaki; Tanaka, Keitaro

    2016-05-01

    Rotavirus gastroenteritis is a highly contagious, acute viral disease that imposes a significant health burden worldwide. In Japan, rotavirus vaccines have been commercially available since 2011 for voluntary vaccination, but vaccine coverage and effectiveness have not been evaluated. In the absence of a vaccination registry in Japan, vaccination coverage in the general population was estimated according to the number of vaccines supplied by the manufacturer, the number of children who received financial support for vaccination, and the size of the target population. Patients with rotavirus gastroenteritis were identified by reviewing the medical records of all children who consulted 6 major hospitals in Saga Prefecture with gastroenteritis symptoms. Vaccination status among these patients was investigated by reviewing their medical records or interviewing their guardians by telephone. Vaccine effectiveness was determined using a screening method. Vaccination coverage increased with time, and it was 2-times higher in municipalities where the vaccination fee was supported. In the 2012/13 season, vaccination coverage in Saga Prefecture was 14.9% whereas the proportion of patients vaccinated was 5.1% among those with clinically diagnosed rotavirus gastroenteritis and 1.9% among those hospitalized for rotavirus gastroenteritis. Thus, vaccine effectiveness was estimated as 69.5% and 88.8%, respectively. This is the first study to evaluate rotavirus vaccination coverage and effectiveness in Japan since vaccination began. PMID:26680277

  1. Rotavirus vaccine-derived shedding and viral reassortants.

    PubMed

    Bowen, Michael D; Payne, Daniel C

    2012-11-01

    EVALUATION OF: Donato CM, Ch’ng LS, Boniface KF et al. Identification of strains of RotaTeq rotavirus vaccine in infants with gastroenteritis following routine vaccination. J. Infect. Dis. 206(3), 377–383 (2012).Two live, attenuated rotavirus vaccines, RotaTeq(®) (Merck) and Rotarix(®) (GlaxoSmithKline), have been used in Australia since July 2007 to prevent severe rotavirus gastroenteritis in children. Using active postvaccination monitoring, passive surveillance and state-of-the-art laboratory techniques, Donato et al. report that RotaTeq rotavirus vaccine and vaccine-derived strains were detected actively in stool samples from 13 out of 61 (21.3%) infants having diarrhea within 2 weeks of rotavirus vaccination, and among three out of 460 (0.7%) cases with acute gastroenteritis captured via the Australian Rotavirus Surveillance Program. Six (37.5%) of these 16 vaccine-derived viral specimens were associated with a G1P[8] strain thought to be the result of genetic reassortment between two component RotaTeq strains. Although nearly half of these reassortant-associated cases had underlying medical conditions, such as severe combined immunodeficiency disorder, further study is needed to understand the relationship between shedding, viral reassortants and underlying medical conditions. PMID:23249230

  2. Rotavirus vaccine: a cost effective control measure for India.

    PubMed

    Kumar, Arun; Goel, Manish K; Jain, Ram Bilas; Khanna, Pardeep; Vibha, Vibha

    2012-04-01

    Globally, rotavirus diarrhea results in 453,000 deaths in children younger than 5 y—37% of deaths attributable to diarrhea and 5% of all deaths in children younger than 5 y. India alone accounts for 22% (~100,000 deaths) of all deaths attributable to rotavirus infection. Two oral rotavirus vaccines are available: Rotarix, a monovalent P1A[8] G1 vaccine (GlaxoSmithKline), and RotaTeq, a pentavalent bovine-human reassortant vaccine (Merck). Rotarix is administered in a 2-dose schedule with the first and second doses of DTP (DTP1, DTP2). RotaTeq requires a 3-dose schedule with DTP1, DTP2 and DTP3 with an interval of 4–10 weeks between doses. The first dose of either vaccine should be administered to infants aged 6–15 weeks irrespective of the history of previous rotavirus infection, and the maximum age for administering the last dose of either vaccine should be 32 weeks. Although India would require funding from international health organizations/GAVI until new indigenous rotavirus vaccine candidates are developed at a cheaper price, introduction of vaccination into the national immunization program would be a cost-effective step toward control of the rotavirus diarrhea-related morbidity and mortality in India. PMID:23071989

  3. Estimating rotavirus vaccine effectiveness in Japan using a screening method

    PubMed Central

    Araki, Kaoru; Hara, Megumi; Sakanishi, Yuta; Shimanoe, Chisato; Nishida, Yuichiro; Matsuo, Muneaki; Tanaka, Keitaro

    2016-01-01

    abstract Rotavirus gastroenteritis is a highly contagious, acute viral disease that imposes a significant health burden worldwide. In Japan, rotavirus vaccines have been commercially available since 2011 for voluntary vaccination, but vaccine coverage and effectiveness have not been evaluated. In the absence of a vaccination registry in Japan, vaccination coverage in the general population was estimated according to the number of vaccines supplied by the manufacturer, the number of children who received financial support for vaccination, and the size of the target population. Patients with rotavirus gastroenteritis were identified by reviewing the medical records of all children who consulted 6 major hospitals in Saga Prefecture with gastroenteritis symptoms. Vaccination status among these patients was investigated by reviewing their medical records or interviewing their guardians by telephone. Vaccine effectiveness was determined using a screening method. Vaccination coverage increased with time, and it was 2-times higher in municipalities where the vaccination fee was supported. In the 2012/13 season, vaccination coverage in Saga Prefecture was 14.9% whereas the proportion of patients vaccinated was 5.1% among those with clinically diagnosed rotavirus gastroenteritis and 1.9% among those hospitalized for rotavirus gastroenteritis. Thus, vaccine effectiveness was estimated as 69.5% and 88.8%, respectively. This is the first study to evaluate rotavirus vaccination coverage and effectiveness in Japan since vaccination began. PMID:26680277

  4. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

    Guerrero, Carlos A.; Guerrero, Rafael A.; Silva, Elver; Acosta, Orlando; Barreto, Emiliano

    2016-01-01

    A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death. PMID:26828934

  5. Scope for rotavirus vaccination in India: revisiting the scientific evidence.

    PubMed

    Neogi, Sutapa Bandyopadhyay; Hasan, Habib; Sheikh, Kabir; Zodpey, Sanjay

    2011-10-01

    Rotavirus vaccines have been developed to prevent deaths resulting from severe diarrhea of rotavirus origin. The use of vaccines as an intervention at scale to prevent and control the burden of rotavirus diarrhea is supported by the argument that prevailing public health measures such as hygiene and sanitation, breast feeding and use of ORS have failed to prevent severe dehydration resulting from diarrhea. The article reviews the existing evidence on the rationale of using rotavirus vaccine as against the feasibility of scaling it up in developing countries like India. The vaccines currently available may not cover the strains circulating in Indian population. The diversity of Rotavirus infection in the country is tremendous and since the safety, immunogenicity and efficacy data has not been collected for India, there is first a need to conduct studies to measure the extent of protection and cross-protection provided by the available vaccines for local strains, before venturing into Rotavirus vaccination program. The potential benefits of immunization have to be first vetted against the risks involved by the policymakers and other stakeholders. PMID:21611712

  6. Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq.

    PubMed

    Matthijnssens, Jelle; Joelsson, Daniel B; Warakomski, Donald J; Zhou, Tingyi; Mathis, Pamela K; van Maanen, Marc-Henri; Ranheim, Todd S; Ciarlet, Max

    2010-08-01

    RotaTeq is a pentavalent rotavirus vaccine that contains five human-bovine reassortant strains (designated G1, G2, G3, G4, and P1) on the backbone of the naturally attenuated tissue culture-adapted parental bovine rotavirus (BRV) strain WC3. The viral genomes of each of the reassortant strains were completely sequenced and compared pairwise and phylogenetically among each other and to human rotavirus (HRV) and BRV reference strains. Reassortants G1, G2, G3, and G4 contained the VP7 gene from their corresponding HRV parent strains, while reassortants G1 and G2 also contained the VP3 gene (genotype M1) from the HRV parent strain. The P1 reassortant contained the VP4 gene from the HRV parent strain and all the other gene segments from the BRV WC3 strain. The human VP7s had a high level of overall amino acid identity (G1: 95-99%, G2: 94-99% G3: 96-100%, G4: 93-99%) when compared to those of representative rotavirus strains of their corresponding G serotypes. The VP4 of the P1 reassortant had a high identity (92-97%) with those of serotype P1A[8] HRV reference strains, while the BRV VP7 showed identities ranging from 91% to 94% to those of serotype G6 HRV strains. Sequence analyses of the BRV or HRV genes confirmed that the fundamental structure of the proteins in the vaccine was similar to those of the HRV and BRV references strains. Sequences analyses showed that RotaTeq exhibited a high degree of genetic stability as no mutations were identified in the material of each reassortant, which undergoes two rounds of replication cycles in cell culture during the manufacturing process, when compared to the final material used to fill the dosing tubes. The infectivity of each of the reassortant strains of RotaTeq, like HRV strains, did not require the presence of sialic acid residues on the cell surface. The molecular and biologic characterization of RotaTeq adds to the significant body of clinical data supporting the consistent efficacy, immunogenicity, and safety of Rota

  7. Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq (registered)

    SciTech Connect

    Matthijnssens, Jelle; Joelsson, Daniel B.; Warakomski, Donald J.; Zhou, Tingyi; Mathis, Pamela K.; Maanen, Marc-Henri van; Ranheim, Todd S.; Ciarlet, Max

    2010-08-01

    RotaTeq (registered) is a pentavalent rotavirus vaccine that contains five human-bovine reassortant strains (designated G1, G2, G3, G4, and P1) on the backbone of the naturally attenuated tissue culture-adapted parental bovine rotavirus (BRV) strain WC3. The viral genomes of each of the reassortant strains were completely sequenced and compared pairwise and phylogenetically among each other and to human rotavirus (HRV) and BRV reference strains. Reassortants G1, G2, G3, and G4 contained the VP7 gene from their corresponding HRV parent strains, while reassortants G1 and G2 also contained the VP3 gene (genotype M1) from the HRV parent strain. The P1 reassortant contained the VP4 gene from the HRV parent strain and all the other gene segments from the BRV WC3 strain. The human VP7s had a high level of overall amino acid identity (G1: 95-99%, G2: 94-99% G3: 96-100%, G4: 93-99%) when compared to those of representative rotavirus strains of their corresponding G serotypes. The VP4 of the P1 reassortant had a high identity (92-97%) with those of serotype P1A[8] HRV reference strains, while the BRV VP7 showed identities ranging from 91% to 94% to those of serotype G6 HRV strains. Sequence analyses of the BRV or HRV genes confirmed that the fundamental structure of the proteins in the vaccine was similar to those of the HRV and BRV references strains. Sequences analyses showed that RotaTeq (registered) exhibited a high degree of genetic stability as no mutations were identified in the material of each reassortant, which undergoes two rounds of replication cycles in cell culture during the manufacturing process, when compared to the final material used to fill the dosing tubes. The infectivity of each of the reassortant strains of RotaTeq (registered) , like HRV strains, did not require the presence of sialic acid residues on the cell surface. The molecular and biologic characterization of RotaTeq (registered) adds to the significant body of clinical data supporting the

  8. Comparative analysis of pentavalent rotavirus vaccine strains and G8 rotaviruses identified during vaccine trial in Africa

    PubMed Central

    Heylen, Elisabeth; Zeller, Mark; Ciarlet, Max; Lawrence, Jody; Steele, Duncan; Van Ranst, Marc; Matthijnssens, Jelle

    2015-01-01

    RotaTeqTM is a pentavalent rotavirus vaccine based on a bovine rotavirus genetic backbone in vitro reassorted with human outer capsid genes. During clinical trials of RotaTeqTM in Sub-Saharan Africa, the vaccine efficacy over a 2-year follow-up was lower against the genotypes contained in the vaccine than against the heterotypic G8P[6] and G8P[1] rotavirus strains of which the former is highly prevalent in Africa. Complete genome analyses of 43 complete rotavirus genomes collected during phase III clinical trials of RotaTeqTM in Sub-Saharan Africa, were conducted to gain insight into the high level of cross-protection afforded by RotaTeqTM against these G8 strains. Phylogenetic analysis revealed the presence of a high number of bovine rotavirus gene segments in these human G8 strains. In addition, we performed an in depth analysis on the individual amino acid level which showed that G8 rotaviruses were more similar to the RotaTeqTM vaccine than non-G8 strains. Because RotaTeqTM possesses a bovine genetic backbone, the high vaccine efficacy against G8 strains might be partially explained by the fact that all these strains contain a complete or partial bovine-like backbone. Altogether, this study supports the hypothesis that gene segments other than VP7 and VP4 play a role in vaccine-induced immunity. PMID:26440913

  9. Rotavirus Genotypes in Belarus, 2008–2012

    PubMed Central

    Semeiko, Galina; Yermalovich, Marina; Poliakova, Nadezhda; Mijatovic-Rustempasic, Slavica; Kerin, Tara; Wasley, Annemarie; Videbaek, Dovile; Gentsch, Jon R.; Bowen, Michael D.; Samoilovich, Elena

    2015-01-01

    This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008–2012. In 2008, data from 3 sites in Belarus (Brest, Mogilev, Minsk) indicated that G4P[8] was the predominant genotype. Data from Minsk (2008–2012) showed that G4P[8] was the predominant RVA genotype in all years except in 2011 when G3P[8] was most frequently detected. Other RVA genotypes common in Europe (G1P[8], G2P[4]) were detected each year of the study. This study reveals the dominance of genotype G4P[8] in Belarus and helps to establish the baseline genotype prevalence prior to RVA vaccine introduction in the country. PMID:25218086

  10. [Isolation and characterization of rotavirus from bat].

    PubMed

    Xia, Le-Le; He, Biao; Hu, Ting-Song; Zhang, Wen-Dong; Wang, Yi-Yin; Xu, Lin; Li, Nan; Qiu, Wei; Yu, Jing; Fan, Quan-Shui; Zhang, Fu-Qiang; Tu, Chang-Chun

    2013-11-01

    Bats are considered as important animal reservoirs for many pathogenic viruses to humans. The viral metagenomic analysis was performed to study gut and lung tissues of 30 insectivorous bats collected in Yunnan Province and 26 reads were noted to group A rotavirus (RVA). Further RT-PCR screening on bat samples and in vitro viral isolation on cell cultures confirmed the presence of a novel RVA, named as RVA/Bat-tc/MYAS33/2013/G3P[10], in one of 30 Stoliczka's trident bats. The VP7 gene of this strain MYAS33 was closely related to that of an equine RVA strain from Argentina and the nucleotide sequence similarity was 93%, while its VP4 gene was a rare P[10] type and obtained the maximum sequence identity (94.8%) with that of a human strain from Thailand. The present study highlights the potential role of bats as reservoirs for RVAs. PMID:24520769

  11. Evidence of Vaccine-related Reassortment of Rotavirus, Brazil, 2008–2010

    PubMed Central

    Marques da Silva, Marcelle Figueira; Goméz, Mariela Martinéz; Resque, Hugo Reis; Ichihara, Maria Yury Travassos; Volotão, Eduardo de Mello; Leite, José Paulo Gagliardi

    2013-01-01

    Analysis of 27 rotavirus strains from vaccinated and unvaccinated children revealed reassortment events in 3 strains: a gene derived from a vaccine; a gene acquired from a circulating strain; and reassortment between circulating strains. Data suggest that the widespread use of this monovalent rotavirus vaccine may introduce vaccine genes into circulating human rotaviruses or vice versa. PMID:24188273

  12. Determinants of Parents' Decision to Vaccinate Their Children against Rotavirus: Results of a Longitudinal Study

    ERIC Educational Resources Information Center

    Dube, E.; Bettinger, J. A.; Halperin, B.; Bradet, R.; Lavoie, F.; Sauvageau, C.; Gilca, V.; Boulianne, N.

    2012-01-01

    Rotavirus disease is a common cause of health care utilization and almost all children are affected by the age of 5 years. In Canada, at the time of this survey (2008-09), immunization rates for rotavirus were less than 20%. We assessed the determinants of a parent's acceptance to have their child immunized against rotavirus. The survey…

  13. Parvovirus particles as platforms for protein presentation.

    PubMed Central

    Miyamura, K; Kajigaya, S; Momoeda, M; Smith-Gill, S J; Young, N S

    1994-01-01

    Empty capsids of the human pathogenic parvovirus B19 can be produced in a baculovirus system. B19 capsids are composed mainly of major capsid protein (VP2) and a small amount of minor capsid protein (VP1); VP1 is identical to VP2 but contains an additional 227-aa N-terminal region ("unique" region). A portion of that region of VP1 is external to the capsid, and VP1 is not required for capsid formation. We substituted the unique region with a sequence encoding the 147 aa of hen egg white lysozyme (HEL) and constructed recombinant baculoviruses with variable amounts of retained VP1 sequence joined to the VP2 backbone. After cotransfection with VP2 baculovirus and expression in insect cells, capsids were purified by density sedimentation. Purified recombinant capsids contained HEL. External presentation of HEL was demonstrated by immunoprecipitation, ELISA, and immune electron microscopy using anti-lysozyme monoclonal antibodies or specific rabbit antisera. Empty particles showed enzymatic activity in a micrococcal cell wall digestion assay. Rabbits inoculated with capsids made antibodies to HEL. Intact heterologous protein can be incorporated in B19 particles and presented on the capsid surface, properties that may be useful in vaccine development, cell targeting, and gene therapy. Images PMID:8078912

  14. Characterization of a monoclonal antibody directed to the surface of MA104 cells that blocks the infectivity of rotaviruses.

    PubMed

    López, S; Espinosa, R; Isa, P; Merchant, M T; Zárate, S; Méndez, E; Arias, C F

    2000-07-20

    Rhesus rotavirus (RRV) binds to sialic acid residues on the surface of target cells, and treatment of these cells with neuraminidase greatly reduces virus binding with the consequent reduction of infectivity. Variants that can efficiently infect neuraminidase-treated cells have been isolated, indicating that attachment to sialic acid is not an essential step for animal rotaviruses to infect cells. To identify and characterize the neuraminidase-resistant receptor for rotaviruses, we have isolated a hybridoma that secrets a monoclonal antibody (MAb) (2D9) that specifically blocks the infectivity of wild-type (wt) RRV and of its sialic acid-independent variant nar3, in untreated as well as in neuraminidase-treated cells. The infectivity of a human rotavirus was also inhibited, although to a lesser extent. MAb 2D9 blocks the binding of the variant to MA104 cells, while not affecting the binding of wt RRV; in addition, this MAb blocked the attachment of a recombinant glutathione S-transferase (GST)-VP5 fusion protein, but did not affect the binding of GST-VP8. Altogether these results suggest that MAb 2D9 is directed to the neuraminidase-resistant receptor. This receptor seems to mediate the direct attachment of the variant to the cell, through VP5, while the receptor is used by wt RRV for a secondary interaction, after its initial binding to sialic acid, through VP8. MAb 2D9 interacts specifically with the cell surface by indirect immunofluorescence, immunoelectron microscopy, and FACS. By a solid-phase immunoisolation technique, MAb 2D9 was found to react with three proteins of ca. 47, 55, and 220 kDa, which might form a complex. PMID:10891418

  15. Characterization and protective efficacy in an animal model of a novel truncated rotavirus VP8 subunit parenteral vaccine candidate.

    PubMed

    Xue, Miaoge; Yu, Linqi; Che, Yaojian; Lin, Haijun; Zeng, Yuanjun; Fang, Mujin; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

    2015-05-21

    The cell-attachment protein VP8* of rotavirus is a potential candidate parenteral vaccine. However, the yield of full-length VP8 protein (VP8*, residues 1-231) expressed in Escherichia coli was low, and a truncated VP8 protein (ΔVP8*, residues 65-231) cannot elicit efficient protective immunity in a mouse model. In this study, tow novel truncated VP8 proteins, VP8-1 (residues 26-231) and VP8-2 (residues 51-231), were expressed in E. coli and evaluated for immunogenicity and protective efficacy, compared with VP8* and ΔVP8*. As well as ΔVP8*, the protein VP8-1 and VP8-2 were successfully expressed in high yield and purified in homogeneous dimeric forms, while the protein VP8* was expressed with lower yield and prone to aggregation and degradation in solution. Although the immunogenicity of the protein VP8*, VP8-1, VP8-2 and ΔVP8* was comparable, immunization of VP8* and VP8-1 elicited significantly higher neutralizing antibody titers than that of VP8-2 and ΔVP8* in mice. Furthermore, when assessed using a mouse maternal antibody model, the efficacy of VP8-1 to protect against rotavirus-induced diarrhea in pups was comparable to that of VP8*, both were dramatically higher than that of VP8-2 and ΔVP8*. Taken together, the novel truncated protein VP8-1, with increased yield, improved homogeneity and high protective efficacy, is a viable candidate for further development of a parenterally administrated prophylactic vaccine against rotavirus infection. PMID:25882173

  16. Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis.

