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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.

  4. 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.

  5. Antigenic characteristics of the complete and truncated capsid protein VP1 of enterovirus 71.

    PubMed

    Zhang, Jianhua; Dong, Min; Jiang, Bingfu; Dai, Xing; Meng, Jihong

    2012-08-01

    The complete VP1 protein of enterovirus 71 (EV71) and a series of truncations were expressed in Escherichia coli and their antigenic characteristics were studied. Immunoblot analysis showed the major immunoreactive region of the VP1 protein was located in the N-terminal portion at position of amino acid (aa) 1-100. The complete VP1 possessed strong cross-reactivity with antisera against coxsackievirus A16 (CA16) and echovirus 6 (Echo6), while the truncated fragment at position 1-100 aa only had weak cross-reactivity. Moreover, an EV71-specific linear epitope at position 94-105 aa was identified using two EV71-specific mAbs (2B9 and 5B7) with indirect ELISA, but could not be recognized by antibodies against EV71 virus particles. The complete and all of truncated VP1 proteins except His-VP1(202-297) and GST-VP1(202-248) failed to elicit a significant neutralizing antibody response in mice. His-VP1(202-297) and GST-VP1(202-248) containing neutralizing epitope(s) could be recognized only by anti-EV71 mouse sera but not rabbit or human sera. These findings may contribute to a further understanding of antigenic characteristics of the capsid protein VP1 and may be helpful to the development of diagnostic reagents and vaccines.

  6. Oral immunization of mice using Bifidobacterium longum expressing VP1 protein from enterovirus 71.

    PubMed

    Yu, Zhijian; Huang, Zhen; Sao, Chongwen; Huang, Yuanjian; Zhang, Fan; Ma, Guihong; Chen, Zhong; Zeng, Zhongming; Qiwen, Deng; Zeng, Weiseng

    2013-05-01

    Bifidobacterium longum is an attractive candidate for delivering biologically active proteins by the mucosal route due to its non-pathogenic and colonizing properties. Enterovirus 71 (EV71) has aroused widespread attention recently due to several epidemics, and great attention should be paid to the fact that there are currently no effective antiviral drugs or vaccines against EV71 infection. In this report, we described a recombinant B. longum that could be used to develop an oral vaccine against EV71 infection. A VP1 expression vector (pBBADs-VP1) was constructed by amplifying the EV71 VP1 gene and inserting it into the E. coli-Bifidobacterium shuttle expression vector pBBAD/Xs. Then, the expression of VP1 protein in pBBADs-VP1-transformed bacteria was demonstrated by western blot. In vivo studies indicated that oral immunization of BALB/c mice with pBBADs-VP1-transformed bacteria induced potent immune responses against EV71 infection, including virus-neutralising titers, anti-EV71-VP1 antibody and the induction of Th1 immune responses in the spleen and Peyer's patches. Importantly, immunization of mother mice with this recombinant VP1-expressing B. longum conferred protection to neonatal mice. These results demonstrate that the novel oral vaccine utilizing B. longum expressing the VP1 protein might successfully elicit a specific immune response against EV71 infection.

  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.

  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. Oral immunization of mice using transgenic tomato fruit expressing VP1 protein from enterovirus 71.

    PubMed

    Chen, Hsuan-Fu; Chang, Meng-Huei; Chiang, Bor-Luen; Jeng, Shih-Tong

    2006-04-05

    Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease associated with fatal neurological complications in young children, and several major outbreaks have occurred recently. This study developed an effective antiviral agent by transforming the gene for VP1 protein, a previously defined epitope and also a coat protein of EV71, into tomato plant. VP1 protein was first fused with sorting signals to enable it to be retained in the endoplasmic reticulum of tomato plant, and its expression level increased to 27 microg/g of fresh tomato fruit. Transgenic tomato fruit expressing VP1 protein was then used as an oral vaccine, and the development of VP1-specific fecal IgA and serum IgG were observed in BALB/c mice. Additionally, serum from mice fed transgenic tomato could neutralize the infection of EV71 to rhabdomyosarcoma cells, indicating that tomato fruit expressing VP1 was successful in orally immunizing mice. Moreover, the proliferation of spleen cells from orally immunized mice was stimulated by VP1 protein, and provided further evidence of both humoral and cellular immunity. Results of this study not only demonstrate the feasibility of using transgenic tomato as an oral vaccine to generate protective immunity in mice against EV71, but also suggest the probability of enterovirus vaccine development.

  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. Treating Progressive Multifocal Leukoencephalopathy With Interleukin 7 and Vaccination With JC Virus Capsid Protein VP1

    PubMed Central

    Sospedra, Mireia; Schippling, Sven; Yousef, Sara; Jelcic, Ilijas; Bofill-Mas, Silvia; Planas, Raquel; Stellmann, Jan-Patrick; Demina, Viktoria; Cinque, Paola; Garcea, Robert; Croughs, Therese; Girones, Rosina; Martin, Roland

    2014-01-01

    Progressive multifocal leukoencephalopathy is a currently untreatable infection of the brain. Here, we demonstrate in 2 patients that treatment with interleukin 7, JC polyomavirus (JCV) capsid protein VP1, and a Toll-like receptor 7 agonist used as adjuvant, was well tolerated, and showed a very favorable safety profile and unexpected efficacy that warrant further investigation. PMID:25214510

  12. Immunogenicity of a truncated enterovirus 71 VP1 protein fused to a Newcastle disease virus nucleocapsid protein fragment in mice.

    PubMed

    Ch'ng, W C; Saw, W T; Yusoff, K; Shafee, N

    2011-01-01

    Enterovirus 71 (EV71) is one of the viruses that cause hand, foot and mouth disease. Its viral capsid protein 1 (VP1), which contains many neutralization epitopes, is an ideal target for vaccine development. Recently, we reported the induction of a strong immune response in rabbits to a truncated VP1 fragment (Nt-VP1t) displayed on a recombinant Newcastle disease virus (NDV) capsid protein. Protective efficacy of this vaccine, however, can only be tested in mice, since all EV71 animal models thus far were developed in mouse systems. In this study, we evaluated the type of immune responses against the protein developed by adult BALB/c mice. Nt-VP1t protein induced high levels of VP1 IgG antibody production in mice. Purified VP1 antigen stimulated activation, proliferation and differentiation of splenocytes harvested from these mice. They also produced significant levels of IFN-γ, a Th1-related cytokine. Taken together, Nt-VP1t protein is a potent immunogen in adult mice and our findings provide the data needed for testing of its protective efficacy in mouse models of EV71 infections.

  13. Expression of enterovirus 71 capsid protein VP1 in Escherichia coli and its clinical application.

    PubMed

    Shi, Mei; Zhou, Yaping; Cao, Limin; Ding, Cuijun; Ji, Yun; Jiang, Qinbo; Liu, Xiping; Li, Xiang; Hou, Xueling; Peng, Hongjun; Shi, Weifeng

    2013-12-01

    The VPl gene of enterovirus 71 (EV71) was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16), A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25%) from CA16-infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD).

  14. Expression of enterovirus 71 capsid protein VP1 in Escherichia coli and its clinical application

    PubMed Central

    Shi, Mei; Zhou, Yaping; Cao, Limin; Ding, Cuijun; Ji, Yun; Jiang, Qinbo; Liu, Xiping; Li, Xiang; Hou, Xueling; Peng, Hongjun; Shi, Weifeng

    2013-01-01

    The VPl gene of enterovirus 71 (EV71) was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16), A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25%) from CA16-infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD). PMID:24688514

  15. High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

    PubMed Central

    Mukherjee, Santanu; Abd-El-Latif, Mahmoud; Bronstein, Michal; Ben-nun-Shaul, Orly; Kler, Stanislav; Oppenheim, Ariella

    2007-01-01

    SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility. PMID:17712413

  16. High cooperativity of the SV40 major capsid protein VP1 in virus assembly.

    PubMed

    Mukherjee, Santanu; Abd-El-Latif, Mahmoud; Bronstein, Michal; Ben-nun-Shaul, Orly; Kler, Stanislav; Oppenheim, Ariella

    2007-08-22

    SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of approximately 6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.

  17. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice.

    PubMed

    Wang, Man; Jiang, Shuai; Wang, Yefu

    2013-01-04

    Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni-NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

  18. Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells.

    PubMed

    Weng, Tsui-Ying; Chen, Lien-Cheng; Shyu, Huey-Wen; Chen, Shun-Hua; Wang, Jen-Ren; Yu, Chun-Keung; Lei, Huan-Yao; Yeh, Trai-Ming

    2005-07-01

    The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-alpha expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.

  19. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

    PubMed Central

    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  20. 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.

  1. The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat.

    PubMed

    Yeo, Wee M; Chow, Vincent T K

    2007-04-01

    Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease (HFMD). Several outbreaks in East Asia were associated with neurological complications and numerous deaths. EV71 possesses four structural proteins VP1-VP4 that are necessary in the formation of the pentameric icosahedral capsid. The viral capsid contributes to virulence, and VP1 is a prime target for EV71 vaccine development. Using yeast two-hybrid analysis, we demonstrated binding affinity between VP1 and three human proteins, i.e. ornithine decarboxylase (ODC1), gene trap ankyrin repeat (GTAR), and KIAA0697 expressed in brain tissue. These interactions were authenticated by co-immunoprecipitation experiments, and by indirect immunofluorescent confocal microscopy of transfected and EV71-infected Vero cells. The significant interaction between VP1 and ODC1 may compromise the latter's activity, and interfere with polyamine biosynthesis, growth and proliferation of EV71-infected cells. The interaction between VP1 and GTAR is noteworthy, since ankyrin proteins are associated with certain neural cell adhesion molecules and with the CRASH neurological syndrome. Given that VP1 is synthesized in large amounts during productive infection, these viral-host protein interactions may provide insights into the role of VP1 in the pathogenesis of EV71 disease and its neurological complications such as acute flaccid paralysis and encephalitis.

  2. Recombinant VP1 protein of duck hepatitis virus 1 expressed in Pichia pastoris and its immunogenicity in ducks.

    PubMed

    Wang, C; Li, X K; Wu, T C; Wang, Y; Zhang, C J; Cheng, X C; Chen, P Y

    2014-01-01

    The VP1 gene of duck hepatitis virus type 1 (DHV-1) strain VJ09 was amplified by reverse transcription PCR from the liver of a duckling with clinical symptoms of viral hepatitis. The resulting VP1 cDNA was 720 bp in length and encoded a 240-amino-acid protein. In VP1 gene-based phylogenetic analysis, the VJ09 strain grouped with DHV-1 genotype C. The VP1 gene was inserted into the expression vector pPICZαA and expressed in Pichia pastoris. The expressed VP1 protein was purified and identified by western blot analysis. To evaluate the recombinant VP1's immunogenic potential in ducklings, the antibodies raised in the immunized ducklings were titrated by ELISA, and lymphocyte proliferation and virus neutralization assays were performed. The results show that the recombinant VP1 protein induced a significant immune response in ducklings and this could be a candidate for the development of a subunit vaccine against DHV-1 genotype C.

  3. Expression of VP1 protein in the milk of transgenic mice: a potential oral vaccine protects against enterovirus 71 infection.

    PubMed

    Chen, Hsiao-Ling; Huang, Jiun-Yan; Chu, Te-Wei; Tsai, Tung-Chou; Hung, Che-Ming; Lin, Chih-Cheng; Liu, Fang-Chueh; Wang, Li-Chung; Chen, Yi-Ju; Lin, Ming-Fong; Chen, Chuan-Mu

    2008-06-02

    Enterovirus 71 (EV71) is the most common etiological agent detected in cases of hand-foot-and-mouth disease (HFMD) resulting in incidences of neurological complications and fatality in recent years. The clinical data have already shown the significant increase in recent EV71 epidemic activity throughout the Asia-Pacific region. Due to the lack of an effective antiviral agent, primary prevention of the disease, including the development of an effective vaccine, has been the top priority in terms of control strategies. In this study, we first generated a transgenic animal system to produce the EV71 VP1 capsid protein under the control of alpha-lactalbumin promoter and alpha-casein leader sequences. A high level of recombinant VP1 protein (2.51 mg/ml) was expressed and secreted into the milk of transgenic mice. Mouse pups that received VP1-transgenic milk orally demonstrated relatively better health conditions after challenge with the respective virus as compared with the non-transgenic milk fed group; moreover, the mice fed with the VP1-milk had body weights similar to those of the PBS placebo control groups. According to the serum-neutralization assay and serum antibody detection, the littermates suckling VP1-milk generated antibodies specific to EV71. Our data suggest that EV71 VP1-containing milk is suitable for development as a potential oral vaccine.

  4. 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

  5. Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity.

    PubMed

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

    2011-11-01

    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.

  6. Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

    PubMed Central

    Wu, Xiaoying; Li, Xiaojun; Zhang, Qingshan; Wulin, Shaozhou; Bai, Xiaofei; Zhang, Tingting; Wang, Yue; Liu, Ming; Zhang, Yun

    2015-01-01

    Background The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. Methods and Results To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. Conclusions and Significance We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1. PMID:25706372

  7. Investigation of host-cell influence on polyomavirus biological activity and the major virus-coded structural protein VP1

    SciTech Connect

    Ludlow, J.W.

    1987-01-01

    The host cell influence on polyomavirus biological activity and modification of the major virus-coded structural protein VP1 was investigated. Polyoma virions resulting from the permissive infections of primary mouse kidney cells, as compared to virus progeny from primary mouse embryo cells, exhibited a ten-fold greater ability to agglutinate guinea pig erythrocytes, a three-fold lower ability to become internalized into monopincytotic vesicles, and a two-fold lower ability to initiate a productive infection based on positive nuclear immunofluorescence when using mouse embryo host cells. Mouse kidney cell progeny were found to bind to host cells less specifically than mouse-embryo cell progeny. Two-dimensional gel electrophoresis of VP1, following in vivo labeling with /sup 32/P, revealed that species D and E of the mouse kidney cell progeny were phosphorylated to the same degree while mouse embryo cell progeny VP1 species E and F were phosphorylated equally. In vivo labeling of polyomavirus with /sup 35/S-sulfate, followed by gel electrophoretic analysis of the structural proteins and two-dimensional chromatographic analysis of the VP1 amino acids revealed that species E and F are modified by sulfation of tyrosine. These data suggest that host cells play a role in modulating biological activity of the virus by affecting the degree and site specific modification of the major capsid protein VP1. Modification of this protein may influence the recognition of virus attachment proteins for specific cellular receptors as well as other biological functions.

  8. Cysteine residues in the major capsid protein, Vp1, of the JC virus are important for protein stability and oligomer formation.

    PubMed

    Kobayashi, Shintaro; Suzuki, Tadaki; Igarashi, Manabu; Orba, Yasuko; Ohtake, Noriko; Nagakawa, Keita; Niikura, Kenichi; Kimura, Takashi; Kasamatsu, Harumi; Sawa, Hirofumi

    2013-01-01

    The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.

  9. Glucocorticoids Prevent Enterovirus 71 Capsid Protein VP1 Induced Calreticulin Surface Exposure by Alleviating Neuronal ER Stress.

    PubMed

    Hu, Dan-Dan; Mai, Jian-Ning; He, Li-Ya; Li, Pei-Qing; Chen, Wen-Xiong; Yan, Jian-Jiang; Zhu, Wei-Dong; Deng, Li; Wei, Dan; Liu, Di-Hui; Yang, Si-Da; Yao, Zhi-Bin

    2017-02-01

    Severe hand-foot-and-mouth disease (HFMD) caused by Enterovirus 71 (EV71) always accompanies with inflammation and neuronal damage in the central nervous system (CNS). During neuronal injuries, cell surface-exposed calreticulin (Ecto-CRT) is an important mediator for primary phagocytosis of viable neurons by microglia. Our data confirmed that brainstem neurons underwent neuronophagia by glia in EV71-induced death cases of HFMD. EV71 capsid proteins VP1, VP2, VP3, or VP4 did not induce apoptosis of brainstem neurons. Interestingly, we found VP1-activated endoplasmic reticulum (ER) stress and autophagy could promote Ecto-CRT upregulation, but ER stress or autophagy alone was not sufficient to induce CRT exposure. Furthermore, we demonstrated that VP1-induced autophagy activation was mediated by ER stress. Meaningfully, we found dexamethasone treatment could attenuate Ecto-CRT upregulation by alleviating VP1-induced ER stress. Altogether, these findings identify VP1-promoted Ecto-CRT upregulation as a novel mechanism of EV71-induced neuronal cell damage and highlight the potential of the use of glucocorticoids to treat severe HFMD patients with CNS complications.

  10. 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.

  11. Molecular Evolution of Hepatitis A Virus: a New Classification Based on the Complete VP1 Protein

    PubMed Central

    Costa-Mattioli, Mauro; Cristina, Juan; Romero, Héctor; Perez-Bercof, Raoul; Casane, Didier; Colina, Rodney; Garcia, Laura; Vega, Ines; Glikman, Graciela; Romanowsky, Victor; Castello, Alejandro; Nicand, Elisabeth; Gassin, Michelle; Billaudel, Sylviane; Ferré, Virginie

    2002-01-01

    Hepatitis A virus (HAV) is a positive-stranded RNA virus in the genus Hepatovirus in the family Picornaviridae. So far, analysis of the genetic variability of HAV has been based on two discrete regions, the VP1/2A junction and the VP1 N terminus. In this report, we determined the nucleotide and deduced amino acid sequences of the complete VP1 gene of 81 strains from France, Kosovo, Mexico, Argentina, Chile, and Uruguay and compared them with the sequences of seven strains of HAV isolated elsewhere. Overall strain variation in the complete VP1 gene was found to be as high as 23.7% at the nucleotide level and 10.5% at the amino acid level. Different phylogenetic methods revealed that HAV sequences form five distinct and well-supported genetic lineages. Within these lineages, HAV sequences clustered by geographical origin only for European strains. The analysis of the complete VP1 gene allowed insight into the mode of evolution of HAV and revealed the emergence of a novel variant with a 15-amino-acid deletion located on the VP1 region where neutralization escape mutations were found. This could be the first antigenic variant of HAV so far identified. PMID:12186933

  12. A novel finding for enterovirus virulence from the capsid protein VP1 of EV71 circulating in mainland China.

    PubMed

    Liu, Yongjuan; Fu, Chong; Wu, Suying; Chen, Xiong; Shi, Yingying; Zhou, Bingfei; Zhang, Lianglu; Zhang, Fengfeng; Wang, Zhihao; Zhang, Yingying; Fan, Chengpeng; Han, Song; Yin, Jun; Peng, Biwen; Liu, Wanhong; He, Xiaohua

    2014-04-01

    Enterovirus 71 (EV71) is a neurotropic virus that causes various clinical manifestations in young children, ranging from asymptomatic to fatal. Different pathotypes of EV71 notably differ in virulence. Several virulence determinants of EV71 have been predicted. However, these reported virulence determinants could not be used to identify the EV71 strains of subgenotype C4, which mainly circulate in China. In this study, VP1 sequences of 37 EV71 strains from severe cases (SC-EV71) and 192 EV71 strains from mild cases (MC-EV71) in mainland China were analyzed to determine the potential virulence determinants in the capsid protein VP1 of EV71. Although most SC-EV71 strains belonged to subgenotype C4a, no specific genetic lineages in C4a were correlated with EV71 virulence. Interestingly, amino acid substitutions at nine positions (H22Q, P27S, N31S/D, E98K, E145G/Q, D164E, T240A/S, V249I, and A289T) were detected by aligning the VP1 sequences of the SC-EV71 and MC-EV71 strains. Moreover, both the constituent ratios of the conservative or mutated residues in the MC-EV71 and SC-EV71 strains and the changes in the VP1 3D structure resulting from these mutations confirmed that the conservative residues (22H, 249V, and 289A) and the mutated residues (27S, 31S/D, 98K, 145G/Q, 164E, and 240A/S) might be potential virulence determinants in VP1 of EV71. Furthermore, these results led to the hypothesis that VP1 acts as a sandwich switch for viral particle stabilization and cellular receptors attachment, and specific mutations in this protein can convert mild cases into severe cases. These findings highlight new opportunities for diagnostic and therapeutic interventions.

  13. Molecular evolution of the major capsid protein VP1 of enterovirus 70.

    PubMed Central

    Takeda, N; Tanimura, M; Miyamura, K

    1994-01-01

    Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate

  14. 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.

  15. Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein.

    PubMed

    Abaitua, F; O'Hare, P

    2008-06-01

    VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.

  16. JC Virus nucleotides 376-396 are critical for VP1 capsid protein expression

    PubMed Central

    Ellis, Laura C.; Koralnik, Igor J.

    2014-01-01

    JC Virus (JCV) infection of the brain can cause Progressive Multifocal Leukoencephalopathy, JCV Granule Cell Neuronopathy and JCV Encephalopathy (JCVE). JCVCPN, isolated from the brain of a patient with JCVE, is a naturally occurring strain of JCV with a 143 base pair deletion in the agnogene. Cell culture studies of JCVCPN have shown that the loss of these nucleotides in the agnogene results in impaired expression of VP1 and infectious virion production. To better understand the role of this DNA sequence in JCV replication, we generated a series of deletions in the agnogene on the backbone of a virus which has a mutated agnoprotein start codon preventing agnoprotein expression. We found that deletion of nucleotides 376-396 results in decreased levels of viral DNA replication and a lack of VP1 expression. These results indicate that these nucleotides play a crucial role in JCV replication. PMID:25142442

  17. 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

    2016-04-14

    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.

  18. 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.

  19. Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides.

    PubMed

    Foo, Damian Guang Wei; Alonso, Sylvie; Phoon, Meng Chee; Ramachandran, N P; Chow, Vincent Tak Kwong; Poh, Chit Laa

    2007-04-01

    Enterovirus 71 (EV71) is the main causative agent of Hand, foot and mouth disease (HFMD) and has been associated with severe neurological diseases resulting in high mortalities. Currently, there is no vaccine available and treatment is limited to palliative care. In this study, antisera were raised in mice against 95 overlapping synthetic peptides spanning the VP1 capsid protein of EV71. Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, respectively, are capable of eliciting neutralizing antibodies against EV71 in the in vitro microneutralization assay. SP70 was identified to be particularly potent in eliciting a neutralizing antibody titer comparable to that obtained with a whole virion-immune serum. Immunization of mice with either SP55 or SP70 triggered an EV71-specific IgG response as high as that obtained with the whole virion as immunogen. The IgG sub-typing revealed that the neutralizing antibodies elicited by both synthetic peptides are likely belonging to the IgG1 sub-type. Alignment with databases showed that the amino acid residues of SP70 are highly conserved amongst the VP1 sequences of EV71 strains from various sub-genogroups. Altogether, these data indicate that SP70 represents a promising candidate for an effective synthetic peptide-based vaccine against EV71.

  20. The VP1 protein of human enterovirus 71 self-associates via an interaction domain spanning amino acids 66-297.

    PubMed

    Lal, Sunil K; Kumar, Purnima; Yeo, Wee M; Kar-Roy, Anindita; Chow, Vincent T K

    2006-05-01

    Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease (HFMD). Several recent outbreaks of HFMD in East Asia were associated with neurological complications and numerous deaths. In 2000, an outbreak in Singapore afflicted thousands of children, resulting in four fatal cases from whom EV71 was isolated. The virus possesses four structural proteins VP1, VP2, VP3, and VP4, each of which is involved in forming the pentameric icosahedral structure of the virus. Here we report that the full-length VP1 structural protein of EV71 is capable of self-association. Dimerization of VP1 was tested using the yeast two-hybrid system, fluorescence resonance energy transfer (FRET) analysis, in vitro coupled transcription-translation binding assays, and mammalian cell transcription-translation experiments. Dimerization of various truncated versions of the VP1 protein was also studied by mutational analysis. Systematic deletions of parts of VP1 revealed that the region spanning amino acids 66-132 of VP1 contains the major dimerization domain. However, the region between amino acids 132 and 297 was indispensable, and contributed largely to increasing the strength of the interaction. This ability of EV71 VP1 to self-associate and to participate significantly in forming the characteristic icosahedral capsid strongly enhances the pathogenicity and stability of the virus to withstand the environment of the gastrointestinal tract.

  1. Characterization of the herpes simplex virus (HSV)-1 tegument protein VP1-2 during infection with the HSV temperature-sensitive mutant tsB7.

    PubMed

    Abaitua, F; Souto, R N; Browne, H; Daikoku, T; O'Hare, P

    2009-10-01

    VP1-2, encoded by the UL36 gene of herpes simplex virus (HSV), is a large structural protein, conserved across the family Herpesviridae, that is assembled into the tegument and is essential for virus replication. Current evidence indicates that VP1-2 is a central component in the tegumentation and envelopment processes and that it also possesses important roles in capsid transport and entry. However, any detailed mechanistic understanding of VP1-2 function(s) remains limited. This study characterized the replication of HSV-1 tsB7, a temperature-sensitive mutant restricted at the non-permissive temperature due to a defect in VP1-2 function. A tsB7 virus expressing green fluorescent protein-fused VP16 protein was used to track the accumulation and location of a major tegument protein. After infection at the permissive temperature and shift to the non-permissive temperature, the production of infectious virus ceased. VP1-2 accumulated in altered cytosolic clusters, together with VP16 and other virion proteins. Furthermore, correlating with the results of immunofluorescence, electron microscopy demonstrated abnormal cytosolic capsid clustering and a block in envelopment. As VP1-2 encompasses a ubiquitin-specific protease domain, the occurrence of ubiquitin-conjugated proteins during tsB7 infection was also examined at the non-permissive temperature. A striking overaccumulation was observed of ubiquitin-specific conjugates in cytoplasmic clusters, overlapping and adjacent to the VP1-2 clusters. These results are discussed in relation to the possible functions of VP1-2 in the assembly pathway and the nature of the defect in tsB7.

  2. The presence of Chlamydia phage PhiCPG1 capsid protein VP1 genes and antibodies in patients infected with Chlamydia trachomatis.

    PubMed

    Ma, Jingyue; Liu, Yuan; Liu, Yuanjun; Li, Lingjie; Hou, Shuping; Gao, Xibo; Qi, Manli; Liu, Quanzhong

    2016-01-01

    Chlamydia phage PhiCPG1 has been found in Chlamydia caviae in a guinea pig model for inclusion conjunctivitis, raising the possibility that Chlamydia phage is also present in patients infected with C. trachomatis (Ct). In the present study, we assayed for presence of Chlamydia phage capsid protein VP1 genes and antibodies in 84 non-Ct controls and 206 Ct patients using an enzyme-linked immunoassay (ELISA), followed by verification with Western blot. None of the subjects were exposed to an antibiotic treatment or had a C. pneumoniae infection. The VP1 antibody test was positive in both, the ELISA and Western blot assay, in 4 Ct patients. PCR amplification experiments revealed presence of the VP1 gene in 5 Ct patients. The results suggest that Chlamydia phage capsid protein VP1 may exist in some Ct patients.

  3. Blue native protein electrophoresis for studies of mouse polyomavirus morphogenesis and interactions between the major capsid protein VP1 and cellular proteins.

    PubMed

    Horníková, Lenka; Man, Petr; Forstová, Jitka

    2011-12-01

    Morphogenesis of the mouse polyomavirus virion is a complex and not yet well understood process. Nuclear lysates of infected cells and cells transiently producing the major capsid protein (VP1) of the mouse polyomavirus and whole-cell lysates were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) to characterize the participation of cellular proteins in virion precursor complexes. Several VP1-specific complexes were found by immunostaining with the anti-VP1 antibody. Some of these complexes contained proteins from the heat shock protein 70 family. The BN-PAGE was found to be a useful tool for the identification of protein complexes by immunostaining of separated cell lysates. However, whole-cell lysates and lysates of isolated nuclei of cells infected with polyomavirus appeared to be too complex for BN-PAGE separation followed by mass spectrometry. No distinct bands specific for cells infected with polyomavirus were detected by Coomassie blue stained gels, hence this method is not suitable for the discovery of new cellular proteins participating in virion assembly. Nevertheless, BN-PAGE can be valuable for the analyses of different types of complexes formed by proteins after their enrichment or isolation by affinity chromatography.

  4. Characterization of a novel monoclonal antibody reactive against the N-terminal region of Enterovirus 71 VP1 capsid protein.

    PubMed

    Lim, Xiao Fang; Jia, Qiang; Chow, Vincent T K; Kwang, Jimmy

    2013-03-01

    Hand, foot and mouth disease (HFMD) is a viral infectious disease caused by human Enterovirus A, particularly Enterovirus 71 (EV71) and Coxsackievirus 16 (CA16) serotypes, with EV71 infection associated with severe neurological complications and mortality. Lots of attention has been placed on elucidating viral epitopes, which is useful for EV71 viral research. In this study, a murine monoclonal antibody (mAb 4) specific for EV71 was generated and mapped to target the N-terminal region of VP1 capsid protein, spanning amino acid residues 12-19 (IGDSVSRA). mAb 4 can cross-react with all the 11 representative EV71 subgenotypes (A, B1-5, C1-5), but not with the representative strain of CA16 as demonstrated by immunofluorescence assay (IFA). BLAST analyses of this epitope against all Enterovirus entries in Genbank also demonstrated that this epitope is unique in EV71, but not other Enterovirus such as CA16 It may be useful for structural study of VP1 morphogenesis during infection and also applications for identification of EV71 infection.

  5. Evolutionary analysis of structural protein gene VP1 of foot-and-mouth disease virus serotype Asia 1.

    PubMed

    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.

  6. Identification of a conserved neutralizing linear B-cell epitope in the VP1 proteins of duck hepatitis A virus type 1 and 3.

    PubMed

    Zhang, Ruihua; Zhou, Guomei; Xin, Yinghao; Chen, Junhao; Lin, Shaoli; Tian, Ye; Xie, Zhijing; Jiang, Shijin

    2015-11-18

    Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection.

  7. 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.

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

    PubMed Central

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

    2016-01-01

    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

  9. Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles.

    PubMed Central

    Poruchynsky, M S; Atkinson, P H

    1991-01-01

    Rotavirus, a double-shelled nonenveloped member of the REoviridae family, becomes transiently membrane enveloped during its maturation process, as single-shelled particles bud from cytoplasmic viroplasm structures into the adjacent endoplasmic reticulum. The present study describes the isolation of these membrane-enveloped viral intermediates from rotavirus SA11-infected Ma104 cells. The enveloped intermediates comprised the proteins VP1, VP2, VP4, VP6, VP7, and NS28 and small amounts of NS35 and NS34. VP7 in the intermediate particles was recognized by either a polyclonal antibody to VP7, which previous studies had shown recognizes the membrane-associated form of VP7, or a monoclonal antibody which recognizes VP7 on mature virus. NS28, VP7, and VP4 could be complexed to a higher-molecular-weight form when the membrane-permeable cross-linker dithiobis(succinimidylproprionate) was used. However, when an impermeable cross-linker was used, the structural proteins, including VP7, were not accessible to cross-linking. Velocity sedimentation of cross-linked immunoisolated enveloped virus particles showed that VP7 and VP4 were located in the same fractions only when the membrane-permeable cross-linker was used, implying their heterooligomeric association during outer capsid formation. When intermediate enveloped virus particles were treated with protease, VP6 and VP7 were protected, but not in the presence of detergent. Taken together, these results support the idea that in the membrane-enveloped intermediate, VP7 is repositioned from its location in the endoplasmic reticulum lumen back across the viral membrane envelope to the inferior of the virus particle during the maturation process. Images PMID:1651404

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

    PubMed Central

    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

  11. Molecular Evolution and Genetic Analysis of the Major Capsid Protein VP1 of Duck Hepatitis A Viruses: Implications for Antigenic Stability.

    PubMed

    Ma, Xiuli; Sheng, Zizhang; Huang, Bing; Qi, Lihong; Li, Yufeng; Yu, Kexiang; Liu, Cunxia; Qin, Zhuoming; Wang, Dan; Song, Minxun; Li, Feng

    2015-01-01

    The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10(-4) per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses.

  12. Display of enterovirus 71 VP1 on baculovirus as a type II transmembrane protein elicits protective B and T cell responses in immunized mice.

    PubMed

    Kolpe, Annasaheb B; Kiener, Tanja K; Grotenbreg, Gijsbert M; Kwang, Jimmy

    2012-09-01

    Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215-219 (Lim et al., 2012). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities.

  13. Enteroviruses and the pathogenesis of type 1 diabetes revisited: cross-reactivity of enterovirus capsid protein (VP1) antibodies with human mitochondrial proteins.

    PubMed

    Hansson, Sara F; Korsgren, Stella; Pontén, Fredrik; Korsgren, Olle

    2013-04-01

    Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes.

  14. 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.

  15. Identification and characterization of a monoclonal antibody recognizing the linear epitope RVADVI on VP1 protein of enterovirus 71.

    PubMed

    Man-Li, Tang; Szyporta, Milene; Fang, Lim Xiao; Kwang, Jimmy

    2012-10-01

    Several large outbreaks of hand-foot-mouth disease (HFMD) have occurred in the Asian-Pacific region since 1997, with Enterovirus 71 (EV71) and/or Coxsackievirus A16 (CAV16) as the main causative agents. Despite the close genetic relationship between the two viruses, only EV71 is associated with severe clinical manifestations and deaths. Effective antiviral treatment and vaccines are not available. High-quality monoclonal antibodies (mAbs) are necessary to improve the accuracy of the diagnosis of EV71. In this study, a mAb (designated 1D9) was generated using EV71 C5 strain virus particles as immunogens. Examined by indirect immunofluorescence assay (IFA) and Western blotting, 1D9 detected successfully all 11 subgenotypes of EV71 and showed no cross-reactivity to the four selected subgenogroups of Coxsackieviruses CAV4, CAV6, CAV10, and CAV16. A linear motif, R(3) VADVI(8), which is located at the N-terminus of the EV71 VP1 protein, was identified as the minimal binding region of 1D9. Alignment and comparison of the 1D9-defined epitope sequence against the listed sequences in the NCBI EV71 database indicated that this epitope R(3) VADVI(8) was highly conserved among EV71 strains, while no significant similarity was observed when blasted against the Coxsackieviruses. This suggests that the mAb 1D9 may be useful for the development of a cost-effective and accurate method for surveillance and early differentiation of EV71 from CAV16 infection.

  16. Virucidal efficacy of glutaraldehyde against enteroviruses is related to the location of lysine residues in exposed structures of the VP1 capsid protein.

    PubMed

    Chambon, Martine; Archimbaud, Christine; Bailly, Jean-Luc; Gourgand, Jeanne-Marie; Charbonné, Françoise; Peigue-Lafeuille, Hélène

    2004-03-01

    Glutaraldehyde (GTA) is a potent virucidal disinfectant whose exact mode of action against enteroviruses is not understood. Earlier reports showed that GTA reacts preferentially with the VP1 capsid protein of echovirus 25 and poliovirus 1 and that GTA has affinity for exposed lysine residues on proteins. To investigate further the inactivation of enteroviruses by GTA, seven strains were selected on the basis of differences in their overall number and the positions of lysine residues in the amino acid sequences of the VP1 polypeptide. Inactivation kinetics experiments were performed with 0.10% GTA. The viruses grouped into three clusters and exhibited significantly different levels of sensitivity to GTA. The results were analyzed in the light of current knowledge of the three-dimensional structure of enteroviruses and the viral life cycle. The differences observed in sensitivity to GTA were related to the number of lysine residues and their locations in the VP1 protein. The overall findings suggest that the BC and DE loops, which cluster at the fivefold axis of symmetry and are the most exposed on the outer surface of the virions, are primary reactive sites for GTA.

  17. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein

    PubMed Central

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer. PMID:24147211

  18. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein.

    PubMed

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  19. Engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (FMDV) VP1 inhibits type I interferon response in cells.

    PubMed

    Li, Xiaying; Wang, Jianchang; Liu, Jue; Li, Zhonghua; Wang, Yongqiang; Xue, Yanfei; Li, Xiaoqi; Cao, Hong; Zheng, Shijun J

    2013-09-27

    Foot-and-mouth disease (FMD) is an acute, highly contagious animal disease caused by FMD virus (FMDV). Although FMDV-induced immunosuppression in host has been well established, the exact molecular mechanism for such induction is not very clear. We report here the identification of FMDV VP1 as an interferon-suppressor by interacting with soluble resistance-related calcium binding protein (sorcin). We found that VP1 suppressed tumor necrosis factor (TNF)-α or Sendai virus (SeV)-induced type I interferon response in HEK293T cells, and that this suppression could be completely abolished by knockdown of sorcin by shRNA. Furthermore, overexpression of sorcin inhibited type I interferon response. Conversely, TNF- or SeV-induced type I interferon response increased when sorcin knocked down, leading to inhibition of vesicular stomatitis virus (VSV) replication. Thus, VP1-induced suppression of type I interferon is mediated by interacting with sorcin, a protein that appears to regulate cell response to viral infections.

  20. Re-emergence of circulatory foot-and-mouth disease virus serotypes Asia1 in Bangladesh and VP1 protein heterogeneity with vaccine strain IND 63/72.

    PubMed

    Ullah, H; Siddique, M A; Al Amin, Md; Das, B C; Sultana, M; Hossain, M A

    2015-02-01

    Foot-and-mouth disease virus (FMDV) serotypes O, A and Asia1 are responsible for significant number of disease outbreaks in Bangladesh; however serotype Asia1 has not been reported in circulation since 1996. The present investigation reports the detection of serotype FMDV Asia1 from local farms in 2012 and 2013 outbreaks. The farms were located in Jessore and Gazipur districts, and one of these farms was under vaccine control programme. Phylogenetic analysis of the complete VP1 gene revealed that FMDV Asia1 is under genetic lineage C having close similarity to the Asia1 sequences of Indian origin. The circulatory genotype Asia1 showed VP1 protein sequence heterogeneity of eight amino acid substitutions within the G-H loop with the vaccine strain [IND 63/72 (AY304994)] used in vaccination programme. ELISA assay revealed that, of seven, only one local field serum sample (cattle vaccinated 38 days earlier) was positive at a titre level of >2.4 (log10) but failed to protect the cattle from infection occurred by the virus. This investigation focused that the eight amino acid substitution in VP1 protein at G-H loop of the locally circulated FMDV serotype Asia1 strain may be a reason for current vaccination failure.

  1. 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.

  2. Rotavirus.

    PubMed Central

    Parashar, U. D.; Bresee, J. S.; Gentsch, J. R.; Glass, R. I.

    1998-01-01

    Rotavirus, the most common diarrheal pathogen in children worldwide, causes approximately one third of diarrhea-associated hospitalizations and 800,000 deaths per year. Because natural infection reduces the incidence and severity of subsequent episodes, rotavirus diarrhea might be controlled through vaccination. Serotypespecific immunity may play a role in protection from disease. Tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) (which contains a rhesus rotavirus with serotype G3 specificity and reassortant rhesus-human rotaviruses with G1, G2, and G4 specificity) provides coverage against the four common serotypes of human rotavirus. In clinical trials in industrialized countries, RRV-TV conferred 49% to 68% protection against any rotavirus diarrhea and 61% to 100% protection against severe disease. This vaccine was licensed by the U.S. Food and Drug Administration on August 31, 1998, and should be cost-effective in reducing diarrheal diseases in industrialized countries. The vaccine's efficacy and cost-effectiveness in developing countries should be evaluated. PMID:9866732

  3. Expression and immunogenic analysis of recombinant polypeptides derived from capsid protein VP1 for developing subunit vaccine material against hepatitis A virus.

    PubMed

    Jang, Kyoung Ok; Park, Jong-Hwa; Lee, Hyun Ho; Chung, Dae Kyun; Kim, Wonyong; Chung, In Sik

    2014-08-01

    Three recombinant polypeptides, VP1-His, VP1-3N-His, and 3D2-His, were produced by Escherichia coli expression system. Recombinant VP1-His, VP1-3N-His, and 3D2-His were expressed as bands with molecular weights of 32, 38, and 30 kDa, respectively. These were purified by affinity chromatography using Ni-NTA Fast-flow resin and/or ion-exchange chromatography using DEAE-Sepharose Fast-flow resin. Intraperitoneal immunizations of recombinant polypeptides successfully elicited the productions of VP1-His, VP1-3N-His, and 3D2-His specific IgG antibodies (IgG subclass distribution of IgG1>IgG2a>IgG2b>IgG3) in sera and induced the secretions of cytokines IFN-γ and IL-6 in spleen cells. Sera from recombinant VP1-His-, VP1-3N-His-, and 3D2-His-immunized mice neutralized the propagation of HAV. The highest neutralizing activity was shown in sera from recombinant VP1-3N-His-immunized mice. These results suggest that recombinant VP1-3N-His can be a useful source for developing hepatitis A virus (HAV) subunit vaccine candidates.

  4. Molecular determinants of enterovirus 71 viral entry: cleft around GLN-172 on VP1 protein interacts with variable region on scavenge receptor B 2.

    PubMed

    Chen, Pan; Song, Zilin; Qi, Yonghe; Feng, Xiaofeng; Xu, Naiqing; Sun, Yinyan; Wu, Xing; Yao, Xin; Mao, Qunyin; Li, Xiuling; Dong, Wenjuan; Wan, Xiaobo; Huang, Niu; Shen, Xinliang; Liang, Zhenglun; Li, Wenhui

    2012-02-24

    Enterovirus 71 (EV71) is one of the major pathogens that cause hand, foot, and mouth disease outbreaks in young children in the Asia-Pacific region in recent years. Human scavenger receptor class B 2 (SCARB2) is the main cellular receptor for EV71 on target cells. The requirements of the EV71-SCARB2 interaction have not been fully characterized, and it has not been determined whether SCARB2 serves as an uncoating receptor for EV71. Here we compared the efficiency of the receptor from different species including human, horseshoe bat, mouse, and hamster and demonstrated that the residues between 144 and 151 are critical for SCARB2 binding to viral capsid protein VP1 of EV71 and seven residues from the human receptor could convert murine SCARB2, an otherwise inefficient receptor, to an efficient receptor for EV71 viral infection. We also identified that EV71 binds to SCARB2 via a canyon of VP1 around residue Gln-172. Soluble SCARB2 could convert the EV71 virions from 160 S to 135 S particles, indicating that SCARB2 is an uncoating receptor of the virus. The uncoating efficiency of SCARB2 significantly increased in an acidic environment (pH 5.6). These studies elucidated the viral capsid and receptor determinants of enterovirus 71 infection and revealed a possible target for antiviral interventions.

  5. Stable expression of foot-and-mouth disease virus protein VP1 fused with cholera toxin B subunit in the potato (Solanum tuberosum).

    PubMed

    He, Dong-Mei; Qian, Kai-Xian; Shen, Gui-Fang; Li, Yi-Nü; Zhang, Zhi-Fang; Su, Zhong-Liang; Shao, Hong-Bo

    2007-04-01

    The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity.

  6. [Protective immune response of guinea pigs against challenge with foot and mouth disease virus by immunization with foliar extracts from transgenic tomato plants expressing the FMDV structural protein VP1].

