Immunogenicity of recombinant Lactobacillus plantarum NC8 expressing goose parvovirus VP2 gene in BALB/c mice.

PubMed

Liu, Yu-Ying; Yang, Wen-Tao; Shi, Shao-Hua; Li, Ya-Jie; Zhao, Liang; Shi, Chun-Wei; Zhou, Fang-Yu; Jiang, Yan-Long; Hu, Jing-Tao; Gu, Wei; Yang, Gui-Lian; Wang, Chun-Feng

2017-06-30

Goose parvovirus (GPV) continues to be a threat to goose farms and has significant economic effects on the production of geese. Current commercially available vaccines only rarely prevent GPV infection. In our study, Lactobacillus (L.) plantarum NC8 was selected as a vector to express the VP2 gene of GPV, and recombinant L. plantarum pSIP409-VP2/NC8 was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 70 kDa. Mice were immunized with a 2 × 10 9 colony-forming unit/200 μL dose of the recombinant L. plantarum strain, and the ratios and numbers of CD11c + , CD3 + CD4 + , CD3 + CD8 + , and interferon gamma- and tumor necrosis factor alpha-expressing spleen lymphocytes in the pSIP409-VP2/NC8 group were higher than those in the control groups. In addition, we assessed the capacity of L. plantarum SIP409-VP2/NC8 to induce secretory IgA production. We conclude that administered pSIP409-VP2/NC8 leads to relatively extensive cellular responses. This study provides information on GPV infection and offers a clear framework of options available for GPV control strategies.

Poster - 24: Characterization of the energy dependence of high-sensitivity MCP-N TLD and Al2O3:C OSLD in-vivo dosimetry systems for 40–100 kVp energies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poirier, Yannick; Kuznetsova, Svetlana; Barajas, E

Purpose: To characterize the energy dependence of high-sensitivity MCP-N TLD and Al{sub 2}O{sub 3}:C OSLD dosimetry systems at low (40–100 kVp) energies for in-vivo dosimetry. Methods: We assessed the variation of response with energy of two detectors in the 40–100 kVp energy range: high-sensitivity MCP-N TLDs (LiF:Mg,Cu,P) and OSLDs (Al{sub 2}O{sub 3}:C). The detectors were irradiated with an XRad 320ix biological irradiator under reference conditions. The delivered dose was 10 cGy for 7 beam qualities ranging from 40–100 kVp, 1.7–4.0 mm Al, and effective energies 26.9–37.9 keV. Both sets of detectors were also irradiated under reference conditions at 6 MVmore » using a Varian Clinac 21Ex to assess the change in response from high-energy beams. Results: The MCP-N high-sensitivity TLDs were relatively insensitive to energies in the kV range, as their response varied by ±5%, i.e. well within the reproducibility limits of these detectors. However, the OSLDs exhibited a linearly-decreasing response with energy with a response 18.7% higher at 40 kVp than at 100 kVp for the same nominal dose. Compared to the 6 MV beams used in conventional radiotherapy, OSLDs responded 3.3–3.9 times higher depending on beam quality while the MCP-N TLD response was unchanged within experimental uncertainty. Conclusions: Unlike the more commonly used TLD-100, the high-sensitivity MCP-N TLDs exhibit little to no energy response. OSLDs are shown to be highly energy-dependent, both from MV to kV and within the kV range.« less

Indirect CT Venography at 80 kVp with Sinogram-Affirmed Iterative Reconstruction Compared to 120 kVp with Filtered Back Projection: Assessment of Image Quality and Radiation Dose

PubMed Central

Song, Inyoung; Yi, Jeong Geun; Park, Jeong Hee; Ko, Sung Min

2016-01-01

Objective To evaluate the image quality and radiation dose of indirect computed tomographic venography (CTV) using 80 kVp with sinogram-affirmed iterative reconstruction (SAFIRE) and 120 kVp with filtered back projection (FBP). Materials and Methods This retrospective study was approved by our institution and informed consent was waived. Sixty-one consecutive patients (M: F = 27: 34, mean age 60 ± 16, mean BMI 23.6 ± 3.6 kg/m2) underwent pelvic and lower extremity CTVs [group A (n = 31, 120 kVp, reconstructed with FBP) vs. group B (n = 30, 80 kVp, reconstructed with SAFIRE)]. The vascular enhancement, image noise, contrast-to-noise ratio (CNR), and signal-to-noise ratio (SNR) were compared. Subjective image analysis for image quality and noise was performed by two radiologists. Radiation dose was compared between the two groups. Results Compared with group A, higher mean vascular enhancement was observed in the group B (group A vs. B, 118.8 ± 15.7 HU vs. 178.6 ± 39.6 HU, p < 0.001), as well as image noise (12.0 ± 3.8 HU vs. 17.9 ± 6.1 HU, p < 0.001) and CNR (5.1 ± 1.9 vs. 7.6 ± 3.0, p < 0.001). The SNRs were not significantly different in both groups (11.2 ± 4.8 vs. 10.8 ± 3.7, p = 0.617). There was no significant difference in subjective image quality between the two groups (all p > 0.05). The subjective image noise was higher in the group B (p = 0.036 in reader 1, p = 0.005 in reader 2). The inter-observer reliability for assessing subjective image quality was good (ICC 0.746~0.784, p < 0.001). The mean CT dose index volume (CTDIvol) and mean dose length product (DLP) were significantly lower in group B than group A [CTDIvol, 6.4 ± 1.3 vs. 2.2 ± 2.2 mGy (p < 0.001); DLP, 499.1 ± 116.0 vs. 133.1 ± 45.7 mGy × cm (p < 0.001)]. Conclusions CTV using 80 kVp combined with SAFIRE provides lower radiation dose and improved CNR compared to CTV using 120 kVp with FBP. PMID:27662618

Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form.

PubMed

Royuela, Enrique; Sánchez-Fauquier, Alicia

2010-01-01

The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.

Crystal Structures of Yellowtail Ascites Virus VP4 Protease

PubMed Central

Chung, Ivy Yeuk Wah; Paetzel, Mark

2013-01-01

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala716) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser633 as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water. PMID:23511637

Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

PubMed Central

Liang, Jingjing; Sagum, Cari A.; Bedford, Mark T.; Sudol, Marius; Han, Ziying

2017-01-01

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. PMID:28076420

Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress.

PubMed

Liang, Jingjing; Sagum, Cari A; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Han, Ziying; Harty, Ronald N

2017-01-01

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.

Evaluation of the immunogenicity of a recombinant HSV-1 vector expressing human group C rotavirus VP6 protein.

PubMed

Rota, Rosana P; Palacios, Carlos A; Temprana, C Facundo; Argüelles, Marcelo H; Mandile, Marcelo G; Mattion, Nora; Laimbacher, Andrea S; Fraefel, Cornell; Castello, Alejandro A; Glikmann, Graciela

2018-06-01

Group C Rotavirus (RVC) has been associated globally with sporadic outbreaks of gastroenteritis in children and adults. RVC also infects animals, and interspecies transmission has been reported as well as its zoonotic potential. Considering its genetic diversity and the absence of effective vaccines, it is important and necessary to develop new generation vaccines against RVC for both humans and animals. The aim of the present study was to develop and characterize an HSV-1-based amplicon vector expressing a human RVC-VP6 protein and evaluate the humoral immune response induced after immunizing BALB/c mice. Local fecal samples positive for RVC were used for isolation and sequencing of the vp6 gene, which phylogenetically belongs to the I2 genotype. We show here that cells infected with the HSV[VP6C] amplicon vector efficiently express the VP6 protein, and induced specific anti-RVC antibodies in mice immunized with HSV[VP6C], in a prime-boost schedule. This work highlights that amplicon vectors are an attractive platform for the generation of safe genetic immunogens against RVC, without the addition of external adjuvants. Copyright © 2018 Elsevier B.V. All rights reserved.

Screening of binding proteins that interact with Chinese sacbrood virus VP3 capsid protein in Apis cerana larvae cDNA library by the yeast two-hybrid method.

PubMed

Fei, Dongliang; Wei, Dong; Yu, Xiaolei; Yue, Jinjin; Li, Ming; Sun, Li; Jiang, Lili; Li, Yijing; Diao, Qingyun; Ma, Mingxiao

2018-03-15

Chinese sacbrood virus (CSBV) causes larval death and apiary collapse of Apis cerana. VP3 is a capsid protein of CSBV but its function is poorly understood. To determine the function of VP3 and screen for novel binding proteins that interact with VP3, we conducted yeast two-hybrid screening, glutathione S-transferase pull-down, and co-immunoprecipitation assays. Galectin (GAL) is a protein involved in immune regulation and host-pathogen interactions. The yeast two-hybrid screen implicated GAL as a major VP3-binding candidate. The assays showed that the VP3 interacted with GAL. Identification of these cellular targets and clarifying their contributions to the host-pathogen interaction may be useful for the development of novel therapeutic and prevention strategies against CSBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

[Use of the recombinant baculovirus BacVP6C for the construction of an internal positive control of rotavirus C].