    PubMed Central

    Yolken, R H; Peterson, J A; Vonderfecht, S L; Fouts, E T; Midthun, K; Newburg, D S

    1992-01-01

    Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens. Images PMID:1331178

  17. Introduction and prolonged circulation of G12 rotaviruses in Sicily.

    PubMed

    Giammanco, G M; Bonura, F; DI Bernardo, F; Cascio, A; Ferrera, G; Dones, P; Saporito, L; Collura, A; Terranova, D M; Valenzise, M; Allù, M T; Casuccio, N; Palermo, M; Bányai, K; Martella, V; DE Grazia, S

    2016-07-01

    Genotype G12 strains are now considered to be the sixth most prevalent human rotaviruses worldwide. In two Sicilian cities, Palermo and Messina, surveillance of rotavirus circulation performed since 1985 and 2009, respectively, did not detect G12 strains until 2012. From 2012 to 2014 rotavirus infection was detected in 29·7% of 1647 stool samples collected from children admitted for acute gastroenteritis to three Sicilian hospitals in Palermo, Messina and Ragusa. In 2012, G12P[8] was first detected in Palermo and then in Messina where it represented the second most frequent genotype (20% prevalence) after G1P[8]. Thereafter, G12 strains continued to circulate in Sicily, showing a marked prevalence in Ragusa (27·8%) in 2013 and in Palermo (21%) and Messina (16·6%) in 2014. All but one of the Sicilian G12 strains carried a P[8] VP4 genotype, whereas the single non-P[8] rotavirus strain was genotyped as G12P[9]. Phylogenetic analysis of the VP7 and VP4 sequences allowed distinction of several genetic lineages and separation of the G12P[8] strains into three cluster combinations. These findings indicate independent introductions of G12 rotavirus strains in Sicily in recent years. PMID:26743189

  18. Effects of wastewater sludge and its detergents on the stability of rotavirus

    SciTech Connect

    Ward, R.L.; Ashley, C.S.

    1980-06-01

    Wastewater sludge reduced the heat required to inactivate rotavirus SA-11, and ionic detergents were identified as the sludge components responsible for this effect. A similar result was found previously with reovirus. The quantitative effects of individual ionic detergents on rotavirus and reovirus were very different, and rotavirus was found to be extremely sensitive to several of these detergents. However, neither virus was destabilized by nonionic detergents. On the contrary, rotavirus was stabilized by a nonionic detergent against the potent destabilizing effects of the ionic detergent sodium dodecyl sulfate. The destabilizing effects of both cationic and anionic detergents on rotavirus were greatly altered by changes in the pH of the medium.

  19. Heat Shock Protein 70 Enhances Mucosal Immunity against Human Norovirus When Coexpressed from a Vesicular Stomatitis Virus Vector

    PubMed Central

    Ma, Yuanmei; Duan, Yue; Wei, Yongwei; Liang, Xueya; Niewiesk, Stefan; Oglesbee, Michael

    2014-01-01

    ABSTRACT Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942–2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 106 PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 106 PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo, thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine. IMPORTANCE Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is

  20. Study on Rotavirus Infection and Its Genotyping in Children Below 5 Years in South West Iran

    PubMed Central

    Azaran, Azarakhsh; Makvandi, Manoochehr; Samarbafzadeh, Alireza; Neisi, Niloofar; Hoseinzadeh, Mohsen; Rasti, Mojtaba; Teymurirad, Majid; Teimoori, Ali; Varnaseri, Mehran; Makvandi, Kamyar

    2016-01-01

    Background Human rotaviruses are the most important agents for severe dehydrating diarrhea in children below 5 years old. Rotaviruses (RV) is a serious public health problem in developing and developed countries. Objectives The aim of this study was to determine the prevalence of rotavirus infection and their genotypes in children younger than 5 years of age with acute diarrhea in Ahvaz, Iran. Materials and Methods For this study, 200 stool samples from children below 5 years of age with acute diarrhea were collected between October 2011 and March 2012. Initially all stool samples were tested for rotavirus antigen by ELISA, and positive samples were confirmed by RT-PCR targeting the VP6 rotavirus gene. Determination of rotavirus genotypes was carried out by performing RT-PCR for G and P types. Altogether, 15 samples were sequenced. Results Out of 200 stool samples, 100 (50%) had rotavirus antigen detected by ELISA and 73 (36.5%) were found positive by RT-PCR. Of the rotavirus strains identified, only 63 (86.3%) were positive for both VP7 and VP4 while 10 (13.7%) strains were found nontypeable. Rotavirus infection accounts for 36.5% of gastroenteritis cases in samples from symptomatic children. The most prevalent rotavirus genotypes were G1P [8] (80%) followed by G2P [4] (20%). Conclusions Our results suggest that group A rotavirus is a major pathogene of acute diarrhea in Ahvaz city. The genotypes circulating are similar with those of other countries. PMID:27307959

  1. Accelerating the introduction of rotavirus immunization in Indonesia.

    PubMed

    Suwantika, Auliya A; Zakiyah, Neily; Lestari, Keri; Postma, Maarten J

    2014-04-01

    The introduction of the rotavirus vaccine in Indonesia is currently in its infancy. Delay in its development might be caused by factors related to the perceived value of the vaccine, health system characteristics and policy considerations. Other factors, which may also interfere with optimizing the introduction, are financial barriers because Indonesia is a lower-middle-income country. Creating fiscal space to finance new immunization programs, such as for the rotavirus immunization, is very important to ensure the sustainability of new programs so that such programs would be financed over the long term and not endanger the sustainability of the Indonesian government's financial position. This article provides an illustration of the various steps needed to accelerate the introduction of the rotavirus immunization. PMID:24580504

  2. Preventing rotavirus gastroenteritis: do you have the facts?

    PubMed

    Davidson, Geoffrey; Elliott, Elizabeth J; Kirkwood, Carl; Pearce, Rodney

    2007-01-01

    From 1 July 2007 two new rotavirus vaccines licensed for use in Australia (RotaTeq CSL Biotherapies/Merck and Rotarix Glaxo Smith Kline) will be funded for the National Immunisation Program. The vaccines differ with respect to their composition and the timing and mode of administration. Both have been evaluated in huge randomised trials and shown to be highly effective in preventing rotavirus gastroenteritis, including severe disease requiring hospital admission. Neither has been associated with an increased rate of intussusception; however, surveillance for adverse effects following vaccination will be important. As rotavirus infection is ubiquitous in young children, funding of this vaccine will significantly decrease the enormous morbidity and costs associated with this disease in our community. PMID:17635688

  3. Demographic variability, vaccination, and the spatiotemporal dynamics of rotavirus epidemics.

    PubMed

    Pitzer, Virginia E; Viboud, Cécile; Simonsen, Lone; Steiner, Claudia; Panozzo, Catherine A; Alonso, Wladimir J; Miller, Mark A; Glass, Roger I; Glasser, John W; Parashar, Umesh D; Grenfell, Bryan T

    2009-07-17

    Historically, annual rotavirus activity in the United States has started in the southwest in late fall and ended in the northeast 3 months later; this trend has diminished in recent years. Traveling waves of infection or local environmental drivers cannot account for these patterns. A transmission model calibrated against epidemiological data shows that spatiotemporal variation in birth rate can explain the timing of rotavirus epidemics. The recent large-scale introduction of rotavirus vaccination provides a natural experiment to further test the impact of susceptible recruitment on disease dynamics. The model predicts a pattern of reduced and lagged epidemics postvaccination, closely matching the observed dynamics. Armed with this validated model, we explore the relative importance of direct and indirect protection, a key issue in determining the worldwide benefits of vaccination. PMID:19608910

  4. Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST)

    PubMed Central

    Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

    2014-01-01

    During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase–polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains. PMID:25424931

  5. Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

    PubMed

    Walther, Ashley; Mohanty, Sujit K; Donnelly, Bryan; Coots, Abigail; Lages, Celine S; Lobeck, Inna; Dupree, Phylicia; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

    2015-09-15

    Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers. PMID:26206856

  6. Different virulence of porcine and porcine-like bovine rotavirus strains with genetically nearly identical genomes in piglets and calves

    PubMed Central

    2013-01-01

    Direct interspecies transmissions of group A rotaviruses (RVA) have been reported under natural conditions. However, the pathogenicity of RVA has never been directly compared in homologous and heterologous hosts. The bovine RVA/Cow-tc/KOR/K5/2004/G5P[7] strain, which was shown to possess a typical porcine-like genotype constellation similar to that of the G5P[7] prototype RVA/Pig-tc/USA/OSU/1977/G5P9[7] strain, was examined for its pathogenicity and compared with the porcine G5P[7] RVA/Pig-tc/KOR/K71/2006/G5P[7] strain possessing the same genotype constellation. The bovine K5 strain induced diarrhea and histopathological changes in the small intestine of piglets and calves, whereas the porcine K71 strain caused diarrhea and histopathological changes in the small intestine of piglets, but not in calves. Furthermore, the bovine K5 strain showed extra-intestinal tropisms in both piglets and calves, whereas the porcine K71 strain had extra-intestinal tropisms in piglets, but not in calves. Therefore, we performed comparative genomic analysis of the K71 and K5 RVA strains to determine whether specific mutations could be associated with these distinct clinical and pathological phenotypes. Full-length sequencing analyses for the 11 genomic segments for K71 and K5 revealed that these strains were genetically nearly identical to each other. Two nucleotide mutations were found in the 5′ untranslated region (UTR) of NSP5 and the 3′ UTR of NSP3, and eight amino acid mutations in VP1-VP4 and NSP2. Some of these mutations may be critical molecular determinants for RVA virulence and/or pathogenicity. PMID:24083947

  7. Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection.

    PubMed Central

    Offit, P A; Cunningham, S L; Dudzik, K I

    1991-01-01

    The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria, viruses, and parasites. The first line of defense against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. In addition, the intestine is a rich source of lymphocytes located within Peyer's patches and the lamina propria. Little is known about the function, memory, trafficking, or origin of intestinal T lymphocytes after intestinal infection. We studied the murine cytotoxic T-lymphocyte (CTL) response to the intestinal pathogen rotavirus (simian strain RRV). Adult mice were inoculated orally or via the hind footpad with RRV; virus-specific cytotoxic activities in intestinal and nonintestinal lymphocyte populations were determined by 51Cr release assays. In addition, virus-specific CTL precursor (CTLp) frequencies were determined by limiting-dilution analysis. IELs containing rotavirus-specific cytotoxic activity were detected after oral but not footpad inoculation and expressed alpha/beta but not gamma/delta cell surface protein; virus-specific CTLs did not appear to arise from CTLp among IELs. In addition, the site at which RRV was presented to the immune system determined the site at which RRV-specific CTLp first appeared. Frequencies of rotavirus-specific CTLp detected in Peyer's patches were 25- to 30-fold greater after oral than after footpad inoculation. However, regardless of the route of inoculation, rotavirus-specific CTLp were distributed throughout the lymphoid system 21 days after infection. Implications of these findings for vaccine design are discussed. PMID:1847457

  8. Whole-genomic analysis of 12 porcine group A rotaviruses isolated from symptomatic piglets in Brazil during the years of 2012-2013.

    PubMed

    Silva, Fernanda D F; Espinoza, Luis R L; Tonietti, Paloma O; Barbosa, Bruna R P; Gregori, Fabio

    2015-06-01

    Group A rotaviruses (RVAs) are leading causes of viral diarrhea in children and in the young of many animal species, particularly swine. In the current study, porcine RVAs were found in fecal specimens from symptomatic piglets on 4 farms in Brazil during the years of 2012-2013. Using RT-PCR, Sanger nucleotide sequencing, and phylogenetic analyses, the whole genomes of 12 Brazilian porcine RVA strains were analyzed. Specifically, the full-length open reading frame (ORF) sequences were determined for the NSP2-, NSP3-, and VP6-coding genes, and partial ORF sequences were determined for the VP1-, VP2-, VP3-, VP4-, VP7-, NSP1-, NSP4-, and NSP5/6-coding genes. The results indicate that all 12 strains had an overall porcine-RVA-like backbone with most segments being designated as genotype 1, with the exception of the VP6- and NSP1-coding genes, which were genotypes I5 and A8, respectively. These results add to our growing understanding of porcine RVA genetic diversity and will provide a platform for monitoring the role of animals as genetic reservoirs of emerging human RVAs strains. PMID:25796358

  9. Novel porcine-like human G26P[19] rotavirus identified in hospitalized paediatric diarrhoea patients in Ho Chi Minh City, Vietnam

    PubMed Central

    My, Phan Vu Tra; Rabaa, Maia A.; Donato, Celeste; Cowley, Daniel; Phat, Voong Vinh; Dung, Tran Thi Ngoc; Anh, Pham Hong; Vinh, Ha; Bryant, Juliet E.; Kellam, Paul; Thwaites, Guy; Woolhouse, Mark E. J.; Kirkwood, Carl D.

    2014-01-01

    During a hospital-based diarrhoeal disease study conducted in Ho Chi Minh City, Vietnam from 2009 to 2010, we identified four symptomatic children infected with G26P[19] rotavirus (RV) – an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P[19] strain, the whole genome of a representative example was characterized, revealing a novel genome constellation: G26–P[19]–I5–R1–C1–M1–A8–N1–T1–E1–H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine RVs (VP1–3 and NSP2–5). We proposed that this G26P[19] strain was the product of zoonotic transmission coupled with one or more reassortment events occurring in human and/or animal reservoirs. The identification of such strains has potential implications for vaccine efficacy in south-east Asia, and outlines the utility of whole-genome sequencing for studying RV diversity and zoonotic potential during disease surveillance. PMID:25121549

  10. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    SciTech Connect

    Zhang, Yang-De Li, Hao; Liu, Hui; Pan, Yi-Feng

    2007-02-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.

  11. Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

    PubMed Central

    Hyser, Joseph M.; Utama, Budi; Crawford, Sue E.; Broughman, James R.

    2013-01-01

    Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections. PMID:24109210

  12. Bovine Fetal Inoculations with Calf Rotavirus

    PubMed Central

    Schlafer, D. H.; Schultz, R. D.; Scott, F. W.; Duncan, J. R.

    1979-01-01

    The serological and histopathological responses of bovine fetuses to in utero inoculation with virulent and attenuated strains of the calf rotavirus (reovirus-like agent of neonatal calf diarrhea) are described. Thirteen bovine fetuses, 63 to 190 days of gestation, were inoculated in utero with attenuated (three fetuses) or field strain virus (nine fetuses) or both (one fetus). Serum-neutralizing antibody titers ranging from 1:16 to > 1:256 were detected in six of eight fetuses tested, demonstrating the ability of the bovine fetus to respond immunologically to this agent. The youngest fetus in the series was inoculated at 63 days of gestation and developed a titer of 128 in 64 days. This represents the earliest stage of gestation at which a bovine fetus has been inoculated with a bovine virus and found to produce antibody to it. Serum neutralizing titers in six of the eight dams tested increased significantly following the inoculations of their fetuses in utero. Histological changes associated with viral replication and antigenic stimulation of the lymphoreticular system were observed. Pneumonic lesions consisting of both local and diffuse lymphoreticular proliferation were present in five of the nine fetuses that were alive at slaughter. Gliosis and perivascular cuffing were noted in the brains of two of these fetuses and meningitis was seen in one. No evidence of teratogenic change was found. ImagesFig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7. PMID:232853

  13. Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

    SciTech Connect

    Hu, Liya; Ramani, Sasirekha; Czako, Rita; Sankaran, Banumathi; Yu, Ying; Smith, David F.; Cummings, Richard D.; Estes, Mary K.; Venkataram Prasad, B. V.

    2015-09-30

    We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.

  14. Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

    DOE PAGESBeta

    Hu, Liya; Ramani, Sasirekha; Czako, Rita; Sankaran, Banumathi; Yu, Ying; Smith, David F.; Cummings, Richard D.; Estes, Mary K.; Venkataram Prasad, B. V.

    2015-09-30

    We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

  15. Interaction of Rotavirus with Human Myeloid Dendritic Cells

    PubMed Central

    Narváez, Carlos F.; Angel, Juana; Franco, Manuel A.

    2005-01-01

    We have previously shown that very few rotavirus (RV)-specific T cells that secrete gamma interferon circulate in recently infected and seropositive adults and children. Here, we have studied the interaction of RV with myeloid immature (IDC) and mature dendritic cells (MDC) in vitro. RV did not induce cell death of IDC or MDC and induced maturation of between 12 and 48% of IDC. Nonetheless, RV did not inhibit the maturation of IDC or change the expression of maturation markers on MDC. After treatment with RV, few IDC expressed the nonstructural viral protein NSP4. In contrast, a discrete productive viral infection was shown in MDC of a subset of volunteers, and between 3 and 46% of these cells expressed NSP4. RV-treated IDC secreted interleukin 6 (IL-6) (but not IL-1β, IL-8, IL-10, IL-12, tumor necrosis factor alpha, or transforming growth factor beta), and MDC released IL-6 and small amounts of IL-10 and IL-12p70. The patterns of cytokines secreted by T cells stimulated by staphylococcal enterotoxin B presented by MDC infected with RV or uninfected were comparable. The frequencies and patterns of cytokines secreted by memory RV-specific T cells evidenced after stimulation of peripheral blood mononuclear cells (PBMC) with RV were similar to those evidenced after stimulation of PBMC with RV-infected MDC. Finally, IDC treated with RV strongly stimulated naive allogeneic CD4+ T cells to secrete Th1 cytokines. Thus, although RV does not seem to be a strong maturing stimulus for DC, it promotes their capacity to prime Th1 cells. PMID:16282452

  16. Design and Construction of Chimeric VP8-S2 Antigen for Bovine Rotavirus and Bovine Coronavirus

    PubMed Central

    Nasiri, Khadijeh; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza; Zibaee, Saeed

    2016-01-01

    Purpose: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Methods: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. Results: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni–NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. Conclusion: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure. PMID:27123423

  17. Detection of emerging rotavirus G12P[8] in Sonora, México.

    PubMed

    González-Ochoa, G; J, G de; Calleja-García, P M; Rosas-Rodríguez, J A; Virgen-Ortíz, A; Tamez-Guerra, P

    2016-06-01

    Rotavirus is the most common cause of gastroenteritis in children up to five years of age worldwide. The aim of the present study was to analyze the genotypes of rotavirus strains isolated from children with gastroenteritis, after the introduction of the rotavirus vaccine in México. Rotavirus was detected in 14/100 (14%) fecal samples from children with gastroenteritis, using a commercial test kit. The viral genome was purified from these samples and used as a template in RT-PCR amplification of the VP4 and VP7 genes, followed by gene cloning and sequencing. Among the rotavirus strains, 4/14 (28.5%) were characterized as G12P[8], 2/14 (14.3%), as G12P (not typed), and 3/14 (21.42%) as G (not typed) P[8]. Phylogenetic analysis of the VP7 gene showed that G12 genotypes clustered in lineage III. Phylogenetic analysis revealed that VP4 genotype P[8] sequences clustered in lineage V, whereas other P[8] sequences previously reported in Mexico (2005-2008) clustered in different lineages. Rotavirus genotype G12 is currently recognized as a globally emerging rotavirus. To our knowledge, this is the first report of this emerging rotavirus strain G12P[8] in México. Ongoing surveillance is recommended to monitor the distribution of rotavirus genotypes and to continually reassess the suitability of currently available rotavirus vaccines. PMID:27265462

  18. Equine rotaviruses--current understanding and continuing challenges.

    PubMed

    Bailey, Kirsten E; Gilkerson, James R; Browning, Glenn F

    2013-11-29

    Equine rotaviruses were first detected in foals over 30 years ago and remain a major cause of infectious diarrhoea in foals. During this time, there has been substantial progress in the development of sensitive methods to detect rotaviruses in foals, enabling surveillance of the genotypes present in various horse populations. However, there has been limited epidemiological investigation into the significance of these circulating genotypes, their correlation with disease and the use of vaccination in these animal populations. Our knowledge of the pathogenesis of rotavirus infection in foals is based on a limited number of studies on a small number of foals and, therefore, most of our understanding in this area has been extrapolated from studies in other species. Questions such as the concentrations of rotavirus particles shed in the faeces of infected foals, both with and without diarrhoea, and factors determining the presence or absence of clinical disease remain to be investigated, as does the relative and absolute efficacy of currently available vaccines. The answer to these questions may help direct research into the development of more effective control measures. PMID:23932076

  19. Rotavirus serotype G5 associated with diarrhea in Brazilian children.

    PubMed

    Gouvea, V; de Castro, L; Timenetsky, M C; Greenberg, H; Santos, N

    1994-05-01

    Rotavirus serotype G5 in fecal specimens of 38 Brazilian children with diarrhea was identified by PCR and enzyme immunoassays. The strains exhibited long RNA electropherotypes and either subgroup II or nonsubgroup I-nonsubgroup II specificities. Serotype G5 has been found in piglets and horses but not yet in humans. PMID:8051281

  20. Dynamic modeling of cost-effectiveness of rotavirus vaccination, Kazakhstan.

    PubMed

    Freiesleben de Blasio, Birgitte; Flem, Elmira; Latipov, Renat; Kuatbaeva, Ajnagul; Kristiansen, Ivar Sønbø

    2014-01-01

    The government of Kazakhstan, a middle-income country in Central Asia, is considering the introduction of rotavirus vaccination into its national immunization program. We performed a cost-effectiveness analysis of rotavirus vaccination spanning 20 years by using a synthesis of dynamic transmission models accounting for herd protection. We found that a vaccination program with 90% coverage would prevent ≈880 rotavirus deaths and save an average of 54,784 life-years for children <5 years of age. Indirect protection accounted for 40% and 60% reduction in severe and mild rotavirus gastroenteritis, respectively. Cost per life year gained was US $18,044 from a societal perspective and US $23,892 from a health care perspective. Comparing the 2 key parameters of cost-effectiveness, mortality rates and vaccine cost at

  1. Impact of vaccination uptake on hospitalizations due to rotavirus acute gastroenteritis in 2 different socioeconomic areas of Spain.