    PubMed

    Pan, Li; Zhang, Yong-Guang; Wang, Yong-Lu; Wang, Bao-Qin; Xie, Qing-Ge

    2006-10-01

    The plant constitutive expression vector pBin438/VP1 for VP1 gene of foot-and-mouth disease virus strain O/ China/99 was constructed. Mediated with Agrobacterium tumefaciens GV3101 harboring pBin438/VP1, VP1 gene was transferred into cotyledons of tomato. After selected by Kanamysin, sixty resistant lines were obtained. The integration and transcription of the VP1 gene in transformed plants was detected by PCR and RT-PCR. After being detected by sandwich-ELISA assays, about 40% transformed plants confirmed to express the recombinant protein. The leave extracts of two positive lines were respectively emulsified in Freund's adjuvant and guinea pigs were intramuscular inoculation at days 0, 15 and 30d. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID50 FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants. Experimental results showed that the plant expression vector was successfully constructed. PCR and RT-PCR analyses confirmed VP1 gene was transformed into tomato plants and got expression at the transcription levels. The expressed VP1 protein of FMDV, which was identified by ELISA and Western blot, can be specifically recognized by polyclonal antibodies against FMDV. Indirect-ELISA antibody titers reached 1:64 twenty-one days after the third inoculation. In the challenge test, the protection against FMDV challenge in two groups was 80% and 40% respectively. The immunization test in guinea pigs indicated that the expression product of transgenic tomato plants had immunogenicity and could effectively induce the specific antibodies against FMDV.

  7. 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.

  8. Mutagenesis of Adeno-Associated Virus Type 2 Capsid Protein VP1 Uncovers New Roles for Basic Amino Acids in Trafficking and Cell-Specific Transduction▿ †

    PubMed Central

    Johnson, Jarrod S.; Li, Chengwen; DiPrimio, Nina; Weinberg, Marc S.; McCown, Thomas J.; Samulski, R. Jude

    2010-01-01

    The N termini of the capsid proteins VP1 and VP2 of adeno-associated virus (AAV) play important roles in subcellular steps of infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (NLSs). To more clearly understand how virion components influence infection, we have generated mutations in these regions and examined their effects on subcellular trafficking, capsid stability, transduction, and sensitivity to pharmacological enhancement. All mutants tested assembled into capsids; retained the correct ratio of VP1, VP2, and VP3; packaged DNA similarly to recombinant AAV2 (rAAV2); and displayed similar stability profiles when heat denatured. Confocal microscopy demonstrated that these mutants trafficked through a perinuclear region in the vicinity of the Golgi apparatus, with a subset of mutants displaying more-diffuse localization consistent with an NLS-deficient phenotype. When tested for viral transduction, two mutant classes emerged. Class I (BR1−, BR2−, and BR2+K) displayed partial transduction, whereas class II (VP3only, 75HD/AN, BR3−, and BR3+K) were severely defective. Surprisingly, one class II mutant (BR3+K) trafficked identically to rAAV2 and accumulated in the nucleolus, a step recently described by our laboratory that occurs with wild-type infection. The BR3+K mutant, containing an alanine-to-lysine substitution in the third basic region of VP1, was 10- to 100-fold-less infectious than rAAV2 in transformed cell lines (such as HEK-293, HeLa, and CV1-T cells), but in contrast, it was indistinguishable from rAAV2 in several nontransformed cell lines, as well as in tissues (liver, brain, and muscle) in vivo. Complementation studies with pharmacological adjuvants or adenovirus coinfection suggested that additional positive charges in NLS regions restrict mobilization in the nucleus and limit transduction in a transformed-cell-specific fashion. Remarkably, besides displaying cell

  9. Mouse neurovirulence determinants of poliovirus type 1 strain LS-a map to the coding regions of capsid protein VP1 and proteinase 2Apro.

    PubMed Central

    Lu, H H; Yang, C F; Murdin, A D; Klein, M H; Harber, J J; Kew, O M; Wimmer, E

    1994-01-01

    Poliovirus type 1 strain LS-a [PV1(LS-a)] is a OV variant adapted to mice by multiple passages through mouse and monkey tissues. To investigate the molecular basis underlying mouse neurovirulence of PV1(LS-a), a cDNA of the viral genome containing nucleotides 112 to 7441 was cloned, and the nucleotide sequence was determined. Compared with that of the mouse avirulent progenitor PV1(Mahoney), 54 nucleotide changes were found in the genome of the PV1(LS-a) virus, resulting in 20 amino acid substitutions in the virus polyprotein. Whereas the nucleotide changes were scattered throughout the genome, the amino acid substitutions were largely clustered in the capsid proteins and, to a certain extent, in the virus proteinase 2Apro. By in vitro mutagenesis, PV1(LS-a)-specific capsid mutations were introduced into a cDNA clone of PV1(Mahoney). We show that neither the individual amino acid mutations nor combinations of mutations in the region encoding VP1 conferred to PV1(Mahoney) the mouse-adapted phenotype of PV1(LS-a). Chimeric cDNA studies demonstrated that a recombinant type 1 virus containing the PV1(LS-a) sequence from nucleotide 2470 to nucleotide 3625 displayed a neurovirulent phenotype in mice. Further dissection of this region revealed that mouse neurovirulence of PV1(LS-a) was determined by multiple mutations in regions encoding both viral proteinase 2Apro and capsid protein VP1. The mouse neurovirulent viruses, PV1(LS-a), W1-M/LS-Pf [nucleotides 496 to 3625 from PV1(LS-a)], and W1-M/LS-NP [nucleotides 2470 to 3625 from PV1(LS-a)], showed increased sensitivity to heat treatment at 45 degrees C for 1 h. Surprisingly, the thermolabile phenotype was also displayed by a recombinant of PV1(Mahoney) carrying a PV1(LS-a) DNA fragment encoding the N-terminal portion of 2Apro. This suggests that base substitutions in the region encoding 2Apro affected capsid stability, thereby contributing to the neurovirulence of the virus in mice. Images PMID:7933134

  10. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    PubMed

    Haolong, Cong; Du, Ning; Hongchao, Tian; Yang, Yang; Wei, Zhang; Hua, Zhang; Wenliang, Zhang; Lei, Song; Po, Tien

    2013-01-01

    Enterovirus 71 (EV71) is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II) which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  11. Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement.

    PubMed

    Huang, Sheng-Wen; Wang, Ya-Fang; Yu, Chun-Keung; Su, Ih-Jen; Wang, Jen-Ren

    2012-01-05

    Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2(149) (VP2(149M)) or glutamine to glutamic acid substitution at VP1(145) (VP1(145E)) showed greater viral titers and apoptosis. Synergistic effect of VP2(149M) and VP1(145E) double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2(149M) and VP1(145E) mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.

  12. 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

  13. Sequence analysis and structural implications of rotavirus capsid proteins.

    PubMed

    Parbhoo, N; Dewar, J B; Gildenhuys, S

    Rotavirus is the major cause of severe virus-associated gastroenteritis worldwide in children aged 5 and younger. Many children lose their lives annually due to this infection and the impact is particularly pronounced in developing countries. The mature rotavirus is a non-enveloped triple-layered nucleocapsid containing 11 double stranded RNA segments. Here a global view on the sequence and structure of the three main capsid proteins, VP2, VP6 and VP7 is shown by generating a consensus sequence for each of these rotavirus proteins, for each species obtained from published data of representative rotavirus genotypes from across the world and across species. Degree of conservation between species was represented on homology models for each of the proteins. VP7 shows the highest level of variation with 14-45 amino acids showing conservation of less than 60%. These changes are localised to the outer surface alluding to a possible mechanism in evading the immune system. The middle layer, VP6 shows lower variability with only 14-32 sites having lower than 70% conservation. The inner structural layer made up of VP2 showed the lowest variability with only 1-16 sites having less than 70% conservation across species. The results correlate with each protein's multiple structural roles in the infection cycle. Thus, although the nucleotide sequences vary due to the error-prone nature of replication and lack of proof reading, the corresponding amino acid sequence of VP2, 6 and 7 remain relatively conserved. Benefits of this knowledge about the conservation include the ability to target proteins at sites that cannot undergo mutational changes without influencing viral fitness; as well as possibility to study systems that are highly evolved for structure and function in order to determine how to generate and manipulate such systems for use in various biotechnological applications.

  14. 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.

  15. 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.

  16. The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.

    PubMed

    Torres-Flores, Jesús M; Silva-Ayala, Daniela; Espinoza, Marco A; López, Susana; Arias, Carlos F

    2015-01-15

    Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.

  17. Changing the surface of a virus shell fusion of an enzyme to polyoma VP1.

    PubMed Central

    Gleiter, S.; Stubenrauch, K.; Lilie, H.

    1999-01-01

    Recent developments on virus-like particles have demonstrated their potential in transfecting eucaryotic cells. In the case of particles based on the major coat protein VP1 of polyoma virus, transfection occurs via binding of VP1 to sialic acids. Since sialic acid is present on almost every eucaryotic cell line, this results in an unspecific cell targeting. Generation of a cell-type specificity of this system would imply the presentation of a new function on the surface of VP1. To analyze whether a new functional protein can be placed on VP1, we inserted dihydrofolate reductase from Escherichia coli as a model protein. The effect of such an insertion on both VP1 and the inserted protein was investigated, respectively. The function of VP1, like the formation of pentameric capsomers and its ability to assemble into capsids, was not influenced by the insertion. The inserted dihydrofolate reductase showed major changes when compared to the wild-type form. The thermal stability of the enzyme was dramatically reduced in the fusion protein; nevertheless, the dihydrofolate reductase proved to be a fully active enzyme with only slightly increased K(M) values for its substrates. This model system provides the basis for further modifications of the VP1 protein to achieve an altered surface of VP1 with new properties. PMID:10631971

  18. Role of the C-Terminal Region of Vervet Monkey Polyomavirus 1 VP1 in Virion Formation

    PubMed Central

    YAMAGUCHI, Hiroki; KOBAYASHI, Shintaro; MARUYAMA, Junki; SASAKI, Michihito; TAKADA, Ayato; KIMURA, Takashi; SAWA, Hirofumi; ORBA, Yasuko

    2014-01-01

    ABSTRACT Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (ΔC VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and ΔC VP1 VLPs were similar in size, but the number of ΔC VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation. PMID:24419975

  19. Recombinant Newcastle Disease virus capsids displaying enterovirus 71 VP1 fragment induce a strong immune response in rabbits.

    PubMed

    Sivasamugham, Lalita Ambigai; Cardosa, Mary Jane; Tan, Wen Siang; Yusoff, Khatijah

    2006-08-01

    The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.

  20. Reassortant rotaviruses as potential live rotavirus vaccine candidates.

    PubMed Central

    Midthun, K; Greenberg, H B; Hoshino, Y; Kapikian, A Z; Wyatt, R G; Chanock, R M

    1985-01-01

    A series of reassortants was isolated from coinfection of cell cultures with a wild-type animal rotavirus and a "noncultivatable" human rotavirus. Wild-type bovine rotavirus (UK strain) was reassorted with human rotavirus strains D, DS-1, and P; wild-type rhesus rotavirus was reassorted with human rotavirus strains D and DS-1. The D, DS-1, and P strains represent human rotavirus serotypes 1, 2, and 3, respectively. Monospecific antiserum (to bovine rotavirus, NCDV strain) or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7 (of the rhesus rotavirus), was used to select for reassortants with human rotavirus neutralization specificity. This selection technique yielded many reassortants which received only the gene segment coding for the major neutralization protein from the human rotavirus parent, whereas the remaining genes were derived from the animal rotavirus parent. Single human rotavirus gene substitution reassortants of this sort represent potential live vaccine strains. Images PMID:2983101

  1. 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.

  2. 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

  3. The cryo-electron microscopy structure of feline calicivirus bound to junctional adhesion molecule A at 9-angstrom resolution reveals receptor-induced flexibility and two distinct conformational changes in the capsid protein VP1.

    PubMed

    Bhella, David; Goodfellow, Ian G

    2011-11-01

    Caliciviridae are small icosahedral positive-sense RNA-containing viruses and include the human noroviruses, a leading cause of infectious acute gastroenteritis and feline calicivirus (FCV), which causes respiratory illness and stomatitis in cats. FCV attachment and entry is mediated by feline junctional adhesion molecule A (fJAM-A), which binds to the outer face of the capsomere, inducing a conformational change in the capsid that may be important for viral uncoating. Here we present the results of our structural investigation of the virus-receptor interaction and ensuing conformational changes. Cryo-electron microscopy and three-dimensional image reconstruction were used to solve the structure of the virus decorated with a soluble fragment of the receptor at subnanometer resolution. In initial reconstructions, the P domains of the capsid protein VP1 and fJAM-A were poorly resolved. Sorting experiments led to improved reconstructions of the FCV-fJAM-A complex both before and after the induced conformational change, as well as in three transition states. These data showed that the P domain becomes flexible following fJAM-A binding, leading to a loss of icosahedral symmetry. Furthermore, two distinct conformational changes were seen; an anticlockwise rotation of up to 15° of the P domain was observed in the AB dimers, while tilting of the P domain away from the icosahedral 2-fold axis was seen in the CC dimers. A list of putative contact residues was calculated by fitting high-resolution coordinates for fJAM-A and VP1 to the reconstructed density maps, highlighting regions in both virus and receptor important for virus attachment and entry.

  4. Rotaviruses: Extraction and Isolation of RNA, Reassortant Strains, and NSP4 Protein.

    PubMed

    Yakshe, Krystle A; Franklin, Zachary D; Ball, Judith M

    2015-05-01

    Rotavirus (RV) contains 11 double-stranded RNA segments that encode for twelve structural and nonstructural proteins. The separation and isolation of viral RNA is a necessary precursor for many experimental techniques and can be useful for rapid RV RNA typing and sequencing of different rotavirus strains. The segmented genome enables RV to recombine easily. These recombinant viruses are essential for many purposes, including generation of potential vaccine strains. Rotavirus gene 10 expresses the viral enterotoxin, NSP4, which has been the focus of several studies due to the influence of NSP4 on rotavirus replication, morphogenesis, and pathogenesis. This unit will describe the isolation and separation of viral RNAs, the production characterization of recombinant RV in culture, and the expression and isolation of NSP4 in mammalian and insect cells.

  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. Prediction of human rotavirus serotype by nucleotide sequence analysis of the VP7 protein gene.

    PubMed Central

    Green, K Y; Sears, J F; Taniguchi, K; Midthun, K; Hoshino, Y; Gorziglia, M; Nishikawa, K; Urasawa, S; Kapikian, A Z; Chanock, R M

    1988-01-01

    Human rotavirus field isolates were characterized by direct sequence analysis of the gene encoding the serotype-specific major neutralization protein (VP7). Single-stranded RNA transcripts were prepared from virus particles obtained directly from stool specimens or after two or three passages in MA-104 cells. Two regions of the gene (nucleotides 307 through 351 and 670 through 711) which had previously been shown to contain regions of sequence divergence among rotavirus serotypes were sequenced by the dideoxynucleotide method with two different synthetic oligonucleotide primers. The resulting nucleotide sequences were compared with the corresponding sequences from rotaviruses of known serotype (serotype 1, 2, 3, or 4). A total of 25 field isolates and 10 laboratory strains examined by this method exhibited marked sequence identity in both areas of the gene with the corresponding regions of 1 of the 4 reference strains. In addition, the predicted serotype from the sequence analysis correlated in each case with the serotype determined when the rotaviruses were examined by plaque reduction neutralization or reactivity with serotype-specific monoclonal antibodies. These data suggest that as a result of the high degree of sequence conservation observed among rotaviruses of the same serotype, it is possible to predict the serotype of a rotavirus isolate by direct sequence analysis of its VP7 gene. PMID:2833626

  7. [Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

    PubMed

    Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

    2016-01-01

    Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

  8. 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.

  9. 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

  10. 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.

  11. Monoclonal antibodies to VP1 recognize a broad range of enteroviruses.

    PubMed

    Miao, Lynn Yihong; Pierce, Christina; Gray-Johnson, Jennifer; DeLotell, Jill; Shaw, Carl; Chapman, Nate; Yeh, Elaine; Schnurr, David; Huang, Yung T

    2009-10-01

    Enteroviruses (EVs) are common seasonal viruses that are associated with a variety of diseases. High-quality monoclonal antibodies (MAbs) are needed to improve the accuracy of EV diagnosis in clinical laboratories. In the present study, the full-length VP1 genes of poliovirus 1 (Polio 1) and coxsackievirus B3 (Cox B3) were cloned, and the encoded proteins were expressed and used as antigens in an attempt to raise broad-spectrum MAbs to EVs. Two pan-EV MAbs were isolated: one raised against Polio 1 VP1 and the other against Cox B3 VP1. The binding sites of both pan-EV MAbs were mapped to an amino acid sequence within a conserved region in the N terminus of Polio 1 VP1 by peptide and competition enzyme-linked immunosorbent assay. Two additional MAbs, an EV70-specific MAb and an EV71/Cox A16-bispecific MAb, developed against EV70 and 71 VP1 proteins, were pooled with the two pan-EV MAbs (pan-EV MAb mix) and tested for their sensitivity and specificity in the staining of various virus-infected cells. The pan-EV MAb mix detected all 40 prototype EVs tested and showed no cross-reactivity to 18 different non-EV human viruses. Compared with two commercially available EV tests, the pan-EV MAb mix exhibited higher specificity than one test and broader spectrum reactivity than the other. Thus, our study demonstrates that full-length Polio 1 VP1 and Cox B3 VP1 can serve as effective antigens for developing a pan-EV MAb and that the pan-EV MAb mix can be used for the laboratory diagnosis of a wide range of EV infections.

  12. Simple and efficient ultrafiltration method for purification of rotavirus VP6 oligomeric proteins.

    PubMed

    Lappalainen, Suvi; Vesikari, Timo; Blazevic, Vesna

    2016-11-01

    Bacterial endotoxins, DNA, live viruses, and viral proteins derived from bacterial and baculovirus (BV) expression vectors employed in recombinant protein production contaminate the final product. Density gradient centrifugation is commonly used to partially purify oligomeric proteins, but impurities from the expression system still remain. We describe a simple and rapid ultrafiltration method for final purification of rotavirus VP6 oligomeric nanotubes and nanospheres. Contamination originating from the BV vector used in VP6 production was undetectable. The method is highly efficient, fast and inexpensive and can be used for a small-scale laboratory purification of VP6 protein to replace technically demanding multi-step chromatographic procedures.

  13. Enterovirus 71 binding to PSGL-1 on leukocytes: VP1-145 acts as a molecular switch to control receptor interaction.

    PubMed

    Nishimura, Yorihiro; Lee, Hyunwook; Hafenstein, Susan; Kataoka, Chikako; Wakita, Takaji; Bergelson, Jeffrey M; Shimizu, Hiroyuki

    2013-01-01

    Some strains of enterovirus 71 (EV71), but not others, infect leukocytes by binding to a specific receptor molecule: the P-selectin glycoprotein ligand-1 (PSGL-1). We find that a single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. Examination of capsid sequences of representative EV71 strains revealed that the PSGL-1-binding viruses had either a G or a Q at residue 145 within the capsid protein VP1 (VP1-145G or Q), whereas PSGL-1-nonbinding viruses had VP1-145E. Using site-directed mutagenesis we found that PSGL-1-binding strains lost their capacity to bind when VP1-145G/Q was replaced by E; conversely, nonbinding strains gained the capacity to bind PSGL-1 when VP1-145E was replaced with either G or Q. Viruses with G/Q at VP1-145 productively infected a leukocyte cell line, Jurkat T-cells, whereas viruses with E at this position did not. We previously reported that EV71 binds to the N-terminal region of PSGL-1, and that binding depends on sulfated tyrosine residues within this region. We speculated that binding depends on interaction between negatively charged sulfate groups and positively charged basic residues in the virus capsid. VP1-145 on the virus surface is in close proximity to conserved lysine residues at VP1-242 and VP1-244. Comparison of recently published crystal structures of EV71 isolates with either Q or E at VP1-145 revealed that VP1-145 controls the orientation of the lysine side-chain of VP1-244: with VP1-145Q the lysine side chain faces outward, but with VP1-145E, the lysine side chain is turned toward the virus surface. Mutation of VP1-244 abolished virus binding to PSGL-1, and mutation of VP1-242 greatly reduced binding. We propose that conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and that VP1-145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1-244K.

  14. Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction

    PubMed Central

    Nishimura, Yorihiro; Lee, Hyunwook; Hafenstein, Susan; Kataoka, Chikako; Wakita, Takaji; Bergelson, Jeffrey M.; Shimizu, Hiroyuki

    2013-01-01

    Some strains of enterovirus 71 (EV71), but not others, infect leukocytes by binding to a specific receptor molecule: the P-selectin glycoprotein ligand-1 (PSGL-1). We find that a single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. Examination of capsid sequences of representative EV71 strains revealed that the PSGL-1-binding viruses had either a G or a Q at residue 145 within the capsid protein VP1 (VP1-145G or Q), whereas PSGL-1-nonbinding viruses had VP1-145E. Using site-directed mutagenesis we found that PSGL-1-binding strains lost their capacity to bind when VP1-145G/Q was replaced by E; conversely, nonbinding strains gained the capacity to bind PSGL-1 when VP1-145E was replaced with either G or Q. Viruses with G/Q at VP1-145 productively infected a leukocyte cell line, Jurkat T-cells, whereas viruses with E at this position did not. We previously reported that EV71 binds to the N-terminal region of PSGL-1, and that binding depends on sulfated tyrosine residues within this region. We speculated that binding depends on interaction between negatively charged sulfate groups and positively charged basic residues in the virus capsid. VP1-145 on the virus surface is in close proximity to conserved lysine residues at VP1-242 and VP1-244. Comparison of recently published crystal structures of EV71 isolates with either Q or E at VP1-145 revealed that VP1-145 controls the orientation of the lysine side-chain of VP1-244: with VP1-145Q the lysine side chain faces outward, but with VP1-145E, the lysine side chain is turned toward the virus surface. Mutation of VP1-244 abolished virus binding to PSGL-1, and mutation of VP1-242 greatly reduced binding. We propose that conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and that VP1-145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1-244K. PMID:23935488

  15. The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

    PubMed Central

    Leisi, Remo; Von Nordheim, Marcus; Ros, Carlos; Kempf, Christoph

    2016-01-01

    Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells. PMID:27690083

  16. Sequence adaptations affecting cleavage of the VP1/2A junction by the 3C protease in foot-and-mouth disease virus-infected cells.

    PubMed

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham J

    2014-11-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited cleavage of this junction and produced 'self-tagged' virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus [Gullberg et al. (2013). J Virol 87: , 11591-11603]. It was shown here that introduction of this E83K change alone into a serotype O virus resulted in the rapid accumulation of a second site substitution within the 2A sequence (L2P), which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained the uncleaved VP1-2A protein. The 2A L2P substitution resulted in the VP1/2A junction being highly resistant to cleavage by the 3C protease, hence it may be a preferred route for 'tagging' virus particles.

  17. Tsg101 Interacts with Herpes Simplex Virus 1 VP1/2 and Is a Substrate of VP1/2 Ubiquitin-Specific Protease Domain Activity

    PubMed Central

    Caduco, Martina; Comin, Alessandra; Toffoletto, Marta; Munegato, Denis; Sartori, Elena; Celestino, Michele; Salata, Cristiano; Parolin, Cristina

    2013-01-01

    Ubiquitination/deubiquitination of key factors represent crucial steps in the biogenesis of multivesicular body (MVB) and sorting of transmembrane proteins. We and others previously demonstrated that MVB is involved in herpes simplex virus 1 (HSV-1) envelopment and budding. Here, we report that the HSV-1 large tegument protein, VP1/2, interacts with and regulates the ubiquitination of Tsg101, a cellular protein essential in MVB formation, thus identifying the first cellular substrate of a herpesviral deubiquitinating enzyme. PMID:23077308

  18. Oral immunization of mice with Lactococcus lactis expressing the rotavirus VP8* protein.

    PubMed

    Rodríguez-Díaz, Jesús; Montava, Rebeca; Viana, Rosa; Buesa, Javier; Pérez-Martínez, Gaspar; Monedero, Vicente

    2011-06-01

    The efficacy of recombinant Lactococcus lactis as a delivery vehicle for a rotavirus antigen was evaluated in a mouse model. The rotavirus VP8* protein was expressed intracellularly and extracellularly in L. lactis wild type and in an alr mutant deficient in alanine racemase activity, necessary for the synthesis of the cell-wall component D: -alanine. When the mucosal immune response was evaluated by measuring VP8*-specific IgA antibody in faeces, wild-type L. lactis triggered a low IgA synthesis only when the secreting strain was used. In contrast, VP8*-specific IgA was detected in faeces of both groups of mice orally given the alr mutant expressing extracellular VP8* and intracellular VP8*, which reached levels similar to that obtained with the wild type secreting strain. However, oral administration of the recombinant strains did not induce serum IgG or IgA responses. L. lactis cell-wall mutants may therefore provide certain advantages when low-antigenic proteins are expressed intracellularly. However, the low immune response obtained by using this antigen-bacterial host combination prompts to the use of new strains and vaccination protocols in order to develop acceptable rotavirus immunization levels.

  19. Supplemental dietary whey protein concentrate reduces rotavirus-induced disease symptoms in suckling mice.

    PubMed

    Wolber, Frances M; Broomfield, Anne M; Fray, Linley; Cross, Martin L; Dey, Debjit

    2005-06-01

    Rotavirus-induced diarrhea is a common infection that results in the death of nearly 500,000 children annually. Currently, no large-scale preventative treatments or vaccines exist. Because some whey protein concentrates (WPC) were shown to contain bioactive ingredients that may activate immune cells and/or prevent infection, the current study was conducted to assess whether the proprietary WPC IMUCARE (WPC-IC) could protect against rotavirus. Suckling BALB/c mice were treated by gavage once daily with WPC-IC or with the control protein bovine serum albumin from the age of 9 to 17 d, and were infected with murine rotavirus at the age of 11 d. Disease symptoms were graded as mild, moderate, or severe, and viral shedding was measured in fecal samples during the postinfection period. Severe diarrhea occurred in 63% of control mice; this was significantly reduced to 36% in WPC-IC-fed mice. Severe diarrhea occurred for a 4-d period in the control group but only for a 2-d period in the WPC-IC group. Although the mean viral load per mouse did not differ between the groups, the proportion of mice shedding high levels of the virus in the feces postinfection was significantly lower in the WPC-IC group on d 13, 16, and 17, and significantly higher on d 14. Rotavirus-specific antibody levels in serum and gut fluid did not differ between groups. Thus, prophylactic treatment with WPC-IC may reduce rotaviral disease by decreasing the prevalence of severe diarrhea and by decreasing the time period during which severe symptoms and high viral shedding occur.

  20. Rotavirus Symptoms

    MedlinePlus

    ... Search The CDC Cancel Submit Search The CDC Rotavirus Note: Javascript is disabled or is not supported ... message, please visit this page: About CDC.gov . Rotavirus Home About Rotavirus Symptoms Transmission Prevention Treatment Photos ...

  1. Rotavirus Treatment

    MedlinePlus

    ... Search The CDC Cancel Submit Search The CDC Rotavirus Note: Javascript is disabled or is not supported ... message, please visit this page: About CDC.gov . Rotavirus Home About Rotavirus Symptoms Transmission Prevention Treatment Photos ...

  2. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

    PubMed

    Zhao, Bingxin; Pan, Xiaoxia; Teng, Yumei; Xia, Wenyue; Wang, Jing; Wen, Yuling; Chen, Yuanding

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  3. 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.

  4. Mutation distribution in the NSP4 protein in rotaviruses isolated from Mexican children with moderate to severe gastroenteritis.

    PubMed

    González-Ochoa, Guadalupe; Menchaca, Griselda E; Hernández, Carlos E; Rodríguez, Cristina; Tamez, Reyes S; Contreras, Juan F

    2013-03-11

    The NSP4 protein is a multifunctional protein that plays a role in the morphogenesis and pathogenesis of the rotavirus. Although NSP4 is considered an enterotoxin, the relationship between gastroenteritis severity and amino acid variations in NSP4 of the human rotavirus remains unclear. In this study, we analyzed the sequence diversity of NSP4 and the severity of gastroenteritis of children with moderate to severe gastroenteritis. The rotavirus-infected children were hospitalized before the rotavirus vaccine program in Mexico. All children had diarrhea within 1-4 days, 44 (88%) were vomiting and 35 (70%) had fevers. The severity analysis showed that 13 (26%) cases had mild gastroenteritis, 23 (46%) moderate gastroenteritis and 14 (28%) severe. NSP4 phylogenetic analysis showed three clusters within the genotype E1. Sequence analysis revealed similar mutations inside each cluster, and an uncommon variation in residue 144 was found in five of the Mexican NSP4 sequences. Most of the amino acid variations were located in the VP4 and VP6 binding site domains, with no relationship to different grades of gastroenteritis. This finding indicates that severe gastroenteritis caused by the rotavirus appears to be related to diverse viral or cellular factors instead of NSP4 activity as a unique pathogenic factor.

  5. Regulation of Norovirus Virulence by the VP1 Protruding Domain Correlates with B Cell Infection Efficiency

    PubMed Central

    Zhu, Shu; Watanabe, Makiko; Kirkpatrick, Ericka; Murray, Akilah B.; Sok, Ryneth

    2015-01-01

    ABSTRACT Human noroviruses are a leading cause of gastroenteritis across the globe, but the pathogenic mechanisms responsible for disease are not well established. The availability of a murine norovirus model system provides the opportunity to elucidate viral and host determinants of virulence in a natural host. For example, previous studies have revealed that the protruding domain of the murine norovirus capsid protein VP1, specifically residue 296 of VP1, regulates virulent infection. We identified a panel of nonsynonymous mutations in the open reading frame 2 (ORF2) gene encoding VP1 that arose in persistently infected mice and tested whether these mutations conferred phenotypic changes to viral replication and virulence. Consistent with previous studies, we demonstrate that a glutamic acid at position 296 results in attenuation. For the first time, we also demonstrate that a lysine at this position is sufficient to confer virulence on an otherwise attenuated murine norovirus strain. Moreover, our studies reveal a direct correlation between the efficiency of viral replication in B cells and virulence. These data are especially striking because mutations causing reduced B cell replication and attenuation had minimal effects on the ability of the virus to replicate in macrophages. Thus, norovirus infection of B cells may directly contribute to disease outcome. IMPORTANCE Human noroviruses are a major global cause of disease, yet we know very little about their pathogenic mechanisms. The availability of a murine norovirus model system facilitates investigation of noroviruses in a natural host organism and the identification of viral and host determinants of pathogenesis. We have identified a panel of mutations arising in the viral capsid protein VP1 during persistent infection of mice. Our data reveal that the protruding domain of VP1 regulates the ability of the virus to replicate in B cells, and this directly correlates with virulence. Importantly, mutations

  6. Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic

    PubMed Central

    Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Hoshino, Yasutaka; Yuan, Lijuan

    2015-01-01

    The two currently available live oral rotavirus vaccines, Rotarix® and RotaTeq®, are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance. PMID:26091081

  7. 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.

  8. 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.

  9. Production of rotavirus-like particles in tomato (Lycopersicon esculentum L.) fruit by expression of capsid proteins VP2 and VP6 and immunological studies.

    PubMed

    Saldaña, Sergio; Esquivel Guadarrama, Fernando; Olivera Flores, Teresa De Jesús; Arias, Nancy; López, Susana; Arias, Carlos; Ruiz-Medrano, Roberto; Mason, Hugh; Mor, Tsafrir; Richter, Liz; Arntzen, Charles J; Gómez Lim, Miguel A

    2006-01-01

    A number of different antigens have been successfully expressed in transgenic plants, and some are currently being evaluated as orally delivered vaccines. Here we report the successful expression of rotavirus capsid proteins VP2 and VP6 in fruits of transgenic tomato plants. By western blot analysis, using specific antibodies, we determined that the VP2 and VP6 produced in plants have molecular weights similar to those found in native rotavirus. The plant-synthesized VP6 protein retained the capacity to form trimers. We were able to recover rotavirus virus-like particles from tomato fruit (i.e., tomatoes) by centrifugation on a sucrose cushion and to visualize them by electron microscopy. This result indicated that VP2/VP6 can self-assemble into virus-like particles (VLPs) in plant cells, even though only a small proportion of VP2/VP6 assembled into VLPs. To investigate immunogenicity, adult mice were immunized intraperitoneally (i.p.) three times with a protein extract from a transgenic tomatoes in adjuvant. We found that the transgenic tomato extract induced detectable levels of anti-rotavirus antibodies in serum; however, we did not determine the contribution of either the free rotavirus proteins or the VLPs to the induction of the antibody response. These results suggest the potential of plant-based rotavirus VLPs for the development of a vaccine against rotavirus infection.

  10. Functional Mapping of Protective Domains and Epitopes in the Rotavirus VP6 Protein

    PubMed Central

    Choi, Anthony H. C.; Basu, Mitali; McNeal, Monica M.; Flint, Jason; VanCott, John L.; Clements, John D.; Ward, Richard L.

    2000-01-01

    The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-μg doses of each of the chimeras and 10 μg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P ≤ 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P ≤ 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-μg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P ≤ 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4+ T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4+ T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4+ T

  11. 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 ...

  12. Antibody-Independent Protection against Rotavirus Infection of Mice Stimulated by Intranasal Immunization with Chimeric VP4 or VP6 Protein

    PubMed Central

    Choi, Anthony H.-C.; Basu, Mitali; McNeal, Monica M.; Clements, John D.; Ward, Richard L.

    1999-01-01

    This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-μg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-μg doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient μMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody. PMID:10438847

  13. Rotavirus (For Parents)

    MedlinePlus

    ... Old Feeding Your 1- to 2-Year-Old Rotavirus KidsHealth > For Parents > Rotavirus Print A A A ... Call the Doctor en español El rotavirus About Rotavirus Almost all kids have had a rotavirus infection ...

  14. Rotavirus vaccines

    PubMed Central

    Yen, Catherine; Tate, Jacqueline E; Hyde, Terri B; Cortese, Margaret M; Lopman, Benjamin A; Jiang, Baoming; Glass, Roger I; Parashar, Umesh D

    2016-01-01

    Rotavirus is the leading cause of severe diarrhea among children <5 years worldwide. Currently licensed rotavirus vaccines have been efficacious and effective, with many countries reporting substantial declines in diarrheal and rotavirus-specific morbidity and mortality. However, the full public health impact of these vaccines has not been realized. Most countries, including those with the highest disease burden, have not yet introduced rotavirus vaccines into their national immunization programs. Research activities that may help inform vaccine introduction decisions include (1) establishing effectiveness, impact, and safety for rotavirus vaccines in low-income settings; (2) identifying potential strategies to improve performance of oral rotavirus vaccines in developing countries, such as zinc supplementation; and (3) pursuing alternate approaches to oral vaccines, such as parenteral immunization. Policy- and program-level barriers, such as financial implications of new vaccine introductions, should be addressed to ensure that countries are able to make informed decisions regarding rotavirus vaccine introduction. PMID:24755452

  15. Missense mutations in the VP1 gene of simian virus 40 that compensate for defects caused by deletions in the viral agnogene.

    PubMed Central

    Barkan, A; Welch, R C; Mertz, J E

    1987-01-01

    Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smaller plaques than does wild-type virus. Within three passages at low multiplicities of infection, virus stocks of several such mutants accumulated variants that synthesized an altered form of the major virion protein, VP1, having a slightly faster mobility in sodium dodecyl sulfate-polyacrylamide gels than did the wild-type protein. Because these variants overgrew the original virus stocks, we consider them to be second-site revertants. By construction and characterization of a series of recombinants, the second-site mutations were shown to map to at least two different regions of the VP1 gene. Nucleotide sequence analysis indicated that single-amino-acid changes were responsible for the rapid mobility of VP1. When combined in cis with either a wild-type or mutant leader region, these VP1 mutations sped up by 10 to 20 h the time course of accumulation of infectious progeny but not of viral DNA or VP1. LP1, the protein encoded by the agnogene, was shown previously to be necessary for the efficient transport of the virion proteins to the nucleus or for their efficient assembly with viral minichromosomes. The VP1 missense mutations reported here compensate for the lack of LP1 by facilitating this process. On the basis of these findings and findings reported previously by us and others, we hypothesize that LP1 facilitates the formation of infectious particles by inhibiting the polymerization of VP1 molecules until the time they interact with viral minichromosomes; the VP1 mutations reported here compensate for the loss of LP1 by lessening the potential of VP1 for self-polymerization. Images PMID:3041040

  16. Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion

    PubMed Central

    Mathieu, Magali; Petitpas, Isabelle; Navaza, Jorge; Lepault, Jean; Kohli, Evelyne; Pothier, Pierre; Prasad, B.V.Venkataram; Cohen, Jean; Rey, Félix A.

    2001-01-01

    The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 Å resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal β-barrel domain and a proximal α-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn2+ ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer. PMID:11285213

  17. 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

  18. 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.

  19. Fine mapping of sequential neutralization epitopes on the subunit protein VP8 of human rotavirus.

    PubMed Central

    Kovacs-Nolan, Jennifer; Yoo, Dongwan; Mine, Yoshinori

    2003-01-01

    The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes. PMID:12901721

  20. Heterogeneity of Raft-Type Membrane Microdomains Associated with VP4, the Rotavirus Spike Protein, in Caco-2 and MA 104 Cells▿

    PubMed Central

    Delmas, Olivier; Breton, Michelyne; Sapin, Catherine; Le Bivic, André; Colard, Odile; Trugnan, Germain

    2007-01-01

    Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-β-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release. PMID:17135322

  1. Molecular evolution of the VP1, VP2, and VP3 genes in human rhinovirus species C.

    PubMed

    Kuroda, Makoto; Niwa, Shoichi; Sekizuka, Tsuyoshi; Tsukagoshi, Hiroyuki; Yokoyama, Masaru; Ryo, Akihide; Sato, Hironori; Kiyota, Naoko; Noda, Masahiro; Kozawa, Kunihisa; Shirabe, Komei; Kusaka, Takashi; Shimojo, Naoki; Hasegawa, Shunji; Sugai, Kazuko; Obuchi, Masatsugu; Tashiro, Masato; Oishi, Kazunori; Ishii, Haruyuki; Kimura, Hirokazu

    2015-02-02

    Human rhinovirus species C (HRV-C) was recently discovered, and this virus has been associated with various acute respiratory illnesses (ARI). However, the molecular evolution of the major antigens of this virus, including VP1, VP2, and VP3, is unknown. Thus, we performed complete VP1, VP2, and VP3 gene analyses of 139 clinical HRV-C strains using RT-PCR with newly designed primer sets and next-generation sequencing. We assessed the time-scale evolution and evolutionary rate of these genes using the Bayesian Markov chain Monte Carlo method. In addition, we calculated the pairwise distance and confirmed the positive/negative selection sites in these genes. The phylogenetic trees showed that the HRV-C strains analyzed using these genes could be dated back approximately 400 to 900 years, and these strains exhibited high evolutionary rates (1.35 to 3.74 × 10(-3) substitutions/site/year). Many genotypes (>40) were confirmed in the phylogenetic trees. Furthermore, no positively selected site was found in the VP1, VP2, and VP3 protein. Molecular modeling analysis combined with variation analysis suggested that the exterior surfaces of the VP1, VP2 and VP3 proteins are rich in loops and are highly variable. These results suggested that HRV-C may have an old history and unique antigenicity as an agent of various ARI.

  2. A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity.

    PubMed

    McKell, Allison O; LaConte, Leslie E W; McDonald, Sarah M

    2017-04-01

    Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP

  3. 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.

  4. 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

  5. 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

    2016-03-31

    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.

  6. Detection of polyomavirus major capsid antigen (VP-1) in human pilomatricomas.

    PubMed

    Sanjuán, Norberto A; Símula, Silvina; Casas, José; Woscoff, Alberto

    2010-01-01

    The family Polyomaviridae is composed of small, non-enveloped, double-stranded DNA viruses widely used to study cell transformation in vitro and tumor induction in vivo. The development of pilomatricomas in mice experimentally infected with polyomavirus led us to detect the viral major capsid protein VP-1 in human pilomatricomas. This tumor, even uncommon, is one of the most frequent benign hair follicle tumors in humans and is composed of proliferating matrix cells that undergo keratinization, and form cystic neoplasms. The detection of VP-1 was performed using the peroxidase-antiperoxidase technique in paraffin-embedded slides with a specific primary serum. Adjacent slides treated with normal rabbit serum as a primary were employed as internal control. Positive and negative controls were also employed as well as slides of lesions caused by human papillomavirus to rule out any unspecific cross-reactivity. In 4 out of 10 cases polyomavirus VP-1 was clearly detected in nuclei of human pilomatricomas proliferating cells, in a patchy pattern of distribution. The controls confirmed the specificity of the immunocytochemical procedure. These results could indicate either an eventual infection of the virus in already developed tumors or alternatively, a direct involvement of polyomavirus in the pathogenesis of some pilomatricomas. The recent discovery of a new human polyomavirus associated with Merkel cell carcinomas has been a strong contribution to better understand the pathogenesis of some human uncommon skin cancers. Hopefully the results reported in this work will encourage further research on the role of polyomavirus in other human skin neoplasms.

  7. A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19

    PubMed Central

    Šimoliūnas, Egidijus; Rinkūnaitė, Ieva; Smalinskaitė, Luka; Podkopajev, Andrej; Bironaitė, Daiva; Weis, Cleo-Aron; Marx, Alexander; Bukelskienė, Virginija; Gretz, Norbert; Grabauskienė, Virginija; Labeit, Dittmar; Labeit, Siegfried

    2016-01-01

    Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage. PMID:27812527

  8. The effect of bovine rotavirus and its nonstructural protein 4 on ER stress-mediated apoptosis in HeLa and HT-29 cells.

    PubMed

    Goodarzi, Zahra; Soleimanjahi, Hoorieh; Arefian, Ehsan; Saberfar, Esmaeil

    2016-03-01

    Endoplasmic reticulum (ER) plays important roles in multiple cellular processes as well as cell survival and apoptosis. Perturbation of ER functions leads to ER stress and unfolded protein response (UPR). The primary goal of this response is cell survival, but severe ER stress can trigger apoptosis signaling. In tumor cells, chronically activated UPR response provides tumor growth. So, apoptosis induced by the ER stress has been the target for anti-cancer therapy. In this in vitro study, we examined the apoptotic effect associated with ER stress of bovine rotavirus and its nonstructural protein 4 (NSP4) alone in two cancer cell lines. The plasmid pcDNA3.1 encoding NSP4 protein of bovine rotavirus transfected with lipofectamine 2000 into the HeLa and HT-29 cells for protein production. MTT, flow cytometry, and Western blot were used to evaluate the cell viability, apoptosis, and expression level of C/EBP-homologous protein (CHOP) and activated caspase-4. In parallel, the apoptotic effect of the bovine rotavirus associated with ER stress in the infected cells was examined too. The cytotoxic and apoptotic effect of NSP4 protein on the cells were statistically significant compared to the control groups. However, Western blot showed that the expression of the NSP4 protein by recombinant plasmid did not lead to high expression of CHOP and activation of caspase-4. Interestingly, rotavirus not only induced significant apoptosis but also caused an increase in CHOP expression and caspase-4 activation in the infected cells compared to control. As a result, NSP4 protein and bovine rotavirus can be considered a potential novel bio-therapeutic strategy for cancer treatment.