PubMed

Abid-Ayadi, I; Guix, S; Pintó, R M; Bosch, A

2011-06-01

Unlike group A, a few studies have interested other groups of the rotavirus, especially in Tunisia. The role of rotavirus C (RVC) infection is underestimated because of its sporadic nature. The aim of our study was to develop rapid diagnostic procedures of RVC by using an internal positive control of reverse transcription PCR (RT-PCR). The internal positive control (386pb) was designed from the recombinant baculovirus BacVP6C containing the full length cDNA of the Cowden strain gene 5 (1353pb). A fragment of 596pb was amplified by PCR using the BacVP6C DNA ds as template. Then, a central part of 210pb was deleted and the remaining fragment (386pb) was cloned into pGEM-3Zf(+) plasmid between SP6 and T7 RNA polymerase promoters. The obtained recombinant plasmid "pIAM1" was then used for the generation of the internal positive control by in vitro transcription. The sensibility of the RT-PCR was about 3.66×10(5) molecules of RNA/μl. The use of a shorter positive control, as compared to the wild type, allows increased specificity of the RT-PCR reaction, and could be used for efficient diagnostic and surveillance of RVC-caused diseases. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

On transform coding tools under development for VP10

NASA Astrophysics Data System (ADS)

Parker, Sarah; Chen, Yue; Han, Jingning; Liu, Zoe; Mukherjee, Debargha; Su, Hui; Wang, Yongzhe; Bankoski, Jim; Li, Shunyao

2016-09-01

Google started the WebM Project in 2010 to develop open source, royaltyfree video codecs designed specifically for media on the Web. The second generation codec released by the WebM project, VP9, is currently served by YouTube, and enjoys billions of views per day. Realizing the need for even greater compression efficiency to cope with the growing demand for video on the web, the WebM team embarked on an ambitious project to develop a next edition codec, VP10, that achieves at least a generational improvement in coding efficiency over VP9. Starting from VP9, a set of new experimental coding tools have already been added to VP10 to achieve decent coding gains. Subsequently, Google joined a consortium of major tech companies called the Alliance for Open Media to jointly develop a new codec AV1. As a result, the VP10 effort is largely expected to merge with AV1. In this paper, we focus primarily on new tools in VP10 that improve coding of the prediction residue using transform coding techniques. Specifically, we describe tools that increase the flexibility of available transforms, allowing the codec to handle a more diverse range or residue structures. Results are presented on a standard test set.

Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands.

PubMed

Sun, Xiaoman; Wang, Lihong; Qi, Jianxun; Li, Dandi; Wang, Mengxuan; Cong, Xin; Peng, Ruchao; Chai, Wengang; Zhang, Qing; Wang, Hong; Wen, Hongling; Gao, George F; Tan, Ming; Duan, Zhaojun

2018-06-01

Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines. IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new

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. Copyright © 2017 Elsevier B.V. All rights reserved.

The 2009-11 SAHKE Experiment: Preliminary 3D Vp imaging across the interseismically locked southern Hikurangi margin, Wellington, New Zealand

NASA Astrophysics Data System (ADS)

Henrys, S. A.; Wech, A.; Sato, H.; Stern, T. A.; Okaya, D. A.; Iwasaki, T.; Savage, M. K.; Mochizuki, K.; Kurashimo, E.; Sutherland, R.

2013-12-01

We present a preliminary 3D Vp model from the Seismic Array HiKurangi Experiment (SAHKE). This joint project involving New Zealand, Japan, and US institutions aims to investigate the subduction zone fault characteristics beneath Wellington. Situated above where the Pacific Plate is subducting beneath the Australian plate at a rate of c. 42 mm/yr, the Wellington region provides a unique opportunity to investigate the frictional properties, geometry, and seismic potential of a shallow, locked megathrust fault. Here the coupled plate interface is 20-30 km deep beneath land and can be sampled with onshore/offshore data from 3 sides. We have published a 2D Vp model [Henrys et al., 2013] incorporating coast-to-coast onshore-offshore transect of 50 stations and utilising first arrivals from 2000 offshore MCS shots on either side. The transect velocity model also combined first arrivals from 800 stations with 100 m spacing recorded from 12 in-line, 500 kg onshore dynamite explosions. We have expanded the transect data to now include (i) first arrivals from the dense temporary array of 50 seismometers with c. 7 km spacing augmented with 25 regional network instruments to record 49 local and 45 teleseismic earthquakes over a four month period and (ii), 69,000 offshore airgun shots from 17 MCS lines crisscrossing two sides of the array. We combine all shot and earthquake recordings to simultaneously invert c. 750,000 first arrivals for velocity structure and hypocenters in the densely sampled volume. First results from 3D, Vp tomography and relocated hypocenters provide improved resolution over previous studies. Our improved velocity model provides a high-resolution geometry of the subducting plate to support interpretation of other phases identified in SAHKE shot gathers and local earthquakes. Henrys, S., A. Wech, R. Sutherland, T. Stern, M. Savage, H. Sato, K. Mochizuki, T. Iwasaki, D. Okaya, A. Seward, B. Tozer, J. Townend, E. Kurashimo, T. Iidaka, and T. Ishiyama (2013

VpRFP1, a novel C4C4-type RING finger protein gene from Chinese wild Vitis pseudoreticulata, functions as a transcriptional activator in defence response of grapevine

PubMed Central

Yu, Yihe; Xu, Weirong; Wang, Shengyi; Xu, Yan; Li, Hui'e; Wang, Yuejin; Li, Shuxiu

2011-01-01

RING finger proteins comprise a large family and play important roles in regulation of growth and development, hormone signalling, and responses to biotic and abiotic stresses in plants. In this study, the identification and functional characterization of a C4C4-type RING finger protein gene from the Chinese wild grapevine Vitis pseudoreticulata (designated VpRFP1) are reported. VpRFP1 was initially identified as an expressed sequence tag (EST) from a cDNA library constructed from leaves of V. pseudoreticulata inoculated with the grapevine powdery mildew Uncinula necator. Sequence analysis of the deduced VpRFP1 protein based on the full-length cDNA revealed an N-terminal nuclear localization signal (NLS) and a C-terminal C4C4-type RING finger motif with the consensus sequence Cys-X2-Cys-X13-Cys-X1-Cys-X4-Cys-X2-Cys-X10-Cys-X2-Cys. Upon inoculation with U. necator, expression of VpRFP1 was rapidly induced to higher levels in mildew-resistant V. pseudoreticulata plants. In contrast, expression of VpRFP1 was down-regulated in mildew-susceptible V. vinifera plants. Western blotting using an antibody raised against VpRFP1 showed that VpRFP1 was also induced to higher levels in V. pseudoreticulata plants at 12–48 hours post-inoculation (hpi). However, there was only slight increase in VpRFP in V. vinifera plants in the same time frame, even though a more significant increase was observed at 96–144 hpi in these plants. Results from transactivation assays in yeast showed that the RING finger motif of VpRFP1 exhibited some activity of transcriptional activation; however, no activity was seen with the full-length VpRFP1. Overexpression of VpRFP1 in Arabidopsis plants was found to enhance resistance to Arabidopsis powdery mildew Golovinomyces cichoracearum, which seemed to be correlated with increased transcript levels of AtPR1 and AtPR2 in the pathogen-infected tissues. In addition, the Arabidopsis transgenic lines showed enhanced resistance to a virulent bacterial

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

PubMed

Saxena, Shikha; Desai, G S; Kumar, G Ravi; Sahoo, A P; Santra, Lakshman; Singh, Lakshya Veer

2015-05-01

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

3-D Vp/Vs Ratio Distribution in the Geothermal Reservoir at Basel, Switzerland, from Microseismic Data

NASA Astrophysics Data System (ADS)

Kummerow, J.; Reshetnikov, A.; Häring, M.; Asanuma, H.

2012-12-01

Thousands of microseismic events occurred during and after the stimulation of the 4.5km deep Basel 1 well at the Deep Heat Mining Project in Basel, Switzerland, in December 2006. The located seismicity extends about 1km in vertical direction and also 1km in NNW-SSE direction, consistent with the orientation of the maximum horizontal stress. In this study, we analyze 2100 events with magnitudes Mw>0.0, which were recorded by six borehole seismometers between December 2, 2006, and June 7, 2007. We first identify event multiplets based on waveform similarity and apply an automatic, iterative arrival time optimization to calculate high-precision P and S time picks for the multiplet events. Local estimates of the Vp/Vs ratio in the stimulated Basel geothermal reservoir are then obtained from the slope of the demeaned differential S versus P arrival times. The average value of Vp/Vs=1.70 is close to the characteristic reservoir value of 1.72, which was determined independently from sonic log measurements. Also, in the vicinity of the borehole, the depth distribution of Vp/Vs correlates well with the low-pass filtered sonic log data: Vp/Vs values are less than 1.70 at the top of the seismicity cloud at <3.9km depth, close to average at 4.0-4.4km depth, and exceed the value of 1.75 at larger depth (4.4-4.6km), consistent with the sonic log data. Furthermore, we observe a correlation of anomalous Vp/Vs values with zones of enhanced seismic reflectivity which were resolved by microseismic reflection imaging. Away from the borehole, increased Vp/Vs ratios also seem to correlate with domains of high event density, possibly indicating fluid migration paths.

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

PubMed Central

Frau, Aldo; Sgarbanti, Marco; Orsatti, Roberto

2018-01-01

The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance. PMID:29495311

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling.

PubMed

Fanunza, Elisa; Frau, Aldo; Sgarbanti, Marco; Orsatti, Roberto; Corona, Angela; Tramontano, Enzo

2018-02-24

The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z'- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feagins, Alicia R.; Basler, Christopher F., E-mail: chris.basler@mssm.edu

Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, andmore » PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV. - Highlights: • Lloviu virus (LLOV) is a new member of the filovirus family. • LLOV VP35 blocks IRF3 phosphorylation, IFN-α/β production and PKR phosphorylation. • LLOV VP24 inhibits IFN responses by targeting phospho-STAT1 KPNA interaction. • Infection by LLOV may block innate immune responses in a manner similar to EBOV.« less

Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene.