    PubMed

    Giménez Sánchez, Francisco; Nogueira, Esperanza Jiménez; Sánchez Forte, Miguel; Ibáñez Alcalde, Mercedes; Cobo, Elvira; Angulo, Raquel; Garrido Fernández, Pablo

    2016-04-01

    Rotavirus is the leading cause of hospitalization due to acute gastroenteritis (AGE) in infants and toddlers. However, rotavirus vaccination has been associated with a decline in hospitalization rates due to rotavirus AGE. A descriptive retrospective study was conducted to analyze the impact of rotavirus vaccination on the rate of hospitalizations due to AGE among children ≤2 years old in 2 areas of the province of Almería, Spain. After eight years of rotavirus vaccination, rates of hospitalizations due to rotavirus AGE are diminished. This decline is closely related to vaccine coverage in the studied areas. PMID:26810147

  2. Pentavalent Rotavirus Vaccine in Infants With Surgical Gastrointestinal Disease

    PubMed Central

    McGrath, Eric J.; Thomas, Ron; Duggan, Christopher; Asmar, Basim I.

    2015-01-01

    Objectives Pentavalent rotavirus vaccine (RV5) has been shown to be well-tolerated and efficacious in preventing rotavirus gastroenteritis in healthy infants. Safety and immunogenicity of RV5 in infants with surgical gastrointestinal disease have not been studied. The aim of the present study was to evaluate the safety and immunogenicity of RV5 in infants with a history of congenital or acquired intestinal disease requiring resection compared with healthy infants. Methods Infants with intestinal resection were matched by gestational age and chronological age to healthy infants (controls). Dose 1 of RV5 was given at 10 to 12 weeks of chronological age. Doses 2 and 3 were given at intervals of 4 to 10 weeks, with all 3 doses given by 32 weeks. All infants were monitored for adverse events (AEs) by telephone calls, clinic visits, and parental written reports during the first 42 days after each dose and monthly thereafter by telephone for 12 months. Serum anti-rotavirus immunoglobulin A (IgA) titers were measured prevaccination and 2 weeks after dose 3. Results A total of 5 infants with surgical gastrointestinal disease and 3 control subjects were enrolled. All participants (100%) mounted a 3-fold increase in serum anti-rotavirus IgA geometric mean titer postvaccination. RV5 administration to surgical infants was well tolerated with a majority of AEs being attributed to the underlying medical condition. Conclusions Postvaccination serum anti-rotavirus IgA levels indicate that RV5 is immunogenic in infants with a history of bowel resection, despite varying lengths of residual bowel. RV5 was well tolerated with few vaccine-related AEs. PMID:24614127

  3. The cost effectiveness of rotavirus vaccination in Iran.

    PubMed

    Mousavi Jarrahi, Yasaman; Zahraei, Seyed Mohsen; Sadigh, Nader; Esmaeelpoor Langeroudy, Keyhan; Khodadost, Mahmoud; Ranjbaran, Mehdi; Sanjari Moghaddam, Ali; Besharat, Mehdi; Mosavi Jarrahi, Alireza

    2016-03-01

    Rotavirus is the most common cause of severe diarrhea leading to hospitalization or disease-specific death among young children. Effective vaccines have recently been approved and successful vaccination program implemented. The aim of this study was to evaluate the cost effectiveness of mass rotavirus vaccination program in Iran. We developed a Markov model that reflects key features of rotavirus natural history. Parameters of the model were assessed by field study or developed through literature search and published data. We applied the model to the 2009 Iranian birth cohort and evaluated the cost-effectiveness of including the rotavirus vaccine (Rotarix®) into Iranian expanded immunization program (EPI). With an estimated hospitalization rate of 0.05 and outpatient rate of 0.23 cases per person-year, vaccinating cohort of 1231735 infants in Iran with 2 doses of (Rotarix®), would prevent 32092 hospitalizations, 158750 outpatient visits, and 1591 deaths during 5 y of follow-up. Under base-case assumption of $10 cost per course of vaccine, the vaccination would incur an extra cost of $1,019,192 from health care perspective and would avert 54680 DALYs. From societal perspective, there would be $15,192,568 saving for the society with the same averted DALYs. The incremental cost effectiveness ratio showed a cost of $19 US dollars per averted DALY from health care perspective and a saving of $278 US dollars for each averted DALY from societal perspective. Introducing rotavirus vaccine into EPI program would be highly cost-effective public health intervention in Iran. PMID:26360331

  4. Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

    PubMed

    Jere, Khuzwayo C; O'Neill, Hester G; Potgieter, A Christiaan; van Dijk, Alberdina A

    2014-01-01

    Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented

  5. Passive immunisation of children with bovine colostrum containing antibodies to human rotavirus.

    PubMed

    Davidson, G P; Whyte, P B; Daniels, E; Franklin, K; Nunan, H; McCloud, P I; Moore, A G; Moore, D J

    1989-09-23

    The efficacy of a 10-day course of bovine colostrum with high antibody titre against the four known human rotavirus serotypes in protecting children against rotavirus infection was examined in patients admitted to hospital. Children aged 3 to 15 months were blocked in pairs according to ward accommodation (ie, isolation or open area). Each block contained 1 treated and 1 control child. The allocation to treatment or control (an artificial infant formula) was randomised. 9 of 65 control children but none of 55 treated children acquired rotavirus infection during the treatment period (p less than 0.001). The importance of protecting against rotavirus infection was highlighted by the fact that parents of symptomatic rotavirus-positive children sought medical attention seven times more often than did parents of symptomatic rotavirus-negative children (p less than 0.05). PMID:2570959

  6. Challenging the Roles of NSP3 and Untranslated Regions in Rotavirus mRNA Translation.

    PubMed

    Gratia, Matthieu; Vende, Patrice; Charpilienne, Annie; Baron, Hilma Carolina; Laroche, Cécile; Sarot, Emeline; Pyronnet, Stéphane; Duarte, Mariela; Poncet, Didier

    2016-01-01

    Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex for rotavirus mRNAs. To further explore the effects of NSP3 and untranslated regions (UTRs) on rotavirus mRNAs translation, we used a quantitative in vivo assay with simultaneous cytoplasmic NSP3 expression (wild-type or deletion mutant) and electroporated rotavirus-like and standard synthetic mRNAs. This assay shows that the last four GACC nucleotides of viral mRNA are essential for efficient translation and that both the NSP3 eIF4G- and RNA-binding domains are required. We also show efficient translation of rotavirus-like mRNAs even with a 5'UTR as short as 5 nucleotides, while more than eleven nucleotides are required for the 3'UTR. Despite the weak requirement for a long 5'UTR, a good AUG environment remains a requirement for rotavirus mRNAs translation. PMID:26727111

  7. Detection of group C rotavirus antigens and antibodies in animals and humans by enzyme-linked immunosorbent assays.

    PubMed Central

    Tsunemitsu, H; Jiang, B; Saif, L J

    1992-01-01

    Enzyme-linked immunosorbent assays (ELISAs) were developed to detect group (gp) C rotavirus antigens and antibodies. Both assays were confirmed to be specific for gp C rotavirus by using serogroup A, B, and C rotaviruses; hyperimmune antisera to these serogroups of rotaviruses; and paired serum specimens from animals infected with gp C rotaviruses. The ELISA for antigen detection reacted not only with porcine gp C rotaviruses but also with human and bovine gp C rotaviruses. Following experimental challenge of gnotobiotic pigs with porcine gp C rotavirus, the virus was found by ELISA in all diarrheic feces. A high prevalence of antibodies to gp C rotaviruses was detected in sera from adult pigs (93 to 97%) and cattle (47 to 56%) in the United States and Japan. However, no antibody to gp C rotavirus was detected in the sera (n = 20) of adult horses in the United States. In human sera from Hokkaido, Japan, 3% of children and 13% of adults possessed antibody to gp C rotaviruses. These results suggest that the ELISA that we developed may be useful for surveying gp C rotavirus infections in animals and humans. On the basis of serology, gp C rotavirus infections are common in pigs and cattle in the United States and Japan, but they occur at lower levels in humans from the Hokkaido area of Japan. PMID:1323577

  8. Group A rotavirus gastroenteritis: post-vaccine era, genotypes and zoonotic transmission

    PubMed Central

    Luchs, Adriana; Timenetsky, Maria do Carmo Sampaio Tavares

    2016-01-01

    ABSTRACT This article provides a review of immunity, diagnosis, and clinical aspects of rotavirus disease. It also informs about the changes in epidemiology of diarrheal disease and genetic diversity of circulating group A rotavirus strains following the introduction of vaccines. Group A rotavirus is the major pathogen causing gastroenteritis in animals. Its segmented RNA genome can lead to the emergence of new or unusual strains in human populations via interspecies transmission and/or reassortment events. PMID:27462899

  9. Catching-up with pentavalent vaccine: Exploring reasons behind lower rotavirus vaccine coverage in El Salvador.

    PubMed

    Suarez-Castaneda, Eduardo; Burnett, Eleanor; Elas, Miguel; Baltrons, Rafael; Pezzoli, Lorenzo; Flannery, Brendan; Kleinbaum, David; de Oliveira, Lucia Helena; Danovaro-Holliday, M Carolina

    2015-11-27

    Rotavirus vaccine was introduced in El Salvador in 2006 and is recommended to be given concomitantly with DTP-HepB-Haemophilus influenzae type b (pentavalent) vaccine at ages 2 months (upper age limit 15 weeks) and 4 months (upper age limit 8 months) of age. However, rotavirus vaccination coverage continues to lag behind that of pentavalent vaccine, even in years when national rotavirus vaccine stock-outs have not occurred. We analyzed factors associated with receipt of oral rotavirus vaccine among children who received at least 2 doses of pentavalent vaccine in a stratified cluster survey of children aged 24-59 months conducted in El Salvador in 2011. Vaccine doses included were documented on vaccination cards (94.4%) or in health facility records (5.6%). Logistic regression and survival analysis were used to assess factors associated with vaccination status and age at vaccination. Receipt of pentavalent vaccine by age 15 weeks was associated with rotavirus vaccination (OR: 5.1; 95% CI 2.7, 9.4), and receipt of the second pentavalent dose by age 32 weeks was associated with receipt of two rotavirus vaccine doses (OR: 5.0; 95% CI 2.1-12.3). Timely coverage with the first pentavalent vaccine dose was 88.2% in the 2007 cohort and 91.1% in the 2008 cohort (p=0.04). Children born in 2009, when a four-month national rotavirus vaccine stock-out occurred, had an older median age of receipt of rotavirus vaccine and were less likely to receive rotavirus on the same date as the same dose of pentavalent vaccine than children born in 2007 and 2008. Upper age limit recommendations for rotavirus vaccine administration contributed to suboptimal vaccination coverage. Survey data suggest that late rotavirus vaccination and co-administration with later doses of pentavalent vaccine among children born in 2009 helped increase rotavirus vaccine coverage following shortages. PMID:26263200

  10. Detection of group A rotavirus strains circulating among children with acute diarrhea in Indonesia.

    PubMed

    Nirwati, Hera; Wibawa, Tri; Aman, Abu Tholib; Wahab, Abdul; Soenarto, Yati

    2016-01-01

    Rotavirus is the major cause of severe diarrhea in children under 5 years old in developed and developing countries. Since improvements in sanitation and hygiene have limited impact on reducing the incidence of rotavirus diarrhea, implementation of a vaccine will be a better solution. We conducted an observational study to determine the disease burden and to identify the genotype of circulating rotavirus in Indonesia. Hospitalized children due to acute diarrhea were enrolled from four teaching hospitals in Indonesia. Stool samples were collected based on WHO protocol and were tested for the presence of group A rotavirus using enzyme immunoassay. Then, rotavirus positive samples were genotyped using RT-PCR. Fisher's Exact tests, Chi square tests and logistic regression were performed to determine differences across hospital and year in rotavirus prevalence and genotype distribution. There were 4235 samples from hospitalized children with diarrhea during 2006, 2009 and 2010. Among them, the rotavirus positive were 2220 samples (52.42 %) and incidence rates varied between hospitals. The G1P[8], G1P[6], and G2P[4] were recognized as the dominant genotypes circulating strains in Indonesia and the proportion of predominant strains changed by year. Our study showed the high incidence of rotavirus infection in Indonesia with G1P[8], G1P[6], and G2P[4] as the dominant strains circulating in Indonesia. These results reinforce the need for a continuing surveillance of rotavirus strain in Indonesia. PMID:26848437

  11. Genetic diversity of porcine group A rotavirus strains in the UK

    PubMed Central

    Chandler-Bostock, Rebecca; Hancox, Laura R.; Nawaz, Sameena; Watts, Oliver; Iturriza-Gomara, Miren; Mellits, Kenneth M.

    2014-01-01

    Rotavirus is endemic in pig farms where it causes a loss in production. This study is the first to characterise porcine rotavirus circulating in UK pigs. Samples from diarrheic pigs with rotavirus enteritis obtained between 2010 and 2012 were genotyped in order to determine the diversity of group A rotavirus (GARV) in UK pigs. A wide range of rotavirus genotypes were identified in UK pigs: six G types (VP7); G2, G3, G4, G5, G9 and G11 and six P types (VP4); P[6], P[7], P[8], P[13], P[23], and P[32]. With the exception of a single P[8] isolate, there was less than 95% nucleotide identity between sequences from this study and any available rotavirus sequences. The G9 and P[6] genotypes are capable of infecting both humans and pigs, but showed no species cross-over within the UK as they were shown to be genetically distinct, which suggested zoonotic transmission is rare within the UK. We identified the P[8] genotype in one isolate, this genotype is almost exclusively found in humans. The P[8] was linked to a human Irish rotavirus isolate in the same year. The discovery of human genotype P[8] rotavirus in a UK pig confirms this common human genotype can infect pigs and also highlights the necessity of surveillance of porcine rotavirus genotypes to safeguard human as well as porcine health. PMID:25123085

  12. Detection of equine rotavirus by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Nemoto, Manabu; Imagawa, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kondo, Takashi; Matsumura, Tomio

    2010-06-01

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to detection of equine rotavirus. Because equine rotavirus of the single P genotype, P[12], is predominant in the equine population worldwide, an RT-LAMP primer set was designed to target the genotype P[12] sequence and thus detect equine rotavirus. The detection limit of the RT-LAMP assay was 10(3) copies of viral RNA, whereas that of semi-nested RT-PCR for genotype P[12] was 10(5) copies. The RT-LAMP assay specifically amplified genotype P[12] but did not amplify the other P genotype strains. The RT-LAMP assay did not amplify any pathogens related to equine intestinal disorder other than rotavirus. Using 96 diarrheal stools, the RT-LAMP assay detected equine rotavirus in 58 samples, whereas semi-nested RT-PCR only detected equine rotavirus in 25 samples. The RT-LAMP assay did not detect equine rotavirus with fecal samples collected from nine healthy foals. These results indicate that the RT-LAMP assay is specific for equine rotavirus and more sensitive than semi-nested RT-PCR. Because it is easy to manipulate without the need for a thermal cycler or gel electrophoresis, the RT-LAMP assay should be applicable to diagnosis of equine rotavirus infections in diagnostic laboratories. PMID:20160420

  13. Measurement of immunoglobulin A, G, and M class rotavirus antibodies in serum and mucosal secretions.

    PubMed Central

    McLean, B; Sonza, S; Holmes, I H

    1980-01-01

    A solid-phase, enzyme-linked immunospecific assay for measurement of different immunoglobulin classes of human rotavirus antibodies is described. The antigen, which was adsorbed directly to polyvinyl microtiter plates, consisted of a clarified cell culture stock of the simian rotavirus SA 11. The assay was sensitive and reproducible and could readily be calibrated to determine concentrations of each class of antibody. The assay was applied to measurements of rotavirus antibodies in serum, colostrum, milk, and fecal samples. It particularly facilitates investigations of the role of immunoglobulin A antibodies in immunity to rotavirus infections. PMID:6260831

  14. Detection of rotavirus in food associated with a gastroenteritis outbreak in a mother and child sanatorium.

    PubMed

    Mayr, Constanze; Strohe, Gerald; Contzen, Matthias

    2009-10-31

    In the course of a rotavirus outbreak in a mother and child sanatorium 74 food samples from the sanatorium kitchen were taken and tested for rotavirus. Rotavirus particles were isolated from 25 g food samples by a simple method including ultrafiltration, originally designed for the detection of norovirus in various food matrices. Rotavirus was successfully detected in a sample of potato stew by conventional RT-seminested-PCR. Sequence comparison of the amplification products obtained from the potato stew and a stool sample from an infected child verified that the two viruses were identical. PMID:19717201

  15. Monitoring of group A rotaviruses in wild-living birds in Hungary.

    PubMed

    Ursu, K; Papp, H; Kisfali, P; Rigó, D; Melegh, B; Martella, V; Bányai, K

    2011-03-01

    Rotavirus is a common pathogen causing gastroenteritis in humans and domesticated animals. The incidence of rotavirus in wild-living animals, particularly in avian species, has not been systematically investigated. In this study 1220 fecal samples and cloacal swabs collected from wild-living birds during 2008 in Hungary were tested for the presence of group A rotaviruses by a VP6 gene-specific reverse-transcription-polymerase-chain-reaction assay. Of the 1220 samples, 276 and 944 were processed as individual and pooled specimens, respectively. Rotavirus was identified in two pooled pheasant (Phasianus colchicus) samples and two individual reed bunting samples (Emberiza schoeniclus). These data indicated a very low prevalence of group A rotaviruses (0.3%) in our sample set. Nonetheless, the present study, together with existing literature data, implies that rotavirus infections occur in a wide spectrum of feral bird species. These findings are exciting and suggest that pursuing rotavirus monitoring is needed to uncover avian rotavirus strain diversity and understand rotavirus ecology in nature. PMID:21500648

  16. SEASONALITY OF ROTAVIRUS IN SOUTH ASIA: A META-ANALYSIS APPROACH ASSESSING ASSOCIATIONS WITH TEMPERATURE, PRECIPTATION, AND VEGETATION INDEX

    EPA Science Inventory

    Background: Rotavirus infection causes a significant proportion of diarrhea in infants and young children worldwide leading to dehydration, hospitalization, and in some cases death. Rotavirus infection represents a significant burden of disease in developing countries, such as th...

  17. Cost effectiveness of a pentavalent rotavirus vaccine in Oman

    PubMed Central

    2014-01-01

    Background Rotavirus gastroenteritis (RGE) is the leading cause of diarrhea in young children in Oman, incurring substantial healthcare and economic burden. We propose to formally assess the potential cost effectiveness of implementing universal vaccination with a pentavalent rotavirus vaccine (RV5) on reducing the health care burden and costs associated with rotavirus gastroenteritis (RGE) in Oman Methods A Markov model was used to compare two birth cohorts, including children who were administered the RV5 vaccination versus those who were not, in a hypothetical group of 65,500 children followed for their first 5 years of life in Oman. The efficacy of the vaccine in reducing RGE-related hospitalizations, emergency department (ED) and office visits, and days of parental work loss for children receiving the vaccine was based on the results of the Rotavirus Efficacy and Safety Trial (REST). The outcome of interest was cost per quality-adjusted life year (QALY) gained from health care system and societal perspectives. Results A universal RV5 vaccination program is projected to reduce, hospitalizations, ED visits, outpatient visits and parental work days lost due to rotavirus infections by 89%, 80%, 67% and 74%, respectively. In the absence of RV5 vaccination, RGE-related societal costs are projected to be 2,023,038 Omani Rial (OMR) (5,259,899 United States dollars [USD]), including 1,338,977 OMR (3,481,340 USD) in direct medical costs. However, with the introduction of RV5, direct medical costs are projected to be 216,646 OMR (563,280 USD). Costs per QALY saved would be 1,140 OMR (2,964 USD) from the health care payer perspective. An RV5 vaccination program would be considered cost saving, from the societal perspective. Conclusions Universal RV5 vaccination in Oman is likely to significantly reduce the health care burden and costs associated with rotavirus gastroenteritis and may be cost-effective from the payer perspective and cost saving from the societal

  18. [THE COMPARATIVE CHARACTERISTIC OF SETS OF REAGENTS FOR IDENTIFICATION OF ANTIGENS OF ROTAVIRUSES USED ON THE TERRITORY OF RUSSIA].