  9. Protein Malnutrition Modifies Innate Immunity and Gene Expression by Intestinal Epithelial Cells and Human Rotavirus Infection in Neonatal Gnotobiotic Pigs

    PubMed Central

    Paim, Francine C.; Kandasamy, Sukumar; Alhamo, Moyasar A.; Fischer, David D.; Langel, Stephanie N.; Deblais, Loic; Kumar, Anand; Chepngeno, Juliet; Shao, Lulu; Huang, Huang-Chi; Candelero-Rueda, Rosario A.; Rajashekara, Gireesh

    2017-01-01

    ABSTRACT Malnutrition affects millions of children in developing countries, compromising immunity and contributing to increased rates of death from infectious diseases. Rotavirus is a major etiological agent of childhood diarrhea in developing countries, where malnutrition is prevalent. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. In this study, we used neonatal gnotobiotic (Gn) pigs transplanted with the fecal microbiota of a healthy 2-month-old infant (HIFM) and fed protein-deficient or -sufficient bovine milk diets. Protein deficiency induced hypoproteinemia, hypoalbuminemia, hypoglycemia, stunting, and generalized edema in Gn pigs, as observed in protein-malnourished children. Irrespective of the diet, human rotavirus (HRV) infection early, at HIFM posttransplantation day 3 (PTD3), resulted in adverse health effects and higher mortality rates (45 to 75%) than later HRV infection (PTD10). Protein malnutrition exacerbated HRV infection and affected the morphology and function of the small intestinal epithelial barrier. In pigs infected with HRV at PTD10, there was a uniform decrease in the function and/or frequencies of natural killer cells, plasmacytoid dendritic cells, and CD103+ and apoptotic mononuclear cells and altered gene expression profiles of intestinal epithelial cells (chromogranin A, mucin 2, proliferating cell nuclear antigen, SRY-Box 9, and villin). Thus, we have established the first HIFM-transplanted neonatal pig model that recapitulates major aspects of protein malnutrition in children and can be used to evaluate physiologically relevant interventions. Our findings provide an explanation of why nutrient-rich diets alone may lack efficacy in malnourished children. IMPORTANCE Malnutrition and rotavirus infection, prevalent in developing countries, individually and in combination, affect the health of millions of children, compromising their immunity and increasing

  10. Protein Malnutrition Modifies Innate Immunity and Gene Expression by Intestinal Epithelial Cells and Human Rotavirus Infection in Neonatal Gnotobiotic Pigs.

    PubMed

    Vlasova, Anastasia N; Paim, Francine C; Kandasamy, Sukumar; Alhamo, Moyasar A; Fischer, David D; Langel, Stephanie N; Deblais, Loic; Kumar, Anand; Chepngeno, Juliet; Shao, Lulu; Huang, Huang-Chi; Candelero-Rueda, Rosario A; Rajashekara, Gireesh; Saif, Linda J

    2017-01-01

    Malnutrition affects millions of children in developing countries, compromising immunity and contributing to increased rates of death from infectious diseases. Rotavirus is a major etiological agent of childhood diarrhea in developing countries, where malnutrition is prevalent. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. In this study, we used neonatal gnotobiotic (Gn) pigs transplanted with the fecal microbiota of a healthy 2-month-old infant (HIFM) and fed protein-deficient or -sufficient bovine milk diets. Protein deficiency induced hypoproteinemia, hypoalbuminemia, hypoglycemia, stunting, and generalized edema in Gn pigs, as observed in protein-malnourished children. Irrespective of the diet, human rotavirus (HRV) infection early, at HIFM posttransplantation day 3 (PTD3), resulted in adverse health effects and higher mortality rates (45 to 75%) than later HRV infection (PTD10). Protein malnutrition exacerbated HRV infection and affected the morphology and function of the small intestinal epithelial barrier. In pigs infected with HRV at PTD10, there was a uniform decrease in the function and/or frequencies of natural killer cells, plasmacytoid dendritic cells, and CD103(+) and apoptotic mononuclear cells and altered gene expression profiles of intestinal epithelial cells (chromogranin A, mucin 2, proliferating cell nuclear antigen, SRY-Box 9, and villin). Thus, we have established the first HIFM-transplanted neonatal pig model that recapitulates major aspects of protein malnutrition in children and can be used to evaluate physiologically relevant interventions. Our findings provide an explanation of why nutrient-rich diets alone may lack efficacy in malnourished children. IMPORTANCE Malnutrition and rotavirus infection, prevalent in developing countries, individually and in combination, affect the health of millions of children, compromising their immunity and increasing the rates

  11. 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

  12. 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

  13. 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...

  14. Rotavirus Vaccine

    MedlinePlus

    ... including a severe allergy to latex. Babies with "severe combined immunodeficiency" (SCID) should not get rotavirus vaccine. Babies who have had a type of bowel blockage called "intussusception" should not get ... with moderate or severe diarrhea or vomiting. Check with your doctor if ...

  15. Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2.

    PubMed

    Berkova, Z; Morris, A P; Estes, M K

    2003-07-01

    The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.

  16. 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

    2014-05-09

    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.

  17. 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.

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

    PubMed

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

    2015-07-01

    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

  19. Structures of B-lymphotropic polyomavirus VP1 in complex with oligosaccharide ligands.

    PubMed

    Neu, Ursula; Khan, Zaigham Mahmood; Schuch, Benjamin; Palma, Angelina S; Liu, Yan; Pawlita, Michael; Feizi, Ten; Stehle, Thilo

    2013-10-01

    B-Lymphotropic Polyomavirus (LPyV) serves as a paradigm of virus receptor binding and tropism, and is the closest relative of the recently discovered Human Polyomavirus 9 (HPyV9). LPyV infection depends on sialic acid on host cells, but the molecular interactions underlying LPyV-receptor binding were unknown. We find by glycan array screening that LPyV specifically recognizes a linear carbohydrate motif that contains α2,3-linked sialic acid. High-resolution crystal structures of the LPyV capsid protein VP1 alone and in complex with the trisaccharide ligands 3'-sialyllactose and 3'-sialyl-N-acetyl-lactosamine (3SL and 3SLN, respectively) show essentially identical interactions. Most contacts are contributed by the sialic acid moiety, which is almost entirely buried in a narrow, preformed cleft at the outer surface of the capsid. The recessed nature of the binding site on VP1 and the nature of the observed glycan interactions differ from those of related polyomaviruses and most other sialic acid-binding viruses, which bind sialic acid in shallow, more exposed grooves. Despite their different modes for recognition, the sialic acid binding sites of LPyV and SV40 are half-conserved, hinting at an evolutionary strategy for diversification of binding sites. Our analysis provides a structural basis for the observed specificity of LPyV for linear glycan motifs terminating in α2,3-linked sialic acid, and links the different tropisms of known LPyV strains to the receptor binding site. It also serves as a useful template for understanding the ligand-binding properties and serological crossreactivity of HPyV9.

  20. Modeling rotavirus-like particles production in a baculovirus expression vector system: Infection kinetics, baculovirus DNA replication, mRNA synthesis and protein production.

    PubMed

    Roldão, António; Vieira, Helena L A; Charpilienne, Annie; Poncet, Didier; Roy, Polly; Carrondo, Manuel J T; Alves, Paula M; Oliveira, R

    2007-03-10

    Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a million deaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection, since they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those based on attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodoptera frugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteins (VPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronic recombinant baculovirus each one coding for viral proteins VP(2), VP(6) and VP(7), or single-infection strategies using a multigene baculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19+/-0.01 h(-1) was obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to be negligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends to decrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates could be ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for the three proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to evaluating different baculovirus constructs for Rota VLPs production.

  1. Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses: characterization of "self-tagged" particles.

    PubMed

    Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J

    2013-11-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

  2. Diversity in VP3, NSP3, and NSP4 of rotavirus B detected from Japanese cattle.

    PubMed

    Hayashi-Miyamoto, Michiko; Murakami, Toshiaki; Minami-Fukuda, Fujiko; Tsuchiaka, Shinobu; Kishimoto, Mai; Sano, Kaori; Naoi, Yuki; Asano, Keigo; Ichimaru, Toru; Haga, Kei; Omatsu, Tsutomu; Katayama, Yukie; Oba, Mami; Aoki, Hiroshi; Shirai, Junsuke; Ishida, Motohiko; Katayama, Kazuhiko; Mizutani, Tetsuya; Nagai, Makoto

    2017-04-01

    Bovine rotavirus B (RVB) is an etiological agent of diarrhea mostly in adult cattle. Currently, a few sequences of viral protein (VP)1, 2, 4, 6, and 7 and nonstructural protein (NSP)1, 2, and 5 of bovine RVB are available in the DDBJ/EMBL/GenBank databases, and none have been reported for VP3, NSP3, and NSP4. In order to fill this gap in the genetic characterization of bovine RVB strains, we used a metagenomics approach and sequenced and analyzed the complete coding sequences (CDS) of VP3, NSP3, and NSP4 genes, as well as the partial or complete CDS of other genes of RVBs detected from Japanese cattle. VP3, NSP3, and NSP4 of bovine RVBs shared low nucleotide sequence identities (63.3-64.9% for VP3, 65.9-68.2% for NSP3, and 52.6-56.2% for NSP4) with those of murine, human, and porcine RVBs, suggesting that bovine RVBs belong to a novel genotype. Furthermore, significantly low amino acid sequence identities were observed for NSP4 (36.1-39.3%) between bovine RVBs and the RVBs of other species. In contrast, hydrophobic plot analysis of NSP4 revealed profiles similar to those of RVBs of other species and rotavirus A (RVA) strains. Phylogenetic analyses of all gene segments revealed that bovine RVB strains formed a cluster that branched distantly from other RVBs. These results suggest that bovine RVBs have evolved independently from other RVBs but in a similar manner to other rotaviruses. These findings provide insights into the evolution and diversity of RVB strains.

  3. 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.

  4. X-ray crystal structure of the rotavirus inner capsid particle at 3.8 Å resolution

    PubMed Central

    McClain, Brian; Settembre, Ethan; Temple, Brenda R.S.; Bellamy, A. Richard; Harrison, Stephen C.

    2010-01-01

    The rotavirus inner capsid particle, known as the “double-layered particle” (DLP), is the “payload” delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of VP2 and VP6, respectively, package the eleven segments of double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter ~700 Å) in the asymmetric unit of the P212121 unit cell, of dimensions a= 740 Å, b= 1198 Å, c= 1345 Å. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular-replacement model for initial phase determination to about 18.5 Å resolution and the sixty-fold redundancy of the icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 Å). The structure of a VP6 trimer (determined previously by others) fits the outer-layer density with very little adjustment. The T=13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus two per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin, principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two “subdomain” boundaries) allow VP2A and B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the fivefold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: λ1 in orthoreoreoviruses, VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a “fivefold hub” that projects into the DLP interior along the fivefold axis; residues 81-100 link the

  5. RoXaN, a Novel Cellular Protein Containing TPR, LD, and Zinc Finger Motifs, Forms a Ternary Complex with Eukaryotic Initiation Factor 4G and Rotavirus NSP3

    PubMed Central

    Vitour, Damien; Lindenbaum, Pierre; Vende, Patrice; Becker, Michelle M.; Poncet, Didier

    2004-01-01

    Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3′ consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation. PMID:15047801

  6. Combined 5' UTR RFLP analysis and VP1 sequencing for epidemic investigation of enteroviruses.

    PubMed

    Kyriakopoulou, Zaharoula; Tsolis, Kostas; Pliaka, Vaia; Tsakogiannis, Dimitris; Ruether, Irina Georgia Anna; Gartzonika, Constantina; Levidiotou-Stefanou, Stamatina; Markoulatos, Panayotis

    2013-01-01

    Enteroviruses, the main cause of aseptic meningitis, consist of 100 serotypes, and many of them have been associated with large outbreaks. In the present study, a comparison of RFLP analysis of the 5' untranslated region (5'UTR) and sequencing of both the 5'UTR and VP1 regions was conducted for epidemiological linkage of 27 clinical enterovirus strains. The clinical enterovirus strains were clustered into five restriction profile groups. Even though the restriction profile clusters of clinical isolates were not related to those of the respective prototype strains, epidemiological relationships between the members of each cluster were observed. The restriction profile clusters in the 5'UTR corresponded to the phylogenetic clusters in the VP1 genomic region. The incongruence between the topology of Gior strain in 5'UTR and VP1 phylogenetic trees indicates a recombination event. The proposed RFLP assay in combination with VP1 sequencing can offer crucial epidemiological information about the circulating enteroviruses.

  7. Partial protection against enterovirus 71 (EV71) infection in a mouse model immunized with recombinant Newcastle disease virus capsids displaying the EV71 VP1 fragment.

    PubMed

    Ch'ng, Wei-Choong; Stanbridge, Eric J; Ong, Kien-Chai; Wong, Kum-Thong; Yusoff, Khatijah; Shafee, Norazizah

    2011-10-01

    Enterovirus 71 (EV71) infection may cause severe neurological complications, particularly in young children. Despite the risks, there are still no commercially available EV71 vaccines. Hence, a candidate vaccine construct, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP1(1-100) ) protein, was evaluated in a mouse model of EV71 infection. Previously, it was shown that this protein construct provoked a strong immune response in vaccinated adult rabbits. That study, however, did not address the issue of its effectiveness against EV71 infection in young animals. In the present study, EV71 viral challenge in vaccinated newborn mice resulted in more than 40% increase in survival rate. Significantly, half of the surviving mice fully recovered from their paralysis. Histological analysis of all of the surviving mice revealed a complete clearance of EV71 viral antigens from their brains and spinal cords. In hind limb muscles, the amounts of the antigens detected correlated with the degrees of tissue damage and paralysis. Findings from this study provide evidence that immunization with the NPt-VP1(1-100) immunogen in a newborn mouse model confers partial protection against EV71 infection, and also highlights the importance of NPt-VP1(1-100) as a possible candidate vaccine for protection against EV71 infections.

  8. Spike Protein VP8* of Human Rotavirus Recognizes Histo-Blood Group Antigens in a Type-Specific Manner

    PubMed Central

    Huang, Pengwei; Xia, Ming; Zhong, Weiming; Wei, Chao; Wang, Leyi; Morrow, Ardythe

    2012-01-01

    Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Leb) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Leb and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells. PMID:22345472

  9. 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

  10. [Sequence analysis of VP1 gene of the duck hepatitis A virus type 3 strains isolated from Shandong Province of China in 2012].

    PubMed

    Xu, Qian; Chen, Lin-lin; Zhang, Rui-hua; Yang, Lei; Xie, Zhi-jing; Zhu, Yan-li; Jiang, Shi-jin; Si, Xing-kui

    2013-09-01

    To reveal the genetic variation of the viral protein 1 (VP1) gene of the duck hepatitis A virus type 3 (DHAV-3), the VP1 gene of 13 virulent DHAV-3 strains isolated from Shandong province of China in 2012 were amplified by RT-PCR, sequenced and analyzed. The results showed that all the VP1 genes of the 13 isolates contained 720 nucleotides encoding 240 amino acids, and shared with nucleotide identities of 94. 6%-99.9% and amino acid identities of 95.0%-100%. The nucleotide and amino acid sequence homologies between the 13 DHAV-3 isolates and other 31 DHAV-3 reference strains were 92.5%-100% and 90. 8%-100%, respectively. Phylogenetic analysis showed that the VP1 gene of DHAV-3 had distinct geographical characteristics. Distribution of genotypes of the 44 DHAV-3 strains was as follows: except the vaccine strain B63, all the other Chinese isolates belonged to genotype I (GI), Vietnamese wild isolates mainly belonged to subtype 1 (S1) of genotype II (GII), and all Korean isolates belonged to subtype 2 (S2) of GII.

  11. 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.

  12. 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.

  13. Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids.

    PubMed

    Ströh, Luisa J; Gee, Gretchen V; Blaum, Bärbel S; Dugan, Aisling S; Feltkamp, Mariet C W; Atwood, Walter J; Stehle, Thilo

    2015-08-01

    Trichodysplasia spinulosa-associated Polyomavirus (TSPyV) was isolated from a patient suffering from trichodysplasia spinulosa, a skin disease that can appear in severely immunocompromised patients. While TSPyV is one of the five members of the polyomavirus family that are directly linked to a human disease, details about molecular recognition events, the viral entry pathway, and intracellular trafficking events during TSPyV infection remain unknown. Here we have used a structure-function approach to shed light on the first steps of TSPyV infection. We established by cell binding and pseudovirus infection studies that TSPyV interacts with sialic acids during attachment and/or entry. Subsequently, we solved high-resolution X-ray structures of the major capsid protein VP1 of TSPyV in complex with three different glycans, the branched GM1 glycan, and the linear trisaccharides α2,3- and α2,6-sialyllactose. The terminal sialic acid of all three glycans is engaged in a unique binding site on TSPyV VP1, which is positioned about 18 Å from established sialic acid binding sites of other polyomaviruses. Structure-based mutagenesis of sialic acid-binding residues leads to reduction in cell attachment and pseudovirus infection, demonstrating the physiological relevance of the TSPyV VP1-glycan interaction. Furthermore, treatments of cells with inhibitors of N-, O-linked glycosylation, and glycosphingolipid synthesis suggest that glycolipids play an important role during TSPyV infection. Our findings elucidate the first molecular recognition events of cellular infection with TSPyV and demonstrate that receptor recognition by polyomaviruses is highly variable not only in interactions with sialic acid itself, but also in the location of the binding site.

  14. Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids

    PubMed Central

    Ströh, Luisa J.; Gee, Gretchen V.; Blaum, Bärbel S.; Dugan, Aisling S.; Feltkamp, Mariet C. W.; Atwood, Walter J.; Stehle, Thilo

    2015-01-01

    Trichodysplasia spinulosa-associated Polyomavirus (TSPyV) was isolated from a patient suffering from trichodysplasia spinulosa, a skin disease that can appear in severely immunocompromised patients. While TSPyV is one of the five members of the polyomavirus family that are directly linked to a human disease, details about molecular recognition events, the viral entry pathway, and intracellular trafficking events during TSPyV infection remain unknown. Here we have used a structure-function approach to shed light on the first steps of TSPyV infection. We established by cell binding and pseudovirus infection studies that TSPyV interacts with sialic acids during attachment and/or entry. Subsequently, we solved high-resolution X-ray structures of the major capsid protein VP1 of TSPyV in complex with three different glycans, the branched GM1 glycan, and the linear trisaccharides α2,3- and α2,6-sialyllactose. The terminal sialic acid of all three glycans is engaged in a unique binding site on TSPyV VP1, which is positioned about 18 Å from established sialic acid binding sites of other polyomaviruses. Structure-based mutagenesis of sialic acid-binding residues leads to reduction in cell attachment and pseudovirus infection, demonstrating the physiological relevance of the TSPyV VP1-glycan interaction. Furthermore, treatments of cells with inhibitors of N-, O-linked glycosylation, and glycosphingolipid synthesis suggest that glycolipids play an important role during TSPyV infection. Our findings elucidate the first molecular recognition events of cellular infection with TSPyV and demonstrate that receptor recognition by polyomaviruses is highly variable not only in interactions with sialic acid itself, but also in the location of the binding site. PMID:26302170

  15. 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

  16. 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

  17. 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.

  18. 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

    2016-06-22

    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.

  19. Building laboratory capacity to support the global rotavirus surveillance network.

    PubMed

    2013-05-24

    In 2001, in anticipation of rotavirus vaccine licensure and introduction, the World Health Organization (WHO) and partners established regional laboratory surveillance networks for rotavirus detection and strain type monitoring among hospitalized children aged <5 years. In 2006, two WHO-prequalified oral rotavirus vaccines were licensed: a 2-dose, single-strain vaccine (Rotarix, GlaxoSmithKline Biologicals) and a 3-dose, multistrain vaccine (RotaTeq, Merck). Both vaccines provide protection against a range of rotavirus strain types, generally classified as G and P types based on specific viral proteins. Based on results of clinical trial data, disease burden data from surveillance networks, and findings from vaccine impact studies, WHO recommends that all countries include rotavirus vaccination in national immunization programs. Vaccination is recommended to help reduce the morbidity and mortality associated with rotavirus, a leading cause of diarrhea in children aged <5 years that was responsible for approximately 450,000 deaths in 2008. This report describes the expansion of the regional rotavirus laboratory surveillance networks to a global surveillance network, the implementation of data quality assurance measures to ensure quality laboratory data reporting to support rotavirus surveillance activities, and data reporting through the surveillance network. Timely, quality surveillance data can provide baseline estimates of rotavirus disease burden to inform decisions regarding rotavirus vaccine introduction in national immunization programs and can help monitor the impact of vaccine introduction on disease trends.

  20. 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.

  1. Supplementing suckling rats with whey protein concentrate modulates the immune response and ameliorates rat rotavirus-induced diarrhea.

    PubMed

    Pérez-Cano, Francisco J; Marín-Gallén, Silvia; Castell, Margarida; Rodríguez-Palmero, María; Rivero, Montserrat; Castellote, Cristina; Franch, Angels

    2008-12-01

    Group A rotaviruses (RV) are the most common causative agents of acute gastroenteritis in children <2 y. The present study was designed to establish the effect of a bovine whey protein concentrate (WPC) in a RV infection model in suckling rats. From d 3 of life, suckling Lewis rats received daily supplements of WPC, WPC plus lactoferrin (LF), standard infant formula (SIF), or water (RV-infected group and an untreated, uninfected reference group). On d 8 of life, heterologous simian RV SA-11 was inoculated orally in the WPC-RV, WPC+LF-RV, SIF-RV, and RV groups. WPC and WPC+LF reduced diarrhea incidence from approximately 90% in RV group to approximately 60% in WPC-RV and WPC+LF-RV groups (P < 0.05), whereas the area under the curve (AUC) of severity along time diminished from approximately 10 AUC in the RV group to approximately 6 AUC in both supplemented groups (P < 0.05). Serum levels of anti-RV antibodies, splenocyte proliferation, and interferon-gamma secretion after specific stimulation were significantly lower in the WPC-RV and WPC+LF-RV groups than in the SIF-RV and RV groups. In the intraepithelial intestinal compartment, RV infection increased the proportion of typical mucosal T cells (IE-T CD8alphaalpha+); however, this modification was controlled by WPC and WPC+LF supplementation. In general, for most of the parameters studied, the SIF-RV and RV groups did not differ. In summary, daily supplementation with WPC or WPC+LF in early life considerably reduces the severity of RV-induced acute gastroenteritis and modulates the immune response against the pathogen.

  2. 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

  3. Development of a subunit vaccine containing recombinant chicken anemia virus VP1 and pigeon IFN-γ.

    PubMed

    Shen, Sin Ying; Chang, Wei Chun; Yi, Hsiang Heng; Tsai, Shinn-Shong; Liu, Hung Jen; Liao, Pei-Chun; Chuang, Kuo Pin

    2015-10-15

    Chicken anemia virus (CAV) is a severe threat to the chicken industry and causes heavy economic losses worldwide. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant VP1 (rVP1) and pigeon interferon-γ (rPiIFN-γ). Results indicated that rPiIFN-γ enhanced humoral immunity elicited by rVP1 as early as 10 day after primary immunization and reach the high titer after secondary immunization. When compared to chickens immunized with rVP1, inactivated vaccine, chickens immunized with rVP1+rPiIFN-γ showed faster and higher levels (p<0.05) of antibody titer. The CAV challenge result showed that the rVP1+rPiIFN-γ vaccine prevent the reducing of hematocrit values in comparison with the rVP1 or inactivated groups. The relative fold inductions of mRNA expression of Th1-type (IFN-γ), but not Th2-type (IL-4) cytokines in splenocytes isolated from chickens immunized with rVP1+rPiIFN-γ were significantly higher than those of the rVP1 or inactivated vaccine groups. In conclusion, our study found that rPiIFN-γ can enhance both humoral and cellular immunity elicited by an rVP1 vaccine. The rVP1+rPiIFN-γ vaccine may provide a new strategy vaccine against CAV in chicken.

  4. Phylogenetic analysis of the VP1 gene of Enterovirus 71 in Guangzhou during the high occurrence period of 2008.

    PubMed

    Yin, Ying-xian; Ou, Zhi-ying; Xu, Yi; Zhou, Rong; Xia, Hui-min

    2014-06-01

    An outbreak of hand, foot, and mouth disease (HFMD) in Guangzhou in 2008 affected over 10,000 children and resulted in high hospital admission rates. To investigate the molecular epidemiological pattern of EV71 infections in Guangzhou, throat swab samples were collected from 102 children clinically diagnosed with HFMD from May to July of 2008 in Guangzhou. Partial VP1 (virus protein 1) fragments of Enterovirus 71 (EV71) isolates were sequenced, and used alongside EV71 sequences entered in GenBank to construct a phylogenetic tree using MEGA5.0. Blast and phylogenetic analyses showed that all 21 sequences belonged to subgenogroup C4 of EV71. In early May, diverse strains were circulating in Guangzhou, but by July, only a small number of these strains could be detected. These results could indicate that geographic and climatic features may affect the epidemic characteristics of EV71, and that some C4 strains might retain their infectivity at higher temperatures.

  5. Clinical trials of live oral rotavirus vaccines: the Finnish experience.

    PubMed

    Vesikari, T

    1993-01-01

    Live oral candidate rotavirus vaccines of bovine (RIT 4237) or rhesus (RRV-1) origin and reassortants of RRV-1 expressing human serotype 1 (DxRRV) or serotype 2 (DS1xRRV) VP7 protein were evaluated for clinical efficacy in young children in successive trials from 1983 to 1989. In each study, the vaccinations were given before a rotavirus epidemic season and the follow-up of vaccinees covered two rotavirus epidemic seasons lasting up to 2-3 years of age. Serotype 1 rotavirus was predominant in each season. Protection rates against all rotavirus-associated diarrhoea ranged from 0 to 67% but were higher, up to 100%, against more severe rotavirus disease. All tested vaccines also showed efficacy for diarrhoea not apparently associated with rotavirus; therefore the clinical benefit of the vaccinations was greater than could be deduced from efficacy rates for rotavirus-associated diarrhoea alone. Each of the candidate vaccines could significantly reduce severe diarrhoea in Finnish children in the first 2 to 3 years of life. For optimal efficacy, the vaccines should be administered in the autumn before the regular epidemic season of rotavirus.

  6. Rotavirus epidemiology: the Asian Rotavirus Surveillance Network.

    PubMed

    Nelson, E A S; Bresee, J S; Parashar, U D; Widdowson, M-A; Glass, R I

    2008-06-19

    Availability of new rotavirus vaccines has highlighted the need to collect local disease and economic burden data to aid decision makers at global, regional and country level. The World Health Organization and the GAVI Alliance recommended that generic protocols be used and that regional surveillance networks be established to collect these data, thereby helping to fast-track the introduction of these new vaccines into developing countries. Nine countries and regions participated in the first phase of the Asian Rotavirus Surveillance Network (ARSN), which collected data over a 2-year period during 2001-2003. Overall 45% of diarrhoea admissions in the region were positive for rotavirus, which was higher than had been anticipated. Significant rotavirus strain diversity was noted during the surveillance period. Data collection for a second phase of the ARSN commenced in 2004 and included a greater proportion of poorer countries that would in future be eligible for funding support for rotavirus immunization from GAVI. Limited economic evaluations in Asia have demonstrated the potential for new rotavirus vaccines to be cost-effective but more local analyses are required. Despite the ARSN's comprehensive data from a mix of developed and developing countries, Asia has lagged the Americas in terms of the introduction of rotavirus vaccines into National Immunization Programmes (NIPs). Lack on rotavirus vaccine efficacy data in Asia, particularly in poorer populations, will have contributed to this delay. Thus ensuring that all global regions are simultaneously involved in the evaluation of new vaccines from the beginning and also encouraging more regional collaborations of Ministry of Health representatives could help to accelerate the introduction of new vaccines into NIPs.

  7. 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

  8. Differential response of maize catalases to abscisic acid: Vp1 transcriptional activator is not required for abscisic acid-regulated Cat1 expression.

    PubMed Central

    Williamson, J D; Scandalios, J G

    1992-01-01

    In this paper we describe the distinctive responses of the maize catalases to the plant growth regulator abscisic acid (ABA). We analyzed RNA and enzyme accumulation in excised maize embryos and found that each catalase responded differently to exogenously applied ABA. Levels of Cat1 transcript and enzyme activity rapidly increased. In contrast, levels of Cat2 transcript and protein decreased, while Cat3 transcript levels were not affected. In developing kernels of the ABA-deficient/biosynthetic viviparous mutant vp5, lower levels of Cat1 RNA correlated with lower endogenous ABA levels when compared to measured levels in comparably aged wild-type siblings from the same ear. The maize vp1 mutant line is morphologically insensitive to normal endogenous levels of ABA. Analysis of the response of Cat1 to exogenously applied ABA in mutant and wild-type vp1 sibling embryos suggests that, unlike other ABA-responsive genes analyzed to date, the Vp1 gene product is not essential for the ABA-mediated regulation of Cat1. The significance of these responses to ABA in defining the roles of the various CATs in maize is discussed. Images PMID:1388272

  9. Dual inhibitor of PDE7 and GSK-3-VP1.15 acts as antipsychotic and cognitive enhancer in C57BL/6J mice.

    PubMed

    Lipina, Tatiana V; Palomo, Valle; Gil, Carmen; Martinez, Ana; Roder, John C

    2013-01-01

    Cognitive deficit is a core of schizophrenia and it is not effectively treated by the available antipsychotic drugs, hence new and more effective therapy is needed. Schizophrenia is considered as a pathway disorder where Disrupted-In-Schizophrenia-1 (DISC1) is important molecular player that regulates multiple cellular cascades. We recently reported synergistic action between phosphodiesterase-4 (PDE4) and glycogen synthase kinase-3 (GSK-3) as DISC1 interacting proteins. In the current study we characterized behavioural effects of a newly developed compound, VP1.15 that inhibits both PDE7 and GSK-3 with main focus on its antipsychotic and cognitive capacities. VP1.15 reduced ambulation in C57BL/6J mice in a dose-dependent manner (7.5 mg/kg and 3 mg/kg, respectively) and, hence, lower dose was chosen for the further analysis. VP1.1.5 facilitated pre-pulse inhibition (PPI), reversed amphetamine- but not MK-801-induced PPI deficit. The drug was able to ameliorate the disrupted latent inhibition (LI) induced by the increased number of conditioning trials and reversed amphetamine-induced LI deficit, supporting further its antipsychotic effects. The drug also significantly improved episodic memory in the spatial object recognition test, facilitated working memory in Y-maze and enhanced cued fear memory, but had no effect on executive function in the Puzzle box and contextual fear conditioning. Taken together, VP1.15 elicited antipsychotic effects and also facilitated cognitive domains in mice, suggesting that multitarget drugs, affecting molecular substrates from the same pathway, perhaps could be antipsychotics of new-generation that open a new possibilities in drug discoveries. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

  10. Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication

    PubMed Central

    Viskovska, Maria; Anish, Ramakrishnan; Hu, Liya; Chow, Dar-Chone; Hurwitz, Amy M.; Brown, Nicholas G.; Palzkill, Timothy; Estes, Mary K.

    2014-01-01

    ABSTRACT Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity to the RNA template entry and double-stranded RNA (dsRNA) exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the intersubunit junctions, suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap that of VP1, and the NSP5-binding sites overlap those of VP2 and VP1, suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated. IMPORTANCE Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving nonstructural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA

  11. Enhanced immune response with foot and mouth disease virus VP1 and interleukin-1 fusion genes.

    PubMed

    Park, Jong Hyeon; Kim, Sun Jin; Oem, Jae Ku; Lee, Kwang Nyeong; Kim, Yong Joo; Kye, Soo Jeong; Park, Jee Yong; Joo, Yi Seok

    2006-09-01

    The capsid of the foot and mouth disease (FMD) virus carries the epitopes that are critical for inducing the immune response. In an attempt to enhance the specific immune response, plasmid DNA was constructed to express VP1/interleukin-1alpha (IL-1alpha) and precursor capsid (P1) in combination with 2A (P1-2A)/IL-1alpha under the control of the human cytomegalovirus (HCMV) immediateearly promoter and intron. After DNA transfection into MA104 (monkey kidney) cells, Western blotting and an immunofluorescence assay were used to confirm the expression of VP1 or P1-2A and IL-1alpha. Mice were inoculated with the encoding plasmids via the intradermal route, and the IgG1 and IgG2a levels were used to determine the immune responses. These results show that although the immunized groups did not carry a high level of neutralizing antibodies, the plasmids encoding the VP1/ IL-1alpha, and P1-2A /IL-1alpha fused genes were effective in inducing an enhanced immune response.

  12. 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.

  13. [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

  14. Expression of rotavirus VP2 produces empty corelike particles.

    PubMed Central

    Labbé, M; Charpilienne, A; Crawford, S E; Estes, M K; Cohen, J

    1991-01-01

    The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis. Images PMID:1851866

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

    PubMed Central

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

    2004-01-01

    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

  16. Genetic variation of the VP1 gene of the virulent duck hepatitis A virus type 1 (DHAV-1) isolates in Shandong province of China.

    PubMed

    Gao, Jiming; Chen, Junhao; Si, Xingkui; Xie, Zhijing; Zhu, Yanli; Zhang, Xingxiao; Wang, Shujing; Jiang, Shijin

    2012-08-01

    To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1 (DHAV-1) isolates, the virulence, cross neutralization assays and the complete sequence of the virion protein 1 (VP1) gene of nine virulent DHAV-1 strains, which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008, were tested. The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses (ELD(50)s) and the median lethal doses (LD(50)s), respectively. The results showed that the ELD(50)s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10(6)/mL to 1.44 × 10(7)/mL, while the LD(50)s were 2.39 × 10(5)/mL to 6.15 × 10(6)/mL. Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates, respectively. Compared with other virulent, moderate virulent, attenuated vaccine and mild strains, the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains. There were three hypervariable regions at the C-terminus (aa 158-160, 180-193 and 205-219) and other variable points in VP1 protein, but which didn't cause virulence of DHAV-1 change.

  17. 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...

  18. Rotavirus open cores catalyze 5'-capping and methylation of exogenous RNA: evidence that VP3 is a methyltransferase.

    PubMed

    Chen, D; Luongo, C L; Nibert, M L; Patton, J T

    1999-12-05

    Rotavirus open cores prepared from purified virions consist of three proteins: the RNA-dependent RNA polymerase, VP1; the core shell protein, VP2; and the guanylyltransferase, VP3. In addition to RNA polymerase activity, open cores have been shown to contain a nonspecific guanylyltransferase activity that caps viral and nonviral RNAs in vitro. In this study, we examined the structure of RNA caps made by open cores and have analyzed open cores for other capping-related enzymatic activities. Utilizing RNase digestion and thin-layer chromatography, we found that the majority ( approximately 70%) of caps made by open cores contain the tetraphosphate linkage, GppppG, rather than the triphosphate linkage, GpppG, found on mRNAs made by rotavirus double-layered particles. Enzymatic analysis indicated that the GppppG caps resulted from the lack of a functional RNA 5'-triphosphatase in open cores, to remove the gamma-phosphate from the RNA prior to capping. RNA 5'-triphosphatases commonly exhibit an associated nucleoside triphosphatase activity, and this too was not detected in open cores. Caps of some RNAs contained an extra GMP moiety (underlined) and had the structure 3'-GpGp(p)ppGpGpC-RNA-3'. The origin of the extra GMP is not known but may reflect the cap serving as a primer for RNA synthesis. Methylated caps were produced in the presence of the substrate, S-adenosyl-l-methionine (SAM), indicating that open cores contain methyltransferase activity. UV cross-linking showed that VP3 specifically binds SAM. Combined with the results of earlier studies, our results suggest that the viral guanylyltransferase and methyltransferase are both components of VP3 and, therefore, that VP3 is a multifunctional capping enzyme.

  19. Identification of mutations in the genome of rotavirus SA11 temperature-sensitive mutants D, H, I and J by whole genome sequences analysis and assignment of tsI to gene 7 encoding NSP3.

    PubMed

    Vende, Patrice; Gratia, Matthieu; Duarte, Mariela D; Charpilienne, Annie; Saguy, Matthieu; Poncet, Didier

    2013-09-01

    The complete coding sequences of the four unassigned temperature-sensitive (ts) Baylor prototype rotavirus mutants (SA11ts D, H, I and J) were sequenced by deep sequencing double-stranded RNA using RNA-seq. Non-silent mutations were assigned to a specific mutant by Sanger sequencing RT-PCR products from each mutant. Mutations that led to amino acid changes were found in all genes except for genes 1 (VP1), 10 (NSP4) and 11 (NSP5/6). Based on these sequence analyses and earlier genetic analyses, the ts mutations in gene 7, which encodes the protein NSP3, were assigned to ts mutant groups I and H, and confirmed by an in vitro RNA-binding assay with recombinant proteins. In addition, ts mutations in gene 6 were assigned to tsJ. The presence of non-conservative mutations in two genes of two mutants (genes 4 and 2 in tsD and genes 3 and 7 in tsH) underscores the necessity of sequencing the whole genome of each rotavirus ts mutant prototype.

  20. The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice.

    PubMed

    Pastor, Ana Ruth; Rodríguez-Limas, William A; Contreras, Martha A; Esquivel, Ernesto; Esquivel-Guadarrama, Fernando; Ramírez, Octavio T; Palomares, Laura A

    2014-05-19

    Viral protein assemblies have shown to be superior immunogens used in commercial vaccines. However, little is known about the effect of protein assembly structure in immunogenicity and the protection conferred by a vaccine. In this work, rotavirus VP6, a polymorphic protein that assembles into nanotubes, icosahedra (dlRLP) or trimers was used to compare the immune response elicited by three different assemblies. VP6 is the most antigenic and abundant rotavirus structural protein. It has been demonstrated that antibodies against VP6 interfere with the replication cycle of rotavirus, making it a vaccine candidate. Groups of mice were immunized with either nanotubes, dlRLP or trimers and the humoral response (IgG and IgA titers) was measured. Immunized mice were challenged with EDIM rotavirus and protection against rotavirus infection, measured as viral shedding, was evaluated. Immunization with nanotubes resulted in the highest IgG titers, followed by immunization with dlRLP. While immunization with one dose of nanotubes was sufficient to reduce viral shedding by 70%, two doses of dlRLP or trimers were required to obtain a similar protection. The results show that the type of assembly of VP6 results in different humoral responses and protection efficacies against challenge with live virus. This information is important for the design of recombinant vaccines in general.

  1. A comparison of the VP1, VP2, and VP4 regions for molecular typing of human enteroviruses.

    PubMed

    Perera, David; Shimizu, Hiroyuki; Yoshida, Hiromu; Tu, Phan Van; Ishiko, Hiroaki; McMinn, Peter C; Cardosa, Mary J

    2010-04-01

    The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.

  2. P[8] and P[4] Rotavirus Infection Associated with Secretor Phenotypes Among Children in South China

    PubMed Central

    Zhang, Xu-Fu; Long, Yan; Tan, Ming; Zhang, Ting; Huang, Qiong; Jiang, Xi; Tan, Wen-Fang; Li, Jian-Dong; Hu, Gui-Fang; Tang, Shixing; Dai, Ying-Chun

    2016-01-01

    Rotaviruses are known to recognize human histo-blood group antigens (HBGAs) as a host ligand that is believed to play an important role in rotavirus host susceptibility and host range. In this study, paired fecal and saliva samples collected from children with viral gastroenteritis, as well as paired serum and saliva samples collected from the general population in south China were studied to evaluate potential association between rotavirus infections and human HBGA phenotypes. Rotavirus was detected in 75 (28%) of 266 fecal samples and P[8] rotaviruses were found to be the predominant genotype. The HBGA phenotypes of the rotavirus-infected children were determined through their saliva samples. Secretor statuses were found to correlate with the risk of rotavirus infection and all P[8]/P[4] rotavirus infected children were secretors. Accordingly, recombinant VP8* proteins of the P[8]/P[4] rotaviruses bound saliva samples from secretor individuals. Furthermore, correlation between serum P[8]/P[4]-specific IgG and host Lewis and secretor phenotypes has been found among 206 studied serum samples. Our study supported the association between rotavirus infection and the host HBGA phenotypes, which would help further understanding of rotavirus host range and epidemiology. PMID:27708367

  3. 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.

  4. Liposome entrapment and immunogenic studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vaccine design.

    PubMed

    Haro, Isabel; Pérez, Silvia; García, Mónica; Chan, Weng C; Ercilla, Guadalupe

    2003-04-10

    Multiple antigen peptides (MAP) have been demonstrated to be efficient immunological reagents for the induction of immune responses to a variety of infectious agents. Several peptide domains of the hepatitis A virus (HAV) capsid proteins, mainly VP1 and VP3, are the immunodominant targets for a protective antibody response. In the present study we analyse the immunogenic properties of a tetrameric heterogeneous palmitoyl-derivatised MAP containing two defined HAV peptide sequences, VP1(11-25) and VP3(102-121), in rabbits immunised with either Freund's adjuvant or multilamellar liposomes. The immune response was evaluated with a specific enzyme immunoassay using MAP[VP1+VP3], VP1 and VP3 as targets. The avidity of the immune response was measured by a non-competitive enzyme-linked immunosorbent assay and by the surface plasmon resonance technology. Antisera raised against the lipo-MAP peptide entrapped in liposomes demonstrated high avidity of binding with affinity rate constants approximately one order of magnitude greater than those obtained with the Freund's protocol.

  5. Frequently Asked Questions about Rotavirus

    MedlinePlus

    ... Disease Mumps Norovirus Pertussis (Whooping Cough) Pneumococcal Disease Polio Respiratory Syncytial Virus (RSV) Rotavirus Rubella (German Measles) ... Disease Mumps Norovirus Pertussis (Whooping Cough) Pneumococcal Disease Polio Respiratory Syncytial Virus (RSV) Rotavirus Rubella (German Measles) ...

  6. 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

    2015-08-05

    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.

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

    PubMed Central

    Xu, Longfa; He, Delei; Yang, Lisheng; Li, Zhiqun; Ye, Xiangzhong; Yu, Hai; zhao, Huan; Li, Shuxuan; Yuan, Lunzhi; Qian, Hongliu; Que, Yuqiong; Kuo Shih, James Wai; 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

  8. Immunogenicity and protective efficacy of yeast extracts containing rotavirus-like particles: a potential veterinary vaccine.