PubMed

Kattoor, Jobin Jose; Saurabh, Sharad; Malik, Yashpal Singh; Sircar, Shubhankar; Dhama, Kuldeep; Ghosh, Souvik; Bányai, Krisztián; Kobayashi, Nobumichi; Singh, Raj Kumar

2017-12-01

Rotavirus C (RVC), a known etiological agent of diarrheal outbreaks, mainly inflicts swine population globally with sporadic incidence in human, cattle, ferret, mink and dog. To demonstrate the presence of RVC in Indian swine population and characterization of its selected structural (VP6) and non-structural (NSP4 and NSP5) genes. A total of 108 diarrheic samples from different regions of India were used. Isolated RNA was loaded onto polyacrylamide gel to screen for the presence of RVs through the identification of specific electrophoretic genomic migration pattern. To characterize the RVC strains, VP6 gene and NSP4 and NSP5 genes were amplified, sequenced and analyzed. Based on VP6 gene specific diagnostic RT-PCR, the presence of RVC was confirmed in 12.0% (13/108) piglet fecal specimens. The nucleotide sequence analysis of VP6 gene, encoding inner capsid protein, from selected porcine RVC (PoRVC) strains revealed more than 93% homologies to human RVC strains (HuRVC) of Eurasian origin. These strains were distant from hitherto reported PoRVCs and clustered with HuRVCs, owning I2 genotype. However, the two non-structural genes, i.e. NSP4 and NSP5, of these strains were found to be of swine type, signifying a re-assortment event that has occurred in the Indian swine population. The findings indicate the presence of human-like RVC in Indian pigs and division of RVC clade with I2 genotype into further sub-clades. To the best of our knowledge, this appears to be the first report of RVC in Indian swine population. Incidence of human-like RVC VP6 gene in swine supports its subsequent zoonotic prospective.

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

PubMed Central

Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

2006-01-01

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

Ebola virus VP35 blocks stress granule assembly.

PubMed

Le Sage, Valerie; Cinti, Alessandro; McCarthy, Stephen; Amorim, Raquel; Rao, Shringar; Daino, Gian Luca; Tramontano, Enzo; Branch, Donald R; Mouland, Andrew J

2017-02-01

Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components for their own benefit. Here, we demonstrate that intracellular Ebola virus (EBOV) replication and transcription-competent virus like particles (trVLP) infection does not lead to SG assembly but leads to a blockade to Arsenite-induced SG assembly. Moreover we show that EBOV VP35 represses the assembly of canonical and non-canonical SGs induced by a variety of pharmacological stresses. This SG blockade requires, at least in part, the C-terminal domain of VP35. Furthermore, results from our co-immunoprecipitation studies indicate that VP35 interacts with multiple SG components, including G3BP1, eIF3 and eEF2 through a stress- and RNA-independent mechanism. These data suggest a novel function for EBOV VP35 in the repression of SG assembly. Copyright © 2016 Elsevier Inc. All rights reserved.

Joint inversion for Vp, Vs, and Vp/Vs at SAFOD, Parkfield, California

USGS Publications Warehouse

Zhang, H.; Thurber, C.; Bedrosian, P.

2009-01-01

We refined the three-dimensional (3-D) Vp, Vs and Vp/Vs models around the San Andreas Fault Observatory at Depth (SAFOD) site using a new double-difference (DD) seismic tomography code (tomoDDPS) that simultaneously solves for earthquake locations and all three velocity models using both absolute and differential P, S, and S-P times. This new method is able to provide a more robust Vp/Vs model than that from the original DD tomography code (tomoDD), obtained simply by dividing Vp by Vs. For the new inversion, waveform cross-correlation times for earthquakes from 2001 to 2002 were also used, in addition to arrival times from earthquakes and explosions in the region. The Vp values extracted from the model along the SAFOD trajectory match well with the borehole log data, providing in situ confirmation of our results. Similar to previous tomographic studies, the 3-D structure around Parkfield is dominated by the velocity contrast across the San Andreas Fault (SAF). In both the Vp and Vs models, there is a clear low-velocity zone as deep as 7 km along the SAF trace, compatible with the findings from fault zone guided waves. There is a high Vp/Vs anomaly zone on the southwest side of the SAF trace that is about 1-2 km wide and extends as deep as 4 km, which is interpreted to be due to fluids and fractures in the package of sedimentary rocks abutting the Salinian basement rock to the southwest. The relocated earthquakes align beneath the northeast edge of this high Vp/Vs zone. We carried out a 2-D correlation analysis for an existing resistivity model and the corresponding profiles through our model, yielding a classification that distinguishes several major lithologies. ?? 2009 by the American Geophysical Union.

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

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 C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

Detection of uncommon G3P[3] rotavirus A (RVA) strain in rat possessing a human RVA-like VP6 and a novel NSP2 genotype.

PubMed

Ianiro, Giovanni; Di Bartolo, Ilaria; De Sabato, Luca; Pampiglione, Guglielmo; Ruggeri, Franco M; Ostanello, Fabio

2017-09-01

Rotavirus is one of the leading causes of acute gastroenteritis in infants and young children. RVAs infect not only humans but also a wide range of mammals including rats, which represent a reservoir of several other zoonotic pathogens. Due to the segmented nature of the RVA genome, animal RVA strains can easily adapt to the human host by reassortment with co-infecting human viruses. This study aims to detect and characterize RVA in the intestinal content of Italian sinantropic rats (Rattus rattus). Out of 40 samples examined following molecular approach, one resulted positive for RVA. The molecular characterization of VP1-4, 6 and 7, and NSP1-5 genes by sequencing revealed the genomic constellation G3-P[3]-I1-R11-C11-M10-A22-N18-T14-E18-H13. This uncommon genomic combination includes: the VP1-4,VP7, the NSP1, 3, 4 and 5 gene segments, closely related to those of RVA from rodents, the N18 novel genotype established for the NSP2 gene segment and the human Wa-like VP6 gene, suggesting interspecies reassortment. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Calibration of coronary calcium scores determined using iterative image reconstruction (AIDR 3D) at 120, 100, and 80 kVp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blobel, Joerg, E-mail: Joerg.Blobel@toshiba-medical.eu; Mews, Juergen; Schuijf, Joanne D.

Purpose: Computed tomography (CT) radiation dose reduction is frequently achieved by applying lower tube voltages and using iterative reconstruction (IR). For calcium scoring, the reference protocol at 120 kVp with filtered back projection (FBP) is still used, because kVp and IR may influence the Agatston score (AS) and volume score (VS). The authors present a two-step method to optimize dose: first, to determine the lowest feasible exposure and highest noise thresholds; second, to define a calibration method that ensures that the AS and VS are similar to the reference protocol. Methods: AS and VS were measured for an anthropomorphic thoracicmore » phantom that includes a calcium calibration module. The phantom was scanned on a 320-row CT scanner, at tube voltages of 120 kVp using FBP, and 120, 100, and 80 kVp using adaptive iterative dose reduction (AIDR 3D) reconstruction. The minimum CTDIs were determined based on three objective quality criteria. Calibration was performed to estimate adjusted CT number thresholds for the lower kVp acquisitions. Finally, the accuracies of the total and individual insert scores at dose level close to the minimum CTDI level were investigated and compared to low (FBP{sub LD} − 120) and high (FBP{sub HD} − 120) dose reference protocols (based on ten repeated acquisitions for each group). Results: IR allows the exposure to be reduced by 69% at 120 kVp, with no significant effect on the total scores when averaged over all included dose steps and compared to FBP-120 (AS: 693 vs 699, p = 0.182; VS: 588 vs 587 mm{sup 3}, p = 0.569). Also when averaged over ten repeated scans and compared to FBP{sub HD} − 120 (AS: 709 vs 704, p = 0.435; VS: 604 vs 601 mm{sup 3}, p = 0.479), there is no statistical significant effect. Reducing the peak tube voltage allows even greater dose reductions: 73% at 100 kVp and 76% at 80 kVp. The calibrated CT number thresholds for analysis at 120, 100, and 80 kVp were, respectively, 130, 133, and 160 HU

Silencing the alarms: innate immune antagonism by rotavirus NSP1 and VP3

PubMed Central

Morelli, Marco; Ogden, Kristen M.; Patton, John T.

2016-01-01

The innate immune response involves a broad array of pathogen sensors that stimulate the production of interferons (IFN) to induce an antiviral state. Rotavirus, a significant cause of childhood gastroenteritis and a member of the Reoviridae family of segmented, double-stranded RNA viruses, encodes at least two direct antagonists of host innate immunity: NSP1 and VP3. NSP1, a putative E3 ubiquitin ligase, mediates the degradation of cellular factors involved in both IFN induction and downstream signaling. VP3, the viral capping enzyme, utilizes a 2H-phosphodiesterase domain to prevent activation of the cellular oligoadenylate synthase (OAS)-RNase L pathway. Computational, molecular, and biochemical studies have provided key insights into the structural and mechanistic basis of innate immune antagonism by NSP1 and VP3 of group A rotaviruses (RVA). Future studies with non-RVA isolates will be essential to understand how other RV species evade host innate immune responses. PMID:25724417

Identification of a novel NLS of herpes simplex virus type 1 (HSV-1) VP19C and its nuclear localization is required for efficient production of HSV-1.

PubMed

Li, You; Zhao, Lei; Wang, Shuai; Xing, Junji; Zheng, Chunfu

2012-09-01

Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of two copies of VP23 and one copy of VP19C. Here, we identified a non-classical NLS of VP19C between aa 50 and 61, and the nuclear import of VP19C was mediated by RanGTP and importin β1-, but not importin α5-, dependent pathway. Additionally, recombinant virus harbouring this NLS mutation (NLSm) replicates less efficiently as wild-type. These data strongly suggested that the nuclear import of VP19C is required for efficient HSV-1 production.