    PubMed

    Podkolzin, A T; Guseva, A N; Veselova, O A; Kurochkina, D E; Shipulin, G A

    2015-06-01

    The study was carried out to evaluate and compare analytical characteristics of reagents kits for identification of antigens of rotaviruses SD BIOLINE Rotavirus (Standard Diagnostics, Korea), RIDA Quick Rotavirus (R-biopharm AG, Germany), RotaStick One-Step Test (Novamed Ltd., Israel), QuickStripe Rotavirus (Savyon doagnostics Ltd., Israel), Rotavirus-antigen-IFA-BEST (Vector-Best, the Russian Federation), Rota-antigen (NPP AKVAPAST, the Russian Federation). The panel included 84 positive and 43 negative samples of rotaviruses group A according their content. The reagents kit "Amplisense OKI screen-FL" with confirming [P]G typing of positive samples was used for comparison. The comparison of analytical sensitivity of reagents kits was implemented on panel characterized by using technique of droplet digital polymerase chain-reaction. The indicators of diagnostic sensitivity and specificity of reagents kits amounted to 84.52% and 100%for SD BIOLINE Rotavirus and RIDA Quick Rotavirus, 71.43% and 100% for RotaStick One-Step Test, 75.00% and 100%for QuickStripe Rotavirus, 83.33% and 100% for Rotavirus-antigen-FA-BEST, 83.33% and 100%for Rota-antigen. The analytical sensitivity of immunochromatographic and immunoenzyme kits amounted to 5 x 106 GE/ml for [P]8G4 genotype of rotaviruses of group A. The reagents kits SD BIOLINE Rotavirus, RIDA Quick Rotavirus and Rotavirus-antigen-IFA-BESTdemonstrated matched high indicators of diagnostic sensitivity and specificity sufficient for etiological diagnostic of rotavirus infection at acute stage of disease. The analytical sensitivity of compared kits does not allow recommending them to apply in analysis of samples characterized by lower concentrations of rotaviruses (asymptomatic agents, objects of environment). PMID:26466453

  19. Rotavirus Controls Activation of the 2′-5′-Oligoadenylate Synthetase/RNase L Pathway Using at Least Two Distinct Mechanisms

    PubMed Central

    Sánchez-Tacuba, Liliana; Rojas, Margarito; Arias, Carlos F.

    2015-01-01

    ABSTRACT The innate immune response is the first line of defense of the host cell against a viral infection. In turn, viruses have evolved a wide variety of strategies to hide from, and to directly antagonize, the host innate immune pathways. One of these pathways is the 2′-5′-oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by double-stranded RNA (dsRNA) to produce 2′-5′ oligoadenylates, which are the activators of RNase L; this enzyme degrades viral and cellular RNAs, restricting viral infection. It has been recently found that the carboxy-terminal domain (CTD) of rotavirus VP3 has a 2′-5′-phosphodiesterase (PDE) activity that is able to functionally substitute for the PDE activity of the mouse hepatitis virus ns2 protein. This particular phosphodiesterase cleaves the 2′-5′-phosphodiester bond of the oligoadenylates, antagonizing the OAS/RNase L pathway. However, whether this activity of VP3 is relevant during the replication cycle of rotavirus is not known. Here, we demonstrate that after rotavirus infection the OAS/RNase L complex becomes activated; however, the virus is able to control its activity using at least two distinct mechanisms. A virus-cell interaction that occurs during or before rotavirus endocytosis triggers a signal that prevents the early activation of RNase L, while later on the control is taken by the newly synthesized VP3. Cosilencing the expression of VP3 and RNase L in infected cells yields viral infectious particles at levels similar to those obtained in control infected cells, where no genes were silenced, suggesting that the capping activity of VP3 is not essential for the formation of infectious viral particles. IMPORTANCE Rotaviruses represent an important cause of severe gastroenteritis in the young of many animal species, including humans. In this work, we have found that the OAS/RNase L pathway is activated during rotavirus infection, but the virus uses two different strategies to prevent the

  20. Analysis of genetic factors related to preferential selection of the NSP1 gene segment observed in mixed infection and multiple passage of rotaviruses.

    PubMed

    Mahbub Alam, M; Kobayashi, N; Ishino, M; Naik, T N; Taniguchi, K

    2006-11-01

    Reassortment is one of the major evolutionary mechanisms of the rotavirus genome. Preferential selection (assortment) of the NSP1 gene segment from either of the parental viruses after coinfection of these viruses has been reported as a notable finding in reassortment. To analyze genetic factors which are associated with preferential selection of the rotavirus NSP1 gene segment into progeny viruses, mixed infection and multiple passages were performed using two panels of rotaviruses, i.e., bovine rotavirus A5 clones, and simian rotavirus SA11 and five strains of SA11-based single NSP1 gene-substitution reassortants. In the first experiment, three A5 clones (A5-10, A5-13, and A5-16) that had genetically distinct NSP1 genes in the same genetic background were used. In coinfection of these A5 clones, it was noted that the A5-10 NSP1 gene, which encodes an incomplete protein product due to presence of a nonsense codon at an unusual position, was selected more preferentially than the A5-13 NSP1 gene with intact length and structure. The A5-16 NSP1 gene, with a deletion of 500 bp, was least efficiently selected. In the second experiment, we prepared two reassortants, SOF and SRF, which have NSP1 genes from rotavirus strains OSU and RRV, respectively, in the genetic background of SA11, which were used together with previously prepared reassortants SKF, SDF, and SNF, which had NSP1 genes from strains KU, DS1, and K9, respectively. Among the 6 NSP1 genes analyzed, the NSP1 gene from SKF was most preferentially selected, followed by SNF, SOF, SDF, SA11, and SRF, in that order. Although SOF exhibited less growth efficacy than SA11, the growth rates of other reassortants were similar to that of SA11. These findings suggest that for the occurrence of preferential selection of the NSP1 gene, production of the intact NSP1 protein may not be involved, but the presence of intact length of the NSP1 gene may be required. Furthermore, it was also found that genetic similarity based on

  1. Predominance of genotype P[9]G3 in rotavirus gastroenteritis in Polish children

    PubMed Central

    Kacerka, Anna; Majda-Stanisławska, Ewa; Jóźwiak, Barbara; Sidorkiewicz, Małgorzata

    2015-01-01

    Introduction Rotavirus (RV) infection is the most common cause of gastroenteritis in children. This paper identifies the most common genotypes of rotaviruses isolated from children hospitalized with gastroenteritis and attempts to determine any relationship between infection with a certain rotavirus genotype. Material and methods The investigated group consisted of 68 consecutive children with rotavirus gastroenteritis (confirmed by an agglutination test). Rotavirus genotype was determined in stool samples obtained from each child. Results The P[9]VP4 genotype was observed in 41/61 positive samples (over 67.2%) that were permanently associated with the G3 VP7 genotype. Moreover, G3 was determined as the most commonly isolated G type (77.94%). As well as the P[9]G3 type, G3 was also found in the P[4] type (5 cases). Twenty-six out of 61 (42.6%) children in whom rotavirus genotype was determined were co-infected with pathogenic bacteria. No statistical correlation was observed between rotavirus P[9]G3 gastroenteritis and digestive tract co-infection with pathogenic bacteria (p > 0.05). Elevated ALT activity was found in 34/59 (57.6%) cases of rotavirus gastroenteritis. Elevated ALT serum level was found to correlate with P[9]G3 rotavirus genotype but concomitant infections did not. Conclusions The most common genotype of rotaviruses observed in our group of children, P[9]G3, has rarely been described. Co-infection of the digestive tract with pathogenic bacteria and elevated serum ALT concentrations were found to be the most frequent phenomena. A correlation between P[9]G3 rotavirus genotype and elevated serum ALT level was found, but no significant relationship was identified between concomitant infections and P[9]G3 genotype. PMID:26170851

  2. Distribution and titres of rotavirus antibodies in different age groups.

    PubMed Central

    Elias, M. M.

    1977-01-01

    Three hundred and fifty-seven sera selected at random from hospital patients of all ages were examined for rotavirus antibodies using indirect immunofluorescence (FA) and complement fixation levels (CFT). Three hundred and fourteen of these were also tested for neutralizing antibodies to human rotavirus. Sera from patients admitted with a diagnosis of acute gastroenteritis were excluded from this survey. FA antibodies were found in newborn infants but fell to undetectable titres at 3 months. The highest titres were found in children between the ages of one and three years. In older age groups, the model titre fell gradually with increasing age until, in sera from those above 70 years of age, FA antibodies were almost undetectable. The same pattern was observed with neutralizing antibodies. A high model titre of CF antibodies was only found in sera from those aged one to three years. PMID:200676

  3. Critical role of cellular cholesterol in bovine rotavirus infection

    PubMed Central

    2014-01-01

    Background Bovine rotavirus (BRV) is a non-enveloped dsRNA virus that cause neonatal calf diarrhea. Lipid rafts are cholesterol-enrich membrane mircodomains that play a vital role in many cellular processes. In this study, the effect of cellular cholesterol depletion on infection of MA-104 cells with bovine rotavirus was investigated. Results We demonstrated that cholesterol depletion of the plasma membrane by MβCD had no effect on BRV binding to cells but significantly impaired BRV entry in a dose-dependent manner and the effect was partially reversed by addition of exogenous cholesterol, suggesting the reduction of BRV infection by MβCD was specifically due to cholesterol depletion. Cholesterol depletion after virus entry did not reduce BRV replication, whereas affected virus assembly. Conclusions Taken together, our results demonstrate that cell membrane cholesterol is essential to BRV infectivity. PMID:24884772

  4. Dielectrophoresis and dielectrophoretic impedance detection of adenovirus and rotavirus

    NASA Astrophysics Data System (ADS)

    Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya

    2016-01-01

    The aim of this study is the electrical detection of pathogenic viruses, namely, adenovirus and rotavirus, using dielectrophoretic impedance measurement (DEPIM). DEPIM consists of two simultaneous processes: dielectrophoretic trapping of the target and measurement of the impedance change and increase in conductance with the number of trapped targets. This is the first study of applying DEPIM, which was originally developed to detect bacteria suspended in aqueous solutions, to virus detection. The dielectric properties of the viruses were also investigated in terms of their dielectrophoretic behavior. Although their estimated dielectric properties were different from those of bacteria, the trapped viruses increased the conductance of the microelectrode in a manner similar to that in bacteria detection. We demonstrated the electrical detection of viruses within 60 s at concentrations as low as 70 ng/ml for adenovirus and 50 ng/ml for rotavirus.

  5. Structural characterization by multistage mass spectrometry (MSn) of human milk glycans recognized by human rotaviruses.

    PubMed

    Ashline, David J; Yu, Ying; Lasanajak, Yi; Song, Xuezheng; Hu, Liya; Ramani, Sasirekha; Prasad, Venkataram; Estes, Mary K; Cummings, Richard D; Smith, David F; Reinhold, Vernon N

    2014-11-01

    We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.(2), describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent

  6. Analysis of the protein-protein interactions in the parvovirus H-1 capsid.

    PubMed Central

    Paradiso, P R

    1983-01-01

    The structure of the icosahedral capsid of the H-1 parvovirus was probed by chemical cross-linking methods. Treatment of empty capsids with high-molecular-weight polyethylene glycols resulted in irreversible aggregation of the minor capsid protein VP1. Multimers of VP1 containing at least five and perhaps six molecules were obtained, but only with empty capsids and not with the full, DNA-containing virus. Cross-linking of the empty capsids with dimethylsuberimidate confirmed the assignments of the products formed after treatment with polyethylene glycol. With dimethylsuberimidate the most abundant product was a heterologous dimer containing VP1 and the major capsid protein VP2'. A small amount of homologous VP2' dimer was also obtained, but the majority of VP2' remained unreacted even at high concentrations of dimethylsuberimidate. The capsid proteins of the full virus, on the other hand, were completely unreactive to dimethylsuberimidate. The data suggest that the minor protein VP1 may be clustered in the capsid and perhaps composes one or two of the morphological units of the icosahedral shell. Images PMID:6827655

  7. Characterization of a G1P[8] rotavirus causing an outbreak of gastroenteritis in the Northern Territory, Australia, in the vaccine era

    PubMed Central

    Donato, Celeste M; Cowley, Daniel; Snelling, Thomas L; Akopov, Asmik; Kirkness, Ewen F; Kirkwood, Carl D

    2014-01-01

    In 2010, a large outbreak of rotavirus gastroenteritis occurred in the Alice Springs region of the Northern Territory, Australia. The outbreak occurred 43 months after the introduction of the G1P[8] rotavirus vaccine Rotarix®. Forty-three infants were hospitalized during the outbreak and analysis of fecal samples from each infant revealed a G1P[8] rotavirus strain. The outbreak strain was adapted to cell culture and neutralization assays were performed using VP7 and VP4 neutralizing monoclonal antibodies. The outbreak strain exhibited a distinct neutralization resistance pattern compared to the Rotarix® vaccine strain. Whole genome sequencing of the 2010 outbreak virus strain demonstrated numerous amino acid differences compared to the Rotarix® vaccine strain in the characterized neutralization epitopes of the VP7 and VP4 proteins. Phylogenetic analysis of the outbreak strain revealed a close genetic relationship to global strains, in particular RVA/Human-wt/BEL/BE0098/2009/G1P[8] and RVA/Human-wt/BEL/BE00038/2008/G1P[8] for numerous genes. The 2010 outbreak strain was likely introduced from a globally circulating population of strains rather than evolving from an endemic Australian strain. The outbreak strain possessed antigenic differences in the VP7 and VP4 proteins compared to the Rotarix® vaccine strain. The outbreak was associated with moderate vaccine coverage and possibly low vaccine take in the population. PMID:26038746

  8. Characterization of a G1P[8] rotavirus causing an outbreak of gastroenteritis in the Northern Territory, Australia, in the vaccine era.

    PubMed

    Donato, Celeste M; Cowley, Daniel; Snelling, Thomas L; Akopov, Asmik; Kirkness, Ewen F; Kirkwood, Carl D

    2014-07-01

    In 2010, a large outbreak of rotavirus gastroenteritis occurred in the Alice Springs region of the Northern Territory, Australia. The outbreak occurred 43 months after the introduction of the G1P[8] rotavirus vaccine Rotarix(®). Forty-three infants were hospitalized during the outbreak and analysis of fecal samples from each infant revealed a G1P[8] rotavirus strain. The outbreak strain was adapted to cell culture and neutralization assays were performed using VP7 and VP4 neutralizing monoclonal antibodies. The outbreak strain exhibited a distinct neutralization resistance pattern compared to the Rotarix(®) vaccine strain. Whole genome sequencing of the 2010 outbreak virus strain demonstrated numerous amino acid differences compared to the Rotarix(®) vaccine strain in the characterized neutralization epitopes of the VP7 and VP4 proteins. Phylogenetic analysis of the outbreak strain revealed a close genetic relationship to global strains, in particular RVA/Human-wt/BEL/BE0098/2009/G1P[8] and RVA/Human-wt/BEL/BE00038/2008/G1P[8] for numerous genes. The 2010 outbreak strain was likely introduced from a globally circulating population of strains rather than evolving from an endemic Australian strain. The outbreak strain possessed antigenic differences in the VP7 and VP4 proteins compared to the Rotarix(®) vaccine strain. The outbreak was associated with moderate vaccine coverage and possibly low vaccine take in the population. PMID:26038746

  9. Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay.

    PubMed Central

    Burns, J W; Welch, S K; Nakata, S; Estes, M K

    1989-01-01

    Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus. Images PMID:2536755

  10. Intussusception following rotavirus vaccination in the Valencia Region, Spain

    PubMed Central

    Pérez-Vilar, Silvia; Díez-Domingo, Javier; Puig-Barberà, Joan; Gil-Prieto, Ruth; Romio, Silvana

    2015-01-01

    Studies have shown high intussusception rates in Spain. We performed a hospital-based retrospective observational study of the intussusception risk following rotavirus vaccinations among infants in Valencia, a region of Spain with an annual birth cohort of approximately 48,000 children, during 2007–2011, using a self-controlled case series design. We performed medical record review of all cases using Brighton Collaboration´s case definition and assessed the positive predictive value (PPV) of the intussusception diagnosis code. Among 151 hospitalized cases discharged as intussusception, we confirmed 136 as Brighton Collaboration's Levels 1 or 2, resulting in a PPV of 93% (95% CI: 87%–96%). Three confirmed cases occurred within days 1–7 following the first rotavirus vaccination. The incidence rate ratio was 9.0 (95% CI: 0.9–86.5) (crude) and 4.7 (95% CI:0.3–74.1)(age adjusted). In this first study in Europe, the intussusception risk point estimate was comparable to other studies, although results were not statistically significant, maybe due to limited power. The high PPV found will facilitate implementation of a larger study without requiring medical record review. Our finding of very few vaccinated cases despite a thorough 5-year investigation in a country that, according to previous studies, may have a large background rate of intussusception is reassuring and should contribute to deliberations about the need to include rotavirus vaccines in the official Spanish calendars. PMID:26083707

  11. Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca2+ Homeostasis Induced by Infection of Cultured Cells▿

    PubMed Central

    Zambrano, José Luis; Díaz, Yuleima; Peña, Franshelle; Vizzi, Esmeralda; Ruiz, Marie-Christine; Michelangeli, Fabián; Liprandi, Ferdinando; Ludert, Juan Ernesto

    2008-01-01

    Rotavirus infection of cells in culture induces major changes in Ca2+ homeostasis. These changes include increases in plasma membrane Ca2+ permeability, cytosolic Ca2+ concentration, and total cell Ca2+ content and a reduction in the amount of Ca2+ released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca2+ homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca2+ permeability of the plasma membrane and cytosolic Ca2+ concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca2+ permeability and Ca2+ pools released by agonist (ATP). In addition, the increase of total Ca2+ content (as measured by 45Ca2+ uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca2+ homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca2+ homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects. PMID:18400845

  12. A novel group A rotavirus G serotype: serological and genomic characterization of equine isolate FI23.

    PubMed Central

    Browning, G F; Fitzgerald, T A; Chalmers, R M; Snodgrass, D R

    1991-01-01

    Equine rotavirus FI23 was shown to be prototypic of a novel G serotype, provisionally G14, by cross-neutralization and VP7 sequence determination. Although distinct, there are as few as six differing amino acid residues (92, 94, 96, 146, 147, and 221) in the VP7 antigenic regions of FI23 and G3 rotaviruses. PMID:1663521

  13. Complete Genomic Sequence for an Avian Group G Rotavirus from South Africa

    PubMed Central

    Stucker, Karla M.; Stockwell, Timothy B.; Nyaga, Martin M.; Halpin, Rebecca A.; Fedorova, Nadia; Akopov, Asmik; Ngoveni, Harry; Peenze, Ina; Seheri, Mapaseka L.; Mphahlele, M. Jeffrey

    2015-01-01

    We report the first complete sequence for an avian group G rotavirus (RVG) genome from Africa, which is the third publically available RVG genome. These RVG genomes are highly diverse, especially in their VP4, VP7, NSP4, and NSP3 segments, indicating that RVG diversity is comparable to that of rotavirus A. PMID:25767240

  14. Complete genomic sequence for an avian group G rotavirus from South Africa.

    PubMed

    Stucker, Karla M; Stockwell, Timothy B; Nyaga, Martin M; Halpin, Rebecca A; Fedorova, Nadia; Akopov, Asmik; Ngoveni, Harry; Peenze, Ina; Seheri, Mapaseka L; Mphahlele, M Jeffrey; Wentworth, David E

    2015-01-01

    We report the first complete sequence for an avian group G rotavirus (RVG) genome from Africa, which is the third publically available RVG genome. These RVG genomes are highly diverse, especially in their VP4, VP7, NSP4, and NSP3 segments, indicating that RVG diversity is comparable to that of rotavirus A. PMID:25767240

  15. Nested reverse transcriptase-polymerase chain reaction for the detection of group A rotaviruses.

    PubMed

    Elschner, M; Prudlo, J; Hotzel, H; Otto, P; Sachse, K

    2002-03-01

    Rotaviruses are important pathogens associated with diarrhoeal diseases in almost all species of mammals. In the present study, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of group A rotaviruses was developed, which is based on a target region in gene segment 6. Rotavirus strains of human, bovine, porcine, canine, feline, equine, and ovine origin were examined. Furthermore several faecal specimens, in which rotavirus had already been detected using other methods than PCR, were included in the study. A nested RT-PCR product was formed with all strains and faecal samples tested. The detection limit for virus-containing cell culture supernatant was 3 x 10(-2) [50% tissue culture infective dose (TCID50)] by RT-PCR and 3 x 10(-3) TCID50) by nested amplification. In order to examine the influence of the sample matrix on sensitivity, a rotavirus-negative faecal specimen was spiked with virus-containing cell culture suspension of the porcine rotavirus OSU. The detection limit of the present PCR procedure was approximately 1.6 x 10(2) TCID50 per g faeces and could be increased by one order of magnitude using nested PCR. The present method for detection and identification of group A rotaviruses represents a powerful diagnostic tool and was shown to be applicable to rotaviruses of different origin, including human sources. PMID:12002423

  16. Detection and distribution of rotavirus in raw sewage and creeks in Sao Paulo, Brazil

    SciTech Connect

    Mehnert, D.U.; Stewien, K.E. )

    1993-01-01

    Rotavirus invection is an important cause of hospitalization and mortality of infants and children in developing countries, especially where the water supply and sewage disposal systems are in precarious conditions. This report describes the detection, quantitation, and distribution of rotaviruses in domestic sewage and sewage polluted creeks in the city of San Paulo. 22 refs., 1 fig., 3 tabs.