    PubMed

    Rodríguez-Limas, William A; Pastor, Ana Ruth; Esquivel-Soto, Ernesto; Esquivel-Guadarrama, Fernando; Ramírez, Octavio T; Palomares, Laura A

    2014-05-19

    Rotavirus is the most common cause of severe diarrhea in many animal species of economic interest. A simple, safe and cost-effective vaccine is required for the control and prevention of rotavirus in animals. In this study, we evaluated the use of Saccharomyces cerevisiae extracts containing rotavirus-like particles (RLP) as a vaccine candidate in an adult mice model. Two doses of 1mg of yeast extract containing rotavirus proteins (between 0.3 and 3 μg) resulted in an immunological response capable of reducing the replication of rotavirus after infection. Viral shedding in all mice groups diminished in comparison with the control group when challenged with 100 50% diarrhea doses (DD50) of murine rotavirus strain EDIM. Interestingly, when immunizing intranasally protection against rotavirus infection was observed even when no increase in rotavirus-specific antibody titers was evident, suggesting that cellular responses were responsible of protection. Our results indicate that raw yeast extracts containing rotavirus proteins and RLP are a simple, cost-effective alternative for veterinary vaccines against rotavirus.

  9. Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    PubMed Central

    Makvandi, Manoochehr; Teimoori, Ali; Neisi, Niloofar; Samarbafzadeh, Alireza

    2016-01-01

    Background The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing. Objectives The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. Methods The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. Then the products were transformed into Escherichia coli DH5α and retransformed in DH10Bac competent cells. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Results The lengths of subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 1500 and 2000bp, respectively. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 4300 and 3800 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. Conclusions The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine. PMID:28138375

  10. VP1 sequencing protocol for foot and mouth disease virus molecular epidemiology.

    PubMed

    Knowles, N J; Wadsworth, J; Bachanek-Bankowska, K; King, D P

    2016-12-01

    Nucleotide sequences of field strains of foot and mouth disease virus (FMDV) contribute to our understanding of the distribution and evolution of viral lineages that circulate in different regions of the world. This paper outlines a practical reversetranscription polymerase chain reaction (RT-PCR) and sequencing strategy that can be used to generate RNA sequences encoding the VP1 (1D) region of FMDV. The protocol contains a panel of PCR and sequencing primers that can be selected to characterise genetically diverse isolates representing all seven FMDV serotypes. A list of sequences is also described, comprising prototype sequences for all proposed FMDV topotypes, in order to provide a framework for phylogenetic analysis. The technical details and prototype sequences provided in this paper can be employed by FMD Reference Laboratories and others in an approach to harmonise the molecular epidemiology of FMDV.

  11. 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.

  12. Molecular characterization of enterovirus 71 and coxsackievirus A16 using the 5' untranslated region and VP1 region.

    PubMed

    Zhou, Fei; Kong, Fanrong; Wang, Bin; McPhie, Kenneth; Gilbert, Gwendolyn L; Dwyer, Dominic E

    2011-03-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the species Human enterovirus A, and are both major and independent aetiological agents of hand-foot-and-mouth disease. The human enterovirus (HEV) 5' untranslated region (UTR) is fundamentally important for efficient virus replication and for virulence, whilst the VP1 region correlates well with antigenic typing by neutralization, and can be used for virus identification and evolutionary studies. A comparison was undertaken of the 5'UTR and VP1 nucleotide sequences of five EV71 clinical isolates and 10 CVA16 clinical isolates from one laboratory with the 5'UTR and VP1 sequences of 104 EV71 strains and 45 CVA16 strains available in GenBank. The genetic relationships were analysed using standard phylogenetic methods. The EV71 phylogenetic analysis showed that the VP1 sequences were clustered into three genogroups, A, B and C, with genogroups B and C further divided into five subgenogroups, B1-B5 and C1-C5, respectively. All EV71 strains were clustered similarly in the 5'UTR and VP1 trees, except for one Taiwanese strain, which demonstrated different clustering in the two trees, suggesting a recombination event in the phylogeny. The CVA16 phylogenetic analysis showed that the VP1 sequences were clustered into two genogroups, A and B, with genogroup B further divided into B1 (B1a and B1b), B2 and a possible B3; and that a similar pattern and grouping of all strains were displayed in the 5'UTR tree. This study demonstrated that comparing the two regions provides evidence of epidemiological linkage of HEV-A strains, and that mutation in the two regions plays a vital role in the evolution of these viruses. The combination of molecular typing and phylogenetic sequence analysis will be beneficial in both individual patient diagnosis and public health measures.

  13. Molecular Comparison and Evolutionary Analyses of VP1 Nucleotide Sequences of New African Human Enterovirus 71 Isolates Reveal a Wide Genetic Diversity

    PubMed Central

    Nougairède, Antoine; Joffret, Marie-Line; Deshpande, Jagadish M.; Dubot-Pérès, Audrey; Héraud, Jean-Michel

    2014-01-01

    Most circulating strains of Human enterovirus 71 (EV-A71) have been classified primarily into three genogroups (A to C) on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D) and 2 African ones (E and F). Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied. PMID:24598878

  14. Inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step.

    PubMed

    Rossen, John W A; Bouma, Janneke; Raatgeep, Rolien H C; Büller, Hans A; Einerhand, Alexandra W C

    2004-09-01

    Elevated levels of prostaglandins (PGs), products of cyclooxygenases (COXs), are found in the plasma and stool of rotavirus-infected children. We sought to determine the role of COXs, PGs, and the signal transduction pathways involved in rotavirus infection to elucidate possible new targets for antiviral therapy. Human intestinal Caco-2 cells were infected with human rotavirus Wa or simian rotavirus SA-11. COX-2 mRNA expression and secreted PGE2 levels were determined at different time points postinfection, and the effect of COX inhibitors on rotavirus infection was studied by an immunofluorescence assay (IFA). To reveal the signal transduction pathways involved, the effect of MEK, protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inhibitors on rotavirus infection was analyzed. In infected Caco-2 cells, increased COX-2 mRNA expression and secreted PGE2 levels were detected. Indomethacin (inhibiting both COX-1 and COX-2) and specific COX-1 and COX-2 inhibitors reduced rotavirus infection by 85 and 50%, respectively, as measured by an IFA. Indomethacin reduced virus infection at a postbinding step early in the infection cycle, inhibiting virus protein synthesis. Indomethacin did not seem to affect viral RNA synthesis. Inhibitors of MEK, PKA, p38 MAPK, and NF-kappaB decreased rotavirus infection by at least 40%. PGE2 counteracted the effect of the COX and PKA inhibitors but not of the MEK, p38 MAPK, and NF-kappaB inhibitors. Conclusively, COXs and PGE2 are important mediators of rotavirus infection at a postbinding step. The ERK1/2 pathway mediated by PKA is involved in COX induction by rotavirus infection. MAPK and NF-kappaB pathways are involved in rotavirus infection but in a PGE2-independent manner. This report offers new perspectives in the search for therapeutic agents in treatment of severe rotavirus-mediated diarrhea in children.

  15. An Inhibitory Motif on the 5’UTR of Several Rotavirus Genome Segments Affects Protein Expression and Reverse Genetics Strategies

    PubMed Central

    Papa, Guido; Eichwald, Catherine; Burrone, Oscar R.

    2016-01-01

    Rotavirus genome consists of eleven segments of dsRNA, each encoding one single protein. Viral mRNAs contain an open reading frame (ORF) flanked by relatively short untranslated regions (UTRs), whose role in the viral cycle remains elusive. Here we investigated the role of 5’UTRs in T7 polymerase-driven cDNAs expression in uninfected cells. The 5’UTRs of eight genome segments (gs3, gs5-6, gs7-11) of the simian SA11 strain showed a strong inhibitory effect on the expression of viral proteins. Decreased protein expression was due to both compromised transcription and translation and was independent of the ORF and the 3’UTR sequences. Analysis of several mutants of the 21-nucleotide long 5’UTR of gs 11 defined an inhibitory motif (IM) represented by its primary sequence rather than its secondary structure. IM was mapped to the 5’ terminal 6-nucleotide long pyrimidine-rich tract 5’-GGY(U/A)UY-3’. The 5’ terminal position within the mRNA was shown to be essentially required, as inhibitory activity was lost when IM was moved to an internal position. We identified two mutations (insertion of a G upstream the 5’UTR and the U to A mutation of the fifth nucleotide of IM) that render IM non-functional and increase the transcription and translation rate to levels that could considerably improve the efficiency of virus helper-free reverse genetics strategies. PMID:27846320

  16. [Analysis on the preference of synonymous codon in VP1 nucleotide sequence of the EV71 based on RSCU method].

    PubMed

    Qi, Bin; Zhao, Jing-Jing; Gao, Lei; Zhu, Ping

    2009-11-01

    Based on RSCU method and by analyzing the preference of codon usage in VP1 nucleotide sequences of EV71 isolated in Chinese mainland and Taiwan region from 1998 to 2008, it is clear that there is an obvious time discrimination in RSCU calculated from EV71 VP1 strain between two different regions of China and it is more obvious in Taiwan region, therefore, according to the diversity of RSCU, the years can be divided into 2 intervals in Chinese mainland and 4 intervals in Taiwan region, especially, the number of intervals in one region have a positive co-relation with the activity of variation of the EV71 in the same region. The change of the preference of codon usage in VP1 nucleotide sequences of EV71 can significantly embody the Variation of the EV71, so we can make use of the analysis on preference of codon usage in VP1 nucleotide sequences of EV71 to predict the possible variation trend of the EV71.

  17. 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.

  18. 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

  19. 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

  20. Analysis of complete genome sequences of G9P[19] rotavirus strains from human and piglet with diarrhea provides evidence for whole-genome interspecies transmission of nonreassorted porcine rotavirus.

    PubMed

    Yodmeeklin, Arpaporn; Khamrin, Pattara; Chuchaona, Watchaporn; Kumthip, Kattareeya; Kongkaew, Aphisek; Vachirachewin, Ratchaya; Okitsu, Shoko; Ushijima, Hiroshi; Maneekarn, Niwat

    2017-01-01

    Whole genomes of G9P[19] human (RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19]) and porcine (RVA/Pig-wt/THA/CMP-015-12/2012/G9P[19]) rotaviruses concurrently detected in the same geographical area in northern Thailand were sequenced and analyzed for their genetic relationships using bioinformatic tools. The complete genome sequence of human rotavirus RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19] was most closely related to those of porcine rotavirus RVA/Pig-wt/THA/CMP-015-12/2012/G9P[19] and to those of porcine-like human and porcine rotaviruses reference strains than to those of human rotavirus reference strains. The genotype constellation of G9P[19] detected in human and piglet were identical and displayed as the G9-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotypes with the nucleotide sequence identities of VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4, and NSP5 at 99.0%, 99.5%, 93.2%, 97.7%, 97.7%, 85.6%, 89.5%, 93.2%, 92.9%, 94.0%, and 98.1%, respectively. The findings indicate that human rotavirus strain RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19] containing the genome segments of porcine genetic backbone is most likely a human rotavirus of porcine origin. Our data provide an evidence of interspecies transmission and whole-genome transmission of nonreassorted G9P[19] porcine RVA to human occurring in nature in northern Thailand.

  1. Molecular evolution of VP3, VP1, 3C(pro) and 3D(pol) coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa, Japan.

    PubMed

    Nidaira, Minoru; Kuba, Yumani; Saitoh, Mika; Taira, Katsuya; Maeshiro, Noriyuki; Mahoe, Yoko; Kyan, Hisako; Takara, Taketoshi; Okano, Sho; Kudaka, Jun; Yoshida, Hiromu; Oishi, Kazunori; Kimura, Hirokazu

    2014-04-01

    A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C(pro) and 3D(pol) coding regions performed. To assess time-scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p-distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10(-3) substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77-0.94. The p-distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

  2. Monoclonal anti-idiotype induces antibodies against bovine Q17 rotavirus.

    PubMed Central

    Cornaglia, E M; Elazhary, Y M; Brodeur, B R; Talbot, B G

    1992-01-01

    This study describes, for the first time, the production and use of an "internal-image" anti-idiotypic monoclonal antibody (MAb) to elicit a rotavirus-specific antibody response. An immunoglobulin G2a MAb, designated RQ31 (MAb1), specific for the outer capsid protein VP4 of bovine Q17 rotavirus and capable of neutralizing viral infection in vitro was used to generate an anti-idiotypic MAb (MAb2). This MAb2, designated RQA2, was selected by enzyme-linked immunosorbent assay (ELISA) using F(ab')2 fragments of RQ31. RQA2 (MAb2) inhibited the binding of RQ31 (MAb1) to the virus but had no effect on the binding of other rotavirus-specific MAbs. The MAb2 also inhibited virus neutralization mediated by MAb1 in a dose-dependent fashion. Naive guinea pigs immunized with the MAb2 produced anti-anti-idiotypic antibodies (Ab3) that reacted with bovine Q17 rotavirus in an ELISA and neutralized rotavirus infection in vitro. The Ab3 response was characterized as MAb1-like because the Ab3 recognizes only the Q17 and neonatal calf diarrhea virus rotavirus strains in ELISA, as did RQ31 (MAb1). The Ab3 response also possessed two other characteristics of RQ31: the abilities to bind the 1.36 (double-capsid) but not the 1.38 (single-capsid) purified rotavirus fraction in ELISA and to immunoprecipitate the VP4 rotavirus protein. Images PMID:1326641

  3. Rotavirus and the Vaccine (Drops) to Prevent It

    MedlinePlus

    ... Resources Related Links Vaccines & Immunizations Rotavirus and the Vaccine (Drops) to Prevent It Language: English Español (Spanish) ... vaccine. Why should my child get the rotavirus vaccine? The rotavirus vaccine: Protects your child from rotavirus, ...

  4. 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.

  5. Visualization of antigens attached to cytoskeletal framework in animal cells: colocalization of simian virus 40 Vp1 polypeptide and actin in TC7 cells.

    PubMed Central

    Kasamatsu, H; Lin, W; Edens, J; Revel, J P

    1983-01-01

    Actin and the simian virus 40 viral structural polypeptide Vp1 are observed to be present on cytoskeletal fibers of virus-infected TC7 cells, when these antigens in detergent-extracted whole cell mounts were labeled by specific antibodies and colloidal gold particles coated with a second antibody. In both cases, actin and Vp1 were found associated with fibers and fiber-associated electron-dense materials. Patches or clusters of colloidal gold particles denoting the presence of either Vp1 or actin were found on fibers uniformly distributed throughout the cytoplasm. By using simultaneous decoration of the two antigens with colloidal gold particles of different diameters, it was shown that the majority of Vp1 appears attached to cytoskeletal fibers in association with cellular actin. When Vp1 and actin were decorated with Imposil and ferritin simultaneously in infected cells that were fixed first and then permeabilized with saponin, both labels were found in the same spatial domain of the cell cytoplasm. Thus, the colocalization of Vp1 and actin on the cytoskeletal framework seems to reflect their actual state in the living cells. The electron-dense material to which colloidal gold particles localize in our cytoskeletal preparations may be the remnants of subcellular structures with which actin and Vp1 are both associated in intact cells. Images PMID:6308616

  6. Rotavirus interaction with isolated membrane vesicles.

    PubMed

    Ruiz, M C; Alonso-Torre, S R; Charpilienne, A; Vasseur, M; Michelangeli, F; Cohen, J; Alvarado, F

    1994-06-01

    To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

  7. Molecular analysis of the VP7 gene of pheasant rotaviruses identifies a new genotype, designated G23.

    PubMed

    Ursu, Krisztina; Kisfali, Péter; Rigó, Dóra; Ivanics, Eva; Erdélyi, Károly; Dán, Adám; Melegh, Béla; Martella, Vito; Bányai, Krisztián

    2009-01-01

    Rotavirus-associated enteritis has been reported in pheasants, but there is no information on the genetic/antigenic features of pheasant rotaviruses. In this study, we sequenced the VP7-encoding genome segment of three pheasant rotavirus strains detected during 2008 in Hungary. The full-length genome segment was 1,070 bp long, while the open reading frame was predicted to encode a 330-aa-long protein. The nucleotide sequence identities among the three pheasant rotavirus strains were high (> or =94%), whereas the range of nucleotide sequence identities to other avian and mammalian rotavirus VP7 genes fell between 68 and 73% and between 60 and 66%, respectively. Our findings indicate that these Hungarian pheasant rotaviruses need to be considered representatives of a new VP7 genotype specificity, designated G23.

  8. H(+) -pyrophosphatase IbVP1 promotes efficient iron use in sweet potato [Ipomoea batatas (L.) Lam.].

    PubMed

    Fan, Weijuan; Wang, Hongxia; Wu, Yinliang; Yang, Nan; Yang, Jun; Zhang, Peng

    2016-11-18

    Iron (Fe) deficiency is one of the most common micronutrient deficiencies limiting crop production globally, especially in arid regions because of decreased availability of iron in alkaline soils. Sweet potato [Ipomoea batatas (L.) Lam.] grows well in arid regions and is tolerant to Fe deficiency. Here, we report that the transcription of type I H(+) -pyrophosphatase (H(+) -PPase) gene IbVP1 in sweet potato plants was strongly induced by Fe deficiency and auxin in hydroponics, improving Fe acquisition via increased rhizosphere acidification and auxin regulation. When overexpressed, transgenic plants show higher pyrophosphate hydrolysis and plasma membrane H(+) -ATPase activity compared with the wild type, leading to increased rhizosphere acidification. The IbVP1-overexpressing plants showed better growth, including enlarged root systems, under Fe-sufficient or Fe-deficient conditions. Increased ferric precipitation and ferric chelate reductase activity in the roots of transgenic lines indicate improved iron uptake, which is also confirmed by increased Fe content and up-regulation of Fe uptake genes, e.g. FRO2, IRT1 and FIT. Carbohydrate metabolism is significantly affected in the transgenic lines, showing increased sugar and starch content associated with the increased expression of AGPase and SUT1 genes and the decrease in β-amylase gene expression. Improved antioxidant capacities were also detected in the transgenic plants, which showed reduced H2 O2 accumulation associated with up-regulated ROS-scavenging activity. Therefore, H(+) -PPase plays a key role in the response to Fe deficiency by sweet potato and effectively improves the Fe acquisition by overexpressing IbVP1 in crops cultivated in micronutrient-deficient soils.

  9. [Genetic characteristics of enterovirus 71 VP1 and epidemiology of hand-foot-mouth disease in Xinxiang in 2011].

    PubMed

    Ma, Jian-Min; Wei, Hai-Yan; Yu, He-Jun; Li, Kun; Huang, Xue-Yong

    2012-11-01

    The study was performed to examine the enterovirus 71(EV71) VP1 genetic feature and the epidemiology of hand-foot-mouth disease (HFMD) in Xinxiang in 2011. Real-time RT-PCR was used for the detection of Pan-enterovirus, Coxsackievirus A 16(CA16) and EV71 from stool specimens of HFMD. The VP1 region was amplified from 10 EV71 positive samples and the products were sequenced. EV71 genotypes were characterized by homology and phylogenetic tree analyses. Additionally, epidemic data of Xinxiang HFMD in 2011 was analyzed. The results revealed that 73% of the specimens from severe cases were determined as EV71 positive, which was significantly higher than CA16-positive ones (19%) (P < 0.01). Ten EV71 strains isolated in Xinxiang belonged to C4a cluster of sub-genotype C4, with 2.8% nucleotide and 0.9% amino acid sequences divergence among them. At position 170 in VP1 gene, an alanine(A) was predominant in 9 isolates, while a valine(V) residue was observed in one isolate. Compared to the representative C4a strains which were closely related to Xinxiang isolates, the amino acid variations of the pre-dominant Xinxiang strains generally occurred at position 292, threonine --> alanine (T --> A). A total of 1118 HFMD cases were reported in Xinxiang in 2011, and 92% of them were younger than 3 years old; the incidence rate peaked in April and December, suggesting that it is very necessary to strengthen HFMD prevention and control even in cold weather.

  10. Genetic diversity of hepatitis A virus in China: VP3-VP1-2A genes and evidence of quasispecies distribution in the isolates.

    PubMed

    Wang, Hao; Zheng, Huihui; Cao, Jingyuan; Zhou, Wenting; Yi, Yao; Jia, Zhiyuan; Bi, Shengli

    2013-01-01

    Hepatitis A virus (HAV) is the most common cause of infectious hepatitis throughout the world, spread largely by the fecal-oral route. To characterize the genetic diversity of the virus circulating in China where HAV in endemic, we selected the outbreak cases with identical sequences in VP1-2A junction region and compiled a panel of 42 isolates. The VP3-VP1-2A regions of the HAV capsid-coding genes were further sequenced and analyzed. The quasispecies distribution was evaluated by cloning the VP3 and VP1-2A genes in three clinical samples. Phylogenetic analysis demonstrated that the same genotyping results could be obtained whether using the complete VP3, VP1, or partial VP1-2A genes for analysis in this study, although some differences did exist. Most isolates clustered in sub-genotype IA, and fewer in sub-genotype IB. No amino acid mutations were found at the published neutralizing epitope sites, however, several unique amino acid substitutions in the VP3 or VP1 region were identified, with two amino acid variants closely located to the immunodominant site. Quasispecies analysis showed the mutation frequencies were in the range of 7.22 x 10(-4) -2.33 x 10(-3) substitutions per nucleotide for VP3, VP1, or VP1-2A. When compared with the consensus sequences, mutated nucleotide sites represented the minority of all the analyzed sequences sites. HAV replicated as a complex distribution of closely genetically related variants referred to as quasispecies, and were under negative selection. The results indicate that diverse HAV strains and quasispecies inside the viral populations are presented in China, with unique amino acid substitutions detected close to the immunodominant site, and that the possibility of antigenic escaping mutants cannot be ruled out and needs to be further analyzed.

  11. 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.

  12. Modeling of the rotavirus group C capsid predicts a surface topology distinct from other rotavirus species.

    PubMed

    Eren, Elif; Zamuda, Kimberly; Patton, John T

    2016-01-01

    Rotavirus C (RVC) causes sporadic gastroenteritis in adults and is an established enteric pathogen of swine. Because RVC strains grow poorly in cell culture, which hinders generation of virion-derived RVC triple-layered-particle (TLP) structures, we used the known Rotavirus A (RVA) capsid structure to model the human RVC (Bristol) capsid. Comparative analysis of RVA and RVC capsid proteins showed major differences at the VP7 layer, an important target region for vaccine development due to its antigenic properties. Our model predicted the presence of a surface extended loop in RVC, which could form a major antigenic site on the capsid. We analyzed variations in the glycosylation patterns among RV capsids and identified group specific conserved sites. In addition, our results showed a smaller RVC VP4 foot, which protrudes toward the intermediate VP6 layer, in comparison to that of RVA. Finally, our results showed major structural differences at the VP8* glycan recognition sites.

  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. 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

    2016-02-26

    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.

  15. Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71.

    PubMed

    Chiu, Cheng-Hsun; Chu, Chishih; He, Chao-Che; Lin, Tzou-Yien

    2006-06-01

    This study describes the potential use of attenuated Salmonella enterica serovar Typhimurium strains to express and deliver VP1 of enterovirus 71 (EV71) as a vaccination strategy to prevent EV71 infection in mice. When orally administered to BALB/c mice, both attenuated carrier strains, CNP101 and SL7207, were able to efficiently invade livers and spleens, while only the virulence plasmid-carrying strain SL7207 persisted for more than 30 days in these organs. A recombinant in vivo-regulated promoter expression plasmid expressing VP1 antigen of EV71 was constructed. The expression of the VP1, directed by the pagC promoter, in attenuated Salmonella was confirmed by Western blot hybridization. Both humoral and cellular immune responses were elicited in mice by oral immunization with such Salmonella-based VP1 vaccines. We evaluated the protective efficacy of the vaccines in mice using a maternal immunization protocol. With a lethal challenge, ICR newborn mice born to dams immunized with Salmonella-based VP1 vaccine showed a 50-60% survival; in contrast, none of the mice in the control group survived the challenge. Our data indicated that Salmonella-based VP1 subunit vaccines are a promising vaccine strategy in the prevention of EV71 infection.

  16. 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.

  17. 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

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

    PubMed

    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.

  19. Molecular characterization of a human group C rotavirus detected first in Turkey.

    PubMed

    Mitui, Marcelo Takahiro; Bozdayi, Gulendam; Dalgic, Buket; Bostanci, Ilknur; Nishizono, Akira; Ahmed, Kamruddin

    2009-10-01

    The present study was done to find out the prevalence of group B and C rotavirus infections in children with diarrhea presented at two major hospitals in Ankara, Turkey. Group B rotavirus was not found in any samples. One of 122 samples was positive for group C rotavirus. Phylogenetic analysis of genes for nonstructural protein NSP4, and structural proteins VP4, VP6, and VP7 confirmed the human origin of this strain. Similar to other human group C rotaviruses, one N-glycosylation site was predicted at amino acid residue 67 on the VP7 of strain GUP188. The genes of strain GUP188 were closely related to those of human group C rotavirus strain from the UK (Bristol) for NSP4, China (208 and Wu82) for VP4 and VP6, and from Colombia (Javeriana) for VP7, indicating that the Turkish group C rotavirus was unique and can serve as an additional reference strain for the molecular epidemiology of group C rotaviruses.

  20. 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

  1. Protect Your Child from Rotavirus Disease

    MedlinePlus

    ... will be protected from severe rotavirus disease. Most vaccinated children will not get sick from rotavirus at all. ... does not cover vaccines for your child, the Vaccines for Children (VFC) Program may be able to help. This ...

  2. Rotavirus vaccine - what you need to know

    MedlinePlus

    ... Vaccine Information Statement (VIS): www.cdc.gov/vaccines/hcp/vis/vis-statements/rotavirus.pdf . CDC review information ... known reason for it. www.cdc.gov/vaccines/hcp/vis/vis-statements/rotavirus.pdf Problems that could ...

  3. Innate cellular responses to rotavirus infection.

    PubMed

    Holloway, Gavan; Coulson, Barbara S

    2013-06-01

    Rotavirus is a leading cause of severe dehydrating diarrhoea in infants and young children. Following rotavirus infection in the intestine an innate immune response is rapidly triggered. This response leads to the induction of type I and type III interferons (IFNs) and other cytokines, resulting in a reduction in viral replication. Here we review the current literature describing the detection of rotavirus infection by pattern recognition receptors within host cells, the subsequent molecular mechanisms leading to IFN and cytokine production, and the processes leading to reduced rotavirus replication and the development of protective immunity. Rotavirus countermeasures against innate responses, and their roles in modulating rotavirus replication in mice, also are discussed. By linking these different aspects of innate immunity, we provide a comprehensive overview of the host's first line of defence against rotavirus infection. Understanding these processes is expected to be of benefit in improving strategies to combat rotavirus disease.

  4. Human rotavirus strain Wa downregulates NHE1 and NHE6 expressions in rotavirus-infected Caco-2 cells.

    PubMed

    Chen, Honglang; Song, Lijun; Li, Guixian; Chen, Wenfeng; Zhao, Shumin; Zhou, Ruoxia; Shi, Xiaoying; Peng, Zhenying; Zhao, Wenchang

    2017-03-13

    Rotavirus (RV) is the most common cause of severe gastroenteritis and fatal dehydration in human infants and neonates of different species. However, the pathogenesis of rotavirus-induced diarrhea is poorly understood. Secretory diarrhea caused by rotavirus may lead to a combination of excessive secretion of fluid and electrolytes into the intestinal lumen. Fluid absorption in the small intestine is driven by Na(+)-coupled transport mechanisms at the luminal membrane, including Na(+)/H(+) exchanger (NHE). Here, we performed qRT-PCR to detect the transcription of NHEs. Western blotting was employed for protein detection. Furthermore, immunocytochemistry was used to validate the NHE's protein expression. Finally, intracellular Ca(2+) concentration was detected by confocal laser scanning microscopy. The results demonstrated that the NHE6 mRNA and protein expressed in the human colon adenocarcinoma cell line (Caco-2). Furthermore, RV-Wa induced decreased expression of the NHE1 and NHE6 in Caco-2 cell in a time-dependent manner. In addition, intracellular Ca(2+) concentration in RV-Wa-infected Caco-2 cells was higher than that in the mock-infected cells. Furthermore, RV-Wa also can downregulate the expression of calmodulin (CaM) and calmodulin kinase II (CaMKII) in Caco-2 cells. These findings provides important insights into the mechanisms of rotavirus-induced diarrhea. Further studies on the underlying pathophysiological mechanisms that downregulate NHEs in RV-induced diarrhea are required.

  5. 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.

  6. Assessment of Stress Tolerance, Productivity, and Forage Quality in T1 Transgenic Alfalfa Co-overexpressing ZxNHX and ZxVP1-1 from Zygophyllum xanthoxylum

    PubMed Central

    Kang, Peng; Bao, Ai-Ke; Kumar, Tanweer; Pan, Ya-Qing; Bao, Zhulatai; Wang, Fei; Wang, Suo-Min

    2016-01-01

    Salinization, desertification, and soil nutrient deprivation are threatening the production of alfalfa (Medicago sativa L.) in northern China. We have previously generated T0 transgenic alfalfa co-overexpressing Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 genes with enhanced salt and drought tolerance. To further develop this excellent breeding material into the new forage cultivar, stress tolerance, productivity, and forage quality of T1 transgenic alfalfa (GM) were assessed in this study. The GM inherited the traits of salt and drought tolerance from T0 generation. Most importantly, co-overexpression of ZxNHX and ZxVP1-1 enhanced the tolerance to Pi deficiency in GM, which was associated with more Pi accumulation in plants. Meanwhile, T1 transgenic alfalfa developed a larger root system with increased root size, root dry weight and root/shoot ratio, which may be one important reason for the improvement of phosphorus nutrition and high biomass accumulation in GM under various conditions. GM also accumulated more crude protein, crude fiber, crude fat, and crude ash than wild-type (WT) plants, especially under stress conditions and in the field. More interestingly, the crude fat contents sharply dropped in WT (by 66-74%), whereas showed no change or decreased less in GM, when subjected to salinity, drought or low-Pi. Our results indicate that T1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 shows stronger stress tolerance, higher productivity and better forage quality. This study provides a solid foundation for creating the alfalfa cultivars with high yield, good quality and wide adaptability on saline, dry, and nutrient-deprived marginal lands of northern China. PMID:27833624

  7. 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.

  8. Immunoglobulin VH and VK genes of the BALB/c anti-foot-and-mouth disease virus (O1) VP1 response: cloning, characterization and transgenic mice.

    PubMed

    Conrad, U; Becker, K; Ziegner, M; Walter, G

    1991-11-01

    Hybridomas producing monoclonal antibodies of different isotypes were isolated from BALB/c antibody responses to the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) strain O1. According to antigen binding measured by ELISA a weak-binding (81D10, IgM) and a strong-binding antibody (113C12, IgG2a) were selected. As RNA sequencing of productive immunoglobulin VH and VK genes turned out, both chains of the weak-binding antibody (81D10) are encoded by germline (i.e. not mutated) genes whereas the gene encoding the strong-binding antibody (113C12) k chain is mutated at several sites. Therefore, rearranged VH and VK genes of 81D10 were cloned, expressed in immunoglobulin non-producing plasmacytoma cells, and mice transgenic for the 81D10 k gene were produced. These mice provide a first step in the development of a transgenic mouse model for genetical investigations in the affinity maturation of anti-viral immunoglobulin variable genes.

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

    PubMed Central

    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

  10. 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

    2016-08-27

    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.

  11. 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.

  12. Monitoring impact and effectiveness of rotavirus vaccination.

    PubMed

    Tate, Jacqueline E; Parashar, Umesh D

    2011-08-01

    Rotavirus infection is the most common cause of severe gastroenteritis in children <5 years of age globally. Since 2009, the WHO has recommended inclusion of rotavirus vaccine in the national immunization programs of all countries. Data regarding rotavirus vaccine impact and effectiveness under conditions of routine use are important for encouraging countries to implement vaccination programs. In the absence of a national rotavirus vaccination program in France, the IVANHOE study was initiated to determine the real-world impact and effectiveness of rotavirus vaccine following introduction in a limited geographic area. This study found a twofold reduction in rotavirus hospitalizations among children <2 years of age who were age-eligible to receive rotavirus vaccine and a 98% vaccine effectiveness, highlighting the health benefits of a vaccination program.

  13. [Hand-foot-mouth disease pathogen separation and EV71 VP1 gene analysis in Sanmenxia City, Henan Province, China].

    PubMed

    Wu, Shu-xing; Wu, Jing-fu; Yang, Jie; Wei, Hai-yan; Xu, Yu-ling; Huang, Xue-yong

    2014-11-01

    The aim of this study was to understand the enterovirus types and biological features of pediatric cases of HFMD in Sanmenxia City during 2011, and compare the latter to a cohort of healthy children. Stool samples of 55 cases of HFMD and 60 healthy children were collected for the isolation and identification of enteroviruses using RNA extraction and real-time RT-PCR assays. EV71 and CA16 were identified by nucleotide sequencing using virus-specific VP1 primers; for the other enteroviruses, 012/011 and 008/013 primers were used for amplification and sequencing. The results were analysed by sequence alignment with known sequences, and the characteristics of the EV71 VP1 gene were also analyzed. The detection rates for enteroviruses in cases of HFMD and healthy children were 52.73% (29/55) and 18.33% (11/60), respectively. Among these, there were 22 cases of EV71, four cases of CA16 and three cases of other enteroviruses in the cases with HFMD. Eleven healthy children had intestinal viruses, of which nine were Coxsackie B virus strains (81.82%, 9/11). Gene sequencing of the 19 EV71 strains illustrated that they were all subgenotype C4a, but the evolutionary tree showed an obvious clustering between cases from Lingbao City and Lushi County. This study demonstrates that the EV71 subgenotype C4a and CA16 strains were the most common cause of HFMD in Sanmenxia City in 2011, and that Coxsackie B strains were prevalent in healthy children. This finding may indicate that there is a widespread source of recessive infection in the community.

  14. 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

  15. 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

  16. New insights into rotavirus vaccines

    PubMed Central

    Mameli, Chiara; Fabiano, Valentina; Zuccotti, Gian Vincenzo

    2012-01-01

    Rotavirus vaccines have shown to be effective and well tolerated in clinical trials. However it’s crucial to point out that immunization occurs in “real-word” conditions different from ideal clinical trial settings. Thus, the impact of rotavirus vaccines in terms of effectiveness and safety needs to be evaluated in real-world conditions. Post-licensure data regarding vaccine impact, effectiveness and safety under routine use are now available and provide a “real-world view.” PMID:22699445

  17. 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

  18. Rotavirus and the Vaccine (Drops) to Prevent It

    MedlinePlus

    ... PARENT S | DISEASES and the VACCINES THAT PREVENT THEM | Rotavirus and the Vaccine (Drops) to Prevent It Last ... February 2014 The best way to protect against rotavirus is by getting the rotavirus vaccine. Doctors recommend ...

  19. Genetic analysis of the VP1 region of enterovirus 71 reveals the emergence of genotype A in central China in 2008.

    PubMed

    Yu, Haiyang; Chen, Wei; Chang, Hongwei; Tang, Renshu; Zhao, Jun; Gan, Lin; Liu, Boyu; Chen, Jason; Wang, Mingli

    2010-08-01

    Enterovirus 71 (EV71) strains from children were characterized by full-length VP1 nucleotide sequencing. Out of 22 clinical specimens, five isolates identified as EV71 were recovered by virus isolation. The VP1 sequences of the five isolates had more than 97.4% sequence identity with prototype virus BrCr, clustering in the genotype A lineage. This represents the first record of genotype A EV71 in China since the BrCr prototype strain was discovered in the USA in 1969.

  20. Acute effects of rotavirus and malnutrition on intestinal barrier function in neonatal piglets

    PubMed Central

    Jacobi, Sheila K; Moeser, Adam J; Blikslager, Anthony T; Rhoads, J Marc; Corl, Benjamin A; Harrell, Robert J; Odle, Jack

    2013-01-01

    AIM: To investigate the effect of protein-energy malnutrition on intestinal barrier function during rotavirus enteritis in a piglet model. METHODS: Newborn piglets were allotted at day 4 of age to the following treatments: (1) full-strength formula (FSF)/noninfected; (2) FSF/rotavirus infected; (3) half-strength formula (HSF)/noninfected; or (4) HSF/rotavirus infected. After one day of adjustment to the feeding rates, pigs were infected with rotavirus and acute effects on growth and diarrhea were monitored for 3 d and jejunal samples were collected for Ussing-chamber analyses. RESULTS: Piglets that were malnourished or infected had lower body weights on days 2 and 3 post-infection (P < 0.05). Three days post-infection, marked diarrhea and weight loss were accompanied by sharp reductions in villus height (59%) and lactase activity (91%) and increased crypt depth (21%) in infected compared with non-infected pigs (P < 0.05). Malnutrition also increased crypt depth (21%) compared to full-fed piglets. Villus:crypt ratio was reduced (67%) with viral infection. There was a trend for reduction in transepithelial electrical resistance with rotavirus infection and malnutrition (P = 0.1). 3H-mannitol flux was significantly increased (50%; P < 0.001) in rotavirus-infected piglets compared to non-infected piglets, but there was no effect of nutritional status. Furthermore, rotavirus infection reduced localization of the tight junction protein, occludin, in the cell membrane and increased localization in the cytosol. CONCLUSION: Overall, malnutrition had no additive effects to rotavirus infection on intestinal barrier function at day 3 post-infection in a neonatal piglet model. PMID:23964143

  1. 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.

  2. Repression of the LEAFY COTYLEDON 1/B3 regulatory network in plant embryo development by VP1/ABSCISIC ACID INSENSITIVE 3-LIKE B3 genes.

    PubMed

    Suzuki, Masaharu; Wang, Heidi H-Y; McCarty, Donald R

    2007-02-01

    Plant embryo development is regulated by a network of transcription factors that include LEAFY COTYLEDON 1 (LEC1), LEC1-LIKE (L1L), and B3 domain factors, LEAFY COTYLEDON 2 (LEC2), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) of Arabidopsis (Arabidopsis thaliana). Interactions of these genes result in temporal progression of overlapping B3 gene expression culminating in maturation and desiccation of the seed. Three VP1/ABI3-LIKE (VAL) genes encode B3 proteins that include plant homeodomain-like and CW domains associated with chromatin factors. Whereas val monogenic mutants have phenotypes similar to wild type, val1 val2 double-mutant seedlings form no leaves and develop embryo-like proliferations in root and apical meristem regions. In a val1 background, val2 and val3 condition a dominant variegated leaf phenotype revealing a VAL function in vegetative development. Reminiscent of the pickle (pkl) mutant, inhibition of gibberellin biosynthesis during germination induces embryonic phenotypes in val1 seedlings. Consistent with the embryonic seedling phenotype, LEC1, L1L, ABI3, and FUS3 are up-regulated in val1 val2 seedlings in association with a global shift in gene expression to a profile resembling late-torpedo-stage embryogenesis. Hence, VAL factors function as global repressors of the LEC1/B3 gene system. The consensus binding site of the ABI3/FUS3/LEC2 B3 DNA-binding domain (Sph/RY) is strongly enriched in the promoters and first introns of VAL-repressed genes, including the early acting LEC1 and L1L genes. We suggest that VAL targets Sph/RY-containing genes in the network for chromatin-mediated repression in conjunction with the PKL-related CHD3 chromatin-remodeling factors.

  3. 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.

  4. Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

    PubMed

    Wen, X J; Cheng, A C; Wang, M S; Jia, R Y; Zhu, D K; Chen, S; Liu, M F; Liu, F; Chen, X Y

    2014-09-01

    Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.

  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. 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.

  7. Temporal and geographical distributions of human rotavirus serotypes, 1983 to 1988.

    PubMed Central

    Beards, G M; Desselberger, U; Flewett, T H

    1989-01-01

    Between 1983 and 1988, subgroups and serotypes were determined for 907 of 1,084 clinical specimens of rotaviruses collected in various countries of Europe, North and South America, Africa, and Asia. Enhanced enzyme immunoassays based on monoclonal antibodies specific for rotavirus proteins VP6 and VP7 were used. Significant differences in the prevalent serotypes were detected from year to year in the United Kingdom and Brazil and also in different countries during the same year. Throughout the study, rotavirus serotype 1 was detected most often (53.8%), followed in frequency by serotype 2 (17.8%), serotype 3 (12.1%), serotype 4 (11.1%), and serotypes other than 1 to 4 (5.1%). No individual serotype was found to predominate consistently in any one location. In the United Kingdom, rotavirus serotypes varied in prevalence in a regular but not predictable way. We suggest that a similar epidemiology might be found in other settings. Seventeen unusual strains were detected. Of these, five strains did not react with reference monoclonal antibodies specific for subgroup I and subgroup II, but they reacted with rotavirus group A-specific polyclonal and monoclonal antibodies; four strains were of subgroup II, serotype 2, and at least one had a "long" electropherotype; two strains were of subgroup I, serotype 2 with a long electropherotype; and one strain was of subgroup I, serotype 3. Five group C rotaviruses were detected. Images PMID:2556435

  8. The impact of rotavirus disease in Venezuela.

    PubMed

    Pérez-Schael, I

    1996-09-01

    Information concerning the disease burden of rotavirus, particularly in developing countries, has important implications for the use and for monitoring the impact of rotavirus vaccines. Although rotavirus has been recognized as the most frequent cause of hospitalization in the world, national estimates and specific information about the incidence of hospitalization for rotavirus gastroenteritis are very limited. Consequently, estimates of the incidence of hospitalization among children during the first 2 years of life in Venezuela were determined by extrapolation of data from a community-based study carried out in Caracas.

  9. Rotavirus Infection: A Disease of the Past?

    PubMed

    Dennehy, Penelope H

    2015-12-01

    Rotavirus infection is the most common cause of severe diarrhea disease in infants and young children worldwide. Vaccination is the only control measure likely to have a significant impact on the incidence of severe disease. Rotavirus vaccines have reduced the burden of disease in the United States and Europe and vaccine programs are being introduced in Asia and Africa where it is hoped that vaccine will have significant impact on severe infection. Long-term monitoring and strain surveillance are needed to assess the effects of rotavirus immunization programs and to determine whether changes in strain ecology will affect rotavirus vaccine effectiveness.

  10. 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

  11. Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates.

    PubMed

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

    2012-09-21

    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 Escherichia 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.