Numerical Analysis Using WRF-SBM for the Cloud Microphysical Structures in the C3VP Field Campaign: Impacts of Supercooled Droplets and Resultant Riming on Snow Microphysics

NASA Technical Reports Server (NTRS)

Iguchi, Takamichi; Matsui, Toshihisa; Shi, Jainn J.; Tao, Wei-Kuo; Khain, Alexander P.; Hao, Arthur; Cifelli, Robert; Heymsfield, Andrew; Tokay, Ali

2012-01-01

Two distinct snowfall events are observed over the region near the Great Lakes during 19-23 January 2007 under the intensive measurement campaign of the Canadian CloudSat/CALIPSO validation project (C3VP). These events are numerically investigated using the Weather Research and Forecasting model coupled with a spectral bin microphysics (WRF-SBM) scheme that allows a smooth calculation of riming process by predicting the rimed mass fraction on snow aggregates. The fundamental structures of the observed two snowfall systems are distinctly characterized by a localized intense lake-effect snowstorm in one case and a widely distributed moderate snowfall by the synoptic-scale system in another case. Furthermore, the observed microphysical structures are distinguished by differences in bulk density of solid-phase particles, which are probably linked to the presence or absence of supercooled droplets. The WRF-SBM coupled with Goddard Satellite Data Simulator Unit (G-SDSU) has successfully simulated these distinctive structures in the three-dimensional weather prediction run with a horizontal resolution of 1 km. In particular, riming on snow aggregates by supercooled droplets is considered to be of importance in reproducing the specialized microphysical structures in the case studies. Additional sensitivity tests for the lake-effect snowstorm case are conducted utilizing different planetary boundary layer (PBL) models or the same SBM but without the riming process. The PBL process has a large impact on determining the cloud microphysical structure of the lake-effect snowstorm as well as the surface precipitation pattern, whereas the riming process has little influence on the surface precipitation because of the small height of the system.

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

PubMed Central

Graham, Kate L.; Halasz, Peter; Tan, Yan; Hewish, Marilyn J.; Takada, Yoshikazu; Mackow, Erich R.; Robinson, Martyn K.; Coulson, Barbara S.

2003-01-01

Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection. PMID:12941907

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine.

PubMed

Del Vecchio, Kathryn; Frick, Cary T; Gc, Jeevan B; Oda, Shun-Ichiro; Gerstman, Bernard S; Saphire, Erica Ollmann; Chapagain, Prem P; Stahelin, Robert V

2018-03-02

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures ( i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys 224, 225 and Lys 274, 275 ). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3

PubMed Central

Martínez-Torrecuadrada, Jorge L.; Lázaro, Beatriz; Rodriguez, José F.; Casal, J. Ignacio

2000-01-01

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions. PMID:10882666

Antigenic properties and diagnostic potential of baculovirus-expressed infectious bursal disease virus proteins VPX and VP3.

PubMed

Martínez-Torrecuadrada, J L; Lázaro, B; Rodriguez, J F; Casal, J I

2000-07-01

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions.

Dependencies of pore pressure on elastic wave velocities and Vp/Vs ratio for thermally cracked gabbro

NASA Astrophysics Data System (ADS)

Nishimura, K.; Uehara, S. I.; Mizoguchi, K.

2015-12-01

Marine seismic refraction have found that there are high Vp/Vs ratio regions in oceanic crusts at subducting oceanic plates (e.g, Cascadia subduction zone (2.0-2.8) (Audet et al., 2009)). Previous studies based on laboratory measurements indicated that Vp/Vs ratio is high when porosity and/or pore pressure is high (Christensen, 1984; Peacock et al., 2011). Although several studies have investigated the relationships between fracture distributions and Vp, Vs (e.g., Wang et al., 2012; Blake et al., 2013), the relationships for rocks (e.g., gabbro and basalt) composing oceanic crust are still unclear. This study reports the results of laboratory measurements of Vp, Vs (transmission method) at controlled confining and pore pressure and estimation of Vp/Vs ratio for thermally cracked gabbro which mimic highly fractured rocks in the high Vp/Vs ratio zone, in order to declare the dependence of fracture distributions on Vp/Vs. For the measurements, we prepared three type specimens; a non-heated intact specimen, specimens heated up to 500 °C and 700 °C for 24 hours. Porosities of intact, 500 °C and 700 °C specimens measured under the atmospheric pressure are 0.5, 3.4 and 3.5%, respectively. Measurements were conducted at a constant confining pressure of 50 MPa, with decreasing pore pressure from 49 to 0.1 MPa and then increasing to 49 MPa. While Vp/Vs for the intact specimen is almost constant at elevated pore pressure, the Vp/Vs values for the thermally cracked ones were 2.0~2.2 when pore pressure was larger than 30 MPa. In future, we will reveal the relationship between the measured elastic wave velocities and the characteristics of the microfracture distribution. This work was supported by JSPS Grant-in-Aid for Scientific Research (Grant Number 26400492).

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.

Decreased LRIG1 in Human Ovarian Cancer Cell SKOV3 Upregulates MRP-1 and Contributes to the Chemoresistance of VP16.

PubMed

Yang, Hua; Yao, Jun; Yin, Jiangpin; Wei, Xuan

2016-05-01

The leucine-rich repeats and immunoglobulin-like domains (LRIG) are used as tumor suppressors in clinical applications. Although the LRIG has been identified to manipulate the cell proliferation via various oncogenic receptor tyrosine kinases in diverse cancers, its role in multidrug resistance needs to be further elucidated, especially in human ovarian cancer. We herein established that the etoposide (VP16)-resistant SKOV3 human ovarian cancer cell clones (SKOV3/VP16 cells) and mRNA expression of LRIG1 were significantly reduced by the treatment of VP16 in a concentration-dependent manner. Moreover, downregulated LRIG1 in SKOV3 could enhance the colony formation and resist the inhibition of proliferation by VP16, leading to the elevated expression of Bcl-2 and decreased apoptosis of SKOV3. Interestingly, our results uncovered that the multidrug resistance-associated protein 1 (MRP-1) was upregulated for the chemoresistance of VP16. To overcome the chemoresistance of SKOV3, SKOV3/VP16 was ectopically expressed of LRIG1. We found that the inhibition of VP16 on colony formation and proliferation was remarkably enhanced with increased apoptosis in SKOV3/VP16. Furthermore, the expression of MRP-1 and Bcl-2 was also inhibited, suggesting that the LRIG1could negatively control MRP-1 and the apoptosis to improve the sensitivity of VP16-related chemotherapy.

Computed tomographic venography for varicose veins of the lower extremities: prospective comparison of 80-kVp and conventional 120-kVp protocols.

PubMed

Cho, Eun-Suk; Kim, Joo Hee; Kim, Sungjun; Yu, Jeong-Sik; Chung, Jae-Joon; Yoon, Choon-Sik; Lee, Hyeon-Kyeong; Lee, Kyung Hee

2012-01-01

To prospectively investigate the feasibility of an 80-kilovolt (peak) (kVp) protocol in computed tomographic venography for varicose veins of the lower extremities by comparison with conventional 120-kVp protocol. Attenuation values and signal-to-noise ratio of iodine contrast medium (CM) were determined in a water phantom for 2 tube voltages (80 kVp and 120 kVp). Among 100 patients, 50 patients were scanned with 120 kVp and 150 effective milliampere second (mAs(eff)), and the other 50 patients were scanned with 80 kVp and 390 mAs(eff) after the administration of 1.7-mL/kg CM (370 mg of iodine per milliliter). The 2 groups were compared for venous attenuation, contrast-to-noise ratio, and subjective degree of venous enhancement, image noise, and overall diagnostic image quality. In the phantom, the attenuation value and signal-to-noise ratio value for iodine CM at 80 kVp were 63.8% and 33.0% higher, respectively, than those obtained at 120 kVp. The mean attenuation of the measured veins of the lower extremities was 148.3 Hounsfield units (HU) for the 80-kVp protocol and 94.8 HU for the 120-kVp protocol. Contrast-to-noise ratio was also significantly higher with the 80-kVp protocol. The overall diagnostic image quality of the 3-dimensional volume-rendered images was good with both protocols. The subjective score for venous enhancement was higher at the 80-kVp protocol. The mean volume computed tomography dose index of the 80-kVp (5.6 mGy) protocol was 23.3% lower than that of the 120-kVp (7.3 mGy) protocol. The use of the 80-kVp protocol improved overall venous attenuation, especially in perforating vein, and provided similarly high diagnostic image quality with a lower radiation dose when compared to the conventional 120-kVp protocol.

Molecular basis for ebolavirus VP35 suppression of human dendritic cell maturation.

PubMed

Yen, Benjamin; Mulder, Lubbertus C F; Martinez, Osvaldo; Basler, Christopher F

2014-11-01

Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA)-binding protein that inhibits RIG-I signaling and alpha/beta interferon (IFN-α/β) responses by both dsRNA-binding-dependent and -independent mechanisms. VP35 also suppresses dendritic cell (DC) maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) that independently ablate dsRNA binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs (MDDCs) using a lentivirus-based expression system. VP35-WT suppressed not only IFN-α/β but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA, or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I · C). The F239A and R322A mutants exhibited greatly reduced suppression of IFN-α/β and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the VP35 mutants impaired IFN-β production induced by Toll-like receptor 3 (TLR3) or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Furthermore, VP35 did not prevent lipopolysaccharide (LPS)-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. Therefore, it may be possible to counteract EBOV immune evasion by using treatments that bypass the VP35-imposed block to DC maturation. The VP35 protein, which is an

[Prokaryotic expression of vp3 gene of Muscovy duck parvovirus, and its antiserum preparation for detection of virus multiplication].