  17. Increased Rotavirus Prevalence in Diarrheal Outbreak Precipitated by Localized Flooding, Solomon Islands, 2014.

    PubMed

    Jones, Forrest K; Ko, Albert I; Becha, Chris; Joshua, Cynthia; Musto, Jennie; Thomas, Sarah; Ronsse, Axelle; Kirkwood, Carl D; Sio, Alison; Aumua, Audrey; Nilles, Eric J

    2016-05-01

    Flooding on 1 of the Solomon Islands precipitated a nationwide epidemic of diarrhea that spread to regions unaffected by flooding and caused >6,000 cases and 27 deaths. Rotavirus was identified in 38% of case-patients tested in the city with the most flooding. Outbreak potential related to weather reinforces the need for global rotavirus vaccination. PMID:27088272

  18. Genetic Diversity of Rotavirus Strains Circulating in Environmental Water and Bivalve Shellfish in Thailand

    PubMed Central

    Kittigul, Leera; Panjangampatthana, Apinya; Rupprom, Kitwadee; Pombubpa, Kannika

    2014-01-01

    Rotavirus is a common cause of acute diarrhea in young children worldwide. This study investigated the prevalence and molecular characterization of rotavirus in environmental water and oyster samples in Thailand. A total of 114 water samples and 110 oyster samples were collected and tested for group A rotavirus using RT-nested PCR. Rotavirus genotype was identified by phylogenetic analysis of the VP7 genetic sequences. Group A rotavirus was detected in 21 water samples (18.4%) and six oyster samples (5.4%). Twenty five rotavirus strains were successfully sequenced and classified into four genotypes; G1, G2, G3, and G9. Rotavirus G1 (three strains), G2 (three strains), and G9 (two strains) demonstrated the genetic sequences similar to human strains (90%–99% nucleotide identity), whereas G3 (17 strains) was closely related to animal strains (84%–98% nucleotide identity). G1 strains belonged to lineages I (sub-lineage c) and II. G2 strains belonged to lineage II. G9 strains belonged to lineages III (sub-lineage b) and IV. G3 strains belonged to lineages I, III (sub-lineage c), and IV with a predominance of lineage I. The present study provides important information on the rotavirus strains circulating in the environment. PMID:24469269

  19. Increased Rotavirus Prevalence in Diarrheal Outbreak Precipitated by Localized Flooding, Solomon Islands, 2014

    PubMed Central

    Jones, Forrest K.; Ko, Albert I.; Becha, Chris; Joshua, Cynthia; Musto, Jennie; Thomas, Sarah; Ronsse, Axelle; Kirkwood, Carl D.; Sio, Alison; Aumua, Audrey

    2016-01-01

    Flooding on 1 of the Solomon Islands precipitated a nationwide epidemic of diarrhea that spread to regions unaffected by flooding and caused >6,000 cases and 27 deaths. Rotavirus was identified in 38% of case-patients tested in the city with the most flooding. Outbreak potential related to weather reinforces the need for global rotavirus vaccination. PMID:27088272

  20. Survival and detection of rotaviruses on environmental surfaces in day care centers.

    PubMed Central

    Keswick, B H; Pickering, L K; DuPont, H L; Woodward, W E

    1983-01-01

    Previously, we demonstrated that children in day care centers commonly experience diarrhea due to rotavirus, giardia, and bacterial pathogens. Multiple agents frequently coexist, and the environment is heavily contaminated with enteric bacteria during outbreaks. A study of environmental surface contamination with rotavirus was performed during three non-outbreak periods. Of 25 samples collected from environmental surfaces and teachers hands at a day care center, 4 (16%) were positive for rotavirus antigen when a fluorescence assay was used. We also examined the survival of two animal viruses, rotavirus SA-11 and poliovirus type 1, and bacteriophage 12 on similar environmental surfaces in a laboratory. Poliovirus type 1 and bacteriophage f2 were more resistant to drying than rotavirus SA-11 and could be recovered after a 90-min exposure on a dry surface. Rotavirus SA-11 could be detected for 30 min. All three viruses survived longer when they were suspended in fecal material than when they were suspended in distilled water. These data suggest that several agents, including rotavirus, can remain viable on contaminated surfaces long enough to be transmitted to susceptible children. This finding helps explain why rotavirus shows a mode of spread like that of parasitic and bacterial agents within day care center settings. PMID:6314896

  1. A universal infant rotavirus vaccine program in two delivery models: Effectiveness and adverse events following immunization

    PubMed Central

    Sanford, Carolyn; Langley, Joanne M; Halperin, Scott A; Zelman, Mitchell; Maritime Universal Rotavirus Vaccination Program, MURVP

    2015-01-01

    Rotavirus is the most common cause of diarrhea leading to hospitalization in young children. Rotavirus vaccines are available in Canada but have not been introduced in all provinces. In a controlled trial, 2 study sites (Prince Edward Island and the Capital District Health Authority (District 9, Nova Scotia) introduced universal rotavirus vaccine programs for infants at 2 and 4 months of age beginning 1 December 2010, using public health nurse or general practitioner-delivery models, respectively. A third site (Saint John, NB) served as the non-intervention control setting. Vaccine coverage, rotavirus hospitalizations, intussusception and all-cause diarrhea were monitored. A universal rotavirus vaccine program with >90% coverage was associated with reductions in rotavirus-associated hospitalizations (from a peak of 52.8 hospitalizations/100,000 population to 0 hospitalizations) in infants < 12 months and 1 to < 2 y of age 12 months after program implementation. No apparent reduction occurred in the site with vaccine coverage of < 40%, or in the non-intervention control site. No cases of intussusception were associated with vaccine receipt, and no increase in all-cause diarrhea was observed. A universal infant rotavirus vaccine program with high coverage was associated with reductions in rotavirus and no safety signals; no reduction was observed in settings with low vaccine coverage. PMID:25746110

  2. Exceptionally low rotavirus incidence in the Netherlands in 2013/14 in the absence of rotavirus vaccination.

    PubMed

    Hahné, S; Hooiveld, M; Vennema, H; van Ginkel, A; de Melker, H; Wallinga, J; van Pelt, W; Bruijning-Verhagen, P

    2014-01-01

    An unexpected drop in rotavirus (RV) detections was observed in the Netherlands in 2014, without RV vaccination. The estimated decrease in RV detections and gastroenteritis consultations in under five year-olds, in January-April 2014, compared to the same months in previous years, was 72% and 36%, respectively. The low birth rate, mild winter, high RV incidence in the previous year and the introduction of RV vaccination in neighbouring countries may have contributed to this decrease. PMID:25375899

  3. Clinical Symptoms of Human Rotavirus Infection Observed in Children in Sokoto, Nigeria

    PubMed Central

    Alkali, B. R.; Daneji, A. I.; Magaji, A. A.; Bilbis, L. S.

    2015-01-01

    Rotavirus has been identified among the most important causes of infantile diarrhoea, especially in developing countries. The present study was undertaken to determine the occurrence and clinical symptoms of human rotavirus disease among children presenting with varying degree of diarrhoea in selected urban hospitals in Sokoto metropolis, Nigeria. Diarrhoea samples were collected from 200 diarrheic children younger than 5 years of age and tested using a commercially available DAKO Rotavirus ELISA kit which detects the presence of human group A rotaviruses. A questionnaire, based on WHO generic protocol, was completed for each child to generate the primary data. Of the total number of samples collected, 51 were found to be positive for human group A rotavirus indicating 25.5% prevalence of the disease in Sokoto state. The symptoms associated with the disease were analyzed and discussed. PMID:26692016

  4. Association of Rotavirus With Seizures Accompanied by Cerebral White Matter Injury in Neonates.

    PubMed

    Oh, Ki Won; Moon, Chang Hoon; Lee, Kyung Yeon

    2015-10-01

    We aimed to identify whether rotavirus, human parechovirus, or enterovirus are causative or associated viral pathogens of seizures accompanied by diffuse cerebral white matter injury in neonates. Thirty neonates who presented with seizures and diffusion-restriction in the widespread bilateral cerebral white matter on diffusion-weighted magnetic resonance imaging (MRI) were included in this study. All patients were tested for rotavirus, human parechovirus, and enterovirus by using reverse transcription PCR. Stool, cerebrospinal fluid, and serum samples were examined in 30, 25, and 20 patients, respectively. Rotavirus was detected in stool samples from all 30 patients (100%). Stool samples from 5 patients (16.7%) were also positive for enterovirus. Rotavirus or human parechovirus were not detected in any cerebrospinal fluid samples from 25 patients, but 1 patient tested positive for enterovirus. No virus was detected in any of 20 patient sera. This study indicated an association between rotavirus and seizures accompanied by diffuse cerebral white matter lesions in neonates. PMID:25637646

  5. Rotavirus - A Retrospective Study of Incidence at the Hospital Universiti Sains Malaysia (HUSM)

    PubMed Central

    Ludin, C.M. Mat; Radzi, J. Md.; Maimunah, A.

    2003-01-01

    The present study, analyzes data from 1991 to 2000 for rotavirus infection among children with diarrhoea and acute gastroenteritis admitted to the Hospital Universiti Sains Malaysia (HUSM). The Latex Slide Agglutination Test was used for the detection of rotavirus antigens. Out of 1097 stool samples tested, 207 samples or 18.8 % were found to be positive for rotavirus. The infection occurred most frequently in infants and young children from 6 months to 2 years of age. The infection was recorded highest in the year of 2000 – 48 cases (34.1%) and the lowest in 1999 – 5 cases (6.6%). Stool examination and cultures from the rotavirus positive samples revealed no parasites and enteropathogenic bacteria. These observations suggested that rotavirus could still remain as an important agent causing diarrhoea and gastroenteritis in young children admitted to HUSM. PMID:23386803

  6. Rotavirus - A Retrospective Study of Incidence at the Hospital Universiti Sains Malaysia (HUSM).

    PubMed

    Ludin, C M Mat; Radzi, J Md; Maimunah, A

    2003-07-01

    The present study, analyzes data from 1991 to 2000 for rotavirus infection among children with diarrhoea and acute gastroenteritis admitted to the Hospital Universiti Sains Malaysia (HUSM). The Latex Slide Agglutination Test was used for the detection of rotavirus antigens. Out of 1097 stool samples tested, 207 samples or 18.8 % were found to be positive for rotavirus. The infection occurred most frequently in infants and young children from 6 months to 2 years of age. The infection was recorded highest in the year of 2000 - 48 cases (34.1%) and the lowest in 1999 - 5 cases (6.6%). Stool examination and cultures from the rotavirus positive samples revealed no parasites and enteropathogenic bacteria. These observations suggested that rotavirus could still remain as an important agent causing diarrhoea and gastroenteritis in young children admitted to HUSM. PMID:23386803

  7. Lipid droplets form complexes with viroplasms and are crucial for rotavirus replication.

    PubMed

    Crawford, Sue E; Desselberger, Ulrich

    2016-08-01

    Recent evidence has demonstrated that a variety of pathogens target cellular lipid metabolism for their replication. Lipid droplets are a major contributor to lipid homeostasis and contain neutral fats but are also recognized as dynamic organelles involved in signal transduction, membrane trafficking and modulation of immune and inflammatory responses. Rotaviruses co-opt lipid droplets for their replication. Rotavirus viroplasms, sites of viral RNA replication and immature particle assembly, form complexes with cellular lipid droplets early in infection. Chemical compounds blocking fatty acid synthesis or interfering with lipid droplet homeostasis decrease viroplasm formation and the yield of infectious viral progeny. Lipid droplets are vital for the replication of rotaviruses as well as various members of the Flaviviridae family and several intracellular bacteria. Chemical compounds decreasing intracellular triglyceride content reduced rotavirus replication in an animal model and should be considered as potential therapeutic agents against disease caused by rotaviruses, flaviviruses and intracellular bacteria. PMID:27341619

  8. Early impact of rotavirus vaccination in a large paediatric hospital in the UK.

    PubMed

    Hungerford, D; Read, J M; Cooke, R P D; Vivancos, R; Iturriza-Gómara, M; Allen, D J; French, N; Cunliffe, N

    2016-06-01

    The impact of routine rotavirus vaccination on community-acquired (CA) and healthcare-associated (HA) rotavirus gastroenteritis (RVGE) at a large paediatric hospital, UK, was investigated over a 13-year period. A total of 1644 hospitalized children aged 0-15 years tested positive for rotavirus between July 2002 and June 2015. Interrupted time-series analysis demonstrated that, post vaccine introduction (July 2013 to June 2015), CA- and HA-RVGE hospitalizations were 83% [95% confidence interval (CI): 72-90%) and 83% (95% CI: 66-92%] lower than expected, respectively. Rotavirus vaccination has rapidly reduced the hospital rotavirus disease burden among both CA- and HA-RVGE cases. PMID:26876744

  9. Viral determinants of rotavirus pathogenicity in pigs: production of reassortants by asynchronous coinfection.

    PubMed

    Tauscher, G I; Desselberger, U

    1997-01-01

    A porcine rotavirus (prv), variant 4F, isolated in tissue culture from the feces of a Chinese pig with diarrhea, was found to have become highly pathogenic when passaged in gnotobiotic piglets (J. C. Bridger, B. Burke, G. M. Beards, and U. Desselberger, J. Gen. Virol. 73:3011-3015, 1992). Comparison with the closely related pig-apathogenic variant prv 4S suggested the outer capsid protein VP4 (encoded by RNA 4) of prv 4F as a determinant for pathogenicity (B. Burke, J. C. Bridger, and U. Desselberger, J. Gen. Virol. 75:2205-2212, 1994; B. Burke, J. C. Bridger, and U. Desselberger, Virology 202:754-759, 1994). In order to provide more direct evidence, the pathogenic prv 4F variant which grows and forms plaques poorly in tissue culture was reassorted with the well-tissue-culture-adapted, pig-apathogenic bovine rotavirus (brv; UK Compton strain). After asynchronous coinfection of cell cultures (first prv 4F, followed by brv 6 to 12 h later), several reassortants were isolated containing RNA 4 of prv 4F either alone (isolate B-F4) or together with one or two other genes of 4F in the genetic background of brv. Backcrossing of the monoreassortant B-F4 with prv 4S yielded a monoreassortant, S-F4, which carries RNA 4 of the 4F variant in the genetic background of prv 4S. The in vitro growth characteristics of these reassortants were analyzed, and the roles of VP4 in plaque formation and growth kinetics in cell culture were confirmed. The monoreassortant S-F4 and the parental viruses prv 4F and prv 4S are currently being tested for pathogenicity in gnotobiotic piglets (J. C. Bridger, G. Tauscher, and U. Desselberger, unpublished data). PMID:8985430

  10. Human milk oligosaccharides shorten rotavirus-induced diarrhea and modulate piglet mucosal immunity and colonic microbiota

    PubMed Central

    Li, Min; Monaco, Marcia H; Wang, Mei; Comstock, Sarah S; Kuhlenschmidt, Theresa B; Fahey Jr, George C; Miller, Michael J; Kuhlenschmidt, Mark S; Donovan, Sharon M

    2014-01-01

    The impact of human milk oligosaccharides (HMO) on mucosal immunity, gut microbiota and response to rotavirus (RV) infection was investigated in the piglet model. Newborn piglets were fed with formula alone (FF) or formula supplemented with 4 g l−1 HMO (HMO) or a prebiotic mixture of 9:1 short-chain galactooligosaccharides (3.6 g l−1) and long-chain fructooligosaccharides (0.4 g l−1) (PRE) (n=19–21 per group) for 15 days. Piglets (n=7–8) in each dietary group were orally infected with porcine rotavirus (RV) OSU strain on d10, and stool consistency was assessed daily. Blood, small intestine and colonic contents were collected at day 15. Serum RV-specific antibody concentrations, intestinal histomorphology, RV non-structural protein-4 (NSP4) and cytokine mRNA expression were assessed. Colonic content pH, dry matter (DM) and short-chain fatty acid concentrations were measured. Ascending colonic microbiota was analyzed by 16S rRNA gene v1-3 region pyrosequencing. HMO- and PRE-fed groups had shorter duration of diarrhea than FF piglets. Infection changed intestinal histomorphology, increased serum RV-specific antibody response and intestinal RV NSP4 expression, and modulated ileal cytokine expression. HMO enhanced T helper type 1 (interferon-gamma) and anti-inflammatory (interleukin-10) cytokines in the ileum, while prebiotics promoted RV-specific immunoglobulin M response to the infection. RV infection and HMO supplementation altered intraluminal environment and gut microbiota. HMO increased pH and lowered DM of colonic contents and enhanced the abundance of unclassified Lachnospiraceae, which contains numerous butyrate-producing bacteria. In conclusion, HMO and prebiotics did not prevent the onset of RV infection but reduced the duration of RV-induced diarrhea in piglets, in part, by modulating colonic microbiota and immune response to RV infection. PMID:24522264

  11. Symptomatic Infection and Detection of Vaccine and Vaccine-Reassortant Rotavirus Strains in 5 Children: A Case Series

    PubMed Central

    Boom, Julie A.; Sahni, Leila C.; Payne, Daniel C.; Gautam, Rashi; Lyde, Freda; Mijatovic-Rustempasic, Slavica; Bowen, Michael D.; Tate, Jacqueline E.; Rench, Marcia A.; Gentsch, Jon R.; Parashar, Umesh D.; Baker, Carol J.

    2015-01-01

    Vaccine or vaccine-reassortant rotavirus strains were detected in fecal specimens from 5 of 106 (4.7%) immunocompetent children who required treatment for rotavirus gastroenteritis at a large pediatric hospital in Texas in 2009–2010. Four strains were related to pentavalent rotavirus vaccine, whereas one was related to monovalent rotavirus vaccine. The contribution of these strains to each patient’s illness was unclear given that 2 patients had prominent respiratory symptoms and 2 were concurrently infected with another pathogen (group F adenovirus and norovirus). Continued monitoring is necessary to assess the role of vaccine strains and vaccine-reassortant strains in pediatric rotavirus infections. PMID:22872730

  12. Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

    PubMed

    An, K; Gillock, E T; Sweat, J A; Reeves, W M; Consigli, R A

    1999-04-01

    The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells. PMID:10211971

  13. Rotavirus mortality in India: estimates based on a nationally representative survey of diarrhoeal deaths

    PubMed Central

    Awasthi, Shally; Khera, Ajay; Bassani, Diego G; Kang, Gagandeep; Parashar, Umesh D; Kumar, Rajesh; Shet, Anita; Glass, Roger I; Jha, Prabhat

    2012-01-01

    Abstract Objective To estimate the number of rotavirus-associated deaths among Indian children younger than five years. Methods We surveyed more than 23 000 child deaths from a nationally representative survey of 1.1 million Indian households during 2001–2003. Diarrhoeal deaths were characterized by region, age and sex and were combined with the proportion of deaths attributable to rotavirus, as determined by hospital microbiologic data collected by the Indian Rotavirus Strain Surveillance Network from December 2005 to November 2007. Rotavirus vaccine efficacy data from clinical trials in developing countries were used to estimate the number of deaths preventable by a national vaccination programme. Data were analysed using Stata SE version 10. Findings Rotavirus caused an estimated 113 000 deaths (99% confidence interval, CI: 86 000–155 000); 50% (54 700) and 75% (85 400) occurred before one and two years of age, respectively. One child in 242 died from rotavirus infection before five years of age. Rotavirus-associated mortality rates overall, among girls and among boys were 4.14 (99% CI: 3.14–5.68), 4.89 (99% CI: 3.75–6.79) and 3.45 (99% CI: 2.58–4.66) deaths per 1000 live births, respectively. Rates were highest in Bihar, Uttar Pradesh and Madhya Pradesh, which together accounted for > 50% of deaths (64 400) nationally. Rotavirus vaccine could prevent 41 000–48 000 deaths among children aged 3–59 months. Conclusion The burden of rotavirus-associated mortality is high among Indian children, highlighting the potential benefits of rotavirus vaccination. PMID:23109739

  14. Development of a method for detection of human rotavirus in water and sewage.

    PubMed Central

    Smith, E M; Gerba, C P

    1982-01-01

    The simian rotavirus SA11 was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA11 adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter-bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SA11 was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SA11 serum which would detect the simian, human bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SA11. The buoyant density of the sewage isolates in CsCl gradients was 1.36 g/cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 +/- 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology. Images PMID:6285825

  15. Solar and temperature treatments affect the ability of human rotavirus wa to bind to host cells and synthesize viral RNA.

    PubMed

    Romero-Maraccini, Ofelia C; Shisler, Joanna L; Nguyen, Thanh H

    2015-06-15

    Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation. PMID:25862222

  16. Solar and Temperature Treatments Affect the Ability of Human Rotavirus Wa To Bind to Host Cells and Synthesize Viral RNA

    PubMed Central

    Shisler, Joanna L.