  12. Lack of impact of rapid identification of rotavirus-infected patients on nosocomial rotavirus infections.

    PubMed

    Dennehy, P H; Tente, W E; Fisher, D J; Veloudis, B A; Peter, G

    1989-05-01

    The efficacy of rapid identification of rotavirus-infected patients in the control of nosocomial rotavirus infections on an infant and young toddler ward by use of a rotavirus antigen detection test on stool from patients with diarrhea was evaluated by comparing the rate of nosocomial rotavirus infection in children during two separate 5-week periods in the winters of 1984 and 1986. In contrast to 1984 rapid rotavirus antigen testing by latex agglutination of stool from patients with diarrhea was instituted in 1986, in addition to testing for rotavirus by enzyme immunoassay, to determine whether use of rapid antigen testing resulted in an increased incidence of appropriate isolation and a decrease in nosocomial infections. In 1986 rapid identification of rotavirus resulted in an increase in hospitalization of rotavirus-infected patients in single bed rooms from 68% to 100% (P = 0.02, chi square test) but no significant increase in the use of enteric precautions for these patients. The total number of cases of nosocomial rotavirus infection in the two periods did not differ. In both periods 11 cases occurred; the nosocomial infection rate in 1984 was 18.9 cases/1000 days of exposure whereas in 1986 it was 20.2 cases/1000 days. These findings indicate that the use of rapid rotavirus antigen testing of patients with diarrhea is not of appreciable benefit in preventing the nosocomial spread of rotavirus to infants on the ward.

  13. Molecular analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele.

    PubMed

    Martella, V; Ciarlet, M; Pratelli, A; Arista, S; Terio, V; Elia, G; Cavalli, A; Gentile, M; Decaro, N; Greco, G; Cafiero, M A; Tempesta, M; Buonavoglia, C

    2003-12-01

    We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.

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

    PubMed Central

    Ward, R L; Ashley, C S

    1980-01-01

    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 1.35 to about 1.37 g/ml and a reduction in their sedimentation coefficients. Sodium dodecyl 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. PMID:6250474

  15. 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.

  16. Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

    PubMed

    Juárez, Paloma; Presa, Silvia; Espí, Joaquín; Pineda, Benito; Antón, María T; Moreno, Vicente; Buesa, Javier; Granell, Antonio; Orzaez, Diego

    2012-04-01

    Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

  17. 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.

  18. Opsoclonus-myoclonus syndrome following rotavirus gastroenteritis.

    PubMed

    Gurkas, Esra; Gucuyener, Kivilcim; Yılmaz, Unsal; Havalı, Cengiz; Demir, Ercan

    2014-12-01

    Opsoclonus-myoclonus syndrome (OMS) is a rare neurologic disorder characterized by opsoclonus, myoclonus, ataxia and behavioral disturbance. In the pathogenesis, an autoimmune process with infectious or paraneoplastic trigger has been suggested. We describe the case of a 22-month-old girl with OMS following rotavirus gastroenteritis. Rotavirus should be considered in the differential diagnosis of OMS in children.

  19. Genetic engineering of rotaviruses by reverse genetics.

    PubMed

    Komoto, Satoshi; Taniguchi, Koki

    2013-07-01

    The rotavirus genome is composed of 11 gene segments of dsRNA. A recent breakthrough in the field of rotaviruses is the development of a reverse genetics system for generating recombinant rotaviruses possessing a gene segment derived from cloned cDNA. Although this approach is a helper virus-driven system that is technically limited and gives low levels of recombinant viruses, it allows alteration of the rotavirus genome, thus contributing to our understanding of these medically important viruses. So far, this approach has successfully been applied to three of the 11 viral segments in our laboratory and others, and the efficiency of recovery of recombinant viruses has been improved. However, we are still waiting for the development of a helper virus-free reverse genetics system for generating an infectious rotavirus entirely from cDNAs, as has been achieved for other members of the Reoviridae family.

  20. Asymptomatic rotavirus infections in day care centers.

    PubMed Central

    Barrón-Romero, B L; Barreda-González, J; Doval-Ugalde, R; Zermeño-Eguia Liz, J; Huerta-Peña, M

    1985-01-01

    Rotaviruses and other enteropathogenic agents were detected in 288 (42.1%) of 684 children in day care centers of Instituto Politecnico Nacional in Mexico City. The same agents were also found in 114 (37.7%) of 302 adults directly involved in the care of the children. The study was carried out from July to December 1982 and from July 1983 to February 1984. Rotaviruses were the main enteropathogenic agents found and were detected in 169 (29.9%) of 564 children without diarrhea and in 34 (28.3%) of 120 children with diarrhea. These viruses were present in 62 (20.5%) of 302 adults without diarrhea. Of all rotavirus-positive individuals, 20% were also positive for other enteropathogens. All these observations indicate that asymptomatic rotavirus infections are not a rare event in children and that diarrhea caused by rotavirus infections is only one of the expressions of their presence. PMID:2991328

  1. [Impact of rotavirus vaccines in developing countries].

    PubMed

    Delacour, H

    2009-08-01

    Rotaviruses discovered in 1973 are the most common cause of severe diarrheal disease in infants and young children world-wide. Annually rotavirus infections are estimated to cause the deaths of more than 600,000 children under the age of 5 years with more than 90% of fatalities occurring in developing countries. In 2006 two live oral attenuated rotavirus vaccines were licensed: the monovalent human rotavirus vaccine (RotarixT) and the pentavalent bovine-human, reassortant vaccine (RotaTeqT). Both vaccines demonstrated excellent safety and protective effectiveness in large pre-licensing trials conducted in Europe, the United States and Latin America. Several countries in Latin and Central America have already decided to include rotavirus vaccines into their national immunization program. African and Asiatic countries have postponed their decisions pending the results of further studies.

  2. 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.

  3. Burden of Norovirus and Rotavirus in Children after Rotavirus Vaccine Introduction, Cochabamba, Bolivia

    PubMed Central

    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

  4. 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

    2015-10-02

    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.

  5. Effectiveness of Monovalent and Pentavalent Rotavirus Vaccine

    PubMed Central

    Immergluck, Lilly Cheng; Held, Melissa; Jain, Shabnam; Chan, Trisha; Grizas, Alexandra P.; Khizer, Saadia; Barrett, Carol; Quaye, Osbourne; Mijatovic-Rustempasic, Slavica; Gautam, Rashi; Bowen, Michael D.; Moore, Jessica; Tate, Jacqueline E.; Parashar, Umesh D.; Vázquez, Marietta

    2013-01-01

    OBJECTIVE: Previous US evaluations have not assessed monovalent rotavirus vaccine (RV1, a G1P[8] human rotavirus strain) effectiveness, because of its later introduction (2008). Using case-control methodology, we measured the vaccine effectiveness (VE) of the 2-dose RV1 and 3-dose pentavalent vaccine (RV5) series against rotavirus disease resulting in hospital emergency department or inpatient care. METHODS: Children were eligible for enrollment if they presented to 1 of 5 hospitals (3 in Georgia, 2 in Connecticut) with diarrhea of ≤10 days’ duration during January through June 2010 or 2011, and were born after RV1 introduction. Stools were collected; immunization records were obtained from providers and state electronic immunization information system (IIS). Case-subjects (children testing rotavirus antigen-positive) were compared with 2 control groups: children testing rotavirus negative and children selected from IIS. RESULTS: Overall, 165 rotavirus-case subjects and 428 rotavirus-negative controls were enrolled. Using the rotavirus-negative controls, RV1 VE was 91% (95% confidence interval [CI] 80 to 95) and RV5 VE was 92% (CI 75 to 97) among children aged ≥8 months. The RV1 VE against G2P[4] disease was high (94%, CI 78 to 98), as was that against G1P[8] disease (89%, CI 70 to 96). RV1 effectiveness was sustained among children aged 12 through 23 months (VE 91%; CI 75 to 96). VE point estimates using IIS controls were similar to those using rotavirus-negative controls. CONCLUSIONS: RV1 and RV5 were both highly effective against severe rotavirus disease. RV1 conferred sustained protection during the first 2 years of life and demonstrated high effectiveness against G2P[4] (heterotypic) disease. PMID:23776114

  6. 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

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

    PubMed

    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.

  8. 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.

  9. 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.

  10. 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

  11. Mean Platelet Volume as a Negative Marker of Inflammation in Children with Rotavirus Gastroenteritis

    PubMed Central

    Tanju, Celik; Ekrem, Güler; Berksoy Emel, Atas; Nur, Arslan

    2014-01-01

    Objective: Mean platelet volume (MPV) is a determinant of inflammation. The aim of the present study was to investigate the MPV levels in children with rotavirus gastroenteritis and to evaluate the possible relationship between MPV and severity of gastroenteritis. Methods: Children diagnosed with acute rotavirus gastroenteritis and healthy controls were enrolled in this study. Patients were classified into three disease severity groups based on their Vesikari score (<7 mild, 7-10 moderate and >11 severe). Rotavirus was determined in fresh stool samples using ELISA test. Leukocyte and platelet counts, MPV and C-reactive protein (CRP) levels were assessed for all children. Findings: A total of 151 patients with rotavirus gastroenteritis (mean age 2.41± 0.14 years) and 80 healthy controls (mean age 2.63±0.22 years, P=0.129) were enrolled. MPV levels of children with rotavirus gastroenteritis were significantly lower than those of healthy peers (7.48±0.04 vs 7.79±0.07 fl, P=0.000). MPV levels were not significantly different among three gastroenteritis groups. Gastroenteritis score was positively correlated with leukocyte (r=0.670, P<0.01) and platelet count (r=0.159, P<0.05) and CRP level (r=0.256, P<0.01) in patients group. MPV was inversely correlated with platelet count. There was no significant correlation between MPV and gastroenteritis score. Conclusion: MPV levels were significantly lower in children with rotavirus gastroenteritis compared to controls. MPV can be used as a negative acute phase reactant in children with rotavirus gastroenteritis. PMID:25793071

  12. 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

  13. 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

    2016-04-30

    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.

  14. Foot-and-mouth disease marker vaccine: cattle protection with a partial VP1 G-H loop deleted virus antigen.

    PubMed

    Fowler, V L; Bashiruddin, J B; Maree, F F; Mutowembwa, P; Bankowski, B; Gibson, D; Cox, S; Knowles, N; Barnett, P V

    2011-10-26

    Contrary to the dogma that the VP1 G-H loop is essential for FMD vaccine efficacy, it has been previously shown that foot-and-mouth disease 146s antigen containing heterologous VP1 G-H loops confers complete protection in pigs and cattle. Moreover, serological evaluation of cattle vaccinated with an antigen lacking a large proportion of the VP1 G-H loop indicated that these animals should be protected against infection with FMD. Absence of this loop provides opportunity for the development of an FMD negative marker vaccine, allowing infection to be detected by antibodies against this missing region. Cattle vaccinated with this negative marker vaccine were fully protected following virus challenge 28 days post vaccination as determined by the absence of generalised lesions on their feet. Furthermore, use of our improved differentiation ELISA identified animals exposed to infection as early as 7 days post-challenge. We thus demonstrate, for the first time, the ability of this FMD negative marker vaccine to fully protect cattle from experimental challenge and rapidly distinguish animals that are subsequently exposed to infection.

  15. Rotavirus infections in Galapagos sea lions.

    PubMed

    Coria-Galindo, Elsa; Rangel-Huerta, Emma; Verdugo-Rodríguez, Antonio; Brousset, Dulce; Salazar, Sandie; Padilla-Noriega, Luis

    2009-07-01

    Group A rotaviruses infect and cause diarrhea in the young of a broad range of terrestrial mammals, but it is unknown, to our knowledge, whether they infect marine mammals. During February and March of 2002 and 2003, we collected 125 serum samples and 18 rectal swab samples from Galapagos sea lion pups (GSL, Zalophus wollebaeki), and 22 serum samples from Galapagos fur seal pups (GFS, Arctocephalus galapagoensis) from nine islands of the Galapagos archipelago, Ecuador. Sera were tested for antibodies (immunoglobulin G [IgG]) to rotavirus by an enzyme immunoassay using rhesus rotavirus as the capture antigen. In addition, rectal swabs were analyzed for the presence of rotavirus genomic double-stranded RNA by silver-stained polyacrylamide gel electrophoresis. Antibodies to rotavirus were detected in 27 GSL pups (22%) and five GFS pups (23%), and rotavirus RNA was detected in the fecal sample from one GSL pup (6%). These results provide the first evidence that rotavirus infections are prevalent at an early age in Galapagos sea lions and Galapagos fur seals.

  16. Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface.

    PubMed Central

    Rosenfeld, S J; Yoshimoto, K; Kajigaya, S; Anderson, S; Young, N S; Field, A; Warrener, P; Bansal, G; Collett, M S

    1992-01-01

    Capsids of the B19 parvovirus are composed of major (VP2; 58 kD) and minor (VP1; 83 kD) structural proteins. These proteins are identical except for a unique 226 amino acid region at the amino terminus of VP1. Previous immunization studies with recombinant empty capsids have demonstrated that the presence of VP1 was required to elicit virus-neutralizing antibody activity. However, to date, neutralizing epitopes have been identified only on VP2. Crystallographic studies of a related parvovirus (canine parvovirus) suggested the unique amino-terminal portion of VP1 assumed an internal position within the viral capsid. To determine the position of VP1 in both empty capsids and virions, we expressed a fusion protein containing the unique region of VP1. Antisera raised to this protein recognized recombinant empty capsids containing VP1 and VP2, but not those containing VP2 alone, in an enzyme-linked immunosorbent assay. The antisera immunoprecipitated both recombinant empty capsids and human plasma-derived virions, and agglutinated the latter as shown by immune electron microscopy. The sera contained potent neutralizing activity for virus infectivity in vitro. These data indicate that a portion of the amino terminus of VP1 is located on the virion surface, and that this region contains intrinsic neutralizing determinants. The external location of the VP1-specific tail may provide a site for engineered heterologous epitope presentation in novel recombinant vaccines. Images PMID:1376332

  17. A Single Mutation in the VP1 of Enterovirus 71 Is Responsible for Increased Virulence and Neurotropism in Adult Interferon-Deficient Mice

    PubMed Central

    Caine, Elizabeth A.; Moncla, Louise H.; Ronderos, Monica D.; Friedrich, Thomas C.

    2016-01-01

    ABSTRACT Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children, who develop symptoms ranging from painful blisters around their mouths and hands to neurological complications. Many members of the genus Enterovirus (family Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse-adapted EV71 (mEV71) capable of infecting 12-week-old interferon receptor-deficient AG129 mice and used the model to evaluate the efficacy of candidate HFMD vaccines. Here, we present data investigating the genetic correlates of EV71 adaptation and characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was shown to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real-time reverse transcription (RT)-PCR confirmed the presence of mEV71 in the sera and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation. IMPORTANCE EV71 is a reemerging pathogen, and little is known about the genetic determinants involved in its pathogenesis. The absence of animal models has contributed to this lack of knowledge. The data presented here improve upon the existing animal models by characterizing a mouse-adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5-day-old BALB/c mice, neurotropic disease did not

  18. Genomic characterization of porcine rotaviruses in Italy.

    PubMed

    Martella, V; Pratelli, A; Greco, G; Tempesta, M; Ferrari, M; Losio, M N; Buonavoglia, C

    2001-01-01

    A total of 23 rotavirus strains isolated from pigs were analyzed. Twenty strains had been isolated from diarrheic piglets from an outbreak that occurred in northern Italy in 1983. Three strains had been isolated in 1984 from swine herds located in distinct areas of northern Italy. All 23 strains were characterized as type G6P[5] by PCR. The isolation from piglets of rotaviruses displaying typical bovine G- and P-type specificities points out the high frequency of rotavirus transmission between cattle and pigs.

  19. Whole genomic constellation of the first human G8 rotavirus strain detected in Japan.

    PubMed

    Agbemabiese, Chantal Ama; Nakagomi, Toyoko; Doan, Yen Hai; Nakagomi, Osamu

    2015-10-01

    Human G8 Rotavirus A (RVA) strains are commonly detected in Africa but are rarely detected in Japan and elsewhere in the world. In this study, the whole genome sequence of the first human G8 RVA strain designated AU109 isolated in a child with acute gastroenteritis in 1994 was determined in order to understand how the strain was generated including the host species origin of its genes. The genotype constellation of AU109 was G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analyses of the 11 genome segments revealed that its VP7 and VP1 genes were closely related to those of a Hungarian human G8P[14] RVA strain and these genes shared the most recent common ancestors in 1988 and 1982, respectively. AU109 possessed an NSP2 gene closely related to those of Chinese sheep and goat RVA strains. The remaining eight genome segments were closely related to Japanese human G2P[4] strains which circulated around 1985-1990. Bayesian evolutionary analyses revealed that the NSP2 gene of AU109 and those of the Chinese sheep and goat RVA strains diverged from a common ancestor around 1937. In conclusion, AU109 was generated through genetic reassortment event where Japanese DS-1-like G2P[4] strains circulating around 1985-1990 obtained the VP7, VP1 and NSP2 genes from unknown ruminant G8 RVA strains. These observations highlight the need for comprehensive examination of the whole genomes of RVA strains of less explored host species.

  20. Evaluation of a human group a rotavirus assay for on-site detection of bovine rotavirus.

    PubMed

    Maes, Roger K; Grooms, Daniel L; Wise, Annabel G; Han, Cunqin; Ciesicki, Valerie; Hanson, Lora; Vickers, Mary Lynne; Kanitz, Charles; Holland, Robert

    2003-01-01

    Neonatal diarrhea induced by bovine group A rotavirus causes significant economic loss in the dairy and beef industry due to increased morbidity and mortality, treatment costs, and reduced growth rates. The objective of this study was to evaluate a human group A rotavirus assay (ImmunoCardSTAT Rotavirus [ICS-RV]) as an on-site diagnostic test for bovine rotavirus. When used with a collection of bovine diarrhea samples submitted to the Virology Section of the Diagnostic Center for Population and Animal Health at Michigan State University and compared to a bovine group A rotavirus-specific reverse transcription-PCR (RT-PCR), the ICS-RV assay had a sensitivity and specificity of 87.0 and 93.6%, respectively. A commercially available group A rotavirus enzyme-linked immunosorbent assay (ELISA) (Pathfinder; Sanofi Diagnostics, Redmond, Wash.), when used with the same fecal sample collection and compared to the same RT-PCR, had a sensitivity and specificity of 78.3 and 67.7%, respectively. Subsequently, the ICS-RV assay, RT-PCR, and a different commercially available group A rotavirus ELISA (Rotaclone; Meridian Diagnostics, Cincinnati, Ohio) were used to evaluate fecal samples collected from neonatal calves experimentally infected with bovine rotavirus. When diarrheic fecal samples that were positive for bovine rotavirus by RT-PCR were evaluated, the ICS-RV assay and the Rotaclone assay detected bovine rotavirus 85 and 95% of the time, respectively. Based on these studies, the ICS-RV assay appears to be an excellent test for detecting group A bovine rotaviruses. This assay may be useful as an on-site diagnostic test for veterinarians as an aid in the management of bovine neonatal diarrhea.

  1. Rotavirus vaccine and health-care utilization for rotavirus gastroenteritis in Tsu City, Japan

    PubMed Central

    Kamiya, Hajime; Suga, Shigeru; Nagao, Mizuho; Ichimi, Ryoji; Fujisawa, Takao; Umemoto, Masakazu; Tanaka, Takaaki; Ito, Hiroaki; Tanaka, Shigeki; Ido, Masaru; Taniguchi, Koki; Ihara, Toshiaki; Nakano, Takashi

    2016-01-01

    Background Rotavirus vaccines were introduced in Japan in November 2011. We evaluated the subsequent reduction of the health-care burden of rotavirus gastroenteritis. Methods We conducted active surveillance for rotavirus gastroenteritis among children under 5 years old before and after the vaccine introduction. We surveyed hospitalization rates for rotavirus gastroenteritis in children in Tsu City, Mie Prefecture, Japan, from 2007 to 2015 and surveyed the number of outpatient visits at a Tsu City clinic from 2010 to 2015. Stool samples were obtained for rotavirus testing and genotype investigation. We assessed rotavirus vaccine coverage for infants living in Tsu City. Results In the pre-vaccine years (2007–2011), hospitalization rates for rotavirus gastroenteritis in children under 5 years old were 5.5, 4.3, 3.1 and 3.9 cases per 1000 person-years, respectively. In the post-vaccine years (2011–2015), the rates were 3.0, 3.5, 0.8 and 0.6 cases per 1000 person-years, respectively. The hospitalization rate decreased significantly in the 2013–2014 and 2014–2015 seasons compared to the average of the seasons before vaccine introduction (P < 0.0001). In one pre-vaccine year (2010–2011), the number of outpatient visits due to the rotavirus infection was 66. In the post-vaccine years (2011–2015), the numbers for each season was 23, 23, 7 and 5, respectively. The most dominant rotavirus genotype shifted from G3P[8] to G1P[8] and to G2P[4]. The coverage of one dose of rotavirus vaccine in Tsu City was 56.5% in 2014. Conclusion After the vaccine introduction, the hospitalization rates and outpatient visits for rotavirus gastroenteritis greatly decreased. PMID:28246579

  2. Effect of monovalent rotavirus vaccine on rotavirus disease burden and circulating rotavirus strains among children in Morocco.

    PubMed

    Benhafid, Mohammed; Elomari, Nezha; Azzouzi Idrissi, Meryem; Rguig, Ahmed; Gentsch, Jon R; Parashar, Umesh; Elaouad, Rajae

    2015-06-01

    Rotarix(TM) vaccine was introduced into the National Program of Immunization of Morocco in October 2010, reaching quickly 87% of the target population of children nationally. The incidence of rotavirus gastroenteritis and the prevalence of circulating rotavirus strains has been monitored in three sentinel hospitals since June 2006. The average percentage of rotavirus positive cases among all children under 5 years old hospitalized for gastroenteritis during the pre-vaccine period (2006-2010) was 44%. This percentage dropped to 29%, 15% and 24% in the 3 years post vaccine introduction (2011, 2012 and 2013), which is a decline of 34%, 66%, and 45%, respectively. Declines in prevalence were greatest among children 0-1 years of age (53%) and were most prominent during the winter and autumn rotavirus season. The prevalence of the G2P[4] and G9P[8] genotype sharply increased in the post vaccine period (2011-2013) compared to the previous seasons (2006-2010). Rotavirus vaccines have reduced greatly the number of children hospitalized due to rotavirus infection at the three sentinel hospitals; it is however unclear if the predominance of G2P[4] and G9P[8] genotypes is related to the vaccine introduction, or if this is attributable to normal genotype fluctuations. Continued surveillance will be pivotal to answer this question in the future.

  3. Characterization of the neutralizing epitopes of VP7 of the Gottfried strain of porcine rotavirus.

    PubMed

    Kang, S Y; Benfield, D A; Gorziglia, M; Saif, L J

    1993-09-01

    The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.

  4. Molecular epidemiology of rotaviruses in India.

    PubMed

    Broor, Shobha; Ghosh, Dhrubaa; Mathur, Purva

    2003-08-01

    Rotaviruses cause an estimated 140 million cases of gastroenteritis and 800,000 deaths in children between the ages of 6 months to 2 yr in developing countries. In India, one of every 250 children or about 100-150,000 children die of rotavirus diarrhoea each year. The prevalence of rotavirus diarrhoea in India has been found to vary from 5-71 per cent in hospitalized children <5 yr of age with acute gastroenteritis. The seasonal variation of rotavirus diarrhoea in India varies in different geographical regions with high incidence in winter months at low relative humidity in north India. The distinctive features of rotavirus infection in India include the occurrence of severe disease at an early age and common neonatal rotavirus infections which are often asymptomatic. Rotavirus shows genetic and antigenic diversity in terms of subgroup, electropherotypes and G and P serotypes/genotypes. There are a few studies in terms of prevalence of different antigenic and genetic variants from various regions of India. In most studies on subgroup distribution from India a higher prevalence of subgroup II was reported compared to subgroup I. Electropherotyping has also demonstrated that a number of multiple electropherotypes co-circulate at one time in a particular community leading to extensive genomic variation and the appearance of new strains which may become the predominant electropherotype during the peak season. The most common G types reported from India are G1 and G2 and P types are P[4] and P[8]. A significant number of children also have mixed rotavirus infections. G9 strains are also quite commonly seen in Indian children. In addition P6 strains of probable bovine origin have been reported from India. A novel neonatal strain P type 11 human rotavirus (116 E) was isolated from neonates in Delhi, the VP4 of which was closely related to the bovine serotype G10P[11] strain B223 and VP7 was closely related to the human serotype G9 strain. Another neonatal strain G10P[11

  5. Detection of antigenically distinct rotaviruses from infants.

    PubMed Central

    Dimitrov, D H; Estes, M K; Rangelova, S M; Shindarov, L M; Melnick, J L; Graham, D Y

    1983-01-01

    Antigenically distinct rotaviruses, i.e., viruses morphologically identical to conventional rotaviruses by electron microscopy, yet lacking the common group antigen(s) detected by an enzyme-linked immunosorbent assay, were found in 2 of 51 fecal samples from Bulgarian infants with rotavirus gastroenteritis. These antigenically distinct viruses contained 11 segments of double-stranded RNA, but they demonstrated a unique RNA migration profile after electrophoresis of the genome RNA in polyacrylamide gels. This report confirms the presence of a new group of rotaviruses in humans. The significance of these viruses is currently unknown, and specific diagnostic tests must be developed for epidemiological studies to determine their role as human and veterinary pathogens and to evaluate their impact on proposed vaccine development programs. Images PMID:6307873

  6. 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

  7. Discovery of rotavirus: Implications for child health.

    PubMed

    Bishop, Ruth

    2009-10-01

    For centuries, acute diarrhea has been a major worldwide cause of death in young children, and until 1973, no infectious agents could be identified in about 80% of patients admitted to hospital with severe dehydrating diarrhea. In 1973 Ruth Bishop, Geoffrey Davidson, Ian Holmes, and Brian Ruck identified abundant particles of a 'new' virus (rotavirus) in the cytoplasm of mature epithelial cells lining duodenal villi and in feces, from such children admitted to the Royal Children's Hospital, Melbourne. Rotaviruses have now been shown to cause 40-50% of severe acute diarrhea in young children worldwide in both developing and developed countries, and > 600 000 young children die annually from rotavirus disease, predominantly in South-East Asia and sub-Saharan Africa. Longitudinal surveillance studies following primary infection in young children have shown that rotavirus reinfections are common. However the immune response that develops after primary infection is protective against severe symptoms on reinfection. This observation became the basis for development of live oral rotavirus vaccines. Two safe and effective vaccines are now licensed in 100 countries and in use in 17 countries (including Australia). Rotarix (GSK) is a single attenuated human rotavirus, representative of the most common serotype identified worldwide (G1P[8]). RotaTeq (Merck) is a pentavalent mixture of naturally attenuated bovine/human rotavirus reassortants representing G1, G2, G3, G4, and P(8) serotypes. Preliminary surveillance of the numbers of children requiring hospitalization for severe diarrhea, in USA, Brazil, and Australia, after introduction of these vaccines, encourages the hope that rotavirus infection need no longer be a threat to young children worldwide.

  8. Impact of Rotavirus Vaccine on Premature Infants

    PubMed Central

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

    2014-01-01

    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

  9. 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.).

  10. Feline Origin of Rotavirus Strain, Tunisia, 2008

    PubMed Central

    Fredj, Mouna Ben Hadj; Heylen, Elisabeth; Zeller, Mark; Fodha, Imene; Benhamida-Rebai, Meriam; Van Ranst, Marc; Matthijnssens, Jelle

    2013-01-01

    In Tunisia in 2008, an unusual G6P[9] rotavirus, RVA/human-wt/TUN/17237/2008/G6P[9], rarely found in humans, was detected in a child. To determine the origin of this strain, we conducted phylogenetic analyses and found a unique genotype constellation resembling rotaviruses belonging to the feline BA222-like genotype constellation. The strain probably resulted from direct cat-to-human transmission. PMID:23631866

  11. 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

  12. Immunogenicity of porcine P[6], P[7]-specific △VP8* rotavirus subunit vaccines with a tetanus toxoid universal T cell epitope.

    PubMed

    Wen, Xiaobo; Wei, Xiaoman; Ran, Xuhua; Ni, Hongbo; Cao, Si; Zhang, Yao

    2015-08-26

    Currently, commercial porcine rotavirus vaccines remain varied limitations. The objective of this study is to develop an alternative porcine rotavirus subunit vaccine candidate by parenteral administration, which enables to elicit robust immune responses against most prevalence porcine rotavirus strains. The bacterially-expressed porcine rotavirus P[6]- or P[7]-specific truncated VP8* (aa 64-223) recombinant protein with or without a universal tetanus toxoid CD4(+) T cell epitope P2 was generated. All the recombinant subunit proteins △VP8*s or P2-△VP8*s were of high solubility and high yields. The immunogenicity of each purified △VP8* and P2-△VP8* was evaluated in mice (10 μg/dose) or guinea pigs (20 μg/dose) immunized IM with 600 μg aluminum hydroxide three times at 2-week interval. The introduction of P2T cell epitope to P[7]-△VP8* elicited significantly higher IgG titer in mice than its absence. Comparatively, P2 epitope slightly enhanced the immunogenicity of P[6]-△VP8*. P2-P[7]△VP8* elicited high titer of neutralizing antibody against heterotypic P[7]-specific rotaviruses with varied G type combination. Our data indicated that two subunit vaccines could be plausible bivalent rotavirus vaccine candidate to provide antigenic coverage of porcine rotavirus strains of global or regional importance.

  13. Protective effects of natural rotavirus infection.

    PubMed

    Velázquez, F Raúl

    2009-03-01

    Rotavirus is a ubiquitous infection that is the leading cause of severe diarrhea worldwide. Severe infections are most commonly observed in the first 2 years of life. Rotavirus-induced diarrhea is associated with substantial morbidity and mortality rates and socioeconomic costs with adverse outcomes particularly prevalent in developing countries. The natural history of rotavirus infection can provide guidance for the development and optimization of an effective vaccine. Epidemiologic studies have demonstrated that children who acquire natural rotavirus infections develop immunity to subsequent infections, with the protective effect increasing with each natural infection. Natural infections also decrease the severity of any subsequent rotavirus infections. Notably, asymptomatic infections provide protection similar to that induced by symptomatic infections. Data also suggest that the antibody response to natural infection is heterotypic, and therefore may provide protection against multiple serotypes. These data suggest that the development of a vaccine that produces asymptomatic infection at an optimal time point may provide effective immunity. An effective vaccine should mimic protection provided by natural infection and provide protection against the most common rotavirus serotypes (ie, G1, G2, G3, G4, G9) and be able to decrease disease severity, reduce hospitalizations, and decrease disease-related costs.

  14. Australian Rotavirus Surveillance Program annual report, 2012.

    PubMed

    Kirkwood, Carl D; Roczo-Farkas, Susie; Bishop, Ruth F; Barnes, Graeme L

    2014-03-31

    This report from the Australian Rotavirus Surveillance Program, together with collaborating laboratories Australia-wide, describes the rotavirus genotypes responsible for the hospitalisation of children with acute gastroenteritis during the period 1 January to 31 December 2012. During the survey period, 1,300 faecal samples were referred to the centre for rotavirus G and P genotype analysis, and of these 748 were confirmed as rotavirus positive. A total of 491 specimens were collected from children under 5 years of age, while 257 were from older children and adults. Genotype analysis revealed that G1P[8] was the dominant type in this reporting period, identified in 35% of strains nationally. Genotype G2P[4] was the second most common strain nationally, representing 28% of samples, followed by genotype G12P[8] (23%). This represents the first report where G12P[8] strains are a major cause of disease in this community. Fluctuations in genotype distribution were also observed based on the vaccine type in use. Genotype G2P[4] was more common in states and territories using Rotarix while G1P[8] was more common in states using RotaTeq. This survey of rotavirus strains circulating in 2012 highlights the continued fluctuations in rotavirus genotypes, with an annual change in dominant genotypes as well as emergence of a previously rare genotype, suggesting a dynamic wild-type population.

  15. Cultivation and characterization of three strains of murine rotavirus.

    PubMed Central

    Greenberg, H B; Vo, P T; Jones, R

    1986-01-01

    Three distinct strains of murine rotavirus were adapted to growth in cell culture. These strains are genetically related but not identical; they are serotypically heterogeneous. The cultivatable strains were substantially more infectious (approximately 10(6)-fold) for suckling mice than heterologous simian rotaviruses were. Homologous murine rotavirus strains spread from inoculated to uninoculated litter mates and caused diarrhea, while heterologous rotaviruses did not spread and cause illness. Images PMID:3003390

  16. 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

  17. Molecular Analysis of VP7 Gene of Rotavirus G1 Strains Isolated from North India.

    PubMed

    Jain, Swapnil; Vashistt, Jitendraa; Gupta, Kanika; Kumar, Ashok; Changotra, Harish

    2016-12-01

    Rotavirus G1 strains are the predominant cause of diarrhoea in children. Universally common rotavirus vaccines (Rotarix and RotaTeq) include G1 as the immunological component. India has recently introduced rotavirus vaccine in Universal Immunization Programme. Therefore, in the present study, VP7 gene of rotavirus G1 strains circulating in Himachal Pradesh, India is analysed to study their phylogenetic characteristics, and further comparative analysis was performed for assessment of their divergence from the vaccine strains. The rotavirus strains (JU-SOL-5, JU-SOL-58, JU-SOL-77, JU-SOL-173 and JU-SHI-14) analysed in the study were isolated from the faeces of diarrhoeic children during active surveillance for rotaviruses. The Himachal strains clustered together in G1-Lineage 1 in the phylogenetic analysis. All five isolates showed 96.4-98.8 % similarity with the other G1-Lineage 1 strains at amino acid level. However, none of them clustered in the pre-defined sublineages within lineage 1. Interestingly, all the strains were distantly related to the vaccine strains having 93.9-94.5 and 91.9-92.6 % similarities at amino acid level with Rotarix and RotaTeq strains, respectively. The comparative sequence and structural analysis of the Himachal strains with vaccine strains revealed differences in amino acids in epitope region of the protein especially at the antibody neutralization sites. The study highlights variations between the G1 strains from Himachal Pradesh, India and Rotarix and RotaTeq vaccine strains. These differences might have an impact on the neutralization efficiency of vaccine and subsequently on vaccine efficacy. This underscores further investigation to study intragenotype antigenic variability and also impact of viral evolution on vaccine effectiveness.

  18. Australian Rotavirus Surveillance Program annual report, 2014.

    PubMed

    Kirkwood, Carl D; Roczo-Farkas, Suzie

    2015-09-30

    The Australian Rotavirus Surveillance Program, together with collaborating laboratories Australia-wide, reports the rotavirus genotypes responsible for the hospitalisation of children with acute gastroenteritis. During the survey period of 1 January to 31 December 2014, 1,022 faecal samples were referred for rotavirus G and P genotype analysis, and of these 733 were confirmed as rotavirus positive. A total of 480 specimens were collected from children under 5 years of age, while 253 were from older children and adults. Genotype analysis of the 733 rotavirus samples collected from both children and adults revealed that G12P[8] was the dominant genotype in this reporting period, identified in 29.6% of strains nationally. Genotype G1P[8] was the 2nd most common strain nationally, representing 22.9% of samples, followed by genotype G3P[8] (14.9%). This report highlights the continued significance of G12P[8] strains as the major cause of disease in this population. The genotype distribution was slightly altered when the analysis was restricted to samples collected from children under 5 years of age, with G1P[8] being the dominant genotype (29%) followed by G12P[8] as the 2nd most common genotype (26%). Fluctuations in genotype distribution were also observed based on the vaccine type in use. Genotype G12P[8] was more common in states and territories using RotaTeq, while G1P[8] was more common in the locations using Rotarix. This survey highlights the yearly fluctuations in rotavirus genotypes observed since vaccine introduction. The continuation of G12P[8] as the dominant genotype further illustrates the dynamic and diversity present in the wild-type rotavirus population evident in the Australian population since vaccine introduction.

  19. Australian Rotavirus Surveillance Program annual report, 2013.

    PubMed

    Kirkwood, Carl D; Roczo-Farkas, Susie

    2014-12-31

    This report from the Australian Rotavirus Surveillance Program, together with collaborating laboratories Australia-wide, describes the rotavirus genotypes responsible for the hospitalisation of children with acute gastroenteritis during the period 1 January to 31 December 2013. During the survey period, 1,035 faecal samples were referred for rotavirus G and P genotype analysis. Of these 828 were confirmed as rotavirus positive. A total of 503 specimens were collected from children under 5 years of age, while 325 were from older children and adults. Genotype analysis of the 828 rotavirus samples collected from both children and adults revealed that G12P[8] was the dominant genotype in this reporting period, identified in 33% of strains nationally. Genotype G3P[8] was the second most common strain nationally, representing 31% of samples, followed by genotype G2P[4] (14%). This represents the first report where G12P[8] strains are the major cause of disease in this population. The genotype distribution was slightly altered when the analysis was restricted to samples collected from children under 5 years of age, with G3P[8] being the dominant genotype (39.2%) followed by G12P[8] as the second most common genotype (31%). Fluctuations in genotype distribution were also observed based on the vaccine type in use. Genotype G12P[8] was more common in states and territories using RotaTeq, while G3P[8] was more common in the locations using Rotarix. This survey highlights the yearly fluctuations in rotavirus genotypes observed since vaccine introduction, with changes in dominant genotypes an annual event. The emergence of G12P[8] as the dominant genotype further illustrates the ongoing changes in the wild type rotavirus population evident in the Australian population since vaccine introduction.

  20. 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

  1. Genetic and serologic surveillance of rotavirus with P[8] and P[4] genotypes in feces from children in the city of Chihuahua, northern Mexico.

    PubMed

    Contreras-Cordero, Juan F; Romo-Sáenz, César I; Menchaca-Rodríguez, Griselda E; Infante-Ramírez, Rocío; Villarreal-Treviño, Licet; Hernández-Luna, Carlos E; Rodríguez-Padilla, Cristina; Tamez-Guerra, Reyes S

    2015-12-01

    Rotavirus vaccine was developed using the most prominent G and P genotypes circulating in children population. Therefore, severe gastroenteritis has been reduced around the world. This study investigated the G and P rotavirus genotypes circulating in children from two hospitals in the city of Chihuahua, Mexico. Additionally, polyclonal antibodies against Rotavirus Wa strain were used to determine their homotypic and heterotypic reactivity to both P[8] and P[4] genotypes. G1, G2, and G3 VP7 genotypes and P[8] and P[4] VP4 genotypes were detected in common and uncommon combinations as well as mixed infectious. The predominant combination was G1P[8]. Phylogenetic analysis of VP4 gene revealed the presence of P[8]-1 and P[8]-3 lineages of P[8] genotype and P[4]-5 lineage of P[4] genotype. All but five G1P[8] rotavirus were detected by polyclonal anti-Rotavirus Wa strain. Mutation analysis revealed differences in three of the four neutralizing epitopes previously reported to VP8* subunit of VP4 protein. Results of this study offer insights over genetic variants of field rotavirus that could be detected in a homotypic and heterotypic way by antibodies elicited to rotavirus with P[8] genotype. [Int Microbiol 2016; 19(1):27-32].

  2. Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5′ GG Sequence for RTPase Activity

    PubMed Central

    Hu, Liya; Chow, Dar-Chone; Patton, John T.; Palzkill, Timothy; Estes, Mary K.

    2012-01-01

    Rotavirus nonstructural protein NSP2, a functional octamer, is critical for the formation of viroplasms, which are exclusive sites for replication and packaging of the segmented double-stranded RNA (dsRNA) rotavirus genome. As a component of replication intermediates, NSP2 is also implicated in various replication-related activities. In addition to sequence-independent single-stranded RNA-binding and helix-destabilizing activities, NSP2 exhibits monomer-associated nucleoside and 5′ RNA triphosphatase (NTPase/RTPase) activities that are mediated by a conserved H225 residue within a narrow enzymatic cleft. Lack of a 5′ γ-phosphate is a common feature of the negative-strand RNA [(−)RNA] of the packaged dsRNA segments in rotavirus. Strikingly, all (−)RNAs (of group A rotaviruses) have a 5′ GG dinucleotide sequence. As the only rotavirus protein with 5′ RTPase activity, NSP2 is implicated in the removal of the γ-phosphate from the rotavirus (−)RNA. To understand how NSP2, despite its sequence-independent RNA-binding property, recognizes (−)RNA to hydrolyze the γ-phosphate within the catalytic cleft, we determined a crystal structure of NSP2 in complex with the 5′ consensus sequence of minus-strand rotavirus RNA. Our studies show that the 5′ GG of the bound oligoribonucleotide interacts extensively with highly conserved residues in the NSP2 enzymatic cleft. Although these residues provide GG-specific interactions, surface plasmon resonance studies suggest that the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2. A novel observation from our studies, which may have implications in viroplasm formation, is that the C-terminal helix of NSP2 exhibits two distinct conformations and engages in domain-swapping interactions, which result in the formation of NSP2 octamer chains. PMID:22811529

  3. Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background Virus-like particles (VLP) have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP) in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly. Results The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp) or episomal (YEp), did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy. Conclusions We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts could lead to the

  4. The origin of two rare human P[10] rotavirus strains.

    PubMed

    Ghosh, Souvik; Urushibara, Noriko; Kawaguchiya, Mitsuyo; Shintani, Tsuzumi; Kobayashi, Nobumichi

    2013-01-01

    The Group A rotavirus (RVA) P[10] is a rare genotype of the RVA VP4 gene. To date, the whole genome sequence of only a single P[10] RVA strain, RVA/Human-tc/IDN/69M/1980/G8P4[10], has been determined, revealing a DS-1-like genotype constellation. Whole genomic analyses of P[10] RVA strains with other VP7 genotypes are essential to obtain conclusive data on the origin and genetic diversity of the P10] RVAs. In the present study, the whole genome of a human G4P[10] RVA strain, RVA/Human-tc/IDN/57M/1980/G4P[10], was analyzed. Strain 57M exhibited an unusual G4-P[10]-I1-R1-C1-M1-A1-N1-T2-E1-H2 genotype constellation, and was found to originate from intergenogroup reassortment events involving acquisition of RVA strain 69M-like VP4, NSP3 and NSP5 genes by a co-circulating Wa-like human G4 RVA strain. Although the reference P[10] strain, 69M, exhibits a DS-1-like genotype constellation, the exact origin of this RVA remains to be elucidated. By detailed phylogenetic analyses, we found that the VP1-VP3, VP6, NSP2 and NSP4 genes of 69M originated from artiodactyl and/or artiodactyl-like human P[14] strains, whilst its NSP1, NSP3 and NSP5 genes were more related to those of typical human DS-1-like strains than those of other RVAs. On the other hand, the origin of the VP4 gene of 69M could not be established. Nevertheless, these observations clearly indicated that strain 69M might have originated from reassortment events involving at least the artiodactyl or artiodactyl-like human RVAs and the typical human DS-1-like strains. The present study provided rare evidence for intergenogroup reassortment events involving co-circulating typical human Wa-like RVAs and unusual RVAs of the DS-1-like genogroup, and revealed the presence of artiodactyl-like genes in a human P[10] strain, highlighting the complex evolutionary patterns of the P[10] RVAs.

  5. Vaccine-Acquired Rotavirus in Infants with Severe Combined Immunodeficiency

    PubMed Central

    Patel, Niraj C.; Hertel, Paula M.; Estes, Mary K.; de la Morena, Maite; Petru, Ann M.; Noroski, Lenora M.; Revell, Paula A.; Celine Hanson, I.; Paul, Mary E.; Rosenblatt, Howard M.; Abramson, Stuart L.