PubMed

Huang, Yu; Zhu, Yumin; Dong, Shijuan; Yu, Ruisong; Zhang, Yuanshu; Li, Zhen

2015-01-01

New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

An Overview of the 3C-STAR project

NASA Astrophysics Data System (ADS)

Zhang, Y.

2009-04-01

Over the past three decades, city clusters have played a leading role in the economic growth of China, owing to their collective economic capacity and interdependency. However, pollution prevention lags behind the economic boom, led to a general decline in air quality in city clusters. As a result, industrial emissions and traffic exhausts together contribute to high levels of ozone (O3) and fine particulate matter (PM2.5) pollution problems ranging from urban to regional scale. Such high levels of both primary and secondary airborne pollutants lead to the development of a (perhaps typically Chinese) "air pollution complex" concept. Air pollution complex is particularly true and significant in Beijing-Tianjin area, Pearl River Delta (PRD) and Yangtze River Delta. The concurrent high concentrations of O3 and PM2.5 in PRD as well as in other China city clusters have led to rather unique pollution characteristics due to interactions between primary emissions and photochemical processes, between gaseous compounds and aerosol phase species, and between local and regional scale processes. The knowledge and experience needed to find solutions to the unique pollution complex in China are still lacking. Starting from 2007, we launch a major project "Synthesized Prevention Techniques for Air Pollution Complex and Integrated Demonstration in Key City-Cluster Region" (3C-STAR) to address those problems scientifically and technically. The purpose of the project is to build up the capacity of regional air pollution control and to establish regional coordination mechanism for joint implementation of pollution control. The project includes a number of key components technically: regional air quality monitoring network and super-sites, regional dynamic emission inventory of multi-pollutants, regional ensemble air quality forecasting model system, and regional management system supported by decision making platform. The 3C-STAR project selected PRD as a core area to have technical

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

PubMed Central

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

2014-01-01

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

The evolution of Vp1 gene in enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

PubMed

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

2014-01-01

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

Targeting of rotavirus VP6 to DEC-205 induces protection against the infection in mice.

PubMed

Badillo-Godinez, O; Gutierrez-Xicotencatl, L; Plett-Torres, T; Pedroza-Saavedra, A; Gonzalez-Jaimes, A; Chihu-Amparan, L; Maldonado-Gama, M; Espino-Solis, G; Bonifaz, L C; Esquivel-Guadarrama, F

2015-08-20

Rotavirus (RV) is the primary etiologic agent of severe gastroenteritis in human infants. Although two attenuated RV-based vaccines have been licensed to be applied worldwide, they are not so effective in low-income countries, and the induced protection mechanisms have not been clearly established. Thus, it is important to develop new generation vaccines that induce long lasting heterotypic immunity. VP6 constitutes the middle layer protein of the RV virion. It is the most conserved protein and it is the target of protective T-cells; therefore, it is a potential candidate antigen for a new generation vaccine against the RV infection. We determined whether targeting the DEC-205 present in dendritic cells (DCs) with RV VP6 could induce protection at the intestinal level. VP6 was cross-linked to a monoclonal antibody (mAb) against murine DEC-205 (αDEC-205:VP6), and BALB/c mice were inoculated subcutaneously (s.c.) twice with the conjugated containing 1.5 μg of VP6 in the presence of polyinosinic-polycytidylic acid (Poly I:C) as adjuvant. As controls and following the same protocol, mice were immunized with ovalbumin (OVA) cross-linked to the mAb anti-DEC-205 (αDEC-205:OVA), VP6 cross-linked to a control isotype mAb (Isotype:VP6), 3 μg of VP6 alone, Poly I:C or PBS. Two weeks after the last inoculation, mice were orally challenged with a murine RV. Mice immunized with α-DEC-205:VP6 and VP6 alone presented similar levels of serum Abs to VP6 previous to the virus challenge. However, after the virus challenge, only α-DEC-205:VP6 induced up to a 45% IgA-independent protection. Memory T-helper (Th) cells from the spleen and the mesenteric lymph node (MLN) showed a Th1-type response upon antigen stimulation in vitro. These results show that when VP6 is administered parenterally targeting DEC-205, it can induce protection at the intestinal level at a very low dose, and this protection may be Th1-type cell dependent. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of ebola virus.

PubMed

Hartman, Amy L; Bird, Brian H; Towner, Jonathan S; Antoniadou, Zoi-Anna; Zaki, Sherif R; Nichol, Stuart T

2008-03-01

Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.

dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.

PubMed

Zinzula, Luca; Esposito, Francesca; Pala, Daniela; Tramontano, Enzo

2012-03-01

The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions. Copyright © 2012 Elsevier B.V. All rights reserved.

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress.

PubMed

Han, Ziying; Sagum, Cari A; Takizawa, Fumio; Ruthel, Gordon; Berry, Corbett T; Kong, Jing; Sunyer, J Oriol; Freedman, Bruce D; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Harty, Ronald N

2017-10-15

Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress. IMPORTANCE Ebola

Structure of the Ebola VP35 interferon inhibitory domain.

PubMed

Leung, Daisy W; Ginder, Nathaniel D; Fulton, D Bruce; Nix, Jay; Basler, Christopher F; Honzatko, Richard B; Amarasinghe, Gaya K

2009-01-13

Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 A and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.

Full protection against African horsesickness (AHS) in horses induced by baculovirus-derived AHS virus serotype 4 VP2, VP5 and VP7.

PubMed

Martínez-Torrecuadrada, J L; Díaz-Laviada, M; Roy, P; Sánchez, C; Vela, C; Sánchez-Vizcaíno, J M; Casal, J I

1996-06-01

African horsesickness virus serotype 4 (AHSV-4) outer capsid protein VP2, or VP2 and VP5 plus inner capsid protein VP7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. When the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. However, purified VP2 or VP2 and VP5 in the absence of VP7 failed to induce neutralizing antibodies and protection. Immunization with non-purified proteins enhanced the titres of neutralizing antibodies. Again, the combination of the three proteins was able to confer total protection to immunized horses, which showed absence of viraemia. The antigenicity of recombinant VP2 was analysed with a collection of 30 MAbs. Both purified and unpurified recombinant VP2 proteins showed different antigenic patterns in comparison to that of VP2 on virions. An immunization experiment with four more horses confirmed these results. The vaccine described here would not only prevent the disease, but would drastically reduce the propagation of the virus by vectors.

Thermoelastic properties of liquid Fe-C revealed by sound velocity and density measurements at high pressure

NASA Astrophysics Data System (ADS)

Shimoyama, Yuta; Terasaki, Hidenori; Urakawa, Satoru; Takubo, Yusaku; Kuwabara, Soma; Kishimoto, Shunpachi; Watanuki, Tetsu; Machida, Akihiko; Katayama, Yoshinori; Kondo, Tadashi

2016-11-01

Carbon is one of the possible light elements in the cores of the terrestrial planets. The P wave velocity (VP) and density (ρ) are important factors for estimating the chemical composition and physical properties of the core. We simultaneously measured the VP and ρ of Fe-3.5 wt % C up to 3.4 GPa and 1850 K by using ultrasonic pulse-echo method and X-ray absorption methods. The VP of liquid Fe-3.5 wt % C decreased linearly with increasing temperature at constant pressure. The addition of carbon decreased the VP of liquid Fe by about 2% at 3 GPa and 1700 K and decreased the Fe density by about 2% at 2 GPa and 1700 K. The bulk modulus of liquid Fe-C and its pressure (P) and temperature (T) effects were precisely determined from directly measured ρ and VP data to be K0,1700 K = 83.9 GPa, dKT/dP = 5.9(2), and dKT/dT = -0.063 GPa/K. The addition of carbon did not affect the isothermal bulk modulus (KT) of liquid Fe, but it decreased the dK/dT of liquid Fe. In the ρ-VP relationship, VP increases linearly with ρ and can be approximated as VP (m/s) = -6786(506) + 1537(71) × ρ (g/cm3), suggesting that Birch's law is valid for liquid Fe-C at the present P-T conditions. Our results imply that at the conditions of the lunar core, the elastic properties of an Fe-C core are more affected by temperature than those of Fe-S core.

Application of bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2.