    2015-01-01

    Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation. PMID:25862222

  17. Comparison of PremierTM Rotaclone®, ProSpecTTM, and RIDASCREEN® Rotavirus Enzyme Immunoassay Kits for Detection of Rotavirus Antigen in Stool Specimens

    PubMed Central

    Gautam, Rashi; Lyde, Freda; Esona, Mathew D.; Quaye, Osbourne; Bowen, Michael D.

    2015-01-01

    Background Rotaviruses are the major cause of severe dehydrating diarrhea in children throughout the world. Enzyme immunoassays (EIA) have been the standard method for detection of rotavirus in stool specimens since the 1980s. The World Health Organization (WHO) Rotavirus Surveillance Network has proposed including three EIA kits in the WHO-GSM (Global Management System/Système Mondial de Gestion ) catalogue for easy procurement of EIA kits by participating rotavirus surveillance network laboratories. Objectives In this study, we conducted a comparative analysis of 3 commercially available enzyme immunoassay kits: PremierTM Rotaclone® (Meridian Bioscience, Inc.), ProSpecTTM (Oxoid, Ltd.) and RIDASCREEN® (R-biopharm AG) for rotavirus diagnostics. Study design Using reverse-transcriptase-PCR (RT-PCR) as the gold standard, the 3 EIA kits were evaluated by testing a stool panel consisting of 56 rotavirus-positive and 54 rotavirus negative samples. Results The sensitivities of the PremierTM Rotaclone®, ProSpecTTM and RIDASCREEN® kits were 76.8%, 75% and 82.1% respectively, but did not differ significantly. The specificity of all the 3 kits was 100%. The use of RT-PCR as a gold standard lowered the observed sensitivity of all 3 EIA kits but helps to reduce equivocal results that can be seen when another EIA or other non-molecular methods are used as the reference assay in comparison studies. Conclusion Our study found that all three kits are suitable for use by rotavirus surveillance programs. PMID:23850415

  18. Detection of Rotavirus Genotypes in Korea 5 Years after the Introduction of Rotavirus Vaccines.

    PubMed

    Chung, Ju-Young; Kim, Min-Sung; Jung, Tae Woong; Kim, Seong Joon; Kang, Jin-Han; Han, Seung Beom; Kim, Sang Yong; Rhim, Jung Woo; Kim, Hwang-Min; Park, Jae Hong; Jo, Dae Sun; Ma, Sang Hyuk; Jeong, Hye-Sook; Cheon, Doo-Sung; Kim, Jong-Hyun

    2015-10-01

    Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations. PMID:26425045

  19. Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines

    PubMed Central

    Trang, Nguyen V; Braeckman, Tessa; Lernout, Tinne; Hau, Vu T B; Anh, Le T K; Luan, Le T; Van Damme, Pierre; Anh, Dang D

    2015-01-01

    Rotavirus (RV) is the most common cause of childhood diarrhea worldwide, and several vaccines have been successfully developed to reduce the burden of disease. However, lower vaccine immunogenicity and efficacy in developing countries might be related to the virus-neutralizing activity of breast milk. We examined possible differences in breast milk antibody levels (total IgA antibody, RV-specific antibodies, and RV-neutralizing antibodies) between healthy mothers living in a rural area (n = 145) and mothers living in an urban area (n = 147) of Vietnam. Total IgA concentration was significantly higher in samples from mothers in the rural region than in samples from mothers in the urban region, whereas urban mothers had significantly higher RV-specific IgA antibody titers than did rural mothers. Neutralizing antibodies against RV strain G1P[8] were undetected in nearly one-half of the breast milk samples (45–48%), whereas the majority of the remaining samples had low antibody titers (2–16). Despite these low titers, the breast milk still reduced vaccine strain titers (2 × 106 plaque forming units/mL) up to 80% or more, even at a milk-to-virus ratio of 1:8. An increase in neutralizing anti-G1P[8] antibody titers (P < 0.05) in rural infants over time suggests a continuous exposure to circulating RV. These results contribute to the understanding of the potential interference of breast milk with RV vaccine efficacy and immunogenicity in Vietnamese infants. PMID:25668672

  20. Intestinal ribosomal p70(S6K) signaling is increased in piglet rotavirus enteritis.

    PubMed

    Rhoads, J Marc; Corl, Benjamin A; Harrell, Robert; Niu, Xiaomei; Gatlin, Lori; Phillips, Oulayvanh; Blikslager, Anthony; Moeser, Adam; Wu, Guoyao; Odle, Jack

    2007-03-01

    Recent identification of the mammalian target of rapamycin (mTOR) pathway as an amino acid-sensing mechanism that regulates protein synthesis led us to investigate its role in rotavirus diarrhea. We hypothesized that malnutrition would reduce the jejunal protein synthetic rate and mTOR signaling via its target, ribosomal p70 S6 kinase (p70(S6K)). Newborn piglets were artificially fed from birth and infected with porcine rotavirus on day 5 of life. Study groups included infected (fully fed and 50% protein calorie malnourished) and noninfected fully fed controls. Initially, in "worst-case scenario studies," malnourished infected piglets were killed on days 1, 3, 5, and 11 postinoculation, and jejunal samples were compared with controls to determine the time course of injury and p70(S6K) activation. Using a 2 x 2 factorial design, we subsequently determined if infection and/or malnutrition affected mTOR activation on day 3. Western blot analysis and immunohistochemistry were used to measure total and phosphorylated p70(S6K); [(3)H]phenylalanine incorporation was used to measure protein synthesis; and lactase specific activity and villus-crypt dimensions were used to quantify injury. At the peak of diarrhea, the in vitro jejunal protein synthetic rate increased twofold (compared with the rate in the uninfected pig jejunum), concomitant with increased jejunal p70(S6K) phosphorylation (4-fold) and an increased p70(S6K) level (3-fold, P < 0.05). Malnutrition did not alter the magnitude of p70(S6K) activation. Immunolocalization revealed that infection produced a major induction of cytoplasmic p70(S6K) and nuclear phospho-p70(S6K), mainly in the crypt. A downregulation of semitendinosus muscle p70(S6K) phosphorylation was seen at days 1-3 postinoculation. In conclusion, intestinal activation of p70(S6K) was not inhibited by malnutrition but was strongly activated during an active state of mucosal regeneration. PMID:17138969

  1. Expression and characterization of human group C rotavirus virus-like particles in insect cells

    SciTech Connect

    Clark, Kristina B.; Lin, S.-C.; Humphrey, Charles; Foytich, Kimberly; Esona, Mathew; Wang Yuhuan; Liu, Merry; Jiang Baoming

    2009-05-10

    Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.

  2. One-step RT-qPCR with an internal control system for the detection of turkey rotaviruses in faecal samples.

    PubMed

    Akimkin, V; Bindel, F; Hoferer, M; Sting, R; Polley, B; Hänel, A; Hafez, H M

    2011-10-01

    Turkey rotaviruses are one of the major pathogens responsible for the poult enteritis syndrome (PES). In this study a one step real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the rotaviral non-structural protein 4 (NSP4) was developed. The NSP4 is a highly conserved gene inside the turkey rotavirus genome and contains an internal control system to monitor any potential RT-qPCR inhibitors. The detection limit of the optimized NSP4-RT-qPCR assay ranged from 8.15 to 8.15 × 10(5) copy numbers. In total 149 faecal samples were collected from eight different flocks of commercial turkey farms. Faecal samples from hens and toms were collected separately at 2-week intervals from the 2nd week of age through the 16th and 20th week of age (age of slaughter for female and male, respectively) and tested. One farm reared only hens. The samples were tested previously using conventional RT-PCR targeting the same gene. When the conventional RT-PCR was compared with the developed NSP4-RT-qPCR, the results revealed that 11% of the samples of the conventional RT-PCR were false negative. The results indicate that this NSP4-RT-qPCR is highly sensitive for the detection of turkey rotaviruses in faeces. In addition, it could be suitable for the development of high-throughput screening. PMID:21816176

  3. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

    PubMed Central

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M. Cruz; Álvarez, Miguel A.; Hammarström, Lennart

    2015-01-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  4. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    PubMed

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  5. The VP5 domain of VP4 can mediate attachment of rotaviruses to cells.

    PubMed

    Zárate, S; Espinosa, R; Romero, P; Méndez, E; Arias, C F; López, S

    2000-01-01

    Some animal rotaviruses require the presence of sialic acid (SA) on the cell surface to infect the cell. We have isolated variants of rhesus rotavirus (RRV) whose infectivity no longer depends on SA. Both the SA-dependent and -independent interactions of these viruses with the cell are mediated by the virus spike protein VP4, which is cleaved by trypsin into two domains, VP5 and VP8. In this work we have compared the binding characteristics of wild-type RRV and its variant nar3 to MA104 cells. In a direct nonradioactive binding assay, both viruses bound to the cells in a saturable and specific manner. When neutralizing monoclonal antibodies directed to both the VP8 and VP5 domains of VP4 were used to block virus binding, antibodies to VP8 blocked the cell attachment of wild-type RRV but not that of the variant nar3. Conversely, an antibody to VP5 inhibited the binding of nar3 but not that of RRV. These results suggest that while RRV binds to the cell through VP8, the variant does so through the VP5 domain of VP4. This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produced in bacteria as fusion products with glutathione S-transferase, were found to bind to MA104 cells in a specific and saturable manner and, when preincubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively. In addition, the VP5 and VP8 recombinant proteins inhibited the infectivity of nar3 and RRV, respectively, confirming the results obtained in the binding assays. Interestingly, when the infectivity assay was performed on neuraminidase-treated cells, the VP5 fusion protein was also found to inhibit the infectivity of RRV, suggesting that RRV could bind to the cell through two sequential steps mediated by the interaction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively. PMID:10623720

  6. Health Impact of Rotavirus Vaccination in Developing Countries: Progress and Way Forward.

    PubMed

    Parashar, Umesh D; Johnson, Hope; Steele, A Duncan; Tate, Jacqueline E

    2016-05-01

    Two rotavirus vaccines have been licensed in >100 countries worldwide since 2006. As of October 2105, these vaccines have been implemented in the national immunization programs of 79 countries, including 36 low-income countries that are eligible for support for vaccine purchase from Gavi, the Vaccine Alliance. Rotavirus vaccines were initially introduced in Australia and countries of the Americas and Europe after completion of successful clinical trials in these regions, and the impact of routine vaccination in reducing the health burden of severe childhood gastroenteritis in these regions has been well documented. Because of concerns around the performance of orally administered rotavirus vaccines in developing countries, vaccine implementation in these settings only began after additional clinical trials were completed and the World Health Organization issued a global recommendation for use of rotavirus vaccines in 2009. This supplementary issue of Clinical Infectious Diseases includes a collection of articles describing the impact and effectiveness of routine rotavirus vaccination in developing countries that were among the early adopters of rotavirus vaccine. The data highlight the benefits of vaccination and should provide valuable evidence to sustain vaccine use in these countries and encourage other countries to adopt routine rotavirus vaccination to reduce the health burden of severe childhood gastroenteritis. PMID:27059361

  7. Prevention of rotavirus infections in vitro with aqueous extracts of Quillaja Saponaria Molina

    PubMed Central

    Roner, Michael R; Tam, Ka Ian; Kiesling-Barrager, Melody

    2010-01-01

    Background Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide, estimated to be responsible for over 300,000 childhood deaths every year, mostly in developing countries. Rotavirus-related deaths represent approximately 5% of all deaths in children younger than 5 years of age worldwide. Saponins are readily soluble in water and are approved by the US FDA for inclusion in beverages intended for human consumption. The addition of saponins to existing water supplies offers a new form of intervention into the cycle of rotavirus infection. We believe that saponins will ‘coat’ the epithelium of the host's small intestine and prevent attachment of rotavirus. Discussion This experiment provides in vitro data for the possibility of including saponin in drinking water to prevent infections of rotavirus. We demonstrate that microgram amounts of extract, while exhibiting no cell cytotoxicity or direct virucidal activity, prevent rotavirus from infecting its host cells. In addition, the presence of residual amounts of extract continue to block viral infection and render cells resistant to infection for at least 16 h after the removal of the extract from the cell culture media. Conclusion We demonstrate that two Quillaja extracts possess strong antiviral activity at concentrations more than 1000-fold lower than concentrations exhibiting cell cytotoxicity. Extract concentrations as high as 1000 μg/ml are not cytotoxic, but concentrations as low as 1.0 μg/ml are able to block rotavirus and reovirus attachment and infection. PMID:20725585

  8. Analysis of factors in response to rotavirus vaccination counselling in a private paediatric clinic.

    PubMed

    Kannan Kutty, P; Pathmanathan, G; Salleh, N M

    2010-06-01

    Rotavirus vaccine is available as an optional vaccine in Malaysia. The counselling of optional vaccines is considered an integral part of the health services offered in a private paediatric clinic. While ensuring that all babies are up-todate with their compulsory immunization, counselling of optional vaccines like the rotavirus vaccine could give parents the choice to make an informed decision on the acceptance of this vaccine. Over a period of two years, we counselled the parents regarding diarrhoea caused by rotavirus disease and the rotavirus vaccine. In this study, the factors that were significantly associated with the acceptance of the rotavirus vaccine were the gender of the baby, the mother's age, the mother's occupation, the mode of payment for the vaccine, the number of previous visits to the clinic by the parents, the number of counselling sessions given to the parents and the pre-counselling awareness or knowledge of rotavirus disease and rotavirus vaccine. It is hoped that these findings may assist busy clinicians in their continuous efforts to provide health education and vaccination counselling to the parents of their patients. PMID:23756797

  9. Detection of Rotavirus in children with acute gastroenteritis in Zagazig University Hospitals in Egypt

    PubMed Central

    Ibrahim, Salwa Badrelsabbah; El-Bialy, Abdallah Abdelkader; Mohammed, Mervat Soliman; El-Sheikh, Azza Omar; Elhewala, Ahmed; Bahgat, Shereen

    2015-01-01

    Introduction: Rotavirus is the major cause of acute gastroenteritis (AGE) in infants and young children all over the world. The objective of the study was to compare different methods for detecting rotavirus and to assess the burden of rotavirus as a causative agent for AGE in children younger than five. Methods: This case control study included 65 children with AGE and 35 healthy control children. They were chosen from the Pediatric Department of Zagazig University Hospitals from October 2014 to March 2015. Stool samples were obtained and assayed for rotavirus by the immunochromatography test (ICT), enzyme linked immunosorbent assay (ELISA) and quantitative real time RT-PCR (qr RT-PCR). Results: Fifty out of the 65 patients (76.9%) were positive for qr RT-PCR. Forty-five (69.2%) and 44 (67.7%) were positive for ICT and ELISA, respectively. There was a significant association between the severity of the disease as determined by the Vesikari score and rotavirus infection. Conclusion: This study demonstrated that ICT is a useful method for the rapid screening of group A rotavirus in fecal specimens, because it is rapid, inexpensive, easy to perform, and requires very little equipment. In addition, this study highlights the substantial health burden of rotavirus AGE among children less than five. PMID:26435821

  10. High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry

    PubMed Central

    Nair, Nitya; Newell, Evan W.; Vollmers, Christopher; Quake, Stephen R.; Morton, John M.; Davis, Mark M.; He, Xiao-Song; Greenberg, Harry B.

    2015-01-01

    In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus double-layered particles with a metal isotope and characterized B cells specific to the rotavirus VP6 major structural protein. We describe the heterogeneity of the intestinal B cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B cell subsets in the intestine and blood, and revealed that IgM+ memory B cells (MBCs) and naïve B cells were phenotypically related as were CD27− MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B cell subsets in immune donors, including naïve B cells, with phenotypes representative of the overall B cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state. PMID:25899688

  11. Preliminary characterization of an epitope involved in neutralization and cell attachment that is located on the major bovine rotavirus glycoprotein.

    PubMed Central

    Sabara, M; Gilchrist, J E; Hudson, G R; Babiuk, L A

    1985-01-01

    The 38,200-molecular weight (unreduced)/41,900-molecular-weight (reduced) glycoprotein of bovine rotavirus, isolate C486, was identified as the major neutralizing antigen. This glycoprotein as well as the corresponding glycoprotein of another bovine rotavirus serotype also specifically attached to cell monolayers under normal conditions for virus adsorption in vitro. Further support for this glycoprotein being directly responsible for virus attachment to cells was that (i) infectious virus of both serotypes could compete with the C486 glycoprotein for cell surface receptors, and (ii) neutralizing monospecific antiserum and neutralizing monoclonal antibodies directed toward the glycoprotein could block this virus-cell interaction. Preliminary epitope mapping of the glycoprotein with monoclonal antibodies further localized the neutralization-adsorption domain to a peptide with an approximate molecular weight of 14,000. The effect of two protein modifications, glycosylation and disulfide bridging, on the reactivity of this peptide with antibodies and cell surface receptors was investigated. It was demonstrated that, whereas glycosylation did not appear to affect these reactivities, disulfide bridging seemed to be essential. Images PMID:2578197

  12. Permissive replication of homologous murine rotavirus in the mouse intestine is primarily regulated by VP4 and NSP1.

    PubMed

    Feng, Ningguo; Yasukawa, Linda L; Sen, Adrish; Greenberg, Harry B

    2013-08-01

    Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1. PMID:23698306

  13. Permissive Replication of Homologous Murine Rotavirus in the Mouse Intestine Is Primarily Regulated by VP4 and NSP1

    PubMed Central

    Feng, Ningguo; Yasukawa, Linda L.; Sen, Adrish

    2013-01-01

    Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1. PMID:23698306

  14. Effect of breastfeeding promotion interventions on cost-effectiveness of rotavirus immunization in Indonesia

    PubMed Central

    2013-01-01

    Background Rotavirus infection has been reported to be responsible for the majority of severe diarrhea in children under-5-years-old in Indonesia. Breast milk is considered to give protection against rotavirus infection. Increasing breastfeeding promotion programs could be an alternative target to reduce the incidence of rotavirus diarrhea. This study aims to investigate the effect of breastfeeding promotion interventions on cost-effectiveness of rotavirus immunization in Indonesia, focusing on breastfeeding education and support interventions. Methods An age-structured cohort model was developed for the 2011 Indonesia birth cohort. We compared four interventions in scenarios: (i) base-case ( I 0 ) reflecting the current situation for the population of under-5-years-old, (ii) with an additional breastfeeding education intervention ( I 1 ), (iii) with a support intervention on initiation and duration ( I 2 ) and (iv) with both of these two interventions combined ( I 3 ). The model applied a 5-years time horizon, with 1 month analytical cycles for children less than 1 year of age and annually thereafter. Monte Carlo simulations were used to examine the economic acceptability and affordability of rotavirus vaccination. Results Rotavirus immunization would effectively reduce severe cases of rotavirus during the first 5 years of a child's life even assuming various breastfeeding promotion interventions. The total yearly vaccine cost would amount to US$ 64 million under the market vaccine price. Cost-effectiveness would increase to US$ 153 per quality-adjusted-life-year (societal perspective) with an optimal breastfeeding promotion intervention. Obviously, this is much lower than the 2011 Gross Domestic Product (GDP) per capita of US$ 3,495. Affordability results showed that at the market vaccine price, rotavirus vaccination could be affordable for the Indonesian health system. Conclusions Rotavirus immunization would be a highly cost-effective public health

  15. Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunized or nonimmunized cows.