    2014-01-01

    SUMMARY Live pentavalent human–bovine reassortant rotavirus vaccine is recommended in the United States for routine immunization of infants. We describe three infants, two with failure to thrive, who had dehydration and diarrhea within 1 month after their first or second rotavirus immunization and subsequently received a diagnosis of severe combined immunodeficiency. Rotavirus was detected, by means of reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay, in stool specimens obtained from all three infants, and gene-sequence analysis revealed the presence of vaccine rotavirus. These infections raise concerns regarding the safety of rotavirus vaccine in severely immunocompromised patients. PMID:20107217

  6. Full Genome Characterization of Novel DS-1-Like G8P[8] Rotavirus Strains that Have Emerged in Thailand: Reassortment of Bovine and Human Rotavirus Gene Segments in Emerging DS-1-Like Intergenogroup Reassortant Strains

    PubMed Central

    Tacharoenmuang, Ratana; Komoto, Satoshi; Guntapong, Ratigorn; Ide, Tomihiko; Sinchai, Phakapun; Upachai, Sompong; Yoshikawa, Tetsushi; Tharmaphornpilas, Piyanit; Sangkitporn, Somchai; Taniguchi, Koki

    2016-01-01

    The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotavirus strains have been recently reported in Asia, Australia, and Europe. During rotavirus surveillance in Thailand in 2013–2014, novel DS-1-like intergenogroup reassortant strains having G8P[8] genotypes (i.e., strains KKL-17, PCB-79, PCB-84, PCB-85, PCB-103, SKT-107, SWL-12, NP-130, PCB-656, SKT-457, SSKT-269, and SSL-55) were identified in stool samples from hospitalized children with severe diarrhea. In this study, we determined and characterized the complete genomes of these 12 strains (seven strains, KKL-17, PCB-79, PCB-84, PCB-85, PCB-103, SKT-107, and SWL-12, found in 2013 (2013 strains), and five, NP-130, PCB-656, SKT-457, SSKT-269, and SSL-55, in 2014 (2014 strains)). On full genomic analysis, all 12 strains showed a unique genotype constellation comprising a mixture of genogroup 1 and 2 genes: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. With the exception of the G genotype, the unique genotype constellation of the 12 strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) was found to be shared with DS-1-like intergenogroup reassortant strains. On phylogenetic analysis, six of the 11 genes of the 2013 strains (VP4, VP2, VP3, NSP1, NSP3, and NSP5) appeared to have originated from DS-1-like intergenogroup reassortant strains, while the remaining four (VP7, VP6, VP1, and NSP2) and one (NSP4) gene appeared to be of bovine and human origin, respectively. Thus, the 2013 strains appeared to be reassortant strains as to DS-1-like intergenogroup reassortant, bovine, bovine-like human, and/or human rotaviruses. On the other hand, five of the 11 genes of the 2014 strains (VP4, VP2, VP3, NSP1, and NSP3) appeared to have originated from DS-1-like intergenogroup reassortant strains, while three (VP7, VP1, and NSP2) and one (NSP4) were assumed to be of bovine and human origin, respectively. Notably, the remaining two genes, VP6 and NSP5, of the 2014 strains appeared to have originated from locally

  7. Rotavirus vaccination within the South African Expanded Programme on Immunisation.

    PubMed

    Seheri, L Mapaseka; Page, Nicola A; Mawela, Mothahadini P B; Mphahlele, M Jeffrey; Steele, A Duncan

    2012-09-07

    Diarrhoeal diseases are ranked the third major cause of childhood mortality in South African children less than 5 years, where the majority of deaths are among black children. Acute severe dehydrating rotavirus diarrhoea remains an important contributor towards childhood mortality and morbidity and has been well documented in South Africa. As the preventive strategy to control rotavirus diarrhoea, South Africa became the first country in the WHO African Region to adopt the rotavirus vaccine in the national childhood immunisation programme in August 2009. The rotavirus vaccine in use, Rotarix, GSK Biologicals, is given at 6 and 14 weeks of age, along with other vaccines as part of Expanded Programme on Immunisation (EPI). Studies which facilitated the introduction of rotavirus vaccine in South Africa included the burden of rotavirus disease and strain surveillance, economic burden of rotavirus infection and clinical trials to assess the safety and efficacy of vaccine candidates. This paper reviews the epidemiology of rotavirus in South Africa, outlines some of the steps followed to introduce rotavirus vaccine in the EPI, and highlights the early positive impact of vaccination in reducing the rotavirus burden of disease based on the post-marketing surveillance studies at Dr George Mukhari hospital, a sentinel site at University of Limpopo teaching hospital in Pretoria, South Africa, which has conducted rotavirus surveillance for >20 years.

  8. Isolation, propagation, and characterization of a second equine rotavirus serotype.

    PubMed Central

    Hoshino, Y; Wyatt, R G; Greenberg, H B; Kalica, A R; Flores, J; Kapikian, A Z

    1983-01-01

    A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses. Images PMID:6309657

  9. 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

  10. Role of Interferon Regulatory Factor 3 in Type I Interferon Responses in Rotavirus-Infected Dendritic Cells and Fibroblasts▿

    PubMed Central

    Douagi, Iyadh; McInerney, Gerald M.; Hidmark, Åsa S.; Miriallis, Vassoula; Johansen, Kari; Svensson, Lennart; Karlsson Hedestam, Gunilla B.

    2007-01-01

    The main pathway for the induction of type I interferons (IFN) by viruses is through the recognition of viral RNA by cytosolic receptors and the subsequent activation of interferon regulatory factor 3 (IRF-3), which drives IFN-α/β transcription. In addition to their role in inducing an antiviral state, type I IFN also play a role in modulating adaptive immune responses, in part via their effects on dendritic cells (DCs). Many viruses have evolved mechanisms to interfere with type I IFN induction, and one recently reported strategy for achieving this is by targeting IRF-3 for degradation, as shown for rotavirus nonstructural protein 1 (NSP1). It was therefore of interest to investigate whether rotavirus-exposed DCs would produce type I IFN and/or mature in response to the virus. Our results demonstrate that IRF-3 was rapidly degraded in rotavirus-infected mouse embryonic fibroblasts (MEFs) and type I IFN was not detected in these cultures. In contrast, rotavirus induced type I IFN production in myeloid DCs (mDCs), resulting in their activation. Type I IFN induction in response to rotavirus was reduced in mDCs from IRF-3−/− mice, indicating that IRF-3 was important for mediating the response. Exposure of mDCs to UV-treated rotavirus induced significantly higher type I IFN levels, suggesting that rotavirus-encoded functions also antagonized the response in DCs. However, in contrast to MEFs, this action was not sufficient to completely abrogate type I IFN induction, consistent with a role for DCs as sentinels for virus infection. PMID:17215281

  11. Australian Rotavirus Surveillance Program annual report, 2015.

    PubMed

    Roczo-Farkas, Susie; Kirkwood, Carl D; Bines, Julie E

    2016-12-24

    The Australian Rotavirus Surveillance Program, together with collaborating laboratories Australia-wide, reports the rotavirus genotypes responsible for the hospitalisation of children with acute gastroenteritis during the period 1 January to 31 December 2015. During the survey period, 1,383 faecal samples were referred for rotavirus G and P genotype analysis, and of these, 1,031 were confirmed as rotavirus positive. A total of 634 specimens had been collected from children under 5 years of age, while 397 were from older children and adults. Genotype analysis of samples from both children and adults revealed that G12P[8] was the dominant genotype in this reporting period, identified in 48.2% of strains nationally. Genotype G3P[8] was the second most common strain nationally, representing 22.8% of samples, followed by G2P[4] and G1P[8] (9% and 8% respectively). G3P[8] was further divided as equine-like G3P[8] (13.2% of all strains) and other wild-type G3P[8] (9.6%). This report highlights the continued predominance of G12P[8] strains as the major cause of disease in this population. Genotype distribution was distinct between jurisdictions using RotaTeq and Rotarix vaccines. Genotype G12P[8] was more common in states using RotaTeq, while equine-like G3P[8] and G2P[4] were more common in the states and territories using Rotarix. This survey highlights the dynamic change in rotavirus genotypes observed since vaccine introduction, including the emergence of a novel equine-like G3P[8] as a major strain. The prolonged dominance of G12P[8] for a 4th consecutive year further illustrates the unexpected trends in the wild type rotaviruses circulating in the Australian population since vaccine introduction.

  12. Reduction in Rotavirus Disease and Sustained Predominance of G2P[4] Rotavirus Strain following Introduction of Rotavirus Vaccine in Recife, Brazil.

    PubMed

    Montenegro, Fernanda Maria Ulisses; Falbo, Ana Rodrigues; Germano, Eliane Mendes; Correia, Nancy Barros; Souza, Edvaldo da Silva; Nakagomi, Osamu; Nakagomi, Toyoko; Cuevas, Luis E; Cunliffe, Nigel A; Correia, Jailson B

    2015-06-01

    Rotavirus vaccination was introduced in Brazil in March 2006. We describe the distribution of rotavirus genotypes in children with acute gastroenteritis in a hospital in Recife, Brazil, during pre- and post-vaccination periods. There was a 43.8% reduction in the proportion of diarrhea episodes due to rotavirus. Nevertheless, we observed a sustained predominance of G2P[4] as the main genotype identified in the post-vaccination period.

  13. 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.

  14. A comparison of the VP7 gene sequences of human and bovine rotaviruses.

    PubMed

    Gerna, G; Steele, A D; Hoshino, Y; Sereno, M; Garcia, D; Sarasini, A; Flores, J

    1994-07-01

    The sequences of the gene encoding VP7 (the major outer capsid protein) from one bovine and three human rotavirus strains were determined because of their unusual VP7 specificities. Two of the human strains (PA 169 and PA 151) had VP7 serotype 6 specificity whereas the two other strains, recovered from a child (HAL 1166) and a calf (678) belonged to VP7 serotype 8. The serotype 8 strains exhibited a high degree of sequence conservation when compared with each other and with other serotype 8 strains previously sequenced. The serotype 6 human strains shared a greater degree of sequence similarity with previously reported serotype 6 bovine strains than with other rotavirus serotypes; however the degree of sequence similarity among PA 169, PA 151 and the bovine strains was lower than had been previously reported for strains belonging to the same serotype. The demonstration of rotavirus serotypes that are shared between human and animal species supports the concept that interspecies transmission occurs and may play a role in rotavirus evolution.

  15. 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

  16. Understanding internalization of rotavirus VP6 nanotubes by cells: towards a recombinant vaccine.

    PubMed

    Rodríguez, Mabel; Wood, Christopher; Sanchez-López, Rosana; Castro-Acosta, Ricardo M; Ramírez, Octavio T; Palomares, Laura A

    2014-05-01

    Rotavirus VP6 nanotubes are an attractive option for a recombinant vaccine against rotavirus disease. Protection against rotavirus infection and an adjuvant effect have been observed upon immunization with VP6 nanotubes. However, little information exists on how VP6 nanotubes interact with cells and trigger an immune response. In this work, the interaction between VP6 nanotubes and different cell lines was characterized. VP6 nanotubes were not cytotoxic to any of the animal or human cell lines tested. Uptake of nanotubes into cells was cell-line-dependent, as only THP1 and J774 macrophage cells internalized them. Moreover, the size and spatial arrangement of VP6 assembled into nanotubes allowed their uptake by macrophages, as double-layered rotavirus-like particles also displaying VP6 in their surface were not taken up. The internalization of VP6 nanotubes was inhibited by methyl-β-cyclodextrin, but not by genistein, indicating that nanotube entry is specific, depends on the presence of cholesterol in the plasma membrane, and does not require the activity of tyrosine kinases. The information generated here expands our understanding of the interaction of protein nanotubes with cells, which is useful for the application of VP6 nanotubes as a vaccine.

  17. Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

    PubMed Central

    Tokuhara, Daisuke; ρlvarez, Beatriz; Mejima, Mio; Hiroiwa, Tomoko; Takahashi, Yuko; Kurokawa, Shiho; Kuroda, Masaharu; Oyama, Masaaki; Kozuka-Hata, Hiroko; Nochi, Tomonori; Sagara, Hiroshi; Aladin, Farah; Marcotte, Harold; Frenken, Leon G.J.; Iturriza-Gómara, Miren; Kiyono, Hiroshi; Hammarström, Lennart; Yuki, Yoshikazu

    2013-01-01

    Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections. PMID:23925294

  18. Spread and predominance in Japan of novel G1P[8] double-reassortant rotavirus strains possessing a DS-1-like genotype constellation typical of G2P[4] strains.

    PubMed

    Fujii, Yoshiki; Nakagomi, Toyoko; Nishimura, Naoko; Noguchi, Atsuko; Miura, Sinobu; Ito, Hisato; Doan, Yen Hai; Takahashi, Tsutomu; Ozaki, Takao; Katayama, Kazuhiko; Nakagomi, Osamu

    2014-12-01

    Rotavirus is a major cause of severe gastroenteritis in children <5 years of age worldwide, and two, live attenuated rotavirus vaccines are globally available. As rotavirus vaccines are introduced into national immunization programs, there is an increasing need to monitor circulating wild-type strains. However, few studies have systematically examined their full genotype constellation. This study was therefore undertaken to characterize the whole genotype constellation of circulating rotavirus strains in three widely-separated locations in Japan during the 2012 rotavirus season when rotavirus vaccines became available in the country for the first time. Of 107 rotavirus-positive specimens, 50 (46.7%) strains collected from all three locations possessed an unusual G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 constellation in which a typical G2P[4] strain appeared to have acquired its two surface protein genes from the most common G1P[8] strain. These G1P[8] double-reassortant strains were shown to possess the 11 genome segments virtually indistinguishable from each other in their nucleotide sequences and phylogenetic lineages except for two strains that underwent further intra-genotype reassortment. Successful spread to and predominance in broad locations across Japan of novel rotavirus strains possessing a genotype constellation that was previously thought not to be preferred suggests unexpected genomic flexibility of the genotype constellation.

  19. Variation in the VP1 gene of foot-and-mouth disease virus serotype A associated with epidemiological characteristics of outbreaks in the 2001 epizootic in Argentina.

    PubMed

    Perez, Andres M; König, Guido; Späth, Ernesto; Thurmond, Mark C

    2008-07-01

    A mixed binomial Bayesian regression model was used to quantify the relation between nucleotide differences in the VP1 gene of Foot-and-mouth disease virus (FMDV) serotype A, and epidemiologic characteristics of the outbreaks from which the viruses were obtained between January and December 2001 in Argentina. An increase in the probability of different nucleotides between isolates was associated with a longer time between isolation dates, a greater distance between isolation locations, an increase in the difference between attack rates, and an increase in the difference in outbreak durations. The farther apart the outbreak herds were in the southerly and easterly directions, the greater the difference in nucleotide changes. The model accurately predicted genetic distances of isolates obtained in 2001 compared with a 2002 isolate (P < 0.01), which suggested that the predictive modeling approach applied in the present study may be useful in understanding the epidemiology of evolution of FMDV and in forensic analysis of disease epidemics.

  20. Spectrum of VP1 region genetic variants in the foot-and-mouth disease virus serotype O populations derived from infected cattle tongue epithelium.

    PubMed

    Sarangi, L N; Mohapatra, J K; Subramaniam, S; Pandey, L K; Das, B; Sanyal, A; Misri, J; Pattnaik, B

    2015-09-01

    RNA virus population exists as a complex distribution of non-identical but closely related sequences known as viral quasispecies. Variant strains are selected from this quasispecies population in response to changing environment. The quasispecies dynamics of a virus existing within an infected host differs from that in a cell culture-adapted population. This study was carried out to explore the genetic variations present in the VP1 coding region of the foot-and-mouth disease (FMD) virus serotype O derived directly from infected cattle tongue epithelium. Molecular clonal populations of two serotype O strains belonging to lineages Ind2001 (IND 30/2011) and PanAsia2 (IND 5/2011) were sequenced at VP1 coding region. For IND 30/2011, 19 clones were sequenced and analysis showed variations at 12 nucleotide positions (nt) resulting in 8 amino acid (aa) replacements. Similarly, for IND 5/2011 virus, 18 clones were sequenced, of which six showed nt variations leading to 3 aa replacements. Most of the variable positions mapped to the surface-exposed loops and some of them were found in the neutralizing antigenic sites (position 81, 149, 169, 186 and 202 of IND 30/2011 and 141 of IND 5/2011), which potentially could be beneficial in rapid adaptive evolution of the virus by giving rise to antigenic variants to overcome neutralizing antibodies. These findings encourage further research into the landscape of the viral quasispecies population in vivo and its implication for viral ecology.

  1. Incidence of Rotavirus and Circulating Genotypes in Northeast Brazil during 7 Years of National Rotavirus Vaccination

    PubMed Central

    Gurgel, Ricardo Q.; Alvarez, Alberto De Juan; Rodrigues, Alda; Ribeiro, Robergson R.; Dolabella, Sílvio S.; Da Mota, Natanael L.; Santos, Victor S.; Iturriza-Gomara, Miren; Cunliffe, Nigel A.; Cuevas, Luis E.

    2014-01-01

    Background and Aims Rotavirus causes severe diarrhoea and Brazil introduced the Rotarix G1P[8] vaccine in 2006. We aimed to describe changes in rotavirus incidence and diarrhoea epidemiology before and after vaccine introduction. Methods Design: (i) hospital-based survey of children with diarrhoea (2006–2012); (ii) diarrhea-mortality and hospitalization surveillance (1999–2012). Setting (i) Aracaju and (ii) state and national level. Results 1841 children were enrolled and 231 (12.5%) had rotavirus. Rotavirus was less frequent from January-June than from July-December (9.4% versus 20.9%, p<0.01), but the seasonal variation was less defined after 2009. Very few rotavirus cases (8–3.9%) were detected in 2011, with an increase in 2012 (13–18.5%). In 2006, unvaccinated children were more likely to have rotavirus, but thereafter unvaccinated and vaccinated children had equally low incidence. Older children and those with rotavirus were more likely to have severe diarrhea episodes. The most frequent genotype from 2006 to 2010 was G2P[4]; except in 2009, when most cases were G1P[8]. Very few G2P[4] were detected from 2011 and 50% cases in 2012 were G8P[4]. Diarrhoea-hospitalizations decreased nationally from 89,934 (2003) to 53,705 (2012; 40.3% reduction) and in the state from 1729 to 748 (56.7% reduction). Diarrhoea-deaths decreased nationally from 4368 in 1999 to 697 in 2012 (84% reduction, p<0.001) and in the state from 132 to 18 (86% reduction). These changes were much larger after vaccine introduction. Conclusions The vaccine was associated with substantial reductions in rotavirus incidence and diarrhoea-hospitalizations and deaths. The G2P[4] genotype predominance disappeared over time and may be replaced by other heterotypic genotypes. PMID:25360784

  2. 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.

  3. A four-nucleotide translation enhancer in the 3'-terminal consensus sequence of the nonpolyadenylated mRNAs of rotavirus.

    PubMed Central

    Chizhikov, V; Patton, J T

    2000-01-01

    The 5' cap and poly(A) tail of eukaryotic mRNAs work synergistically to enhance translation through a process that requires interaction of the cap-associated eukaryotic initiation factor, eIF-4G, and the poly(A)-binding protein, PABP. Because the mRNAs of rotavirus, and other members of the Reoviridae, contain caps but lack poly(A) tails, their translation may be enhanced through a unique mechanism. To identify translation-enhancement elements in the viral mRNAs that stimulate translation in vivo, chimeric RNAs were prepared that contained an open reading frame for luciferase and the 5' and 3' untranslated regions (UTRs) of a rotavirus mRNA or of a nonviral mRNA. Transfection of the chimeric RNAs into rotavirus-infected cells showed that the viral 3' UTR contained a translation-enhancement element that promoted gene expression. The element did not enhance gene expression in uninfected cells and did not affect the stability of the RNAs. Mutagenesis showed that the conserved sequence GACC located at the 3' end of rotavirus mRNAs operated as an enhancement element. The 3'-GACC element stimulated protein expression independently of the sequence of the 5' UTR, although efficient expression required the RNA to contain a cap. The results indicate that the expression of viral proteins in rotavirus-infected cells is specifically up-regulated by the activity of a novel 4-nt 3' translation enhancer (TE) common to the 11 nonpolyadenylated mRNAs of the virus. The 4-nt sequence of the rotavirus 3' TE represents by far the shortest of any of the sequence enhancers known to stimulate translation. PMID:10864041

  4. Rotavirus Seasonal Distribution and Prevalence Before and After the Introduction of Rotavirus Vaccine in a Peri-Urban Community of Lima, Peru

    PubMed Central

    Chang, Millie R.; Velapatiño, Grace; Campos, Miguel; Chea-Woo, Elsa; Baiocchi, Nelly; Cleary, Thomas G.; Ochoa, Theresa J.

    2015-01-01

    We evaluated the monthly distribution of rotavirus diarrhea in a cohort of children 12–24 months of age followed as part of a diarrhea clinical trial in a peri-urban community of Lima. We observed a peak of rotavirus diarrhea in the winter months and a decrease in rotavirus prevalence after the introduction of the rotavirus vaccine in Peru. PMID:25778507

  5. Epidemiological and clinical studies of rotavirus-induced diarrhea in China from1994–2013

    PubMed Central

    Nan, Xiao; Jinyuan, Wu; Yan, Zhou; Maosheng, Sun; Hongjun, Li

    2015-01-01

    Group A rotavirus infections cause diarrhea in infants and primarily occur in winter. These viruses are characterized by the viral structural proteins VP7 (G subtypes) and VP4 (P subtypes). Current treatments employ vaccines combined with symptomatic treatments. Through a review of published papers from 2003–2013, which included articles, theses, dissertations and academic conference proceedings in PubMed and CNKI (China National Knowledge Infrastructure), we examined the epidemiological and clinical studies conducted in the 7 regional administrative geographical divisions of the People's Republic of China. Binning of the clinical studies provided a quantitative synthesis of the published incidences of rotavirus-positive numbers, the infection rates and the distributions of different subtypes in the provinces. In an investigation of the 32 provincial administrative regions from 1994–2013, the number of positive infections in children, primarily ≤ 5 years of age, was highest in Guangdong (187,000 cases), Zhejiang (133,000 cases) and Hubei (11,000 cases) Provinces; the provinces that displayed the highest positive rates were Henan (75.20%), Jilin (64.94%) and the Ningxia Hui Autonomous Region (60.16%). Furthermore, the virus, which causes diarrhea, can also infect adults. Statistics regarding the rotavirus subtypes indicated that the top 3 G-subtypes reported were G3 (8,509 cases), G1 (6,490 cases) and G2 (1,601 cases); the top 3 P-subtypes reported were P[8] (8,483 cases), P[4] (2,017 cases) and P[6] (740 cases). The majority of clinical treatments for these infections comprised a combination of Chinese traditional and Western medicines, together with nursing and the administration of a variety of oral liquids to relieve symptoms. The top 3 positive rates were identified in provinces at higher latitude, which confirms increases in humidity and ambient temperature reduce the rotavirus infection rate and the number of rotavirus infections tends to be highest

  6. Specific primer amplification of the VP1 region directed by 5' UTR sequence analysis: enterovirus testing and identification in clinical samples from hand-foot-and-mouth disease patients.

    PubMed

    Ge, Shengxiang; Yan, Qiang; He, Shuizhen; Zhuang, Sijie; Niu, Jianjun; Xia, Ningshao

    2013-11-01

    Many genotypes of the enterovirus (EV) pathogens can cause clinical hand-foot-and-mouth disease (HFMD). Therefore, rapid identification and monitoring of HFMD pathogens can be difficult, especially from the original clinical specimens. In this study, both universal pan-enterovirus and EV71/CA16 VP1-specific primer sets were designed and used to examine clinical specimens from HFMD patients. Based on the initial sequence analysis of the 5'-untanslated region (5'-UTR) and VP1 amplification products, additional primers for the VP1 region were redesigned for further genotyping of the remaining small portion non-EV71/non-CA16 specimens. With a known panel, it was possible to identify 15 out of 16 members using 5'-UTR sequence typing and VP1 typing, suggesting good detectability and genotyping of this method. One strain that was not typed by 5'-UTR was shown to be a recombinant virus. When this method was applied to examine clinical specimens from 44 suspected HFMD patients, 41 were detected as EV positive. In only one case, the VP1 sequence could not be identified. Four types of EVs, including CA16 (26/41, 63.4%), EV71-C4 (6/41, 14.6%), CA6 (5/41, 12.2%) and CA10 (3/41, 7.3%), were detected. In conclusion, 5' UTR amplification sequencing and subsequent VP1 specific primer amplification ensures a high detection rate and good genotyping accuracy in the examination of clinical samples. This detection strategy can be used for routine evaluation and monitoring of HFMD to follow local trends of EV infection.

  7. Rotavirus Vaccine: What You Need to Know

    MedlinePlus

    ... including a severe allergy to latex. Babies with “severe combined immunodeficiency” (SCID) should not get rotavirus vaccine. Babies who have had a type of bowel blockage called “intussusception” should not get ... moderate or severe diarrhea or vomiting. Check with your doctor if ...

  8. 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

  9. Virus protein assembly in microgravity

    NASA Astrophysics Data System (ADS)

    Chang, D.; Paulsen, A.; Johnson, T. C.; Consigli, R. A.

    1993-07-01

    The coat of polyomavirus is composed of three proteins that can self-assemble to form an icosahedral capsid. VP1 represents 75% of the virus capsid protein and the VP1 capsomere subunits are capable of self assembly to form a capsid-like structure. Ground-based and orbiter studies were conducted with VP1 protein cloned in an expression vector and purified to provide ample quantities for capsomere-capsid assembly. Flight studies were conducted on STS-37 on April 5-9, 1991. Assembly initiated when a VP1 protein solution was interfaced with a Ca+2 buffer solution (pH 5.0). After four days a second alignment terminated the assembly process and allowed for glutaraldehyde fixation. Flight and ground-based samples were analyzed by electron microscopy. Ground-based experiments revealed the assembly of VP1 into capsid-like structures and a heterogenous size array of capsomere subunits. Samples reacted in microgravity, however, showed capsomeres of a homogenous size, but lack of capsid-like assembly.

  10. 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.

  11. Options for improving effectiveness of rotavirus vaccines in developing countries.

    PubMed

    Tissera, Marion S; Cowley, Daniel; Bogdanovic-Sakran, Nada; Hutton, Melanie L; Lyras, Dena; Kirkwood, Carl D; Buttery, Jim P

    2016-11-11

    Rotavirus gastroenteritis is a leading global cause of mortality and morbidity in young children due to diarrhea and dehydration. Over 85% of deaths occur in developing countries. In industrialised countries, 2 live oral rotavirus vaccines licensed in 2006 quickly demonstrated high effectiveness, dramatically reducing severe rotavirus gastroenteritis admissions in many settings by more than 90%. In contrast, the same vaccines reduced severe rotavirus gastroenteritis by only 30-60% in developing countries, but have been proven life-saving. Bridging this "efficacy gap" offers the possibility to save many more lives of children under the age of 5. The reduced efficacy of rotavirus vaccines in developing settings may be related to differences in transmission dynamics, as well as host luminal, mucosal and immune factors. This review will examine strategies currently under study to target the issue of reduced efficacy and effectiveness of oral rotavirus vaccines in developing settings.

  12. A study on neonatal calf diarrhea induced by rotavirus.

    PubMed

    Castrucci, G; Ferrari, M; Frigeri, F; Traldi, V; Angelillo, V

    1994-01-01

    This review summarizes the results of a study on rotaviruses isolated from calves affected by neonatal diarrhea. The results indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. Differences in virulence among bovine rotaviruses appeared also to be confirmed. Conventionally reared calves were fully susceptible to the experimental infection induced by rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits. When rotavirus strains of bovine, simian and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain. Finally, it was proven that feeding newborn calves with colostrum and first milk of their dams, previously vaccinated with an inactivated adjuvanted rotavirus vaccine, could prevent the neonatal diarrhea from occurring.

  13. Rotavirus vaccines: safety, efficacy and public health impact.

    PubMed

    Gray, J

    2011-09-01

    Rotaviruses are the cause of acute gastroenteritis, and disease is widespread amongst infants and young children throughout the world. Also, rotavirus is associated with significant mortality in developing countries with more than 500 000 children dying each year as a result of the severe dehydration associated with rotavirus disease. Efforts have been ongoing for more than 30 years to develop a safe and effective rotavirus vaccine. Currently, two vaccines, RotaRix and RotaTeq, have been licensed for use in many countries throughout the world following comprehensive safety and efficiency trials. Monitoring their effectiveness after licensure has confirmed that their incorporation into early childhood vaccination schedules can significantly prevent severe rotavirus diarrhoea, which would have resulted in hospitalizations, emergency room visits or increased diarrhoea-related mortality. Although the efficacy of both vaccines is lower at approximately 40-59% in developing countries, their use could significantly reduce the mortality associated with rotavirus disease that is concentrated in these countries.

  14. Structural characterization of rotavirus-directed synthesis and assembly of metallic nanoparticle arrays.

    PubMed

    Plascencia-Villa, Germán; Medina, Ariosto; Palomares, Laura A; Ramírez, Octavio T; Ascencio, Jorge A

    2013-08-01

    Self-assembled structures derived of viral proteins display sophisticated structures that are difficult to obtain with even advanced synthesis methods and the use of protein nanotubes for synthesis and organization of inorganic nanoarrays into well-defined architectures are here reported. Nanoparticle arrays derived of rotavirus VP6 nanotubes were synthesized by in situ functionalization with silver and gold nanoparticles. The size and morphology of metal nanoparticles were characterized by transmission electron microscopy (TEM) and high resolution TEM (HR-TEM). Processing of micrographs to obtain fast Fourier transforms (FFT) patterns of nanoparticles shown that the preferred morphologies are fcc-like and multiple twinned ones. Micrographs were used to assign structure and orientation, and the elemental composition analysis was performed with energy dispersive spectroscopy (EDS). Structural characterization of functionalized rotavirus VP6 demonstrated its utility for directed construction of hybrid anisotropic nanomaterials formed by arrays of metallic nanoparticles.

  15. Clinical characteristics of rotavirus diarrhea in hospitalized Romanian infants.

    PubMed

    Lesanu, Gabriela; Becheanu, Cristina Adriana; Vlad, Raluca Maria; Pacurar, Daniela; Tincu, Iulia Florentina; Smadeanu, Roxana Elena

    2013-01-01

    Clinical characteristics of rotavirus enteritis were evaluated by comparison with acute diarrhea of other etiologies. We reviewed the medical records of children (aged 0-12 months) admitted with acute diarrhea in our hospital between January and December 2011. Of the 839 patients, 49.3% had rotavirus diarrhea. The incidence of severe disease was significantly higher for rotavirus diarrhea (65.2%, P < 0.01) than for other types of diarrheal disease.

  16. 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.

  17. RNA electropherotypes of human rotaviruses from North and South America*

    PubMed Central

    Dimitrov, D. H.; Graham, D. Y.; Lopez, J.; Muchinik, G.; Velasco, G.; Stenback, W. A.; Estes, M. K.

    1984-01-01

    Between April 1979 and December 1982, viral agents were found in 231 of 695 children admitted to the Texas Children's Hospital with gastroenteritis. Electron microscopic analysis showed that rotaviruses were the most common viral agents, and a seasonal pattern of rotavirus disease was observed. The migration patterns of the RNA segments of these rotaviruses on electrophoresis in polyacrylamide gels were compared with those of rotaviruses collected from other areas of the United States of America and from Argentina, Colombia and Mexico. A number of different RNA electropherotypes were found, including some patterns not previously reported. ImagesFig. 2Fig. 3Fig. 4Fig. 5 PMID:6329538

  18. [A rare cause of afebrile convulsion: rotavirus gastroenteritis].

    PubMed

    Karabel, Müsemma; Karabel, Duran; Kara, Semra; Halıcı Taş, Tuğba; Türkay, Sadi

    2013-04-01

    Rotavirus is the most common infectious diarrhea that causes important mortality and morbidities in small children, severe dehydration and electrolyte imbalance. Extraintestinal signs are rare in rotavirus infections. Recently, afebrile seizures associated with rotavirus gastroenteritis but without encephalopathy, dehydration, electrolyte imbalance or hypoglycemia have being reported. In this article, the fact that rotavirus, which is seen commonly in our country, can be confronted with various clinical manifestations was emphasized by reminding that it can be seen not only in infants with neurologic and systemic disease but also in healthy infants.

  19. Interactions among the major and minor coat proteins of polyomavirus.

    PubMed Central

    Barouch, D H; Harrison, S C

    1994-01-01

    Murine polyomavirus contains two related minor coat proteins, VP2 and VP3, in addition to the major coat protein, VP1. The sequence of VP3 is identical to that of the carboxy-terminal two-thirds of VP2. VP2 may serve a role in uncoating of the virus, and both minor coat proteins may be important for viral assembly. In this study, we show that VP3 and a series of deletion mutants of VP3 can be expressed in Escherichia coli as fusion proteins to glutathione S-transferase and partially solubilized with a mild detergent. Using an in vitro binding assay, we demonstrate that a 42-amino-acid fragment near the carboxy terminus of VP3 (residues 140 to 181) is sufficient for binding to purified VP1 pentamers. This binding interaction is rapid, saturable, and specific for the common carboxy terminus of VP2 and VP3. The VP1-VP3 complex can be coimmunoprecipitated with an antibody specific to VP1, and a purified VP3 fragment can selectively extract VP1 from a crude cell lysate. The stoichiometry of the binding reaction suggests that each VP1 pentamer in the virus binds either one VP2 or one VP3, with the VP1-VP2/3 complex stabilized by hydrophobic interactions. These results, taken together with studies from other laboratories on the expression of polyomavirus capsid proteins in mouse and insect cells (S. E. Delos, L. Montross, R. B. Moreland, and R. L. Garcea, Virology, 194:393-398, 1993; J. Forstova, N. Krauzewicz, S. Wallace, A. J. Street, S. M. Dilworth, S. Beard, and B. E. Griffin, J. Virol. 67:1405-1413, 1993), support the idea that a VP1-VP2/3 complex forms in the cytoplasm and, after translocation into the nucleus, acts as the unit for viral assembly. Images PMID:8189532

  20. 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

  1. 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

  2. Genetic diversity of the VP1 gene of duck hepatitis virus type I (DHV-I) isolates from southeast China is related to isolate attenuation.

    PubMed

    Liu, Guangqing; Wang, Fei; Ni, Zheng; Yun, Tao; Yu, Bin; Huang, Jionggang; Chen, Jianping

    2008-10-01

    The complete sequence of an isolate (ZJ-V) of Duck hepatitis virus I (DHV-I), originally taken from the field in southeast China was determined. It was 7691 nucleotides long and had 5'- and 3'-terminal non-coding regions of 626 and 315 nucleotides, respectively. The poly(A) tail contained at least 22 residues and the single open reading frame encoded a polypeptide of 2249 amino acids. The VP1 gene was also sequenced from nine southeast China field isolates and three attenuated DHV-I vaccine strains. In phylogenetic analysis of the isolates and other published sequences, attenuated and tissue-adapted isolates (including ZJ-V) clustered as genotypes significantly different from the field isolates that had not been passaged in chicken/duck embryos. There were two consistent amino acid substitutions (E(129)-->V(129) and A(142)-->S(142)) between all the field isolates and all the tissue-adapted ones. The carboxyl terminal region was generally the most variable and here the four attenuated Chinese isolates showed six consistent differences from the field isolates (S(181)-->L(181), H(183)K(184)-->R(183)G(1841), N(193)-->D(193), E(205)-->K(205), R(217)-->K(217), N(235)-->D(235)). It seems likely that at least some of these differences result from mutations leading to isolate attenuation.

  3. 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

  4. Characterization of rotavirus genotypes before and after the introduction of a monovalent rotavirus vaccine in Colombia.

    PubMed

    Peláez-Carvajal, Dioselina; Cotes-Cantillo, Karol; Paternina-Caicedo, Angel; Gentsch, Jon; de la Hoz-Restrepo, Fernando; Patel, Manish

    2014-06-01

    Strain monitoring for emergence of novel strains after the introduction of rotavirus vaccine is an integral component of routine rotavirus immunization programs. Using a laboratory based strain surveillance system between 2008 and 2012, a wide variation in strain pattern in Colombia was founded both before and after the introduction of a monovalent rotavirus vaccine in 2009. G2P[4], a strain fully heterotypic to the vaccine was predominant before vaccine introduction in 2008 (47%) and after vaccine introduction in 2010 (54%), 2011 (86%), and 2012 (32%). The presence of this strain before the introduction of vaccine and decreasing prevalence during the most recent surveillance year suggests secular variation rather than vaccine pressure as a cause for this fluctuation. While strain monitoring can be valuable after vaccine introduction, these surveillance data alone without information on disease incidence or strain specific vaccine effectiveness can be prone to misinterpretation with regard to the role of vaccine pressure on emergence of new or persistent strains.

  5. 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.

  6. New approaches in oral rotavirus vaccines.

    PubMed

    Kuate Defo, Zenas; Lee, Byong

    2016-05-01

    Rotavirus is the leading cause of severe dehydrating diarrhea worldwide, and affects primarily developing nations, in large part because of the inaccessibility of vaccines and high rates of mortality present therein. At present, there exist two oral rotaviral vaccines, Rotarix™ and RotaTeq™. These vaccines are generally effective in their actions: however, associated costs often stymie their effectiveness, and they continue to be associated with a slight risk of intussusception. While different programs are being implemented worldwide to enhance vaccine distribution and monitor vaccine administration for possible intussusception in light of recent WHO recommendation, another major problem persists: that of the reduced efficacy of the existing rotaviral vaccines in developing countries over time. The development of new oral rotavirus vaccine classes - live-attenuated vaccines, virus-like particles, lactic acid bacteria-containing vaccines, combination therapy with immunoglobulins, and biodegradable polymer-encapsulated vaccines - could potentially circumvent these problems.

  7. Porcine rotaviruses antigenically related to human rotavirus serotypes 1 and 2.

    PubMed Central

    Bellinzoni, R B; Mattion, N M; Matson, D O; Blackhall, J; La Torre, J L; Scodeller, E A; Urasawa, S; Taniguchi, K; Estes, M K

    1990-01-01

    Fecal samples from rotavirus-infected piglets were characterized by a serotyping enzyme-linked immunosorbent assay (ELISA) by using monoclonal antibodies (MAbs) specific to human serotypes 1, 2, 3, and 4 (D. O. Matson, M. K. Estes, J. W. Burns, H. B. Greenberg, K. Taniguchi, and S. Urasawa, submitted for publication). Rotavirus in 19 of 25 specimens tested from two herds of pigs from Buenos Aires province, Argentina, were classified antigenically as follows: one serotype 1, four serotype 2, two serotype 3, and no serotype 4. Six specimens reacted with both serotype 1 and 2 MAbs, and viruses in six specimens probably belonged to other serotypes because they reacted only with a VP7 common epitope MAb. Two porcine rotavirus fecal samples found to contain both serotype 1 and 2 viruses by the MAb-based test and one found to contain a serotype 2 virus were grown in tissue culture. When plaque-purified preparations of these tissue culture-adapted viruses were analyzed in the serotyping ELISA, the C60 and C86 preparations reacted only as serotype 1 viruses, indicating that the original fecal samples, which showed multiple VP7 reactivities, were heterogeneous and apparently contained two types of viruses. Testing of plaque-purified C134 virus confirmed its serotype 2 reactivity. The MAb-based serotype designations of these viruses also were confirmed by using a neutralization immunoperoxidase focus reduction assay. This is the first report of the occurrence of serotype 1 and 2 rotaviruses in animals. The MAbs originally developed to serotype human rotaviruses can be utilized to type animal rotaviruses. PMID:2157739

  8. Impact of rotavirus vaccination on hospitalizations for rotavirus diarrhea: the IVANHOE study.

    PubMed

    Gagneur, Arnaud; Nowak, Emmanuel; Lemaitre, Thomas; Segura, Jean-Francois; Delaperrière, Nadège; Abalea, Lydie; Poulhazan, Elise; Jossens, Anne; Auzanneau, Lucie; Tran, Adissa; Payan, Christopher; Jay, Nadine; de Parscau, Loic; Oger, Emmanuel

    2011-05-12

    The aim of the IVANHOE study was to determine the real-world impact of the rotavirus vaccine, controlling for epidemic-to-epidemic variation in disease burden. A population-based prospective cohort study was conducted in Brest City and 7 suburban districts (CUB area), North-western Brittany, France (210,000 inhabitants; 5500 births per year). The vaccination program started in May 2007 for a 2-year period for all infants born in the Brest birth zone through pediatricians, public outpatient clinics and general practitioners. To determine vaccine impact we monitored trends in hospitalizations for rotavirus-specific diarrhea using an active hospital-based surveillance system initiated 5 years before vaccine introduction. The number of hospitalizations for rotavirus-specific diarrhea during the 2008/2009 epidemic in infants less than 2 years of age whose parents lived within the CUB area was modelled as a function of (1) the number of hospitalizations in infants 2-5 years of age to control for epidemic-to-epidemic variation and (2) vaccine introduction. A total of 4684 infants received at least one dose. Of these, 2635 lived within the CUB area. Vaccine coverage for a complete schedule in the CUB area was 47.1%. Poisson modelling revealed a reduction by a factor of 2.04 (1.56-2.66) in the number of hospitalizations during the last epidemic season (2008/2009), the number of observed cases being equal to 30, against an expected number of 61. Relative risk reduction for hospitalizations for rotavirus diarrhea was 98% (95% CI: 83-100%). We observed a noticeable impact of vaccination on rotavirus diarrhea hospitalizations within 2 years of vaccine introduction integrating for the first time rotavirus epidemics variation. The trial is registered with ClinicalTrials.gov, number, NCT00740935.

  9. 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.

  10. Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain.

    PubMed

    Martella, V; Ciarlet, M; Bányai, K; Lorusso, E; Cavalli, A; Corrente, M; Elia, G; Arista, S; Camero, M; Desario, C; Decaro, N; Lavazza, A; Buonavoglia, C

    2006-03-15

    Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15 shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6 genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.

  11. Development of improved vaccine cell lines against rotavirus

    PubMed Central

    Wu, Weilin; Orr-Burks, Nichole; Karpilow, Jon; Tripp, Ralph A.