PubMed

Feenstra, Femke; Pap, Janny S; van Rijn, Piet A

2015-02-04

Bluetongue is a disease of ruminants caused by the bluetongue virus (BTV). Bluetongue outbreaks can be controlled by vaccination, however, currently available vaccines have several drawbacks. Further, there are at least 26 BTV serotypes, with low cross protection. A next-generation vaccine based on live-attenuated BTV without expression of non-structural proteins NS3/NS3a, named Disabled Infectious Single Animal (DISA) vaccine, was recently developed for serotype 8 by exchange of the serotype determining outer capsid protein VP2. DISA vaccines are replicating vaccines but do not cause detectable viremia, and induce serotype specific protection. Here, we exchanged VP2 of laboratory strain BTV1 for VP2 of European serotypes 2, 4, 8 and 9 using reverse genetics, without observing large effects on virus growth. Exchange of VP2 from serotype 16 and 25 was however not possible. Therefore, chimeric VP2 proteins of BTV1 containing possible immunogenic regions of these serotypes were studied. BTV1, expressing 1/16 chimeric VP2 proteins was functional in virus replication in vitro and contained neutralizing epitopes of both serotype 1 and 16. For serotype 25 this approach failed. We combined VP2 exchange with the NS3/NS3a negative phenotype in BTV1 as previously described for serotype 8 DISA vaccine. DISA vaccine with 1/16 chimeric VP2 containing amino acid region 249-398 of serotype 16 raised antibodies in sheep neutralizing both BTV1 and BTV16. This suggests that DISA vaccine could be protective for both parental serotypes present in chimeric VP2. We here demonstrate the application of the BT DISA vaccine platform for several serotypes and further extend the application for serotypes that are unsuccessful in single VP2 exchange. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

PubMed Central

Xue, Qiao

2017-01-01

Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects domestic and wild cloven-hoofed animals. The structural protein VP1 plays an important role in FMDV pathogenesis. However, the interacting partners of VP1 in host cells and the effects of these interactions in FMDV replication remain incompletely elucidated. Here, we identified a porcine cell protein, serine/threonine kinase 3 (STK3), which interacts with FMDV VP1 using the yeast two-hybrid system. The VP1-STK3 interaction was further confirmed by coimmunoprecipitation experiments in human embryonic kidney 293T and porcine kidney 15 (PK-15) cells. The carboxyl-terminal region (amino acids 180–214) of VP1 was essential for its interaction with STK3. The effects of overexpression and underexpressing of STK3 in PK-15 cells were assessed, and the results indicated that STK3 significantly inhibited FMDV replication. Our data expand the role of STK3 during viral infection, provide new information regarding the host cell kinases that are involved in viral replication, and identify potential targets for future antiviral strategies. PMID:29226127

VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis.

PubMed

Xu, G; Wilson, W; Mecham, J; Murphy, K; Zhou, E M; Tabachnick, W

1997-07-01

The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis of Fe3O4@P4VP@ZIF-8 core-shell microspheres and their application in a Knoevenagel condensation reaction

NASA Astrophysics Data System (ADS)

Miao, Zongcheng; Yang, Fengxia; Luan, Yi; Shu, Xin; Ramella, Daniele

2017-12-01

In this work, a core-shell magnetic composite Fe3O4@P4VP@ZIF-8 microspheres were successfully designed and synthesized. A polymerization approach on the surface of pre-made Fe3O4 microspheres was employed for the synthesis of Fe3O4@P4VP. The zinc-derived Zeolite Imidazolate Framework (ZIF) shell was introduced through a layer-by-layer strategy. The obtained Fe3O4@P4VP@ZIF-8 core-shell structure was employed as an efficient Knoevenagel condensation catalyst for a variety of aldehydes. Furthermore, the inner P4VP layer also served as a basic additive in the condensation reaction process, while much less homogeneous basic additive was used. High catalytic reaction efficiency was achieved when the P4VP layer was utilized in combination with a Lewis acidity bearing ZIF-8 layer. The Fe3O4@P4VP@ZIF-8 catalyst was tested for recyclability and no drop in the catalytic activity was observed after more than five cycles.

The ebolavirus VP24 interferon antagonist

PubMed Central

Zhang, Adrianna P.P.; Abelson, Dafna M.; Bornholdt, Zachary A.; Liu, Tong; Woods, Jr, Virgil L.; Saphire, Erica Ollmann

2012-01-01

Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways.1,2 The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation.3 A more recent discovery is that VP24 also binds STAT1 directly,4 suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses. PMID:23076242

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwarz, Toni M.; Edwards, Megan R.; Diederichs, Audrey

ABSTRACT Zaire ebolavirus(EBOV),Bundibugyo ebolavirus(BDBV), andReston ebolavirus(RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA bindingmore » affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCEThe interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of theEbolavirusgenus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability.

PubMed

Schwarz, Toni M; Edwards, Megan R; Diederichs, Audrey; Alinger, Joshua B; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

2017-02-15

Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

PubMed

Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H

2000-04-01

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Integrated Medical Model (IMM) Project Verification, Validation, and Credibility (VVandC)

NASA Technical Reports Server (NTRS)

Walton, M.; Boley, L.; Keenan, L.; Kerstman, E.; Shah, R.; Young, M.; Saile, L.; Garcia, Y.; Meyers, J.; Reyes, D.

2015-01-01

The Integrated Medical Model (IMM) Project supports end user requests by employing the Integrated Medical Evidence Database (iMED) and IMM tools as well as subject matter expertise within the Project. The iMED houses data used by the IMM. The IMM is designed to forecast relative changes for a specified set of crew health and mission success risk metrics by using a probabilistic model based on historical data, cohort data, and subject matter expert opinion. A stochastic approach is taken because deterministic results would not appropriately reflect the uncertainty in the IMM inputs. Once the IMM was conceptualized, a plan was needed to rigorously assess input information, framework and code, and output results of the IMM, and ensure that end user requests and requirements were considered during all stages of model development and implementation, as well as lay the foundation for external review and application. METHODS: In 2008, the Project team developed a comprehensive verification and validation (VV) plan, which specified internal and external review criteria encompassing 1) verification of data and IMM structure to ensure proper implementation of the IMM, 2) several validation techniques to confirm that the simulation capability of the IMM appropriately represents occurrences and consequences of medical conditions during space missions, and 3) credibility processes to develop user confidence in the information derived from the IMM. When the NASA-STD-7009 (7009) [1] was published, the Project team updated their verification, validation, and credibility (VVC) project plan to meet 7009 requirements and include 7009 tools in reporting VVC status of the IMM. Construction of these tools included meeting documentation and evidence requirements sufficient to meet external review success criteria. RESULTS: IMM Project VVC updates are compiled recurrently and include updates to the 7009 Compliance and Credibility matrices. Reporting tools have evolved over the lifetime of

The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.

Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures ofmore » the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.« less

Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

PubMed Central

Watanabe, Marika; Phamduong, Ellen; Huang, Chu-Han; Itoh, Noriko; Bernal, Janie; Nakanishi, Akira; Rundell, Kathleen; Gjoerup, Ole

2013-01-01

The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1ΔC, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1ΔC in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1ΔC. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins. PMID:23427157

A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

PubMed

Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

2016-01-01

Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

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

PubMed

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

2016-01-01

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

[Effects of canine IL-2 and IL-7 genes on enhancing immunogenicity of canine parvovirus VP2 gene vaccine in mice].

PubMed

Chen, Huihui; Zhong, Fei; Li, Xiujin; Wang, Lu; Sun, Yan; Neng, Changai; Zhang, Kao; Li, Wenyan; Wen, Jiexia

2012-11-04

To investigate the effects of canine interleukin-2 (cIL-2) and cIL-7 genes on enhancing the immunogenicity of canine parvovirus (CPV) VP2 DNA vaccine. The bicistronic vectors of cIL-2 and cIL-7 genes were constructed using the eukaryotic expression vector containing internal ribosome entry site (IRES). The cIL-2/ cIL-7 dicistronic vector plus previously constructed vectors, including CPV VP2 DNA vaccine vector, cIL-2 vector and cIL-7 vector, were used to co-immunize mice with different combinations, consisting of VP2 alone, VP2 + cIL-2, VP2 + cIL-7 and VP2 + cIL-2/cIL-7. The VP2-specific antibody levels in immunized mice were measured by ELISA at different time post-immunization. The proliferation indices and interferon-gamma expression were measured by lymphocyte proliferation assay and ELISA, respectively. The cIL-2/cIL-7 bicistronic vector was correct and could mediate cIL-2 and cIL-7 gene expression in eukaryotic cells. Immunization results revealed that the antibody titers and the neutralizing antibody levels of the mice co-immunized with VP2 + cIL-7/cIL-2 vectors were significantly higher than that with either VP2 + cIL-2 vectors or VP2 + cIL-7 vectors (P < 0.05). The lymphocyte proliferation indices of VP2 + cIL-7/cIL-2 vector-immunized mice were also higher than that of other two groups although not statistically significant. However, the IFN-gamma expression levels of VP2 + cIL-7/cIL-2 vector-immunized mice were significantly higher than other immunized mice (P < 0.05). The cIL-2 and cIL-7 genes showed the significant synergic effects on enhancing the immunogenecity of CPV VP2 DNA vaccine.

Species-Specific and Cross-Reactive IgG1 Antibody Binding to Viral Capsid Protein 1 (VP1) Antigens of Human Rhinovirus Species A, B and C

PubMed Central

Iwasaki, Jua; Smith, Wendy-Anne; Stone, Shane R.; Thomas, Wayne R.; Hales, Belinda J.

2013-01-01

Background Human rhinoviruses (HRV) are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects. Methods Recombinant polypeptides of viral capsid protein 1 (VP1) from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST) fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults. Results Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P<0.0001). A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies. Conclusions High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined. PMID:23950960

RNA binding specificity of Ebola virus transcription factor VP30.

PubMed

Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

2016-09-01

The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

Kobuvirus VP3 protein restricts the IFN-β-triggered signaling pathway by inhibiting STAT2-IRF9 and STAT2-STAT2 complex formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Qianqian; Lan, Xi; Wang, Chen

Emerged porcine kobuvirus (PKV) has adversely affected the global swine industry since 2008, but the etiological biology of PKV is unclear. Screening PKV-encoded structural and non-structural proteins with a type I IFN-responsive luciferase reporter showed that PKV VP3 protein inhibited the IFN-β-triggered signaling pathway, resulting in the decrease of VSV-GFP replication. QPCR data showed that IFN-β downstream cytokine genes were suppressed without cell-type specificity as well. The results from biochemical experiments indicated that PKV VP3 associated with STAT2 and IRF9, and interfered with the formation of the STAT2-IRF9 and STAT2-STAT2 complex, impairing nuclear translocation of STAT2 and IRF9. Taken together,more » these data reveal a new mechanism for immune evasion of PKV. - Highlights: •PKV VP3 inhibits the IFN-β-triggered signaling pathway. •VP3 associates with STAT2 and IRF9. •VP3 blocks the STAT2-IRF9 nuclear translocation. •VP3 utilizes a novel strategy for innate immune evasion.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.