    PubMed Central

    Saif, L J; Redman, D R; Smith, K L; Theil, K W

    1983-01-01

    Colostrum was collected and pooled from each of five cows in three experimental groups: group I cows received intramuscular and intramammary inoculations of adjuvanted modified live Ohio Agricultural Research and Development Center rotavirus vaccine; group II cows were injected intramuscularly with a commercial modified-live rota-coronavirus vaccine; and group III cows were uninoculated controls. Pooled colostrum from group I cows had higher (P less than 0.05) enzyme-linked immunosorbent assay (ELISA) immunoglobulin G (IgG1) and virus neutralization (VN) rotavirus antibody titers (ELISA IgG1 = 2,413,682; VN = 360,205) than did colostrum from group II (ELISA IgG1 = 8,192; VN = 4,395) or group III cows (ELISA IgG1 = 5,916; VN = 2,865). The antibody titers of these last two colostrum pools did not differ (P greater than 0.05). Samples of these colostrum pools were fed as daily supplements (percent [vol/vol] in cow's milk infant formula) to 28 newborn, unsuckled, antibody-seronegative, male Holstein calves. Eight calves received no supplemental colostrum. The calves were orally challenged with virulent bovine rotavirus and monitored daily for diarrhea and fecal rotavirus shedding. Diarrhea and rotavirus shedding occurred in the eight calves fed no supplemental colostrum and persisted longest in this group. The pooled colostrum from group I cows protected eight of eight calves from both rotavirus diarrhea and shedding when fed as a 1% supplement. The pooled colostrum from neither group II nor group III cows protected 12 other calves against rotavirus diarrhea or shedding when fed at the same concentration (1%). Six rotavirus-challenged calves fed 0.1% supplemental colostrum from group I cows and two calves fed 10 and 50% supplemental colostrum from control cows displayed partial passive immunity, exemplified by delayed onset and shortened duration of rotavirus-associated diarrhea and virus shedding. PMID:6309660

  16. Evaluation of the Intussusception Risk after Pentavalent Rotavirus Vaccination in Finnish Infants

    PubMed Central

    Leino, Tuija; Ollgren, Jukka; Strömberg, Nina; Elonsalo, Ulpu

    2016-01-01

    Background An association between rotavirus immunisation and intussusception (IS) has been suggested with present rotavirus vaccines in post-licensure studies. In Finland, rotavirus vaccination programme was implemented in September 2009 using a 2, 3, and 5 months schedule with the pentavalent rotavirus vaccine. By the end of 2013, it is estimated that 719 000 rotavirus vaccine doses have been given in the national programme of which 240 000 were first doses. Nationwide register allows us to evaluate the association between rotavirus vaccination and IS. Methods and Materials Cases of IS diagnosed during 1999–2013 were identified from National Hospital Discharge Register. All cases under 250 days of age diagnosed during 2009–2013 were confirmed by reviewing medical charts. Self-controlled case-series method was used to assess the risk of IS during 1–21 days compared to 22–42 days post vaccination. Findings In register data the relative incidence of IS at 2 months of age between the post and pre vaccination era was 9.1 (95%CI 2.0–84.3). We identified 22 verified cases with date of admission less than 43 days after any of the three rotavirus vaccine doses. The incidence of IS in the risk period after the 1st dose relative to the control period was 2.0 (95% CI 0.5–8.4; p = 0.34.) Number of excess IS cases per 100 000 first vaccine doses was therefore estimated to be 1.04 (95% CI 0.0–2.5), i.e. one additional IS case per 96 000 first doses of rotavirus vaccine (95% CI 54 600 to ∞). There was no risk detected after 2nd and 3rd doses. Conclusion The finding is in line with the recent published estimates. The benefits of rotavirus immunisation programme outweigh possible small risks of intussusception. PMID:26950702

  17. Cost-effectiveness of rotavirus vaccination in the Netherlands; the results of a consensus model

    PubMed Central

    2011-01-01

    Background Each year rotavirus gastroenteritis results in thousands of paediatric hospitalisations and primary care visits in the Netherlands. While two vaccines against rotavirus are registered, routine immunisation of infants has not yet been implemented. Existing cost-effectiveness studies showed inconsistent results for these vaccines because of lack of consensus on the impact. We aimed to investigate which factors had a major impact on cost-effectiveness and were primarily responsible for the large differences in previously estimated cost-effectiveness ratios. Methods Based on updated data on health outcomes and cost estimates, we re-assessed the cost-effectiveness of routine paediatric rotavirus vaccination within the National Immunization Program for the Netherlands. Two consensus meetings were organised with national and international experts in the field to achieve consensus and resolve potential controversies. Results It was estimated that rotavirus vaccination in the Netherlands could avert 34,214 cases of rotavirus gastroenteritis in children aged less than 5 years. Notably, 2,779 hospitalisations were averted of which 315 were extensions of existing hospital stays due to nosocomial rotavirus infection. With a threshold varying from 20K€ - 50K€ per QALY and according to the base-case scenario, the full vaccination costs per child leading to cost-effectiveness was €57.76 -€77.71. Results were sensitive to the inclusion of potential vaccine induced herd protection, QALY losses and number of deaths associated with rotavirus gastroenteritis. Conclusions Our economic analysis indicates that inclusion of rotavirus vaccination in the Dutch National Immunization Program might be cost-effective depending on the cost of the vaccine and the impact of rotavirus gastroenteritis on children's quality of life. PMID:21663620

  18. Quantification of human infection risk caused by rotavirus in surface waters from Córdoba, Argentina.

    PubMed

    Prez, V E; Gil, P I; Temprana, C F; Cuadrado, P R; Martínez, L C; Giordano, M O; Masachessi, G; Isa, M B; Ré, V E; Paván, J V; Nates, S V; Barril, P A

    2015-12-15

    Fecal contamination of water is a worrying problem because it is associated with the transmission of enteric pathogenic microorganisms that can cause many infectious diseases. In this study, an environmental survey was conducted to assess the level of viral contamination by viable enterovirus and rotavirus genome in two recreational rivers (Suquía and Xanaes) of Córdoba, Argentina. Quantitative microbial risk assessment (QMRA) was calculated to estimate the risk of rotavirus infection. Water sampling was carried out during a one-year period, the presence of total and fecal coliforms was determined and water samples were then concentrated for viral determination. Cell culture and indirect immunofluorescence were applied for enterovirus detection and RT-qPCR for rotavirus quantification. Coliform bacteria levels found in Suquía River often far exceeded the guideline limits for recreational waters. The Xanaes exhibited a lower level of bacterial contamination, frequently within the guideline limits. Enterovirus and rotavirus were frequently detected in the monitoring rivers (percentage of positive samples in Suquía: 78.6% enterovirus, 100% rotavirus; in Xanaes: 87.5% enterovirus, 18.7% rotavirus). Rotavirus was detected at a media concentration of 5.7×10(5) genome copies/L (gc/L) in the Suquía and 8.5×10(0)gc/L in the Xanaes. QMRA revealed high risk of rotavirus infection in the Suquía, at sampling points with acceptable and non-acceptable bacteria numbers. The Xanaes showed significantly lower health risk of rotavirus infection but it proved to be a public health hazard. The viral occurrence was not readily explained by the levels of bacteria indicators, thus viral monitoring should be included to determine microbiological water quality. These findings provide the first data of QMRA for recreational waters in Argentina and reveal the need for public awareness of the health implications of the use of the river waters. PMID:26311578

  19. Sentinel Hospital-Based Surveillance for Assessment of Burden of Rotavirus Gastroenteritis in Children in Pakistan

    PubMed Central

    Kazi, Abdul Momin; Warraich, Gohar Javed; Qureshi, Shahida; Qureshi, Huma; Khan, Muhammad Mubashir Ahmad; Zaidi, Anita Kaniz Mehdi

    2014-01-01

    Objectives To determine the burden and molecular epidemiology of rotavirus gastroenteritis in children hospitalized with severe acute watery diarrhea in Pakistan prior to introduction of rotavirus vaccine. Methods A cross-sectional study was carried out over a period of two years from 2006 – 2008 at five sentinel hospitals in the cities of Karachi, Lahore, Rawalpindi, and Peshawar. Stool samples collected from children under five years of age hospitalized with severe acute watery diarrhea were tested for rotavirus antigen via enzyme immunoassay (EIA) (IDEA REF K6020 Oxoid Ltd (Ely), Cambridge, United Kingdom). A subset of EIA positive stool samples were further processed for genotyping. Results 6679 children were enrolled and stool specimens of 2039 (30.5%) were positive for rotavirus. Rotavirus positivity ranged from 16.3% to 39.4% in the 5 hospitals with highest positivity in Lahore. 1241 (61%) of all rotavirus cases were in infants under one year of age. Among the strains examined for G-serotypes, the occurrence of G1, G2, G9 and G4 strains was found to be 28%, 24%, 14% and 13%, respectively. Among P-types, the most commonly occurring strains were P6 (31.5%) followed by P8 (20%) and P4 (12%). Prevalent rotavirus genotype in hospitalized children of severe diarrhea were G1P[8] 11.6% (69/593), followed by G2P[4] 10.4% (62/593), and G4P[6] 10.1% (60/593). Conclusions Approximately one third of children hospitalized with severe gastroenteritis in urban centers in Pakistan have rotavirus. Introduction of rotavirus vaccine in Pakistan's national immunization program could prevent many severe episodes and diarrheal deaths. PMID:25295613

  20. Sustained low rotavirus activity and hospitalisation rates in the post-vaccination era in Belgium, 2007 to 2014.

    PubMed

    Sabbe, Martine; Berger, Nicolas; Blommaert, Adriaan; Ogunjimi, Benson; Grammens, Tine; Callens, Michiel; Van Herck, Koen; Beutels, Philippe; Van Damme, Pierre; Bilcke, Joke

    2016-07-01

    In 2006, Belgium was the first country in the European Union to recommend rotavirus vaccination in the routine infant vaccination schedule and rapidly achieved high vaccine uptake (86-89% in 2007). We used regional and national data sources up to 7 years post-vaccination to study the impact of vaccination on laboratory-confirmed rotavirus cases and rotavirus-related hospitalisations and deaths. We showed that (i) from 2007 until 2013, vaccination coverage remained at 79-88% for a complete course, (ii) in children 0-2 years, rotavirus cases decreased by 79% (95% confidence intervals (CI): 68--89%) in 2008-2014 compared to the pre-vaccination period (1999--2006) and by 50% (95% CI: 14-82%) in the age group ≥ 10 years, (iii) hospitalisations for rotavirus gastroenteritis decreased by 87% (95% CI: 84-90%) in 2008--2012 compared to the pre-vaccination period (2002--2006), (iv) median age of rotavirus cases increased from 12 months to 17 months and (v) the rotavirus seasonal peak was reduced and delayed in all post-vaccination years. The substantial decline in rotavirus gastroenteritis requiring hospitalisations and in rotavirus activity following introduction of rotavirus vaccination is sustained over time and more pronounced in the target age group, but with evidence of herd immunity. PMID:27418466

  1. Whole genome characterization of a G6P[5] rotavirus A strain isolated from a stray cat in Japan.

    PubMed

    Kaneko, Miho; Mochizuki, Masami; Nakagomi, Osamu; Nakagomi, Toyoko

    2016-05-30

    The whole genome of an unusual G6P[5] rotavirus A strain named FRV537, which was isolated from a stray cat in Japan, was characterized to determine its species of origin. The genotype constellation of FRV537 was G6-P[5]-I2-R2-C2-M2-A13-N2- T6-E2-H3. No known feline rotavirus has this genotype constellation; the Japanese equine strain OH-4 is the only known strain that does. While FRV537 shares the same genotype with some feline rotaviruses in all genes except those encoding VP4 and NSP1, none of these genes are closely related to those of known feline rotaviruses. By contrast, G6P[5] is almost exclusively present in bovine rotaviruses. The VP7 and VP4 genes of FRV537 formed a lineage with typical bovine rotaviruses with high bootstrap values. As to the internal capsid and nonstructural gene constellation, three bovine rotavirus strains had a constellation identical to that of FRV537. Moreover, each of the genotypes of FRV537 was found to be a common bovine genotype. In addition to the high nucleotide sequence identities between FRV537 and bovine rotaviruses in each genome segment (≥95%), phylogenetic analysis revealed a close relationship to bovine/artiodactyl rotaviruses. Thus, the molecular and phylogenetic evidence suggests that FRV537 isolated from a stray cat was of bovine rotavirus origin. PMID:27139026

  2. Role of the Enteric Nervous System in the Fluid and Electrolyte Secretion of Rotavirus Diarrhea

    NASA Astrophysics Data System (ADS)

    Lundgren, Ove; Peregrin, Attila Timar; Persson, Kjell; Kordasti, Shirin; Uhnoo, Ingrid; Svensson, Lennart

    2000-01-01

    The mechanism underlying the intestinal fluid loss in rotavirus diarrhea, which often afflicts children in developing countries, is not known. One hypothesis is that the rotavirus evokes intestinal fluid and electrolyte secretion by activation of the nervous system in the intestinal wall, the enteric nervous system (ENS). Four different drugs that inhibit ENS functions were used to obtain experimental evidence for this hypothesis in mice in vitro and in vivo. The involvement of the ENS in rotavirus diarrhea indicates potential sites of action for drugs in the treatment of the disease.

  3. Uptake of oral rotavirus vaccine and timeliness of routine immunization in Brazil’s National Immunization Program

    PubMed Central

    Flannery, Brendan; Samad, Samia; de Moraes, José Cássio; Tate, Jacqueline E.; Danovaro-Holliday, M. Carolina; de Oliveira, Lúcia Helena; Rainey, Jeanette J.

    2015-01-01

    Introduction In March, 2006, oral rotavirus vaccine was added to Brazil’s infant immunization schedule with recommended upper age limits for initiating (by age 14 weeks) and completing (by age 24 weeks) the two-dose series to minimize age-specific risk of intussusception following rotavirus vaccination. Several years after introduction, estimated coverage with rotavirus vaccine (83%) was lower compared to coverage for other recommended childhood immunizations (≥94%). Methods We analyzed data from Brazil’s national immunization program on uptake of oral rotavirus vaccine by geographic region and compared administrative coverage estimates for first and second doses of oral rotavirus vaccine (Rota1 and Rota2) with first and second doses of diphtheria-tetanus-pertussis-Haemophilus influenzae type b vaccine (DTP-Hib1 and DTP-Hib2). For 27 Brazilian cities, we compared differences between estimated rotavirus and DTP-Hib coverage in 2010 with delayed receipt of DTP-Hib vaccine among a cohort of children surveyed before rotavirus introduction. Results In 2010, infant vaccination coverage was 99.0% for DTP-Hib1 versus 95.2% for Rota1 (3.8% difference), and 98.4% for DTP-Hib2 versus 83.0% for Rota2 (15.4% difference), with substantial regional variation. Differences between DTP-Hib and rotavirus vaccination coverage in Brazilian cities correlated with delay in DTP-Hib vaccination among children surveyed. Age restrictions for initiating and completing the rotavirus vaccination series likely contributed to lower coverage with rotavirus vaccine in Brazil. Conclusion To maximize benefits of rotavirus vaccination, strategies are needed to improve timeliness of routine immunizations; monitoring rotavirus vaccine uptake and intussusception risk is needed to guide further recommendations for rotavirus vaccination. PMID:23313652

  4. Burden of paediatric Rotavirus Gastroenteritis (RVGE) and potential benefits of a universal Rotavirus vaccination programme with a pentavalent vaccine in Spain

    PubMed Central

    2010-01-01

    Background Rotavirus is the most common cause of gastroenteritis in young children worldwide. The aim of the study was to assess the health outcomes and the economic impact of a universal rotavirus vaccination programme with RotaTeq, the pentavalent rotavirus vaccine, versus no vaccination programme in Spain. Methods A birth cohort was followed up to the age of 5 using a cohort model. Epidemiological parameters were taken from the REVEAL study (a prospective epidemiological study conducted in Spain, 2004-2005) and from the literature. Direct and indirect costs were assessed from the national healthcare payer and societal perspectives by combining health care resource utilisation collected in REVEAL study and unit costs from official sources. RotaTeq per protocol efficacy data was taken from a large worldwide rotavirus clinical trial (70,000 children). Health outcomes included home care cases, General Practioner (GP)/Paediatrician, emergency department visits, hospitalisations and nosocomial infections. Results The model estimates that the introduction of a universal rotavirus vaccination programme with RotaTeq (90% coverage rate) would reduce the rotavirus gastroenteritis (RVGE) burden by 75% in Spain; 53,692 home care cases, 35,187 GP/Paediatrician visits, 34,287 emergency department visits, 10,987 hospitalisations and 2,053 nosocomial infections would be avoided. The introduction of RotaTeq would avoid about 76% of RVGE-related costs from both perspectives: €22 million from the national health system perspective and €38 million from the societal perspective. Conclusions A rotavirus vaccination programme with RotaTeq would reduce significantly the important medical and economic burden of RVGE in Spain. PMID:20698958

  5. Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells

    PubMed Central

    Richards, James E.; Desselberger, Ulrich; Lever, Andrew M.

    2013-01-01

    At present the ability to create rationally engineered mutant rotaviruses is limited because of the lack of a tractable helper virus-free reverse genetics system. Using the cell culture adapted bovine RV RF strain (G6P6 [1]), we have attempted to recover infectious RV by co-transfecting in vitro transcribed ssRNAs which are identical in sequence to the positive sense strand of each of the 11 dsRNA genomic segments of the RF strain. The RNAs were produced either from cDNAs cloned by a target sequence-independent procedure, or from purified double layered RV particles (DLPs). We have validated their translational function by in vitro synthesis of 35S-labelled proteins in rabbit reticulocyte lysates; all 11 proteins encoded by the RV genome were expressed. Transfection experiments with DLP- or cDNA-derived ssRNAs suggested that the RNAs do not act independently as mRNAs for protein synthesis, once delivered into various mammalian cell lines, and exhibit cytotoxicity. Transfected RNAs were not infectious since a viral cytopathic effect was not observed after infection of MA104 cells with lysates from transfected cells. By contrast, an engineered mRNA encoding eGFP was expressed when transfected under identical conditions into the same cell lines. Co-expression of plasmids encoding NSP2 and NSP5 using a fowlpox T7 polymerase recombinant virus revealed viroplasm-like structure formation, but this did not enable the translation of transfected RV ssRNAs. Attempts to recover RV from ssRNAs transcribed intracellularly from transfected cDNAs were also unsuccessful and suggested that these RNAs were also not translated, in contrast to successful translation from a transfected cDNA encoding an eGFP mRNA. PMID:24019962

  6. Improved immunogenicity of biodegradable polymer particles entrapped rotavirus vaccine.

    PubMed

    Nayak, Bismita; Ray, Alok R; Panda, Amulya K; Ray, Pratima

    2011-01-01

    Rotavirus (RV) entrapped in polylactide (PLA) and polylactide-coglycolide (PLGA) polymer particles were formulated and evaluated in mice for improved immunogenicity using oral, intranasal (IN), and intramuscular (IM) routes of administration. Microparticles of size ranges between 1 and 8 µm were prepared using double emulsion solvent evaporation technique. Stabilizers like mouse serum albumin, sucrose, and sodium bicarbonate that were used during particle formulation helped in minimizing the denaturation of the entrapped antigen. Immunization with 20 µg of antigen entrapped in polymeric particles through various routes of administration elicited measurable amount of antibody titer in mice. The immunoglobulin A (IgA) and immunoglobulin G (IgG) titer (≥4-fold rise between pre and post immunized sera) was analyzed by the use of enzyme-linked immunosorbent assay. PLGA encapsulated RV microparticles elicited better antibody response through IN route (90%) where as PLA encapsulated RV microparticles showed improved response when administrated through oral route (83.3%). Overall, the performance of IN route based immunization was significantly higher than oral and IM route ( p<0.001) with both the polymers. The results are of indication that, PLGA encapsulated RV microparticles have greater potential for vaccine formulation to combat rotavirus infection. PMID:20207774

  7. Inactivation of human and simian rotaviruses by chlorine dioxide

    SciTech Connect

    Chen, Yu-Shiaw ); Vaughn, J.M. )

    1990-05-01

    The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4{degree}C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10{sup 5}-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate a neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.

  8. Rotavirus NSP1 Mediates Degradation of Interferon Regulatory Factors through Targeting of the Dimerization Domain

    PubMed Central

    Arnold, Michelle M.; Barro, Mario

    2013-01-01

    Rotavirus nonstructural protein NSP1 can inhibit expression of interferon (IFN) and IFN-stimulated gene products by inducing proteasome-mediated degradation of IFN-regulatory factors (IRFs), including IRF3, IRF5, and IRF7. All IRF proteins share an N-terminal DNA-binding domain (DBD), and IRF3, IRF5, and IRF7 contain a similar C-proximal IRF association domain (IAD) that mediates IRF dimerization. An autoinhibitory domain (ID) at the extreme C terminus interacts with the IAD, burying residues necessary for IRF dimerization. Phosphorylation of serine/threonine residues in the ID induces charge repulsions that unmask the IAD, enabling IRF dimerization and subsequent nuclear translocation. To define the region of IRF proteins targeted for degradation by NSP1, we generated IRF3 and IRF7 truncation mutants and transiently expressed each with simian SA11-4F NSP1. These assays indicated that the IAD represented a necessary and sufficient target for degradation. Because NSP1 did not mediate degradation of truncated forms of the IAD, NSP1 likely requires a structurally intact IAD for recognition and targeting of IRF proteins. IRF9, which contains an IAD-like region that directs interactions with signal inducer and activator of transcription (STAT) proteins, was also targeted for degradation by NSP1, while IRF1, which lacks an IAD, was not. Analysis of mutant forms of IRF3 unable to undergo dimerization or that were constitutively dimeric showed that both were targeted for degradation by NSP1. These results indicate that SA11-4F NSP1 can induce degradation of inactive and activated forms of IAD-containing IRF proteins (IRF3 to IRF9), allowing a multipronged attack on IFN-based pathways that promote antiviral innate and adaptive immune responses. PMID:23824805

  9. Repeated circulation over 6 years of intergenogroup mono-reassortant G2P[4] rotavirus strains with genotype N1 of the NSP2 gene.