    2017-01-01

    Rotavirus is a major cause of severe gastroenteritis among very young children. In developing countries, rotavirus is the major cause of mortality in children under five years old, causing up to 20% of all childhood deaths in countries with high diarrheal disease burden, with more than 90% of these deaths occurring in Africa and Asia. Rotavirus vaccination mimics the first infection without causing illness, thus inducing strong and broad heterotypic immunity against prospective rotavirus infections. Two live vaccines are available, Rotarix and RotaTeq, but vaccination efforts are hampered by high production costs. Here, we present a dataset containing a genome-wide RNA interference (RNAi) screen that identified silencing events that enhanced rotavirus replication. Evaluated against several rotavirus vaccine strains, hits were validated in a Vero vaccine cell line as well as CRISPR/Cas9 generated cells permanently and stably lacking the genes that affect RV replication. Knockout cells were dramatically more permissive to RV replication and permitted an increase in rotavirus replication. These data show a means to improve manufacturing of rotavirus vaccine. PMID:28248921

  12. Hospitalizations associated with rotavirus gastroenteritis in Spain, 2001–2005

    PubMed Central

    López-de-Andrés, Ana; Jiménez-García, Rodrigo; Carrasco-Garrido, Pilar; Alvaro-Meca, Alejandro; Galarza, Patricia Graciela; de Miguel, Ángel Gil

    2008-01-01

    Background This study aims to describe and analyze hospital admissions in Spain due to rotavirus infections among children aged 5 years or under during the period 2001–2005, along with the associated health cost. Methods To update estimates of rotavirus hospitalizations rates in Spain, we conducted a retrospective study of 5 years of national hospitalization data associated with acute gastroenteritis using the Minimum Basic Data Set. Results During the study period, a total of 17.1% of all admissions due to acute gastroenteritis of any etiology in children aged ≤ 5 years were attributable to rotavirus infection as determined by the rotavirus-specific International Classification of Diseases, ninth revision, Clinical Modification code. A mean incidence of 135 hospital admissions attributable to rotavirus per 100,000 children aged ≤ 5 years was found. Hospitalizations associated with rotavirus had a marked winter-time seasonality. The estimated cost of hospital admission attributable to rotavirus has risen from 3 million euros estimated for 2001 to almost 7 million euros estimated in 2005. Conclusion Rotavirus gastroenteritis remains an important cause of hospitalizations in Spanish children, mostly during the winter season. PMID:18397512

  13. Modeling rotavirus infection and antiviral therapy using primary intestinal organoids.

    PubMed

    Yin, Yuebang; Bijvelds, Marcel; Dang, Wen; Xu, Lei; van der Eijk, Annemiek A; Knipping, Karen; Tuysuz, Nesrin; Dekkers, Johanna F; Wang, Yijin; de Jonge, Jeroen; Sprengers, Dave; van der Laan, Luc J W; Beekman, Jeffrey M; Ten Berge, Derk; Metselaar, Herold J; de Jonge, Hugo; Koopmans, Marion P G; Peppelenbosch, Maikel P; Pan, Qiuwei

    2015-11-01

    Despite the introduction of oral vaccines, rotavirus still kills over 450,000 children under five years of age annually. The absence of specific treatment prompts research aiming at further understanding of pathogenesis and the development of effective antiviral therapy, which in turn requires advanced experimental models. Given the intrinsic limitations of the classical rotavirus models using immortalized cell lines infected with laboratory-adapted strains in two dimensional cultures, our study aimed to model infection and antiviral therapy of both experimental and patient-derived rotavirus strains using three dimensional cultures of primary intestinal organoids. Intestinal epithelial organoids were successfully cultured from mouse or human gut tissues. These organoids recapitulate essential features of the in vivo tissue architecture, and are susceptible to rotavirus. Human organoids are more permissive to rotavirus infection, displaying an over 10,000-fold increase in genomic RNA following 24h of viral replication. Furthermore, infected organoids are capable of producing infectious rotavirus particles. Treatment of interferon-alpha or ribavirin inhibited viral replication in organoids of both species. Importantly, human organoids efficiently support the infection of patient-derived rotavirus strains and can be potentially harnessed for personalized evaluation of the efficacy of antiviral medications. Therefore, organoids provide a robust model system for studying rotavirus-host interactions and assessing antiviral medications.

  14. Whole-genome characterization of a Peruvian alpaca rotavirus isolate expressing a novel VP4 genotype.

    PubMed

    Rojas, Miguel; Gonçalves, Jorge Luiz S; Dias, Helver G; Manchego, Alberto; Pezo, Danilo; Santos, Norma

    2016-11-30

    The SA44 isolate of Rotavirus A (RVA) was identified from a neonatal Peruvian alpaca presenting with diarrhea, and the full-length genome sequence of the isolate (designated RVA/Alpaca-tc/PER/SA44/2014/G3P[40]) was determined. Phylogenetic analyses showed that the isolate possessed the genotype constellation G3-P[40]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which differs considerably from those of RVA strains isolated from other species of the order Artiodactyla. Overall, the genetic constellation of the SA44 strain was quite similar to those of RVA strains isolated from a bat in Asia (MSLH14 and MYAS33). Nonetheless, phylogenetic analyses of each genome segment identified a distinct combination of genes. Several sequences were closely related to corresponding gene sequences in RVA strains from other species, including human (VP1, VP2, NSP1, and NSP2), simian (VP3 and NSP5), bat (VP6 and NSP4), and equine (NSP3). The VP7 gene sequence was closely related to RVA strains from a Peruvian alpaca (K'ayra/3368-10; 99.0% nucleotide and 99.7% amino acid identity) and from humans (RCH272; 95% nucleotide and 99.0% amino acid identity). The nucleotide sequence of the VP4 gene was distantly related to other VP4 sequences and was designated as the reference strain for the new P[40] genotype. This unique genetic makeup suggests that the SA44 strain emerged from multiple reassortment events between bat-, equine-, and human-like RVA strains.

  15. Rabbit colony infected with a bovine-like G6P[11] rotavirus strain.

    PubMed

    Schoondermark-van de Ven, Esther; Van Ranst, Marc; de Bruin, Wieke; van den Hurk, Patrick; Zeller, Mark; Matthijnssens, Jelle; Heylen, Elisabeth

    2013-09-27

    Group A rotaviruses (RVAs) are the main etiological agent of infantile diarrhea in both humans and animals worldwide. A limited number of studies have investigated the molecular characteristics of RVA strains in stool specimens of rabbits, with only a few lapine RVA strains isolated and (partially) characterized to date. The most common G/P-genotype combinations found in rabbits are G3P[14] and G3P[22]. In this study a RVA strain was isolated from the small intestine of a 9-week-old rabbit from an infected laboratory rabbit colony. The RVA strain RVA/Rabbit-tc/NLD/K1130027/2011/G6P[11] was shown to possess the typical bovine G6 and P[11] genotypes. The complete genome of this unusual lapine strain was sequenced and characterized. Phylogenetic analyses of all 11 gene segments revealed the following genotype constellation: G6-P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3. The VP1, VP2, VP3, VP6, NSP2 and NSP4 genes all belonged to DS-1-like genotype 2, but clustered more closely to bovine RVA strains than to lapine RVA strains. The NSP1 genotype A13 is typically associated with bovine RVAs, while the NSP3 genotype T6 and the NSP5 genotype H3 have been found in a wide variety of species. However, the isolated strain clustered within bovine(-like) T6 and H3 subclusters. Overall, the data indicate that the RVA strain is most closely related to bovine-like RVA strains and most likely represents a direct interspecies transmission from a cow to a rabbit. Altogether, these findings indicate that a RVA strain with an entirely bovine genome constellation was able to infect and spread in a laboratory rabbit colony.

  16. Possible sex differences in the susceptibility of calves to rotavirus infection.

    PubMed Central

    Hasso, S A; Pandey, R

    1986-01-01

    The agar gel precipitation test was used to detect rotavirus antibodies in the serum of 562 calves and bovine rotavirus antigen in the feces of 347 calves. Significantly more females had rotavirus antibodies in the serum (P less than 0.01) and rotavirus antigen in the feces (P less than 0.1) than did male calves. Female buffalo calves were also found to be more susceptible than male buffalo calves to rotavirus infection. PMID:3019501

  17. 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

  18. Characteristics of a foot-and-mouth disease virus with a partial VP1 G-H loop deletion in experimentally infected cattle.

    PubMed

    Fowler, Veronica; Bashiruddin, John B; Belsham, Graham J; Stenfeldt, Carolina; Bøtner, Anette; Knowles, Nick J; Bankowski, Bartlomiej; Parida, Satya; Barnett, Paul

    2014-02-21

    Previous work in cattle illustrated the protective efficacy and negative marker potential of a A serotype foot-and-mouth disease virus (FMDV) vaccine prepared from a virus lacking a significant portion of the VP1 G-H loop (termed A(-)). Since this deletion also includes the arginine-glycine-aspartate (RGD) motif required for virus attachment to the host cell in vivo, it was hypothesised that this virus would be attentuated in naturally susceptible animals. The A(-) virus was passaged three times in cattle via needle inoculation of virus suspension delivered into the intradermal space of the tongue (intradermolingual: IDL). Included in the study were three direct contact cattle, two of which were used for the third cattle passage (by inoculation) after direct contact exposure for three days. Cattle were monitored for clinical signs and samples were collected for sequencing as well as antibody and viral genome detection by ELISA and qRT-PCR. Following needle inoculation with the A(-) virus, naïve cattle developed typical clinical signs of FMDV infection, diagnostic assays also provided positive serological and virological results. However, the contact cattle did not develop clinical signs or generate serological or virological markers indicative of FMDV infection even when the cattle were subsequently needle inoculated with 10(5) TCID50 A(-) FMDV delivered IDL following three days of direct contact exposure. The results suggest that the A(-) virus is not attentuated in cattle when inoculated IDL. This virus could be useful as a tool to understand further the natural pathogenesis, receptor usage and internalisation pathways of FMDV.

  19. Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

    PubMed

    Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

    2014-08-01

    JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment.

  20. Molecular characterization of the porcine group A rotavirus NSP2 and NSP5/6 genes from São Paulo State, Brazil, in 2011/12.

    PubMed

    Barbosa, Bruna Rocha Passos; Bernardes, Nara Thiers Cacciatori Galleti; Beserra, Laila Andreia Rodrigues; Gregori, Fábio

    2013-01-01

    Rotaviruses are responsible for the acute diarrhea in various mammalian and avian species. The nonstructural proteins NSP2 and NSP5 are involved in the rotavirus replication and the formation of viroplasm, cytoplasmic inclusion bodies within which new viral particles morphogenesis and viral RNA replication occur. There are few studies on the genetic diversity of those proteins; thus this study aims at characterizing the diversity of rotavirus based on NSP2 and NSP5 genes in rotaviruses circulating in Brazilian pig farms. For this purpose, 63 fecal samples from pig farms located in six different cities in the São Paulo State, Brazil, were screened by nested RT-PCR. Seven strains had the partial nucleotide sequencing for NSP2, whereas in six, the total sequencing for NSP5. All were characterized as genotype H1 and N1. The nucleotide identity of NSP2 genes ranged from 100% to 86.4% and the amino acid identity from 100% to 91.5%. For NSP5, the nucleotide identity was from 100% to 95.1% and the amino acid identity from 100% to 97.4%. It is concluded that the genotypes of the strains circulating in the region of study are in agreement with those reported in the literature for swine and that there is the possibility of interaction between human and animal rotaviruses.

  1. Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells.

    PubMed

    Gilbert, Leona; Välilehto, Outi; Kirjavainen, Sanna; Tikka, Päivi J; Mellett, Mark; Käpylä, Pirjo; Oker-Blom, Christian; Vuento, Matti

    2005-01-17

    A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

  2. 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.

  3. Analysis of gene selection in reassortant formation between canine rotavirus K9 and human rotaviruses with different antigenic specificities.

    PubMed

    Kobayashi, N; Taniguchi, K; Urasawa, T; Urasawa, S

    1993-01-01

    A number of antigenic mosaic reassortants which have neutralization proteins VP4 and VP7 derived from different parental strains were analysed in order to study gene selection in reassortant formation between animal and human rotaviruses (HRV). These reassortants were isolated from mixed infection of MA-104 cells with canine rotavirus strain K9 (subgroup I and G serotype 3) and HRV strains (with subgroup I or II antigen and G serotype 1-4, 9 or 12 antigen), through repeated selections with anti-VP4 and anti-VP7 neutralizing monoclonal antibodies directed specifically at HRV and K9, respectively. By serological and genomic analyses, all the isolated clones were found to be antigenic mosaic reassortants possessing VP4 of K9 and VP7 of HRV. In the reassortants between strain K9 and one of the six strains of subgroup II HRV, a single or a few genotypes with particular constellations of RNA segments were predominant, with only a few RNA segments including gene 4 (encoding VP4) being derived from K9. In contrast, in the reassortants between strain K9 and any one of the subgroup I HRV, more than nine different genotypes were identified and various RNA segments, except for segments 8 and 10, were derived from K9. These findings indicated that the RNA segments of K9 might be reassorted more readily with those of subgroup I HRV than with those of subgroup II HRV, suggesting the possible existence of functional mechanisms which determine the extent of diversity of genome selection depending on the pairs of parent strains in the reassortant formation.

  4. [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.

  5. Rotavirus-specific subclass antibody and cytokine responses in Bangladeshi children with rotavirus diarrhoea.

    PubMed

    Azim, Tasnim; Zaki, M Hasan; Podder, Goutam; Sultana, Novera; Salam, M Abdus; Rahman, S Moshfiqur; Sefat-e-Khuda; Sack, David A

    2003-02-01

    Rotavirus-specific subclass antibody responses and cytokines, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-8 (IL-8), and IL-10, were measured in children 7-24 months of age with rotavirus diarrhoea (n = 29); the responses were compared with children with watery diarrhoea from whom no enteric pathogens were isolated (controls; n = 11). All children had diarrhoea for < 5 days and were enrolled from the Dhaka Hospital of the Centre for Health and Population Research. Samples of blood and stools were collected on the day of enrollment and 18-21 days after the onset of diarrhoea. Children showing a > or = 4-fold rise in antibody titre between the acute and convalescent stages were considered to have a response. The numbers of children with rotavirus-specific IgA and IgA1 responses in stool were similar in the two groups of children. In the plasma, more children with rotavirus diarrhoea had rotavirus-specific IgA, IgA1, IgG, IgG1, and IgG3 responses than did control children (P = 0.049, 0.007, 0.001, 0.002, and 0.012, respectively). IgA2 was not detectable. Among cytokines measured in supernatants from peripheral blood mononuclear cells (PBMCs) cultured for 6 and 24 hr, IFN-gamma was the only cytokine that was higher in children with rotavirus diarrhoea compared with controls (P = 0.013). Severity of illness did not correlate with nutritional status or antibody titres, but severity did correlate with TNF-alpha during the acute stage of illness. IFN-gamma correlated positively with IgG1 titres. These findings suggest a role for IFN-gamma in the pathogenesis of rotavirus infection, but this needs confirmation by other studies. The immune responses described are relevant to future vaccine trials, as immune responses in vaccinees should mimic those in natural infection.

  6. 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.

  7. Australian Rotavirus Surveillance Program annual report, 2007/08.

    PubMed

    Kirkwood, Carl D; Cannan, David; Boniface, Karen; Bishop, Ruth F; Barnes, Graeme L

    2008-12-01

    The National Rotavirus Reference Centre together with collaborating laboratories Australia-wide conducts a laboratory based rotavirus surveillance program. This report describes the types of rotavirus strains responsible for the hospitalisation of children with acute gastroenteritis during the period 1 July 2007 to 30 June 2008, the first complete year of surveillance following introduction of rotavirus into the National Immunisation Program. Six hundred faecal samples from across Australia were examined using a combined approach of monoclonal antibody immunoassays and reverse transcription-polymerase chain reaction. Of the 419 confirmed as rotavirus positive, serotype G1 was the dominant serotype nationally, representing 52% of specimens, followed by serotype G2 (19.8%), serotype G9 (12.2%), and serotype G3 (11%). No serotype G4 strains were identified. All G1, G3 and G9 strains assayed for P genotype contained the P[8] genotype, while all G2 strains contained the P[4] genotype, except one G2 strain which possessed a P[8]. Uncommon rotavirus genotypes, G8 (n = 2) and P[9] (n = 2) were identified during this study period. There was no evidence of unexpected changes in serotype distribution during the first 12 months of rotavirus vaccine use in the National Immunisation Program.

  8. Rotavirus-associated immune thrombocytopenic purpura in children: A retrospective study.

    PubMed

    Ai, Qi; Yin, Jing; Chen, Sen; Qiao, Lijin; Luo, Na

    2016-10-01

    Certain studies have previously indicated that an association may exist between rotavirus infection and primary immune thrombocytopenic purpura (ITP). The present retrospective study aimed to investigate whether rotavirus may cause ITP in children. Firstly, the incidence of ITP in children with or without rotavirus diarrhea was compared. A 14.58% incident rate was observed in children with rotavirus diarrhea compared with a 7.22% incident rate in children without rotavirus diarrhea. Subsequently, the clinical features of ITP children with or without rotavirus infection were compared. The results indicated that ITP children with rotavirus infection were significantly younger, showed significantly decreased mean platelet volume (MPV) levels and presented a significantly higher frequency of bleeding score of 3 against ITP children without rotavirus infection. In conclusion, these findings suggest that rotavirus serves a causative role in ITP.

  9. The molecular biology of rotaviruses X: intercellular dissemination of rotavirus NSP4 requires glycosylation and is mediated by direct cell-cell contact through cytoplasmic extrusions.

    PubMed

    Yang, Weiming; McCrae, Malcolm A

    2012-02-01

    The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions, which, through site-directed mutagenesis of the N-linked glycosylation sites near the amino terminus of the protein, were shown to be dependent on its ability to become fully glycosylated. Real-time confocal microscopy was used to follow the appearance of similar cytoplasmic extrusions in virus-infected cells and revealed them to grow at a rate of ~2 microns/min to more than three cell diameters and to have a lifespan of 30-60 minutes. CellTracker dyes were used to label cell populations and facilitate the monitoring of the transfer of cytoplasm from virus-infected to surrounding uninfected cells through cytoplasmic extrusions that broke down to vesicles, seen both on the surface of and within uninfected cells in mixed cell populations. Staining of these tagged cell mixtures with monospecific antibody to NSP4 revealed the presence of the protein on uninfected cells, suggesting that the cytoplasmic extrusions formed by virus-infected cells facilitates the direct cell-cell spread of NSP4. This direct cell-cell transfer of infected cell material triggered by expression of glycosylated NSP4 in virus-infected cells may contribute to viral pathogenesis and facilitate host invasion by rotaviruses.

  10. Species-specific but not genotype-specific primary and secondary isotype-specific NSP4 antibody responses in gnotobiotic calves and piglets infected with homologous host bovine (NSP4[A]) or porcine (NSP4[B]) rotavirus.

    PubMed

    Yuan, Lijuan; Honma, Shinjiro; Ishida, Shin-Ichi; Yan, Xiao-Yi; Kapikian, Albert Z; Hoshino, Yasutaka

    2004-12-05

    Using recombinant baculoviruses expressing rotavirus NSP4 [A], [B], [C], and [D] genotypes of bovine, porcine, human, simian, or murine origin, we analyzed serum antibody responses to NSP4s in gnotobiotic calves and piglets infected by the oral/alimentary or intraamniotic route with bovine (NSP4[A]) (Wyatt, R.G., Mebus, C.A., Yolken, R.H., Kalica, A.R., James, H.D., Jr., Kapikian, A.Z., Chanock, R.M., 1979. Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus. Science 203(4380), 548-550) or porcine (NSP4[B]) (Hoshino, Y., Saif, L.J., Sereno, M.M., Chanock, R.M., Kapikian, A.Z., 1988. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 62(3), 744-748) rotaviruses. Following primary infection and challenge with virulent rotaviruses, the animals developed higher or significantly higher antibody titers to homologous host homotypic NSP4s than to heterologous host homotypic or heterologous host heterotypic NSP4s, indicating that antibody responses were species specific rather than genotype specific. Antibody responses to NSP4s corresponded closely with the phylogenetic relationships of NSP4s within a species-specific region of amino acids (aa) 131-141. In contrast, NSP4 genotypes determined by amino acid full-length sequence identity predicted poorly their "serotypes". In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus in primary infection. Thus, in an approach to immunization with a live oral rotavirus vaccine, the NSP4 protein does not appear to play an important role in protection against rotavirus disease and infection.

  11. 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.

  12. Intrahepatic portal-vein gas associated with rotavirus infection.

    PubMed

    Esposito, F; Senese, R; Salvatore, P; Vallone, G

    2011-03-01

    Rotavirus enteritis is an infectious disease of the small bowel caused by an RNA reovirus. It is manifested by cytotoxic diarrhea [1]. Rotavirus is the most common viral cause of enteritis (incidence 15-35%) [2]. In infants and children with abdominal pain and diarrhea, ultrasonography is the diagnostic study of choice, and its use has increased significantly in young patients. We describe two cases in which portal-vein gas was detected on abdominal ultrasound scans in children with severe dehydration secondary to rotavirus gastroenteritis, which resolved rapidly after treatment.

  13. 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

  14. Direct isolation in cell culture of human rotaviruses and their characterization into four serotypes.

    PubMed Central

    Wyatt, R G; James, H D; Pittman, A L; Hoshino, Y; Greenberg, H B; Kalica, A R; Flores, J; Kapikian, A Z

    1983-01-01

    Of 73 rotavirus-positive fecal specimens tested, 39 yielded a human rotavirus that could be cultivated serially in MA104 or primary African green monkey kidney cells or both; 18 were serotyped. Four distinct serotypes were identified by plaque reduction or tube neutralization assay or both, and three of these serotypes were the same as those established previously by plaque reduction, using human rotaviruses cultivated by genetic reassortment with a cultivable bovine rotavirus. Ten human rotavirus strains received from Japan were found to be similar, if not identical, to our candidate prototype strains representing these four human rotavirus serotypes. Images PMID:6311872

  15. 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.

  16. Whole genomic analysis of bovine group A rotavirus strains A5-10 and A5-13 provides evidence for close evolutionary relationship with human rotaviruses.

    PubMed

    Komoto, Satoshi; Pongsuwanna, Yaowapa; Tacharoenmuang, Ratana; Guntapong, Ratigorn; Ide, Tomihiko; Higo-Moriguchi, Kyoko; Tsuji, Takao; Yoshikawa, Tetsushi; Taniguchi, Koki

    2016-11-15

    Bovine group A rotavirus (RVA) is an important cause of acute diarrhea in calves worldwide. In order to obtain precise information on the origin and evolutionary dynamics of bovine RVA strains, we determined and analyzed the complete nucleotide sequences of the whole genomes of six archival bovine RVA strains; four Thai strains (RVA/Cow-tc/THA/A5-10/1988/G8P[1], RVA/Cow-tc/THA/A5-13/1988/G8P[1], RVA/Cow-tc/THA/61A/1989/G10P[5], and RVA/Cow-tc/THA/A44/1989/G10P[11]), one American strain (RVA/Cow-tc/USA/B223/1983/G10P[11]), and one Japanese strain (RVA/Cow-tc/JPN/KK3/1983/G10P[11]). On whole genomic analysis, the 11 gene segments of strains A5-10, A5-13, 61A, A44, B223, and KK3 were found to be considerably genetically diverse, but to share a conserved non-G/P genotype constellation except for the NSP1 gene (I2-R2-C2-M2-(A3/11/13/14)-N2-T6-E2-H3), which is commonly found in RVA strains from artiodactyls such as cattle. Furthermore, phylogenetic analysis revealed that most genes of the six strains were genetically related to bovine and bovine-like strains. Of note is that the VP1, VP3, and NSP2 genes of strains A5-10 and A5-13 exhibited a closer relationship with the cognate genes of human DS-1-like strains than those of other RVA strains. Furthermore, the VP6 genes of strains A5-10 and A5-13 appeared to be equally related to both human DS-1-like and bovine strains. Thus, strains A5-10 and A5-13 were suggested to be derived from the same evolutionary origin as human DS-1-like strains, and were assumed to be examples of bovine RVA strains that provide direct evidence for a close evolutionary relationship between bovine and human DS-1-like strains. Our findings will provide important insights into the origin of bovine RVA strains, and into evolutionary links between bovine and human RVA strains.

  17. Methods for recovering poliovirus and rotavirus from oysters.

    PubMed Central

    Speirs, J I; Pontefract, R D; Harwig, J

    1987-01-01

    Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters. PMID:2827573

  18. 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

  19. Extracellular self-assembly of virus-like particles from secreted recombinant polyoma virus major coat protein.

    PubMed

    Ng, J; Koechlin, O; Ramalho, M; Raman, D; Krauzewicz, N

    2007-12-01

    Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.

  20. Novel G10P[14] Rotavirus Strain, Northern Territory, Australia

    PubMed Central

    Donato, Celeste M.; Roczo-Farkas, Susie; Kirkwood, Carl D.

    2013-01-01

    We identified a genotype G10P[14] rotavirus strain in 5 children and 1 adult with acute gastroenteritis from the Northern Territory, Australia. Full genome sequence analysis identified an artiodactyl-like (bovine, ovine, and camelid) G10-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genome constellation. This finding suggests artiodactyl-to-human transmission and strengthens the need to continue rotavirus strain surveillance. PMID:23876354

  1. Haze is an important medium for the spread of rotavirus.

    PubMed

    Ye, Qing; Fu, Jun-Feng; Mao, Jian-Hua; Shen, Hong-Qiang; Chen, Xue-Jun; Shao, Wen-Xia; Shang, Shi-Qiang; Wu, Yi-Feng

    2016-09-01

    This study investigated whether the rotavirus infection rate in children is associated with temperature and air pollutants in Hangzhou, China. This study applied a distributed lag non-linear model (DLNM) to assess the effects of daily meteorological data and air pollutants on the rotavirus positive rate among outpatient children. There was a negative correlation between temperature and the rotavirus infection rate. The impact of temperature on the detection rate of rotavirus presented an evident lag effect, the temperature change shows the greatest impact on the detection rate of rotavirus approximate at lag one day, and the maximum relative risk (RR) was approximately 1.3. In 2015, the maximum cumulative RR due to the cumulative effect caused by the temperature drop was 2.5. Particulate matter (PM) 2.5 and PM10 were the primary air pollutants in Hangzhou. The highest RR of rotavirus infection occurred at lag 1-1.5 days after the increase in the concentration of these pollutants, and the RR increased gradually with the increase in concentration. Based on the average concentrations of PM2.5 of 53.9 μg/m(3) and PM10 of 80.6 μg/m(3) in Hangzhou in 2015, the cumulative RR caused by the cumulative effect was 2.5 and 2.2, respectively. The current study suggests that temperature is an important factor impacting the rotavirus infection rate of children in Hangzhou. Air pollutants significantly increased the risk of rotavirus infection, and dosage, lag and cumulative effects were observed.

  2. Serotype G9 Rotavirus Infections in Adults in Sweden

    PubMed Central

    Rubilar-Abreu, Elba; Hedlund, Kjell-Olof; Svensson, Lennart; Mittelholzer, Christian

    2005-01-01

    Rotavirus is a major cause of acute gastroenteritis. By examining 1,517 stool samples collected in 2001 and 2002 from Swedish adults with acute diarrhea, rotavirus was found in 3.2%, with the emerging G9P[8] serotype being the one most commonly identified (42.9%). This is the first documentation of G9 infections in adults in Europe. PMID:15750111

  3. Unexpectedly high burden of rotavirus gastroenteritis in very young infants

    PubMed Central

    2010-01-01

    Background The highest incidence of rotavirus gastroenteritis has generally been reported in children 6-24 months of age. Young infants are thought to be partially protected by maternal antibodies acquired transplacentally or via breast milk. The purpose of our study was to assess the age distribution of children with confirmed community-acquired rotavirus gastroenteritis presenting to an urban referral hospital. Methods Children presenting to The Children's Hospital of Philadelphia with acute gastroenteritis have been monitored for the presence of rotavirus antigen in the stool by ELISA (followed by genotyping if ELISA-positive) since the 1994-95 epidemic season. Results Over the last 12 rotavirus seasons prior to the introduction of the pentavalent rotavirus vaccine in 2006, stool specimens from 1646 patients tested positive for community-acquired rotavirus infection. Gender or age was not recorded in 6 and 5 cases, respectively. Overall, 58% of the cases occurred in boys. G1 was the predominant VP7 serotype, accounting for 72% of cases. The median (IQR) age was 11 (5-21) months. A total of 790 (48%) cases occurred in children outside the commonly quoted peak age range, with 27% in infants <6 months of age and 21% in children >24 months of age. A total of 220 (13%) cases occurred during the first 3 months of life, and the highest number of episodes per month of age [97 (6%)] was observed during the second month of life. Conclusions The incidence of community-acquired rotavirus gastroenteritis monitored over 12 seasons in the prevaccine era at a major university hospital was nearly constant for each month of age during the first year of life, revealing an unexpectedly high incidence of symptomatic rotavirus disease in infants <3 months old. A sizeable fraction of cases occurred in children too young to have been vaccinated according to current recommendations. PMID:20540748

  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. 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.

  6. Economic Impact of a Rotavirus Vaccine in Brazil

    PubMed Central

    Constenla, Dagna O.; Linhares, Alexandre C.; Rheingans, Richard D.; Antil, Lynn R.; Waldman, Eliseu A.; da Silva, Luiz J.

    2008-01-01

    The study was done to evaluate the cost-effectiveness of a national rotavirus vaccination programme in Brazilian children from the healthcare system perspective. A hypothetical annual birth-cohort was followed for a five-year period. Published and national administrative data were incorporated into a model to quantify the consequences of vaccination versus no vaccination. Main outcome measures included the reduction in disease burden, lives saved, and disability-adjusted life-years (DALYs) averted. A rotavirus vaccination programme in Brazil would prevent an estimated 1,804 deaths associated with gastroenteritis due to rotavirus, 91,127 hospitalizations, and 550,198 outpatient visits. Vaccination is likely to reduce 76% of the overall healthcare burden of rotavirus-associated gastroenteritis in Brazil. At a vaccine price of US$ 7-8 per dose, the cost-effectiveness ratio would be US$ 643 per DALY averted. Rotavirus vaccination can reduce the burden of gastroenteritis due to rotavirus at a reasonable cost-effectiveness ratio. PMID:19069617

  7. Characteristics of Rotavirus Diarrhea in Hospitalized Children in Kosovo

    PubMed Central

    Ismaili-Jaha, Vlora; Shala, Muje; Azemi, Mehmedali; Hoxha-Kamberi, Teuta; Avdiu, Muharrem; Spahiu, Shqipe; Jaha, Luan

    2014-01-01

    Background: Diarrhea is a leading cause of child mortality worldwide. Rotavirus is one of the most common causes of severe diarrhea and dehydration in children. Authors reviewed epidemiological and clinical data of the rotavirus diarrhea in Kosovo. Methods: This is a prospective study carried between January 1st and December 31st 2011. All data, comprising demographics, nutrition, clinical presentation, laboratory findings, management and outcome of the rotavirus diarrhea are collected on the specially designed form. Results: 116 children with rotavirus diarrhea are included in the study. The majority boys (74.4%) and children aged 0 – 12 months (82.75%). Mean age of children in the study was 16.38 months. Almost every third child in the study was hypotrophic (29.2%). More than half of the infants (55.2%) were on mixed food, somewhat more than every third was breast feeding (36.45%), and every twelfth (8.33%) was on artificial milk (animal or formula). Apart from diarrhea, present in all patients, vomiting (97.41%) and fever (43.96%) were characteristics of the clinical presentation of the diarrhea. Two thirds of the children had mild grade dehydration (70.7%). All patients recovered with no sequels. Conclusion: Rotavirus continues to be responsible for a significant portion of acute diarrhea in Kosovo. Clinical features, epidemiological data and the agglutination test are safe enough to establish the diagnosis. Treated correctly rotavirus diarrhea has a favorable outcome. PMID:25568634

  8. Safety and Immunogenicity of Sequential Rotavirus Vaccine Schedules

    PubMed Central

    Libster, Romina; McNeal, Monica; Walter, Emmanuel B.; Shane, Andi L.; Winokur, Patricia; Cress, Gretchen; Berry, Andrea A.; Kotloff, Karen L.; Sarpong, Kwabena; Turley, Christine B.; Harrison, Christopher J.; Pahud, Barbara A.; Marbin, Jyothi; Dunn, John; El-Khorazaty, Jill; Barrett, Jill

    2016-01-01

    BACKGROUND AND OBJECTIVES: Although both licensed rotavirus vaccines are safe and effective, it is often not possible to complete the schedule by using the same vaccine formulation. The goal of this study was to investigate the noninferiority of the immune responses to the 2 licensed rotavirus vaccines when administered as a mixed schedule compared with administering a single vaccine formulation alone. METHODS: Randomized, multicenter, open-label study. Healthy infants (6–14 weeks of age) were randomized to receive rotavirus vaccines in 1 of 5 different schedules (2 using a single vaccine for all doses, and 3 using mixed schedules). The group receiving only the monovalent rotavirus vaccine received 2 doses of vaccine and the other 4 groups received 3 doses of vaccine. Serum for immunogenicity testing was obtained 1 month after the last vaccine dose and the proportion of seropositive children (rotavirus immunoglobulin A ≥20 U/mL) were compared in all the vaccine groups. RESULTS: Between March 2011 and September 2013, 1393 children were enrolled and randomized. Immune responses to all the sequential mixed vaccine schedules were shown to be noninferior when compared with the 2 single vaccine reference groups. The proportion of children seropositive to at least 1 vaccine antigen at 1 month after vaccination ranged from 77% to 96%, and was not significantly different among all the study groups. All schedules were well tolerated. CONCLUSIONS: Mixed schedules are safe and induced comparable immune responses when compared with the licensed rotavirus vaccines given alone. PMID:26823540

  9. Rotavirus landscape in Africa-Towards prevention and control: A report of the 8th African rotavirus symposium, Livingstone, Zambia.

    PubMed

    Rudd, Cheryl; Mwenda, Jason; Chilengi, Roma

    2015-06-26

    The 8th African Rotavirus Symposium was held in Livingstone, Zambia from the 12-13 June 2014. Over 130 delegates from 35 countries - 28 from African nations - participated in this symposium, which included scientists, clinicians, immunisation managers, public health officials, policymakers and vaccine manufacturers. The theme for the symposium was Rotavirus Landscape in Africa-Towards Prevention and Control. At the time of the symposium, a total of 21 African countries had introduced the rotavirus vaccine into their national immunisation schedules. This meeting was particularly timely and relevant to review early data on vaccine adoption and impact from these countries. The concluding panel discussion proposed several recommendations for areas of focus moving forward in rotavirus advocacy and research.

  10. FoxP3+ regulatory T cells are not important for rotavirus clearance or the early antibody response to rotavirus.

    PubMed

    Miller, Amber D; Blutt, Sarah E; Conner, Margaret E

    2014-01-01

    Regulatory T cells produce TGF-β that contributes to IgA induction by intestinal commensal bacteria but their importance in IgA responses to pathogens has not been determined. Immunity against the enteropathogen, rotavirus, is dependent on intestinal IgA, but whether FoxP3(+) regulatory T cells contribute to this IgA is unknown. Infection with rotavirus increased the numbers of intestinal FoxP3(+) regulatory T cells. Depletion of FoxP3(+) regulatory T cells altered leukocyte activation but did not significantly alter rotavirus clearance or specific antibody levels. These data suggest FoxP3(+) regulatory T cells are not critical for the early antibody response to rotavirus infection.

  11. Monovalent rotavirus vaccine provides protection against an emerging fully heterotypic G9P[4] rotavirus strain in Mexico.

    PubMed

    Yen, Catherine; Figueroa, Jesùs Reyna; Uribe, Edgar Sánchez; Carmen-Hernández, Luz Del; Tate, Jacqueline E; Parashar, Umesh D; Patel, Manish M; Richardson López-Collado, Vesta

    2011-09-01

    After the introduction of monovalent rotavirus vaccine (RV1) in Mexico in 2006-2007, diarrhea mortality and morbidity declined substantially among Mexican children under 5 years of age. In January 2010, surveillance identified the emergence of a novel G9P[4] rotavirus strain nationwide. We conducted a case-control study to assess the field effectiveness of RV1 against severe rotavirus gastroenteritis caused by this unusual strain and to determine whether the G9P[4] emergence was related to vaccine failure or failure to vaccinate. RV1 was 94% effective (95% confidence interval, 16%-100%) against G9P[4] rotavirus-related hospitalization, indicating that its emergence was likely unrelated to vaccine pressure.

  12. 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

  13. Report of the 7th African Rotavirus Symposium, Cape Town, South Africa, 8th November 2012.

    PubMed

    Seheri, L M; Mwenda, J M; Page, N

    2014-11-12

    The 7th African Rotavirus Symposium was held in Cape Town, South Africa, on the 8th November 2012 as a Satellite Symposium at the First International African Vaccinology Conference. Over 150 delegates participated in this symposium including scientists, clinicians, health officials, policymakers and vaccine manufacturers from across Africa. Key topics discussed included rotavirus surveillance, rotavirus vaccine introduction, post rotavirus vaccine impact analysis and intussusception data and surveillance in Africa. The symposium provided early rotavirus vaccine adopter countries in Africa (South Africa, Ghana and Botswana) an opportunity to share up-to-date information on vaccine introduction, and allowed colleagues to share experiences in establishing routine rotavirus surveillance (Tanzania, Niger and Rwanda). Overall, the symposium highlighted the high burden of rotavirus in Africa, and the need to continue to strengthen efforts in preventing rotavirus diarrhoea in Africa.

  14. Genomic characterization and molecular investigation of VP7 epitopes of uncommon G10P[8] group A rotavirus strains detected in Italy in 2009.

    PubMed

    Ianiro, Giovanni; Delogu, Roberto; Fiore, Lucia; Ruggeri, Franco M

    2015-07-01

    Rotavirus strains with the uncommon genotype G10 have been detected sporadically in cases of acute gastroenteritis in humans and are thought to be transmitted zoonotically. During 2009, 10 G10P[8] rotavirus strains were detected in the stools of children hospitalized with acute diarrhoea in several paediatric hospitals in Italy. The phylogenetic analysis of the VP7 gene of the Italian G10P[8] strains analysed revealed nucleotide identities ranging from 94 to 99 %. Molecular characterization of the 11 genomic segments was performed for one of the G10 strains, which displayed a complete genomic constellation 1 for the non-G genes. The analysis of the deduced amino acid sequences of the G10 VP7 epitopes revealed low amino acid identity with common human strains of different G genotype and with the VP7 proteins included in both anti-rotavirus commercial vaccines (Rotarix and RotaTeq). Amongst the common G genotypes, the VP7 amino acid sequence of the G10 strains showed a high similarity with sequences from G9 strains. A hydrophobic cluster analysis (HCA) of the VP7 protein including aa 20-298 was performed for the G10 Italian sequences in comparison with the major human group A rotavirus G genotypes. The HCA analysis confirmed the findings obtained previously by amino acid analysis of the VP7 epitopes, detecting a genotype-specific pattern of hydrophobicity in the hypervariable regions of the major outer capsid protein.

  15. Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

    PubMed Central

    2012-01-01

    Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule. PMID:22953695

  16. Immune response of pregnant cows to bovine rotavirus immunization.

    PubMed

    Saif, L J; Smith, K L; Landmeier, B J; Bohl, E H; Theil, K W; Todhunter, D A

    1984-01-01

    Fifteen pregnant Holstein cows were freely assigned to 3 experimental groups (5 cows in each group). Cows in group I were inoculated IM and intramammarily (IMm) with Ohio Agricultural Research and Development Center (OARDC) tissue culture-propagated modified-live Nebraska calf diarrhea bovine rotavirus with added adjuvant (OARDC vaccine-immunized cows). Group II cows were given IM injections of a commercial modified-live rotavirus-coronavirus vaccine (commercial vaccine-immunized cows), and the remaining 5 cows were noninoculated controls (group III). Rotavirus antibody in colostrum and milk was mainly associated with immunoglobulin (Ig)G1, and less so with IgG2, IgA, and IgM, as analyzed by the enzyme-linked immunosorbent assay (ELISA), using monospecific anti-bovine IgG1, IgG2, IgM, and IgA sera. In serum, the rotavirus antibody was distributed almost equally between IgG1 and IgG2. The same relationships appeared in both immunized and nonvaccinated cows. All OARDC vaccine-injected cows had virus-neutralization (VN) and ELISA IgG1 rotavirus antibody titers in serum and mammary secretions at significantly increased levels (at least 100-fold; P less than 0.05) compared with the titers in groups II (commercial vaccine-immunized cows) and III (controls). Serum, colostrum, and milk antibody titers from these latter 2 groups did not differ statistically. The ELISA IgG2, IgA, and IgM rotavirus antibody titers also were significantly greater in mammary secretions from OARDC vaccine-immunized cows than in groups II and III cows. There was a high correlation between ELISA IgG1 and VN rotavirus antibody titers for all samples tested (r = 0.97, P less than 0.001), but ELISA IgG1 antibody titers were consistently higher than VN titers. The ELISA IgG1 and VN antibody titers of milk samples collected from cows 30 days after parturition were higher from the OARDC vaccine-immunized cows (ELISA IgG1, geometric mean titer (GMT) = 3,511; VN GMT = 1,689) than were titers from the

  17. Rotavirus genotypes in Belarus, 2008-2012.

    PubMed

    Semeiko, Galina V; Yermalovich, Marina A; Poliakova, Nadezhda; Mijatovic-Rustempasic, Slavica; Kerin, Tara K; Wasley, Annemarie; Videbaek, Dovile; Gentsch, Jon R; Bowen, Michael D; Samoilovich, Elena O

    2014-12-01

    This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008 to 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.

  18. A Dual Chicken IgY Against Rotavirus and Norovirus

    PubMed Central

    Dai, Ying-Chun; Zhang, Xu-Fu; Tan, Ming; Huang, Pengwei; Lei, Wen; Fang, Hao; Zhong, Weiming; Jiang, Xi

    2013-01-01

    Rotavirus (RV) and norovirus (NoV) are the two most important causes of viral gastroenteritis. While vaccine remains an effective prophylactic strategy, development of other approaches, such as passive immunization to control and treat clinical infection and illness of the two pathogens, is necessary. Previously we demonstrated that high titers of NoV-specific IgY were readily developed by immunization of chickens with the NoV P particles. In this study, we developed a dual IgY against both RV and NoV through immunization of chickens with a divalent vaccine comprising neutralizing antigens of both RV and NoV. This divalent vaccine, named P-VP8* particle, is made of the NoV P particle as a carrier with the RV spike protein VP8* as a surface insertion. Approximately 45 mg of IgY were readily obtained from each yolk with high titers of anti-P particle and anti-VP8* antibodies detected by ELISA, Western blot, HBGA blocking (NoV and RV) and neutralization (RV) assays. Reductions of RV replication were observed with viruses treated with the IgY before and after inoculation into cells, suggesting an application of the IgY as both prophylactic and a therapeutic treatment. Collectively, our data suggested that the P-VP8* based IgY could serve as a practical approach against both NoV and RV. PMID:23267830

  19. 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…

  20. Rotavirus activates dendritic cells derived from umbilical cord blood monocytes.

    PubMed

    Rosales-Martinez, D; Gutierrez-Xicotencatl, L; Badillo-Godinez, O; Lopez-Guerrero, D; Santana-Calderon, A; Cortez-Gomez, R; Ramirez-Pliego, O; Esquivel-Guadarrama, F

    2016-10-01

    Rotavirus is the most common cause of acute infectious diarrhea in human neonates and infants. However, the studies aimed at dissecting the anti-virus immune response have been mainly performed in adults. Dendritic cells (DCs) play a crucial role in innate and acquired immune responses. Therefore, it is very important to determine the response of neonatal and infant DCs to rotavirus and to compare it to the response of adult DCs. Thus, we determined the response of monocyte-derived DCs from umbilical cord blood (UCB) and adult peripheral blood (PB) to rotavirus in vitro. It was found that the rotavirus and its genome, composed of segmented doubled stranded RNA (dsRNA), induced the activation of neonatal DCs, as these cells up-regulated the levels of CD40, CD86, MHC II, TLR-3 and TLR-4, the production of cytokines IL-6, IL-12/23p40, IL-10, TGF-β (but not of IL-12p70), and the message for TNF-α and IFN-β. This activation enabled the neonatal DCs to induce a strong proliferation of allogeneic CD4(+) T cells and the production of IFN-γ. Moreover, neonatal DCs could be infected by rotavirus and sustain its replication. Neonatal DCs had a similar response as adult DCs towards rotavirus and its genome. However, adult DCs had a biased pro-inflammatory response compared to neonatal DCs, which showed a biased regulatory profile, as they produced higher levels of IL-10 and TGF-β, and were less efficient in inducing a Th1 type response. So it can be concluded that rotavirus and its genome can induce the activation of neonatal DCs in spite of their tolerogenic bias.