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 themore » 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.« less

Prevalence and genetic heterogeneity of porcine group C rotaviruses in nursing and weaned piglets in Ohio, USA and identification of a potential new VP4 genotype.

PubMed

Amimo, J O; Vlasova, A N; Saif, L J

2013-05-31

Swine fecal samples collected from seven farms were screened for group C rotaviruses (RVCs) using a reverse transcription-polymerase chain reaction assay. A total of 380 samples were tested and 19.5% were positive. Of the 128 samples collected in 2012, 23.5% from nursing piglets and 8.5% from weaned piglets were RVC positive, with a higher RVC frequency in diarrheic (28.4%) than in non-diarrheic (6.6%) piglets. Two strains (RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px) from two different farms were characterized genetically to gain information on virus diversity based on full length sequences of the inner capsid VP6, enterotoxin NSP4 and the outer capsid VP7 and VP4 (partial for RV0104) genes. The VP6 gene of the two strains showed high (99%) nucleotide identity to one another, 84-91% identity to other porcine RVCstrains and 81-82% identity to human and bovine RVC strains. The NSP4 gene analysis revealed that RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px strains were not closely related to each other (87% identity), but shared higher identity with prototype RVC/Pig-wt/USA/Cowden/1980/G1Px strain (93% and 89%, respectively) and were more distantly related to human strains (72-76% identity). The VP7 gene analysis indicated that the two strains were distantly related to one another (72% identity). RVC/Pig-wt/USA/RV0143/2012/G6Px was most closely related to porcine RVC G6 strains (82-86% identity), whereas RVC/Pig-wt/USA/RV0104/2011/G3PX was most closely related to porcine HF (G3) strain (94% identity). Analysis of the full length nucleotide sequence of the VP4 gene revealed that RVC/Pig-wt/USA/RV0143/2012/G6Px was distantly related to porcine (75%), bovine (74%) and human (70%) strains. The deduced amino acid identities (69.5-75.6%) of VP4 between RVC/Pig-wt/USA/RV0143/2012/G6Px and other RVCs were low; hence, we propose that this strain comprises a new VP4 genotype. Our results indicate high genetic heterogeneity in RVCs

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.

Joint 3-D tomographic imaging of Vp, Vs and Vp/Vs and hypocenter relocation at Sinabung volcano, Indonesia from November to December 2013

USGS Publications Warehouse

Nugraha, Andri Dian; Indrastuti, Novianti; Kusnandar, Ridwan; Gunawan, Hendra; McCausland, Wendy A.; Aulia, Atin Nur; Harlianti, Ulvienin

2018-01-01

We conducted travel time tomography using P- and S-wave arrival times of volcanic-tectonic (VT) events that occurred between November and December 2013 to determine the three-dimensional (3D) seismic velocity structure (Vp, Vs, and Vp/Vs) beneath Sinabung volcano, Indonesia in order to delineate geological subsurface structure and to enhance our understanding of the volcanism itself. This was a time period when phreatic explosions became phreatomagmatic and then magma migrated to the surface forming a summit lava dome. We used 4846 VT events with 16,138 P- and 16,138 S-wave arrival time phases recorded by 6 stations for the tomographic inversion. The relocated VTs collapse into three clusters at depths from the surface to sea level, from 2 to 4 km below sea level, and from 5 to 8.5 km below sea level. The tomographic inversion results show three prominent regions of high Vp/Vs (~ 1.8) beneath Sinabung volcano at depths consistent with the relocated earthquake clusters. We interpret these anomalies as intrusives associated with previous eruptions and possibly surrounding the magma conduit, which we cannot resolve with this study. One anomalous region might contain partial melt, at sea level and below the eventual eruption site at the summit. Our results are important for the interpretation of a conceptual model of the “plumbing system” of this hazardous volcano.

Measurement and validation of benchmark-quality thick-target tungsten X-ray spectra below 150 kVp.

PubMed

Mercier, J R; Kopp, D T; McDavid, W D; Dove, S B; Lancaster, J L; Tucker, D M

2000-11-01

Pulse-height distributions of two constant potential X-ray tubes with fixed anode tungsten targets were measured and unfolded. The measurements employed quantitative alignment of the beam, the use of two different semiconductor detectors (high-purity germanium and cadmium-zinc-telluride), two different ion chamber systems with beam-specific calibration factors, and various filter and tube potential combinations. Monte Carlo response matrices were generated for each detector for unfolding the pulse-height distributions into spectra incident on the detectors. These response matrices were validated for the low error bars assigned to the data. A significant aspect of the validation of spectra, and a detailed characterization of the X-ray tubes, involved measuring filtered and unfiltered beams at multiple tube potentials (30-150 kVp). Full corrections to ion chamber readings were employed to convert normalized fluence spectra into absolute fluence spectra. The characterization of fixed anode pitting and its dominance over exit window plating and/or detector dead layer was determined. An Appendix of tabulated benchmark spectra with assigned error ranges was developed for future reference.

[Escherichia coli heat-labile enterotoxin B subunit enhances the immune response against canine parvovirus VP2 in mice immunized by VP2 DNA vaccine].

PubMed

Han, Dongmei; Zhong, Fei; Li, Xiujin; Wang, Wei; Wang, Xingxing; Pan, Sumin

2011-01-01

To investigate the effect of Escherichia coli heat-labile enterotoxin (LT) B subunit (LTB) gene on canine parvovirus (CPV) VP2 gene vaccine. The LTB gene was amplified by PCR from genomic DNA of E. coli 44815 strain. The VP2-70 fragment (210 bp) encoding major epitope of VP2 (70 amino acids) was amplified by PCR from a plasmid encoding VP2 gene. VP2-70 and LTB genes were inserted into the eukaryotic vector to construct VP2-70 gene,LTB gene and VP2-70-LTB fused gene vectors. The mice were immunized with VP2-70 vector, VP2-70-LTB fused vector, or VP2-70 vector plus LTB vector, respectively. The antibody titers at the different time were measured by using ELISA method. The spleen lymphocyte proliferation activity was analyzed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The sequence of VP2-70 and LTB genes was identified. The recombinant VP2-70 and LTB proteins could be expressed in HEK293T cells in a secretory manner. The mice immunized with VP2-70 vector, VP2-70-LTB vector or VP2-70 vector plus LTB vector could generate the specific antibody against VP2 protein. The antibody titer immunized with VP2-70-LTB vector reached 1:5120 at 35 d post immunization, significantly higher than that of other two groups (P < 0.01). For antibody isotype analysis, the IgG1 isotype antibody titers in all test groups were significantly higher than of IgG2a (P < 0.01). The high-level spleen lymphocyte stimulation index was observed in the three test groups under the stimulation with Con A, higher than that in control groups (P < 0.01). LTB gene could enhance the humoral immune response of CPV VP2 gene vaccine in mice.

Preliminary molecular detection of the somatic embryogenesis receptor-like kinase (VpSERK) and knotted-like homeobox (VpKNOX1) genes during in vitro morphogenesis of Vanilla planifolia Jacks.

PubMed

Ramírez-Mosqueda, Marco A; Iglesias-Andreu, Lourdes G; Sáenz, Luis; Córdova, Iván

2018-02-01

This work aimed to evaluate the embryogenic competence of different tissues from different stages (friable callus, bud-regenerating callus, and whole buds) of Vanilla planifolia , through the molecular detection of the somatic embryogenesis receptor-like kinase ( VpSERK ) and knotted-like homeobox ( VpKNOX1 ) genes. RNA was extracted with Trizol ® , cDNA was obtained, and the studied transcripts were amplified. Using non-specific primers, VpSERK and VpSTM gene expression was detected in the three stages evaluated. This study might contribute to providing an explanation for the recalcitrance of this Vanilla species to somatic embryogenesis.

80-kVp CT Using Iterative Reconstruction in Image Space Algorithm for the Detection of Hypervascular Hepatocellular Carcinoma: Phantom and Initial Clinical Experience

PubMed Central

Hur, Saebeom; Kim, Soo Jin; Park, Ji Hoon; Han, Joon Koo; Choi, Byung Ihn

2012-01-01

Objective To investigate whether the low-tube-voltage (80-kVp), intermediate-tube-current (340-mAs) MDCT using the Iterative Reconstruction in Image Space (IRIS) algorithm improves lesion-to-liver contrast at reduced radiation dosage while maintaining acceptable image noise in the detection of hepatocellular carcinomas (HCC) in thin (mean body mass index, 24 ± 0.4 kg/m2) adults. Subjects and Methods A phantom simulating the liver with HCC was scanned at 50-400 mAs for 80, 100, 120 and 140-kVp. In addition, fifty patients with HCC who underwent multiphasic liver CT using dual-energy (80-kVp and 140-kVp) arterial scans were enrolled. Virtual 120-kVP scans (protocol A) and 80-kVp scans (protocol B) of the late arterial phase were reconstructed with filtered back-projection (FBP), while corresponding 80-kVp scans were reconstructed with IRIS (protocol C). Contrast-to-noise ratio (CNR) of HCCs and abdominal organs were assessed quantitatively, whereas lesion conspicuity, image noise, and overall image quality were assessed qualitatively. Results IRIS effectively reduced image noise, and yielded 29% higher CNR than the FBP at equivalent tube voltage and current in the phantom study. In the quantitative patient study, protocol C helped improve CNR by 51% and 172% than protocols A and B (p < 0.001), respectively, at equivalent radiation dosage. In the qualitative study, protocol C acquired the highest score for lesion conspicuity albeit with an inferior score to protocol A for overall image quality (p < 0.001). Mean effective dose was 2.63-mSv with protocol A and 1.12-mSv with protocols B and C. Conclusion CT using the low-tube-voltage, intermediate-tube-current and IRIS help improve lesion-to-liver CNR of HCC in thin adults during the arterial phase at a lower radiation dose when compared with the standard technique using 120-kVp and FBP. PMID:22438682

W 2 and Q 2 dependence of charged hadron and pion multiplicities in vp andbar vp charged current interactionscharged current interactions

NASA Astrophysics Data System (ADS)

Jones, G. T.; Jones, R. W. L.; Morrison, D. R. O.; Mobayyen, M. M.; Wainstein, S.; Aderholz, M.; Hantke, D.; Hoffmann, E.; Katz, U. F.; Kern, J.; Schmitz, N.; Wittek, W.; Allport, P.; Borner, H. P.; Myatt, G.; Radojicic, D.; Bullock, F. W.; Burke, S.