    PubMed

    Doan, Yen Hai; Nakagomi, Toyoko; Nakagomi, Osamu

    2012-08-01

    Human rotavirus A, a major cause of acute diarrhoea in infants and young children worldwide, contains the genome of 11 segments of double-stranded RNA, which is grouped into three major genotype constellations: the Wa genogroup (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1), the DS-1 genogroup (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2), and the AU-1 genogroup (G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3). Recently, a G2P[4] strain detected in Kenya in 1982 was reported to be a nonstructural protein 2 (NSP2) gene mono-reassortant between the Wa and DS-1 genogroups. While NSP2 induces heterotypic antibody responses in children, thereby being the likely target of immune selective pressure, it was not known how frequently these NSP2 mono-reassortants circulated among children. In a previous 10 year epidemiological study, out of 100 G2P[4] rotavirus specimens that were typed into 22 distinct electropherotypes (i.e., strains), RNA-RNA hybridization identified that 12 strains were mono-reassortants involving either the NSP2 or the NSP3 gene. The aim of this study was therefore to determine the nucleotide sequences of all 11 genes of the representative mono-reassortant strain (AU605) and the sequences of the reassorted genes of the other mono-reassortant strains. The genome constellation of AU605 was G2-P[4]-I2-R2-C2-M2-A2-N1-T2-E2-H2, indicating that it was a mono-reassortant strain carrying a Wa-like NSP2 genotype on the DS-1 genogroup background. The reassorted gene of the other 11 strains also had the genotype N1 in the NSP2 gene. Given that electrophoretically-identical rotaviruses represent a single strain, the 12 NSP2 mono-reassortants detected in six rotavirus seasons accounted for 36% (36 of 100) of G2P[4] rotavirus specimens in the 10 year collection. The circulation of NSP2 mono-reassortant strains was observed in epidemic seasons when the G2P[4] genotype was not dominant. Taken together with their repeated occurrence at higher-than-expected frequencies, the identification of NSP2 mono

  10. Transient Creatine Kinase Elevation Followed by Hypocomplementemia in a Case of Rotavirus Myositis

    PubMed Central

    Rokugo, Yuka; Onuma, Ryoichi; Noguchi, Rie; Suzuki, Saeko; Kusaka, Natsuko; Watanabe, Yohei; Kitaoka, Setsuko

    2016-01-01

    We report an infant case of rotavirus myositis, a rare complication of rotavirus infection. Complement levels of the patient were normal when serum creatine kinase (CK) level was at its peak and then decreased when the CK level became normalized. In a previous case report of rotavirus myositis, transient decrease of serum albumin, immunoglobulin, and complement levels was reported. The authors speculated that intravascular complement activation was caused by rotavirus and resulted in the pathogenesis of myositis, although complement levels at onset were not measured by the authors. In this report, however, we demonstrate that the complement activation of our patient is a result of, rather than the cause of, skeletal muscle damage. PMID:27034874

  11. Exploring the cost effectiveness of an immunization programme for rotavirus gastroenteritis in the United Kingdom.

    PubMed

    Lorgelly, P K; Joshi, D; Iturriza Gómara, M; Gray, J; Mugford, M

    2008-01-01

    Rotavirus is the most common cause of gastroenteritis in children aged <5 years old, two new vaccines have recently been developed which can prevent associated morbidity and mortality. While apparently safe and efficacious, it is also important to establish whether rotavirus immunization is cost effective. A decision analytical model which employs data from a review of published evidence is used to determine the cost effectiveness of a rotavirus vaccine. The results suggest that some of the health sector costs, and all of the societal costs, of rotavirus gastroenteritis in children can be avoided by an immunization programme. The additional cost to the health sector may be considered worthwhile if there is a sufficient improvement in the quality-of-life of children and parents affected by gastroenteritis; this study did not find any evidence of research which has measured the utility gains from vaccination. PMID:17335631

  12. Exploring the cost effectiveness of an immunization programme for rotavirus gastroenteritis in the United Kingdom

    PubMed Central

    LORGELLY, P. K.; JOSHI, D.; ITURRIZA GÓMARA, M.; GRAY, J.; MUGFORD, M.

    2008-01-01

    SUMMARY Rotavirus is the most common cause of gastroenteritis in children aged <5 years old, two new vaccines have recently been developed which can prevent associated morbidity and mortality. While apparently safe and efficacious, it is also important to establish whether rotavirus immunization is cost effective. A decision analytical model which employs data from a review of published evidence is used to determine the cost effectiveness of a rotavirus vaccine. The results suggest that some of the health sector costs, and all of the societal costs, of rotavirus gastroenteritis in children can be avoided by an immunization programme. The additional cost to the health sector may be considered worthwhile if there is a sufficient improvement in the quality-of-life of children and parents affected by gastroenteritis; this study did not find any evidence of research which has measured the utility gains from vaccination. PMID:17335631

  13. ENZYME IMMUNOASSAY WITH MONOCLONAL ANTIBODIES FOR THE DETECTION OF ROTAVIRUS IN STOOL SPECIMENS

    EPA Science Inventory

    Rotavirus infections are generally recognized as a major problem in young children; however, they have also been associated with severe gastroenteritis in adults and neonates. Infections in neonates are usually asymptomatic, although the incidence of infection may be high. Adult ...

  14. [Genetic diversity of group A rotavirus isolates found in Western Siberia in 2007-2011].

    PubMed

    Zhirakovskaia, E V; Aksanova, R Kh; Gorbunova, M G; Tikunov, A Iu; Kuril'shchikov, A M; Sokolov, S N; Netesov, S V; Tikunova, N V

    2012-01-01

    Genetic analysis of group A rotavirus recovered from fecal samples of children admitted to hospitals in Novosibirsk and Omsk during four epidemic seasons 2007, 2007/2008, 2009/2010, 2010/2011 was performed. A total of 1416 rotavirus isolates were genotyped using multiplex PCR. The isolates of the most common rotavirus genotypes G1P[8], G4P[8], G2P[4], G3P[8] co-circulated in Western Siberia during 2007-2011. In isolated cases G9P[8], G2P[8], G3P[9], and G4P[6] genotypes were detected. Change of dominant genotype from G1P[8] to G4P[8] occurred in 2008 in Omsk and in Novosibirsk in 2009 as well. Incidence and distribution of rotavirus genotypes differed and changed every epidemic season in both cities. The phylogenetic analysis based on VP4 (VP8*), VP7, and VP6 gene sequences showed that the majority of isolates from Novosibirsk and Omsk were clustered together and demonstrated high level homology with rotavirus isolates found in other regions of Eurasia. In addition, a rare P[8]b (OP354-like) subtype of the VP4 gene was identified in fourteen isolates (G9, G1, and G4) in Novosibirsk and in a single isolate Omsk08-381/G9P[8]b in Omsk. The results obtained in this study demonstrate the necessity of long-term monitoring of rotavirus isolates in Western Siberia. This is important for selection of rotavirus vaccine for immunization of infants, improvement of diagnostic kits and understanding of the epidemiology and the evolution of group A rotaviruses. PMID:23248851

  15. Molecular characterizations of human and animal group a rotaviruses in the Netherlands.

    PubMed

    van der Heide, R; Koopmans, M P G; Shekary, N; Houwers, D J; van Duynhoven, Y T H P; van der Poel, W H M

    2005-02-01

    To gain more insight into interspecies transmission of rotavirus group A, human and animal fecal samples were collected between 1997 and 2001 in The Netherlands. A total of 110 human stool samples were successfully P and G genotyped by reverse transcriptase PCR. All strains belonged to the main human rotavirus genotypes G1 to G4, G9, [P4], [P6], [P8], and [P9]. [P8]G1 was predominant, and 5.5% belonged to the G9 genotype. Eleven percent of all P[8] genotypes could be genotyped only by a recently published modified primer. Rotavirus-positive fecal samples from 28 calf herds were genotyped by DNA sequencing. Genotypes G6 and G10 predominated; G6 and G10 were detected in 22 (78.6%) and 16 (57.1%) of the rotavirus-positive calf herds, respectively. In 12 (42.9%) calf herds, we found mixed infections. Genotype G8 was not found. Genotype G6 bovine rotaviruses were divided into three clusters: UK-like, VMRI-29-like, and Hun4-like. DNA sequencing of a part of the VP7 gene was shown to be useful as a quick determination of uncommon or novel strains of which the genotyping cannot be done by genotyping PCR. Of equine strains, both VP4 and VP7 genes could be used for genotyping: two [P12]G3 and four [P12]G14 equine rotaviruses were determined. We did not find indications for rotavirus interspecies transmissions, although the recently published human G6-Hun4 is genetically related to our G6 bovine isolates. All bovine, porcine, and equine rotaviruses were within genotypes previously reported for these animal species. PMID:15695662

  16. Rotavirus seasonality in urban sewage from Argentina: effect of meteorological variables on the viral load and the genetic diversity.

    PubMed

    Barril, P A; Fumian, T M; Prez, V E; Gil, P I; Martínez, L C; Giordano, M O; Masachessi, G; Isa, M B; Ferreyra, L J; Ré, V E; Miagostovich, M; Pavan, J V; Nates, S V

    2015-04-01

    In Argentina, the rotavirus disease exhibits seasonal variations, being most prevalent in the fall and winter months. To deepen the understanding of rotavirus seasonality in our community, the influence of meteorological factors on the rotavirus load and the genetic diversity in urban raw sewage from Córdoba city, Argentina were evaluated. Wastewater samples were collected monthly during a three-year study period and viral particles were concentrated by polyethylene glycol precipitation. RT-nested PCR was applied for rotavirus detection, and VP7/VP4 characterization and real-time PCR for rotavirus quantification. Both molecular techniques showed relatively similar sensitivity rates and revealed rotavirus presence in urban wastewater in cold and warm seasons, indicating its circulation in the local community all year round. However, a slight trend for rotavirus circulation was noted by real-time PCR in the fall and winter seasons, showing a significantly higher peak of rotavirus concentration at mean temperatures lower than 18°C and also higher, although not statistically different during drier weather. VP7 and VP4 gene characterization showed that G1 and P[8] genotypes were dominant, and temporal variations in genotype distribution were not observed. Rotavirus spread is complex and our results point out that weather factors alone cannot explain the seasonal quantitative pattern of the rotavirus disease. Therefore, alternative transmission routes, changes in human behavior and susceptibility, and the stability and survivability of the virus might all together contribute to the seasonality of rotavirus. The results obtained here provide evidence regarding the dynamics of rotavirus circulation and maintenance in Argentina. PMID:25777068

  17. Seasonality of rotavirus disease in the tropics: a systematic review and meta-analysis

    PubMed Central

    Levy, Karen; Hubbard, Alan E; Eisenberg, Joseph NS

    2009-01-01

    Background To date little conclusive evidence exists on the seasonality of rotavirus incidence in the tropics. We present a systematic review and meta-analysis on the seasonal epidemiology of rotavirus in the tropics, including 26 studies reporting continuous monthly rotavirus incidence for which corresponding climatological data was available. Methods Using linear regression models that account for serial correlation between months, monthly rotavirus incidence was significantly negatively correlated with temperature, rainfall and relative humidity in 65%, 55% and 60% of studies, respectively. We carried out pooled analyses using a generalized estimating equation (GEE) that accounts for correlation from between-study variation and serial correlation between months within a given study. Results For every 1°C (1.8°F) increase in mean temperature, 1 cm (0.39 in.) increase in mean monthly rainfall, and 1% increase in relative humidity (22%) this analysis showed reductions in rotavirus incidence of 10% (95% CI: 6–13%), 1% (95% CI: 0–1%), and 3% (95% CI:0–5%), respectively. Conclusions On the basis of the evidence, we conclude that rotavirus responds to changes in climate in the tropics, with the highest number of infections found at the colder and drier times of the year. PMID:19056806

  18. Estimated hospitalizations attributed to norovirus and rotavirus infection in Canada, 2006-2010.

    PubMed

    Morton, V K; Thomas, M K; McEwen, S A

    2015-12-01

    Enteric viruses including norovirus and rotavirus are leading causes of gastroenteritis in Canada. However, only a small number of clinical cases are actually tested for these pathogens leading to systematic underestimation of attributed hospitalizations in administrative databases. The objective of this analysis was to estimate the number of hospitalizations due to norovirus and rotavirus in Canada. Hospitalization records for acute gastroenteritis-associated discharges at all acute-care hospitals in Canada between 2006 and 2011 were analysed. Cause-unspecified gastroenteritis hospitalizations were modelled using age-specific negative binomial models with cause-specified gastroenteritis admissions as predictors. The coefficients from the models were used to estimate the number of norovirus and rotavirus admissions. The total annual hospitalizations for rotavirus were estimated to be between 4500 and 10 000. Total annual hospitalizations for norovirus were estimated to be between 4000 and 11 000. The mean total annual cost associated with these hospitalizations was estimated to be at least $16 million for rotavirus and $21 million for norovirus (all figures in Canadian dollars). This study is the first comprehensive analysis of norovirus and rotavirus hospitalizations in Canada. These estimates provide a more complete assessment of the burden and economic costs of these pathogens to the Canadian healthcare system. PMID:25991407

  19. Clinic-based surveillance for bacterial- and rotavirus-associated diarrhea in Egyptian children.

    PubMed

    Wierzba, Thomas F; Abdel-Messih, Ibrahim Adib; Abu-Elyazeed, Remon; Putnam, Shannon D; Kamal, Karim A; Rozmajzl, Patrick; Ahmed, Salwa F; Fatah, Abdel; Zabedy, Khaled; Shaheen, Hind I; Sanders, John; Frenck, Robert

    2006-01-01

    To identify enteropathogens for vaccine development, we implemented clinic-based surveillance for severe pediatric diarrhea in Egypt's Nile River Delta. Over 2 years, a physician clinically evaluated and obtained stool samples for microbiology from patients with diarrhea and less than 6 years of age. In the first (N = 714) and second clinic (N = 561), respectively, 36% (N = 254) and 46% (N = 260) of children were infected with rotavirus, enterotoxigenic Escherichia coli (ETEC), Campylobacter, or Shigella. When excluding mixed rotavirus-bacterial infections, for the first and second clinic, 23% and 10% had rotavirus-associated diarrhea, and 14% and 17% had ETEC-associated diarrhea, respectively. Campylobacter-associated diarrhea was 1% and 3%, and Shigella-associated diarrhea was 2% and 1%, respectively, for the two clinics. Rotavirus-associated diarrhea peaked in late summer to early winter, while bacterial agents were prevalent during summer. Rotavirus-associated cases presented with dehydration, vomiting, and were often hospitalized. Children with Shigella- or Campylobacter-associated diarrhea reported as watery diarrhea and rarely dysentery. ETEC did not have any clinically distinct characteristics. For vaccine development and/or deployment, our study suggests that rotavirus is of principle concern, followed by ETEC, Shigella, and Campylobacter. PMID:16407360

  20. Detection of bovine group B rotaviruses in feces by polymerase chain reaction.

    PubMed

    Chinsangaram, J; Akita, G Y; Osburn, B I

    1994-07-01

    A pair of primers designed from the sequence of genome segment 9 of group B rat rotavirus (IDIR) were employed to amplify genome segment 9 of a group B bovine rotavirus in a polymerase chain reaction (PCR) and to sequence the derived PCR products. A new pair of primers were synthesized from the obtained sequence data and used in a PCR detection assay for group B bovine rotavirus in fecal samples. In addition, another pair of primers were designed to produce a PCR-derived internal probe. This probe was used in a chemiluminescent hybridization to confirm the specificity and to increase the sensitivity of the assay. This assay could detect 0.1 fg of target double-stranded RNA. It was specific to group B bovine rotavirus and did not detect group B rat (IDIR) and porcine rotaviruses, group A bovine (NCDV), simian (SA-11), equine (H-2), porcine (OSU), human (DS-1), deer, and avian rotaviruses, coronavirus, or other enteric organisms tested in this study. PMID:7948199

  1. Seroepidemiology of group A rotavirus in suburban São Paulo, Brazil.

    PubMed

    Cox, M J; Azevedo, R S; Nokes, D J; Beards, G M; McCrae, M A; Massad, E; Medley, G F

    1998-06-01

    Age-specific patterns of rotavirus infection were investigated using a randomly selected and representative sample of sera from a suburban community of São Paulo, Brazil screened for class-specific antibodies to group A rotavirus. Age-serology of anti-rotavirus IgG showed primary infection predominant in young infants with a median age of around 18 months consistent with IgM serology suggesting highest rates of recent infection between ages 4 and 48 months. Anti-rotavirus serum IgA prevalence increased gradually with age. Paired samples from infants, collected 1 month apart, indicated high exposure rates with seroconversion occurring in several infants during the reported low transmission season. Between 5 and 10% of adults had elevated IgM levels indicative of recent infection and, potentially, of an important contribution adults may play to rotavirus transmission. Further understanding of the dynamics of rotavirus transmission within populations, at group and serotype level, would benefit the design and monitoring of future immunization programmes. PMID:9692612

  2. Seroepidemiology of group A rotavirus in suburban São Paulo, Brazil.

    PubMed Central

    Cox, M. J.; Azevedo, R. S.; Nokes, D. J.; Beards, G. M.; McCrae, M. A.; Massad, E.; Medley, G. F.

    1998-01-01

    Age-specific patterns of rotavirus infection were investigated using a randomly selected and representative sample of sera from a suburban community of São Paulo, Brazil screened for class-specific antibodies to group A rotavirus. Age-serology of anti-rotavirus IgG showed primary infection predominant in young infants with a median age of around 18 months consistent with IgM serology suggesting highest rates of recent infection between ages 4 and 48 months. Anti-rotavirus serum IgA prevalence increased gradually with age. Paired samples from infants, collected 1 month apart, indicated high exposure rates with seroconversion occurring in several infants during the reported low transmission season. Between 5 and 10% of adults had elevated IgM levels indicative of recent infection and, potentially, of an important contribution adults may play to rotavirus transmission. Further understanding of the dynamics of rotavirus transmission within populations, at group and serotype level, would benefit the design and monitoring of future immunization programmes. PMID:9692612

  3. Economic and psychosocial impact of rotavirus infection in Spain: a literature review.

    PubMed

    Álvarez Aldeán, Javier; Aristegui, Javier; López-Belmonte, Juan Luis; Pedrós, Montse; Sicilia, José García

    2014-06-24

    Severe rotavirus gastroenteritis is common in children under 5 years of age. A literature review was performed to investigate the economic and psychosocial impact of rotavirus infection in children in this age group. We retrieved 56 articles on the economic burden of the disease in Europe, 18 of them reported data from Spain; 8 articles were retrieved analysing its psychosocial impact. In Spain, rotavirus is responsible for 14% to 30% of all cases of gastroenteritis, and a quarter of these require hospitalisation. It is also associated with high use of health care resources (emergency and primary care visits). Rotavirus gastroenteritis costs the Spanish national health system EUR 28 million a year and causes productivity loss in two-thirds of parents (mean of 4 days). Taking into account these costs, it was estimated that implementing universal vaccination could prevent 76% to 95% of hospital admissions due to rotavirus gastroenteritis, as well as reduce emergency and paediatric visits, nosocomial infections, and days missed from work (77% reduction). Rotavirus gastroenteritis also has a considerable psychosocial impact on the family, although it is difficult to compare results due to the diversity of study designs and the low specificity of the measurement tools used. It also causes high stress among parents, adding to their workload and adversely affecting their quality of life. PMID:24837768

  4. Cost-effectiveness analysis of rotavirus vaccination among Libyan children using a simple economic model

    PubMed Central

    Alkoshi, Salem; Maimaiti, Namaitijiang; Dahlui, Maznah

    2014-01-01

    Background Rotavirus infection is a major cause of childhood diarrhea in Libya. The objective of this study is to evaluate the cost-effectiveness of rotavirus vaccination in that country. Methods We used a published decision tree model that has been adapted to the Libyan situation to analyze a birth cohort of 160,000 children. The evaluation of diarrhea events in three public hospitals helped to estimate the rotavirus burden. The economic analysis was done from two perspectives: health care provider and societal. Univariate sensitivity analyses were conducted to assess uncertainty in some values of the variables selected. Results The three hospitals received 545 diarrhea patients aged≤5 with 311 (57%) rotavirus positive test results during a 9-month period. The societal cost for treatment of a case of rotavirus diarrhea was estimated at US$ 661/event. The incremental cost-effectiveness ratio with a vaccine price of US$ 27 per course was US$ 8,972 per quality-adjusted life year gained from the health care perspective. From a societal perspective, the analysis shows cost savings of around US$ 16 per child. Conclusion The model shows that rotavirus vaccination could be economically a very attractive intervention in Libya. PMID:25499622

  5. Seasonal fluctuations in the occurrence of enterotoxigenic bacteria and rotavirus in paediatric diarrhoea in Addis Ababa.

    PubMed

    Stintzing, G; Bäck, E; Tufvesson, B; Johnsson, T; Wadström, T; Habte, D

    1981-01-01

    This study (March 1977-February 1978) was performed at the Ethio-Swedish Pediatric Clinic, Addis Ababa, to determine whether there were any seasonal fluctuations in the occurrence of diarrhoea associated with enterotoxigenic enterobacteria (ETEB), rotavirus and two parasites (Giardia lamblia and Entamoeba histolytica).A total of 1161 children (962 patients and 199 controls) were investigated. ETEB were isolated in 12.2% of the patients and 4.5% of the controls, rotavirus in 27.8% and 8%, and parasites in 6.8% and 1%, respectively. There is a statistically significant difference in the isolation rates betwe