  1. Australian Rotavirus Surveillance Program: annual report, 2009/2010.

    PubMed

    Kirkwood, Carl D; Boniface, Karen; Bishop, Ruth F; Barnes, Graeme L

    2010-12-01

    The Australian Rotavirus Surveillance Program together with 15 collaborating laboratories Australia-wide conducts a laboratory based rotavirus surveillance program. This report describes the genotypes of rotavirus strains responsible for the hospitalisation of children with acute gastroenteritis during the period 1 July 2009 to 30 June 2010, the 3rd year of surveillance following introduction of rotavirus vaccines into the National Immunisation Program. Seven hundred and seventy-eight faecal samples were referred to the centre for G and P genotype analysis using hemi-nested multiplex reverse transcription-polymerase chain reaction. Of the 422 confirmed as rotavirus positive, genotype G1P[8] was the dominant type nationally, representing 49.3%, followed by genotype G2P[4] (21.1%). Genotypes G3P[8], G4P[8] and G9P[8] each represented less than 3% of circulating strains nationally. The dominance of G1P[8] was in part associated with a large outbreak of severe gastroenteritis in the Northern Territory in 2010. The identification of uncommon rotavirus genotype combinations has increased since vaccine introduction, with G1P[4], G2P[8] and G9P[4] identified during this survey. Single strains of G1P[6] and G4P[6] were identified during this study period. This survey continues to highlight the fluctuations in rotavirus genotypes, and results from this survey suggest there is limited genotype selection based on vaccine usage. However, the large G1P[8] outbreak of gastroenteritis in the Northern Territory may have resulted from vaccine pressure on wild-type strains.

  2. Australian Rotavirus Surveillance Program annual report, 2010/11.

    PubMed

    Kirkwood, Carl D; Roczo, Susie; Boniface, Karen; Bishop, Ruth F; Barnes, Graeme L

    2011-12-01

    The Australian Rotavirus Surveillance Program together with collaborating laboratories Australia-wide conducts a laboratory based rotavirus surveillance program. This report describes the genotypes of rotavirus strains responsible for the hospitalisation of children with acute gastroenteritis during 1 July 2010 to 30 June 2011. This report represents the fourth year of surveillance following introduction of rotavirus vaccines into the National Immunisation Program. One thousand one hundred and twenty-seven faecal samples were referred to the centre for G and P genotype analysis using hemi-nested multiplex reverse transcription-polymerase chain reaction. Eight hundred and sixteen samples were confirmed as rotavirus positive. Of these, 551 were collected from children under 5 years of age, while 265 were from older children and adults. Genotype analysis revealed that a change in the dominant type occurred in this reporting period, such that genotype G2P[4] was the dominant type nationally, representing 51% of samples, followed by genotype G1P[8] (26.1%). Genotypes G3P[8] represented 11% of samples while G4P[8] re-emerged as an important genotype, and was identified in 6% of samples. Uncommon rotavirus G and P combinations continue to be identified, with G2P[8] and G9P[4] identified during this survey. Differences in genotype distribution based on vaccine usage continue to be evident in Australian states. This survey continues to highlight the fluctuations in rotavirus genotypes, with an annual change in dominant genotypes suggesting a more dynamic wild-type population.

  3. Surveillance of rotavirus strains in the United States: identification of unusual strains. The National Rotavirus Strain Surveillance System collaborating laboratories.

    PubMed

    Griffin, D D; Kirkwood, C D; Parashar, U D; Woods, P A; Bresee, J S; Glass, R I; Gentsch, J R

    2000-07-01

    Rotavirus strains from 964 fecal specimens collected from children at 11 U.S. hospital laboratories from November 1997 to March 1998 and from samples collected at 12 laboratories from November 1998 to March 1999 were typed for G and P proteins. Serotype G1 was the predominant serotype in 1997-1998 (88%), followed by G2 (6.2%), G9 (3.3%), and G3 (1.5%). This pattern was similar to that seen in 1998-1999: G1 (79%), G2 (15%), G9 (3.0%), G4 (1.6%), and G3 (0.3%). Novel P[9] strains were identified in both seasons, and analysis of a 364-nucleotide fragment from gene segment 4 of one of the strains demonstrated 97.3% nucleotide identity with the prototype P3[9],G3 strain, AU1, isolated in Japan. This is the first report of a human AU1-like strain in the United States. These results reinforce our initial findings that serotype G9 persists in the United States but has not become a predominant strain and that the common serotypes G1 to G4 account for almost 90% of strains in circulation. Other uncommon strains exist in the United States but may have been overlooked before because of their low prevalence and the use of inadequate diagnostic tools.

  4. Comparison of pathogenicities and nucleotide changes between porcine and bovine reassortant rotavirus strains possessing the same genotype constellation in piglets and calves.

    PubMed

    Park, Jun-Gyu; Kim, Deok-Song; Matthijnssens, Jelle; Kwon, Hyoung-Jun; Zeller, Mark; Alfajaro, Mia Madel; Son, Kyu-Yeol; Hosmillo, Myra; Ryu, Eun-Hye; Kim, Ji-Yun; Lee, Ju-Hwan; Park, Su-Jin; Kang, Mun-Il; Kwon, Joseph; Choi, Jong-Soon; Cho, Kyoung-Oh

    2014-08-06

    Although reassortment is one of the most important characteristics of group A rotavirus (RVA) evolution, the host range restriction and/or virulence of reassortant RVAs remain largely unknown. The porcine 174-1 strain isolated from a diarrheic piglet was identified as a reassortant strain, harboring the same genotype constellation as the previously characterized bovine strain KJ56-1. Owing to its same genotype constellation, the pathogenicity of the porcine strain 174-1 in piglets and calves was examined for comparison with that of the bovine reassortant KJ56-1 strain, whose pathogenicity has already been demonstrated in piglets and calves. The porcine 174-1 strain induced diarrhea and histopathological changes in the small intestine of piglets and calves, whereas KJ56-1 had been reported to be virulent only in piglets, but not in calves. Therefore, full genomic sequences of 174-1 and KJ56-1 strains were analyzed to determine whether specific mutations might be associated with clinical and pathological phenotypes. Sequence alignment between the 174-1 and KJ56-1 strains detected one nucleotide substitution at the 3' untranslated region of the NSP3 gene and 16 amino acid substitutions at the VP7, VP4, VP1, VP3, NSP1 and NSP4 genes. These mutations may be critical molecular determinants for different virulence and/or pathogenicity of each strain. This study presents new insights into the host range restriction and/or virulence of RVAs.

  5. Different virulence of porcine and porcine-like bovine rotavirus strains with genetically nearly identical genomes in piglets and calves.

    PubMed

    Park, Jun-Gyu; Kim, Hyun-Jeong; Matthijnssens, Jelle; Alfajaro, Mia Madel; Kim, Deok-Song; Son, Kyu-Yeol; Kwon, Hyoung-Jun; Hosmillo, Myra; Ryu, Eun-Hye; Kim, Ji-Yun; Cena, Rohani B; Lee, Ju-Hwan; Kang, Mun-Il; Park, Sang-Ik; Cho, Kyoung-Oh

    2013-10-01

    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.

  6. Genomic constellation and evolution of Ghanaian G2P[4] rotavirus strains from a global perspective.

    PubMed

    Agbemabiese, Chantal Ama; Nakagomi, Toyoko; Doan, Yen Hai; Do, Loan Phuong; Damanka, Susan; Armah, George E; Nakagomi, Osamu

    2016-11-01

    Understanding of the genetic diversity and evolution of Rotavirus A (RVA) strains, a common cause of severe diarrhoea in children, needs to be based on the analysis at the whole genome level in the vaccine era. This study sequenced the whole genomes of six representative G2P[4] strains detected in Ghana from 2008 to 2013, and analysed them phylogenetically with a global collection of G2P[4] strains and African non-G2P[4] DS-1-like strains. The genotype constellation of the study strains was G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Strains from the same season were highly identical across the whole genome while strains from different seasons were more divergent from each other. The VP7, VP4, VP2, NSP1, and NSP5 genes belonged to lineage IVa; the VP6, VP1, NSP2, and NSP3 genes belonged to lineage V, and all these genes evolved in the same fashion as the global strains. In the NSP4 gene, lineages V (2008) and X (2009) were replaced by VI (2012/2013) whereas in the VP3 gene, lineage V (2008/2009) was replaced by VII (2012/2013) and these replacements coincided with the vaccine introduction period (2012). The evolutionary rate of the NSP4 gene was 1.2×10(-3) substitutions/site/year and was rather comparable to that of the remaining 10 genes. The multiple NSP4 lineages were explained by intra-genotype reassortment with co-circulating African human DS-1-like strains bearing G2[6], G3P[6], G6[6] and G8. There was no explicit evidence of the contribution of animal RVA strains to the genome of the Ghanaian G2P[4] strains. In summary, this study revealed the dynamic evolution of the G2P[4] strains through intra-genotype reassortment events leading to African specific lineages such IX and X in the NSP4 gene. So far, there was no evidence of a recent direct involvement of animal RVA genes in the genome diversity of African G2P[4] strains.

  7. Whole genome analysis provides evidence for porcine-to-simian interspecies transmission of rotavirus-A.

    PubMed

    Navarro, Ryan; Aung, Meiji Soe; Cruz, Katalina; Ketzis, Jennifer; Gallagher, Christa Ann; Beierschmitt, Amy; Malik, Yashpal Singh; Kobayashi, Nobumichi; Ghosh, Souvik

    2017-04-01

    We report here whole genome analysis of a porcine rotavirus-A (RVA) strain RVA/Pig-wt/KNA/ET8B/2015/G5P[13] detected in a diarrheic piglet, and nearly whole genome (except for VP4 gene) analysis of a simian RVA strain RVA/Simian-wt/KNA/08979/2015/G5P[X] detected in a non-diarrheic African green monkey (AGM) on the island of St. Kitts, Caribbean region. Strain ET8B exhibited a G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 genotype constellation that was identical to those of Brazilian porcine RVA G5P[13] strains RVA/Pig-wt/BRA/ROTA01/2013/G5P[13] and RVA/Pig-wt/BRA/ROTA07/2013/G5P[13], the only porcine G5P[13] RVAs that have been analyzed for the whole genome so far. Phylogenetically, all the 11 gene segments of ET8B were closely related to those of porcine and porcine-like human RVAs within the respective genotypes. Although the porcine G5P[13] RVAs exhibited identical genotype constellations, ET8B did not appear to share common evolutionary pathways with the Brazilian porcine G5P[13] RVAs. Interestingly, the VP2, VP3, VP6, VP7, and NSP1-NSP5 genes of simian RVA strain 08979 were closely related to those of porcine and porcine-like human RVA strains, exhibiting 99%-100% nucleotide sequence identities to cognate genes of co-circulating porcine RVA strain ET8B. On the other hand, the VP1 of 08979 appeared to be genetically divergent from porcine and human RVAs within the R1 genotype, and its exact origin could not be ascertained. Taken together, these observations suggested that simian strain 08979 might have been derived from interspecies transmission events involving transmission of ET8B-like RVAs from pigs to AGMs. In St. Kitts, AGMs often stray from the wild into livestock farms. Therefore, it may be possible that the AGM acquired the infection from a pig farm on the island. To our knowledge, this is the first report on detection of porcine-like RVAs in monkeys. Also, the present study is the first to report whole genomic analysis of a porcine RVA strain from the Caribbean

  8. 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.

  9. 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.

  10. 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.

  11. Rotavirus in organ transplantation: drug-virus-host interactions.

    PubMed

    Yin, Y; Metselaar, H J; Sprengers, D; Peppelenbosch, M P; Pan, Q

    2015-03-01

    Although rotavirus is usually recognized as the most common etiology of diarrhea in young children, it can in fact cause severe diseases in organ transplantation recipients irrespective of pediatric or adult patients. This comprehensive literature analysis revealed 200 cases of rotavirus infection with 8 related deaths in the setting of organ transplantation been recorded. Based on published cohort studies, an average incidence of 3% (187 infections out of 6176 organ recipients) was estimated. Rotavirus infection often causes severe gastroenteritis complications and occasionally contributes to acute cellular rejection in these patients. Immunosuppressive agents, universally used after organ transplantation to prevent organ rejection, conceivably play an important role in such a severe pathogenesis. Interestingly, rotavirus can in turn affect the absorption and metabolism of particular immunosuppressive medications via several distinct mechanisms. Even though rotaviral enteritis is self-limiting in general, infected transplantation patients are usually treated with intensive care, rehydration and replacement of nutrition, as well as applying preventive strategies. This article aims to properly assess the clinical impact of rotavirus infection in the setting of organ transplantation and to disseminate the interactions among the virus, host and immunosuppressive medications.

  12. Efficiency of human rotavirus propagation in cell culture.

    PubMed Central

    Ward, R L; Knowlton, D R; Pierce, M J

    1984-01-01

    This study was designed to find methods to reproducibly propagate human rotaviruses from fecal specimens and to determine the relationship between particle numbers and infectivity. Growth of virus was initially compared in primary and continuous lines of monkey kidney cells. Primary cells (African green and cynomolgus monkey kidney) supported virus growth directly from fecal specimens much more efficiently than did continuous lines of African green (CV-1) or rhesus (MA104) monkey kidney cells. Rotaviruses were grown in primary cells from 14 of 14 fecal specimens of different individuals collected over a 3-year period. Although rotaviruses in fecal samples could not always be grown in the continuous cell lines, two passages in primary cells appeared to fully adapt the viruses for propagation in the continuous cell line tested (MA104). The efficiency of rotavirus growth was quantified with five of the fecal isolates. It was calculated that, on the average, 1 out of every 46,000 particles in fecal specimens infected monkey kidney cells. After three passages in primary cells, an average of 1 out of every 6,600 progeny virus particles appeared to be infectious. Thus, rotaviruses in fecal specimens were consistently grown in primary cells, and passage in these cells both increased virus infectivity and adapted the viruses for growth in continuous cell lines. PMID:6088569

  13. Molecular epidemiology of bovine rotaviruses. Characterization of rotaviruses isolated from diarrhoeic calves by genome profile analysis.

    PubMed

    Legrottaglie, R; Rizzi, V; Agrimi, P

    1995-04-01

    Fifteen bovine rotavirus group A strains were isolated in several Italian regions over the period 1981-1989 from calves in ten neonatal diarrhoea outbreaks. The electrophoretical analysis of the genoma showed genomic variations and five different profiles were observed, including one with thirteen dsRNA segments. The finding of extra RNA fragments, with respect to the regular eleven genome segments, suggests the possibility of simultaneous or sequential infection by more than one electropherotype or a modification in the length of RNA segments during infection.

  14. Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

    PubMed Central

    Lutz, Lindy M.; Pace, Chandler R.

    2016-01-01

    ABSTRACT The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity. IMPORTANCE The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin

  15. Hospital-based Surveillance for Rotavirus Gastroenteritis Among Young Children in Bangladesh

    PubMed Central

    Gastanaduy, Paul A.; Islam, Khaleda; Rahman, Mahmudur; Rahman, Mustafizur; Luby, Stephen P.; Heffelfinger, James D.; Parashar, Umesh D.; Gurley, Emily S.

    2017-01-01

    Background: In anticipation of introduction of a rotavirus vaccine into the national immunization program of Bangladesh, active hospital-based surveillance was initiated to provide prevaccine baseline data on rotavirus disease. Methods: Children 5 years of age and younger admitted with acute gastroenteritis (AGE) (≥3 watery or looser-than-normal stools or ≥1 episode of forceful vomiting) at 7 hospitals throughout Bangladesh were identified. Clinical information and stool specimens were collected from every 4th patient. Specimens were tested for rotavirus antigen by enzyme immunoassays; 25% of detected rotaviruses were genotyped. Results: From July 2012 to June 2015, rotavirus was detected in 2432 (64%) of 3783 children hospitalized for AGE. Eight enrolled children died, including 4 (50%) who were rotavirus positive. Rotavirus was detected year-round in Bangladesh with peak detection rates of >80% during November–February. Most (86%) rotavirus AGE cases were 6–23 months of age. Sixty-nine percent of children with rotavirus had severe disease (Vesikari score, ≥11). Among 543 strains genotyped, G1P[8] (31%) and G12P[8] (29%) were the most common. Conclusions: Rotavirus is a major cause of morbidity in Bangladeshi children, accounting for nearly two-thirds of AGE hospitalizations. These data highlight the potential value of rotavirus vaccination in Bangladesh, and will be the key for future measurement of vaccine impact. PMID:27798545

  16. Multi-reassortant G3P[3] group A rotavirus in a horseshoe bat in Zambia.

    PubMed

    Sasaki, Michihito; Orba, Yasuko; Sasaki, Satoko; Gonzalez, Gabriel; Ishii, Akihiro; Hang'ombe, Bernard M; Mweene, Aaron S; Ito, Kimihito; Sawa, Hirofumi

    2016-10-01

    Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events.

  17. Survey of rotavirus infection in a Hungarian paediatric hospital. A short communication.

    PubMed

    Bányai, K; Sas, Yvette; Varga, L; Szucs, G

    2004-01-01

    In anticipation of a future vaccination program against rotavirus disease, a longitudinal survey has been set up to evaluate the epidemiologic features of rotavirus infections. In this report hospitalisation data and serotyping results are compiled from an epidemiologic survey conducted in Baranya County, Hungary. It was found that rotavirus-associated hospitalisation constituted a major part of infectious gastroenteritis cases (range, 14.9% to 28.5%). A higher proportion of rotavirus-positive cases was recorded when the serotype of predominant strains changed from G1 (1996-1999) to G4 (1999-2000), however, due to the short time period it was not possible to demonstrate a firm association between serotype prevalence and rotavirus-associated hospitalisation rate. In the future, such studies might help to understand if serotype-specific immunity against rotavirus infection plays an important role at the population level and if (re-)emerging rotavirus strains make an impact on the annual disease burden.

  18. Effectiveness and impact of rotavirus vaccines in Europe, 2006-2014.

    PubMed

    Karafillakis, Emilie; Hassounah, Sondus; Atchison, Christina

    2015-04-27

    Prior to the introduction of rotavirus vaccines in 2006, rotavirus was the leading cause of severe gastroenteritis among European children <5 years of age. We conducted a systematic review of the published literature to examine the effectiveness and impact of rotavirus vaccines in Europe following the first eight years of routine use. Four publication databases were searched, yielding 276 unique citations from February 1st, 2006 to July 31st, 2014. Twenty four studies on effectiveness (n=9) and impact (n=15) met the inclusion criteria. Across Europe, vaccine effectiveness against rotavirus-related healthcare utilisation ranged from 68% to 98%, consistent with efficacy data from clinical trials. Reductions in rotavirus hospitalisations ranged from 65% to 84%, consistent with findings from post-marketing studies from the US and Latin America. We confirm the significant public health benefit of rotavirus vaccination in Europe and provide further evidence to support implementation of universal rotavirus vaccination in all European countries.

  19. Detection of two atypical rotaviruses in the province of Misiones, Argentina.

    PubMed

    Basnec, S N; Giordano, M O; Bennun, F R; Nates, S V; Vergara, M; Depetris, A R

    1991-09-01

    Out of 317 human gastroenteritis cases studied between August 1988-August 1989, two atypical antigenically distinct rotaviruses (pararotaviruses) were detected in faecal samples among 19 rotaviruses shedding children from Misiones province, North-Eastern Argentina. A 1 3/4 year old girl a 3 years old boy, both with vomiting and normal temperature, shed these atypical rotaviruses. Their morphology by electron microscopy was identical to other rotaviruses; they contained 11 double-stranded RNA segments detected by polyacrylamide gel electrophoresis and failed to react with the antibody directed against the rotavirus group specific antigen (Rotazyme II ELISA). The electrophoretic migration of these RNAs (electropherotype) in polyacrylamide gels did not coincide with the typical pattern of distinct size classes observed in most human rotaviruses reported, instead, they appeared to be related to patterns of rotaviruses group C.

  20. Fulfilling the promise of rotavirus vaccines: how far have we come since licensure?

    PubMed

    Patel, Manish M; Glass, Roger; Desai, Rishi; Tate, Jacqueline E; Parashar, Umesh D

    2012-07-01

    Rotavirus is the most common cause of fatal and severe childhood diarrhoea worldwide. Two new rotavirus vaccines have shown efficacy against severe rotavirus disease in large clinical trials. Between 2006 and 2010, 27 countries introduced rotavirus vaccination into national immunisation programmes and, subsequently, the burden of severe rotavirus disease in these countries has decreased substantially in both vaccinated and unvaccinated children. Rotavirus vaccination has led to large, sustained declines in childhood deaths from diarrhoea in Brazil and Mexico, which supports estimates that rotavirus was the leading cause of diarrhoeal deaths in these countries. Studies after licensing have provided new insights into these vaccines, such as the duration of protection, relative effectiveness in poor populations, and strain evolution after vaccine introduction. The challenge for policy makers worldwide is to analyse the effect of vaccination in early adopter countries and to assess whether the benefits outweigh the costs and encourage wider dissemination of these vaccines.

  1. Higher proportion of G2P[4] rotaviruses in vaccinated hospitalized cases compared with unvaccinated hospitalized cases, despite high vaccine effectiveness against heterotypic G2P[4] rotaviruses.

    PubMed

    Matthijnssens, J; Zeller, M; Heylen, E; De Coster, S; Vercauteren, J; Braeckman, T; Van Herck, K; Meyer, N; Pirçon, J-Y; Soriano-Gabarro, M; Azou, M; Capiau, H; De Koster, J; Maernoudt, A-S; Raes, M; Verdonck, L; Verghote, M; Vergison, A; Van Damme, P; Van Ranst, M

    2014-10-01

    The overall vaccine effectiveness of the monovalent rotavirus vaccine in an observational, prospective, multicentre, hospital-based case-control study in Belgium (RotaBel) was 90%. However, rotavirus genotype and co-infecting pathogens are important parameters to take into account when assessing vaccine effectiveness. In this study we specifically investigated the effect of rotavirus genotypes and co-infecting pathogens on vaccine effectiveness of the monovalent vaccine. In addition, we also investigated the effect of co-infecting pathogens on disease severity. From February 2008 to June 2010 stool samples of rotavirus gastroenteritis cases of a random sample of 39 Belgian hospitals were collected and subsequently genotyped. Fisher's exact tests were performed to investigate the relationships between rotavirus genotype, co-infecting pathogens and disease severity. The vaccine effectiveness of a full series of the monovalent rotavirus vaccine against hospitalized rotavirus gastroenteritis caused by G1P[8] rotavirus strains was 95% (95% CI 77.5-98.7). Against G2P[4], the vaccine effectiveness was 85% (95% CI: 63.7-93.8). G4P[8]- and G3P[8]-specific vaccine effectiveness was 90% (95% CI 19.2-98.7) and 87% (95% CI -5.2 to 98.4), respectively. A post-hoc analysis showed that the genotype distribution was significantly related to the vaccination status (p <0.001), whereby G2P[4] strains were proportionally more prevalent in vaccinated cases than in unvaccinated cases. No statistical associations were found between co-infection status and vaccination status, Vesikari severity score or rotavirus genotype. The high vaccine effectiveness against the individual genotypes implies robust protection of the monovalent rotavirus vaccine against hospitalized rotavirus gastroenteritis caused by the major human rotavirus genotypes. The prevalence of G2P[4] requires continued monitoring.

  2. Effectiveness of rotavirus vaccination in prevention of hospital admissions for rotavirus gastroenteritis among young children in Belgium: case-control study

    PubMed Central

    Braeckman, Tessa; Van Herck, Koen; Meyer, Nadia; Pirçon, Jean-Yves; Soriano-Gabarró, Montse; Heylen, Elisabeth; Zeller, Mark; Azou, Myriam; Capiau, Heidi; De Koster, Jan; Maernoudt, Anne-Sophie; Raes, Marc; Verdonck, Lutgard; Verghote, Marc; Vergison, Anne; Matthijnssens, Jelle; Van Ranst, Marc

    2012-01-01

    Objective To evaluate the effectiveness of rotavirus vaccination among young children in Belgium. Design Prospective case-control study. Setting Random sample of 39 Belgian hospitals, February 2008 to June 2010. Participants 215 children admitted to hospital with rotavirus gastroenteritis confirmed by polymerase chain reaction and 276 age and hospital matched controls. All children were of an eligible age to have received rotavirus vaccination (that is, born after 1 October 2006 and aged ≥14 weeks). Main outcome measure Vaccination status of children admitted to hospital with rotavirus gastroenteritis and matched controls. Results 99 children (48%) admitted with rotavirus gastroenteritis and 244 (91%) controls had received at least one dose of any rotavirus vaccine (P<0.001). The monovalent rotavirus vaccine accounted for 92% (n=594) of all rotavirus vaccine doses. With hospital admission as the outcome, the unadjusted effectiveness of two doses of the monovalent rotavirus vaccine was 90% (95% confidence interval 81% to 95%) overall, 91% (75% to 97%) in children aged 3-11 months, and 90% (76% to 96%) in those aged ≥12 months. The G2P[4] genotype accounted for 52% of cases confirmed by polymerase chain reaction with eligible matched controls. Vaccine effectiveness was 85% (64% to 94%) against G2P[4] and 95% (78% to 99%) against G1P[8]. In 25% of cases confirmed by polymerase chain reaction with eligible matched controls, there was reported co-infection with adenovirus, astrovirus and/or norovirus. Vaccine effectiveness against co-infected cases was 86% (52% to 96%). Effectiveness of at least one dose of any rotavirus vaccine (intention to vaccinate analysis) was 91% (82% to 95%). Conclusions Rotavirus vaccination is effective for the prevention of admission to hospital for rotavirus gastroenteritis among young children in Belgium, despite the high prevalence of G2P[4] and viral co-infection. PMID:22875947

  3. Characterization of the antibody response against EV71 capsid proteins in Chinese individuals by NEIBM-ELISA.

    PubMed

    Ding, Yingying; Chen, Xuguang; Qian, Baohua; Wu, Guorong; He, Ting; Feng, Jiaojiao; Gao, Caixia; Wang, Lili; Wang, Jinhong; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Zhao, Chunyan; Pan, Wei

    2015-05-29

    Human enterovirus 71 (EV71) has become the major pathogen of hand, foot, and mouth disease (HFMD) worldwide, while the anti-EV71 antibody responses other than neutralizing epitopes have not been characterized. In this study, EV71 capsid proteins VP1, VP3, VP0 and various VP1 antigens were constructed to analyze anti-EV71 response in severe HFMD cases, non-HFMD outpatient children and normal adults using a novel evolved immunoglobulin-binding molecule (NEIBM)-based ELISA. The high prevalence of antibody responses against all three capsid proteins was demonstrated, and anti-EV71 VP1 showed the main antibody response. Anti-EV71 VP1 antibody response was found to predominantly target to epitopes based on the common enterovirus cross-reactive sequence. Moreover, inhibition pattern against anti-EV71 VP1 reactions in three groups was obviously different. Taken together, these results firstly characterized the anti-EV71 antibody responses which are predominantly against VP1 epitopes based on common enterovirus cross-reactive sequence. This finding could be helpful for the better understanding of anti-EV71 humoral immunity and useful for seroepidemiological surveillance.

  4. Characterization of the antibody response against EV71 capsid proteins in Chinese individuals by NEIBM-ELISA

    PubMed Central

    Ding, Yingying; Chen, Xuguang; Qian, Baohua; Wu, Guorong; He, Ting; Feng, Jiaojiao; Gao, Caixia; Wang, Lili; Wang, Jinhong; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Zhao, Chunyan; Pan, Wei

    2015-01-01

    Human enterovirus 71 (EV71) has become the major pathogen of hand, foot, and mouth disease (HFMD) worldwide, while the anti-EV71 antibody responses other than neutralizing epitopes have not been characterized. In this study, EV71 capsid proteins VP1, VP3, VP0 and various VP1 antigens were constructed to analyze anti-EV71 response in severe HFMD cases, non-HFMD outpatient children and normal adults using a novel evolved immunoglobulin-binding molecule (NEIBM)-based ELISA. The high prevalence of antibody responses against all three capsid proteins was demonstrated, and anti-EV71 VP1 showed the main antibody response. Anti-EV71 VP1 antibody response was found to predominantly target to epitopes based on the common enterovirus cross-reactive sequence. Moreover, inhibition pattern against anti-EV71 VP1 reactions in three groups was obviously different. Taken together, these results firstly characterized the anti-EV71 antibody responses which are predominantly against VP1 epitopes based on common enterovirus cross-reactive sequence. This finding could be helpful for the better understanding of anti-EV71 humoral immunity and useful for seroepidemiological surveillance. PMID:26023863

  5. Rotavirus VP6 preparations as a non-replicating vaccine candidates.

    PubMed

    Jalilvand, Somayeh; Marashi, Sayed Mahdi; Shoja, Zabihollah

    2015-06-26

    Rotavirus (RV) structural proteins VP4 and VP7, located on the surface of viral particles, elicit neutralizing antibodies (Abs) and are therefore considered to be important components of RV vaccines. However, despite inducing neutralizing Abs, limits of cross-neutralizing activity and lack of full correlation with protection limit the usefulness of these proteins as protective agents against RV disease. VP6 protein, which forms the middle layer of RV particles, is discussed as an alternative vaccine candidate since it can induce cross-protective immune responses against different RV strains although the Ab raised is not neutralizing. This report reviews different functions of VP6 that can lead to considering it as an alternative vaccine against RV disease.

  6. Vaccines: the pentavalent rotavirus vaccine: discovery to licensure and beyond.

    PubMed

    Heaton, Penny M; Ciarlet, Max

    2007-12-15

    Twenty-five years passed between the discovery of the parent strain (WC3) of the pentavalent human-bovine reassortant rotavirus vaccine (PRV) in 1981 and the licensure of PRV in 2006. This orally administered liquid vaccine, which is given as a 3-dose series, is indicated for the prevention of rotavirus gastroenteritis, caused by the G serotypes contained in the vaccine, in infants and children. PRV is recommended by the Advisory Committee on Immunization Practices and the American Academy of Pediatrics for administration to all infants in the United States. These recommendations are supported by the results of the phase III studies, which demonstrated that PRV is well tolerated and efficacious. PRV reduced rotavirus-related hospitalizations by 96% and was not associated with an increased incidence of serious adverse events, including intussusception. This report focuses on the safety and efficacy data from the late-phase studies of PRV and discusses plans for providing this vaccine to the developing world.

  7. Risk factors of rotavirus diarrhea in hospitalized children in Sanglah Hospital, Denpasar: a prospective cohort study

    PubMed Central

    2014-01-01

    Background Diarrhea is a major public health concern throughout the world because the prevalence of morbidity of diarrhea has not changed significantly in the past decade. It remains the third leading cause of death among children less than 5 years of age. Recent surveillance studies have shown that rotavirus is a significant cause of pediatric hospitalization and death due to diarrhea. Indonesia has limited data on risk factors, disease burden, and deaths in children due to rotavirus diarrhea. The objective of this study was to examine the above mentioned factors related to rotavirus diarrhea in hospitalized children in Sanglah Hospital, Denpasar. Methods A prospective cohort study was conducted at Sanglah Hospital Denpasar from April 2009 to December 2011. The present study was part of a nationwide study on Extension for Hospital-based Surveillance and Strain Characterization of Rotavirus Diarrhea Indonesia involving four hospitals throughout Indonesia as a part of the Asian Rotavirus Surveillance Network. We studied children aged <5 years who were hospitalized with acute diarrhea, and analyzed their stool samples using an immunoassay that detects the rotavirus antigen. Results A total of 656 patients met the inclusion criteria for this study. Of 5805 patients under the age of 5 who were hospitalized between April 2009 and December 2011, the prevalence of diarrhea among hospitalized pediatric patients was 11.3% and the prevalence of rotavirus diarrhea was 49.8%. The male to female ratio of those affected by rotavirus was 1.6:1. The occurrence of vomiting was significantly higher in rotavirus diarrhea than in non-rotavirus diarrhea (RR, 1.4; 95% CI, 1.08 to 1.70; p = 0.004). Conclusions Diarrhea remains an important cause of hospitalization in children, and rotavirus was the most important etiology. We found that boys had a greatest risk of rotavirus infection than girls. Good nutritional status and breastfeeding provided the same protection against rotavirus and

  8. 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.

  9. Selection of superior bifidobacteria in the presence of rotavirus

    PubMed Central

    Alp Avci, G.

    2016-01-01

    The main purpose of this study was to investigate bifidobacteria flora in fecal samples from children with rotavirus infection and determine the significance of their selected probiotic properties for improvement of health status. Enzyme-linked immunosorbent assay was used to identify rotavirus antigen in fecal samples from 94 patients with gastroenteritis and from 30 without gastroenteritis. Bifidobacteria were identified by selective media, gram reaction, colony morphology, fructose-6-phosphate phosphoketolase enzyme activity and classical identification tests. Exopolysaccharide (EPS) production was identified by phenol-sulphuric acid method. The modified method was then used to identify the quantity of taurocholic and glycocholic acid deconjugation and cholesterol elimination of the strains. Thirty-five of the 94 fecal samples were found positive for rotavirus antigen (37.23%). Bifidobacteria were identified in 59 of the samples. The EPS production ranges were 29.56-102.21 mg/L. The cholesterol elimination rates ranged between 8.36-39.22%. Furthermore, a positive and strong correlation was determined between EPS production and the presence of cholesterol (r=0.984, P<0.001). The deconjugation rates for the sodium glycocholate group was higher than the sodium taurocholate group. Rotavirus (+) bifidobacteria strains had higher EPS production, deconjugation rate and cholesterol elimination compared to bifidobacteria strains isolated from children in the rotavirus (-) sample and without gastroenteritis. Significant differences were observed among groups in all parameters (P<0.05). Given the increased number of rotavirus cases in Turkey and worldwide, it is very important to add superior bifidobacteria in the diets of infected children to improve the intestinal and vital functions. PMID:27849251

  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. Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

    DOE PAGES

    Hu, Liya; Ramani, Sasirekha; Czako, Rita; ...

    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

  12. 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.

  13. Rotavirus infections and vaccines: burden of illness and potential impact of vaccination.

    PubMed

    Grimwood, Keith; Lambert, Stephen B; Milne, Richard J

    2010-08-01

    Rotaviruses are the most common cause of severe gastroenteritis in children. By 5 years of age virtually every child worldwide will have experienced at least one rotavirus infection. This leads to an enormous disease burden, where every minute a child dies because of rotavirus infection and another four are hospitalized, at an annual societal cost in 2007 of $US2 billion. Most of the annual 527 000 deaths are in malnourished infants living in rural regions of low and middle income countries. In contrast, most measurable costs arise from medical expenses and lost parental wages in high income countries. Vaccines are the only public health prevention strategy likely to control rotavirus disease. They were developed to mimic the immunity following natural rotavirus infection that confers protection against severe gastroenteritis and consequently reduces the risk of primary healthcare utilization, hospitalization and death. The two currently licensed vaccines--one a single human strain rotavirus vaccine, the other a multiple strain human-bovine pentavalent reassortant rotavirus vaccine--are administered to infants in a two- or three-dose course, respectively, with the first dose given at 6-14 weeks of age. In various settings they are safe, immunogenic and efficacious against many different rotavirus genotypes. In high and middle income countries, rotavirus vaccines confer 85-100% protection against severe disease, while in low income regions of Africa and Asia, protection is less, at 46-77%. Despite this reduced efficacy in low income countries, the high burden of diarrheal disease in these regions means that proportionately more severe cases are prevented by vaccination than elsewhere. Post-licensure effectiveness studies show that rotavirus vaccines not only reduce rotavirus activity in infancy but they also decrease rates of rotavirus diarrhea in older and unimmunized children. A successful rotavirus vaccination program will rely upon sustained vaccine efficacy

  14. Rotavirus Replication: Plus-Sense Templates for Double-Stranded RNA Synthesis Are Made in Viroplasms

    PubMed Central

    Silvestri, Lynn S.; Taraporewala, Zenobia F.; Patton, John T.

    2004-01-01

    Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus. PMID:15220450

  15. 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

  16. 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.

  17. Both Lewis and Secretor Status Mediate Susceptibility to Rotavirus Infections in a Rotavirus Genotype–Dependent Manner

    PubMed Central

    Nordgren, Johan; Sharma, Sumit; Bucardo, Filemon; Nasir, Waqas; Günaydın, Gökçe; Ouermi, Djeneba; Nitiema, Leon W.; Becker-Dreps, Sylvia; Simpore, Jacques; Hammarström, Lennart; Larson, Göran; Svensson, Lennart

    2014-01-01

    Background. The live oral rotavirus (RV) vaccines have shown a reduced efficacy in Africa. Recent in vitro studies have shown binding of the RV surface protein (VP4) to histo–blood group antigens (HBGAs) in an RV genotype–dependent manner, suggesting them to be putative receptors for RV. The diversity of HBGA phenotypes in different ethnic populations, combined with prevalence/absence of specific RV genotypes, led us to hypothesize whether the genetic variations in HBGAs in a population limit susceptibility to certain RV genotypes, plausibly leading to reduced vaccine efficacy. Methods. Association between HBGAs status and susceptibility to RV P genotypes was investigated in children in Burkina Faso and Nicaragua. In total, 242 children with diarrhea in Burkina Faso and Nicaragua were investigated, 93 of whom were RV positive. Results. In Burkina Faso, the P[8] RV strains (n = 27) infected only Lewis- and secretor-positive children (27/27; P < .0001), but no Lewis-negative children. In contrast, the P[6] strains (n = 27) infected predominantly Lewis-negative children (n = 18; P < .0001) but also Lewis-positive children, irrespective of their secretor status. The results from Nicaragua confirmed that all P[8]-infected children (n = 22) were secretor Lewis positive. Conclusions. As VP4 of genotype P[8] is a component of current RV vaccines, our finding that Lewis-negative children are resistant to P[8] strains provides a plausible explanation for the reduced vaccine efficacy in populations with a high percentage of Lewis-negative individuals, such as in Africa. Furthermore, our findings provide a plausible explanation as to why P[6] RV strains are more common in Africa. PMID:25097083

  18. Impact of vaccination uptake on hospitalizations due to rotavirus acute gastroenteritis in 2 different socioeconomic areas of Spain

    PubMed Central

    Giménez Sánchez, Francisco; Nogueira, Esperanza Jiménez; Sánchez Forte, Miguel; Ibáñez Alcalde, Mercedes; Cobo, Elvira; Angulo, Raquel; Garrido Fernández, Pablo

    2016-01-01

    ABSTRACT 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

  19. 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

    2016-04-02

    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.

  20. 75 FR 48706 - Proposed Vaccine Information Materials for Rotavirus Vaccine

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-11

    ... HUMAN SERVICES Centers for Disease Control and Prevention Proposed Vaccine Information Materials for Rotavirus Vaccine AGENCY: Centers for Disease Control and Prevention (CDC), Department of Health and Human Services (HHS). ACTION: Notice with comment period. SUMMARY: Under the National Childhood Vaccine...

  1. Hydrophobic polycationic coatings disinfect poliovirus and rotavirus solutions.

    PubMed

    Larson, Alyssa M; Hsu, Bryan B; Rautaray, Debabrata; Haldar, Jayanta; Chen, Jianzhu; Klibanov, Alexander M

    2011-03-01

    Coating surfaces with N-alkylated polyethylenimines (PEIs), namely branched N,N-hexyl,methyl-PEI via covalent attachment to glass or linear N,N-dodecyl,methyl-PEI by physical deposition ("painting") onto polyethylene, enables the resultant materials to quickly and efficiently disinfect aqueous solutions of (non-enveloped) poliovirus and rotavirus.

  2. The Control of Rotavirus Gastroenteritis in The United States

    PubMed Central

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

    2012-01-01

    Since 2006, two new vaccines have been licensed to prevent rotavirus, the cause of 20% to 50% of severe acute gastroenteritis in young children worldwide. These vaccines have been implemented in national immunization programs in about 30 high- and middle-income countries, including the United States, and vaccine use has led to substantial decreases in diarrhea-related health care visits. In addition to reductions in diarrhea burden in vaccinated children, decreases have been observed in older, unvaccinated age groups in many settings, suggesting indirect benefits (i.e., herd immunity) from vaccination. Although the efficacy of these oral rotavirus vaccines is expectedly lower in developing countries in Asia and Africa, the public health benefits of vaccination in these settings, where more than 90% of the estimated 453,000 annual deaths from rotavirus occur, are likely to be substantial. Efforts continue to develop alternative rotavirus vaccines that could have a better efficacy and safety profile and may be less expensive. PMID:23303967

  3. Australian Rotavirus Surveillance Program: annual report, 2006-07.

    PubMed

    Kirkwood, Carl D; Cannan, David; Bogdanovic-Sakran, Nada; Bishop, Ruth F; Barnes, Graeme L

    2007-12-01

    The National Rotavirus Reference Centre, together with collaborating laboratories Australia-wide, conducts a laboratory based rotavirus surveillance program. This report describes the serotypes of rotavirus strains responsible for the hospitalisation of children with acute gastroenteritis during the period 1 July 2006 to 30 June 2007. One thousand and two faecal samples from across Australia were examined using a combined approach of monoclonal antibody immunoassays, reverse transcription-polymerase chain reaction and polyacrylamide gel analysis. Serotype G1 was the dominant serotype nationally, representing 36.7% of all strains, followed by serotype G9 (31.1%), and serotype G3 (23.3%). Serotype G2 represented less than 5% of strains, while no serotype G4 strains were identified. All G1, G3 and G9 strains assayed for P genotype contained the P[8] genotype, bar one G1 strain, which possess