1990-03-01

Using data on vp andbar vp charged current interactions from a bubble chamber experiment with BEBC at CERN, the average multiplicities of charged hadrons and pions are determined as functions of W 2 and Q 2. The analysis is based on ˜20000 events with incident v and ˜10000 events with incidentbar v. In addition to the known dependence of the average multiplicity on W 2 a weak dependence on Q 2 for fixed intervals of W is observed. For W>2 GeV and Q 2>0.1 GeV2 the average multiplicity of charged hadrons is well described by =a 1+ a 2ln( W 2/GeV2)+ a 3ln( Q 2/GeV2) with a 1=0.465±0.053, a 2=1.211±0.021, a 3=0.103±0.014 for the vp and a 1=-0.372±0.073, a 2=1.245±0.028, a 3=0.093±0.015 for thebar vp reaction.

VP24 Is a Chitin-Binding Protein Involved in White Spot Syndrome Virus Infection

PubMed Central

Li, Zaipeng; Han, Yali; Xu, Limei

2015-01-01

ABSTRACT Oral ingestion is the major route of infection for the white spot syndrome virus (WSSV). However, the mechanism by which virus particles in the digestive tract invade host cells is unknown. In the present study, we demonstrate that WSSV virions can bind to chitin through one of the major envelope proteins (VP24). Mutagenesis analysis indicated that amino acids (aa) 186 to 200 in the C terminus of VP24 were required for chitin binding. Moreover, the P-VP24186–200 peptide derived from the VP24 chitin binding region significantly inhibited the VP24-chitin interaction and the WSSV-chitin interaction, implying that VP24 participates in WSSV binding to chitin. Oral inoculation experiments showed that P-VP24186–200 treatment reduced the number of virus particles remaining in the digestive tract during the early stage of infection and greatly hindered WSSV proliferation in shrimp. These data indicate that binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection and provide new ideas for preventing WSSV infection in shrimp farms. IMPORTANCE In this study, we show that WSSV can bind to chitin through the envelope protein VP24. The chitin-binding domain of VP24 maps to amino acids 186 to 200 in the C terminus. Binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection. These findings not only extend our knowledge of WSSV infection but also provide new insights into strategies to prevent WSSV infection in shrimp farms. PMID:26512091

Generation and evaluation of a recombinant genotype VII Newcastle disease virus expressing VP3 protein of Goose parvovirus as a bivalent vaccine in goslings.

PubMed

Wang, Jianzhong; Cong, Yanlong; Yin, Renfu; Feng, Na; Yang, Songtao; Xia, Xianzhu; Xiao, Yueqiang; Wang, Wenxiu; Liu, Xiufan; Hu, Shunlin; Ding, Chan; Yu, Shengqing; Wang, Chunfeng; Ding, Zhuang

2015-05-04

Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

High Resolution Vp and Vp/Vs Local Earthquake Tomography of the Val d'Agri Region (Southern Apennines, Italy).

NASA Astrophysics Data System (ADS)

Improta, L.; Bagh, S.; De Gori, P.; Pastori, M.; Piccinini, D.; Valoroso, L.; Anselmi, M.; Buttinelli, M.; Chiarabba, C.

2015-12-01

The Val d'Agri (VA) Quaternary basin in the southern Apennines extensional belt hosts the largest oilfield in onshore Europe and normal-fault systems with high (up to M7) seismogenic potential. Frequent small-magnitude swarms related to both active crustal extension and anthropogenic activity have occurred in the region. Causal factors for induced seismicity are a water impoundment with severe seasonal oscillations and a high-rate wastewater injection well. We analyzed around 1200 earthquakes (ML<3.3) occurred in the VA and surrounding regions between 2001-2014. We integrated waveforms recorded at 46 seismic stations belonging to 3 different networks: a dense temporary network installed by INGV in 2005-2006, the permanent national network of INGV, and the trigger-mode monitoring network managed by the local operator ENI petroleum company. We used local earthquake tomography to investigate static and transient features of the crustal velocity structure and to accurately locate earthquakes. Vp and Vp/Vs models are parameterized by a 3x3x2 km spacing and well resolved down to about 12 km depth. The complex Vp model illuminates broad antiformal structures corresponding to wide ramp-anticlines involving Mesozoic carbonates of the Apulia hydrocarbon reservoir, and NW-SE trending low Vp regions related to thrust-sheet-top clastic basins. The VA basin corresponds to shallow low-Vp region. Focal mechanisms show normal faulting kinematics with minor strike slip solutions in agreement with the local extensional regime. Earthquake locations and focal solutions depict shallow (< 5 km depth) E-dipping extensional structures beneath the artificial lake located in the southern sector of the basin, and along the western margin of the VA. A few swarms define relatively deep transfer structures accommodating the differential extension between main normal faults. The spatio-temporal distribution of around 220 events correlates with wastewater disposal activity, illuminating a NE

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

PubMed

Johnson, Britney; Li, Jing; Adhikari, Jagat; Edwards, Megan R; Zhang, Hao; Schwarz, Toni; Leung, Daisy W; Basler, Christopher F; Gross, Michael L; Amarasinghe, Gaya K

2016-08-28

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Britney; Li, Jing; Adhikari, Jagat

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulatemore » nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.« less

The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation.

PubMed

Grzela, Renata; Nusbaum, Julien; Fieulaine, Sonia; Lavecchia, Francesco; Bienvenut, Willy V; Dian, Cyril; Meinnel, Thierry; Giglione, Carmela

2017-09-08

Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra

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.more » 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.« less

Intermolecular cleavage of hepatitis A virus (HAV) precursor protein P1-P2 by recombinant HAV proteinase 3C.

PubMed Central

Kusov, Y Y; Sommergruber, W; Schreiber, M; Gauss-Müller, V

1992-01-01

Active proteinase 3C of hepatitis A virus (HAV) was expressed in bacteria either as a mature enzyme or as a protein fused to the entire polymerase 3D or to a part of it, and their identities were shown by immunoblot analysis. Intermolecular cleavage activity was demonstrated by incubating in vitro-translated and radiolabeled HAV precursor protein P1-P2 with extracts of bacteria transformed with plasmids containing recombinant HAV 3C. Identification of cleavage products P1, VP1, and VPO-VP3 by immunoprecipitation clearly demonstrates that HAV 3C can cleave between P1 and P2 as well as within P1 and thus shows an activity profile similar to that of cardiovirus 3C. Images PMID:1328690

Identification of VP1/2A and 2C as Virulence Genes of Hepatitis A Virus and Demonstration of Genetic Instability of 2C

PubMed Central

Emerson, Suzanne U.; Huang, Ying K.; Nguyen, Hanh; Brockington, Alicia; Govindarajan, Sugantha; St. Claire, Marisa; Shapiro, Max; Purcell, Robert H.

2002-01-01

Fourteen different chimeric virus genomes were constructed from two infectious cDNA clones encoding a virulent and an attenuated isolate, respectively, of the HM175 strain of hepatitis A virus. The ability of each recombinant virus to infect tamarins and to cause acute hepatitis was determined. Comparisons of the genotype and phenotype of each virus suggested that VP1/2A and 2C genes were responsible for virulence. The 2C gene derived from the attenuated parent virus was unstable, and one or more mutations arose in this gene during the first passage in tamarins. PMID:12163575

Structural basis for Marburg virus VP35-mediated immune evasion mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramanan, Parameshwaran; Edwards, Megan R.; Shabman, Reed S.

2013-07-22

Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen–associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFNmore » production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.« less

The study of the intercellular trafficking of the fusion proteins of herpes simplex virus protein VP22.

PubMed

Xue, Xiaodong; Huang, Jianhua; Wang, Huishan

2014-01-01

Genetic modifications can improve the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in myocardial infarction. However, so far, the efficiency of MSC modification is very low. Seeking for a more efficient way of MSC modification, we investigated the possibility of employing the intercellular trafficking capacity of the herpes simplex virus type-1 tegument protein VP22 on the enhancement of MSC modification. Plasmids pVP22-myc, pVP22-EGFP, pEGFP-VP22, pVP22-hBcl-xL and phBcl-xL-VP22 were constructed for the expressions of the myc-tagged VP22 and the fusion proteins VP22-EGFP, EGFP-VP22, VP22-hBcl-xL and hBcl-xL-VP22. MSCs were isolated from rat bone marrow and the surface markers were identified by Flowcytometry. COS-1 cells were transfected with the above plasmids and co-cultured with untransfected MSCs, the intercellular transportations of the constructed proteins were studied by immunofluorescence. The solubility of VP22-hBcl-xL and hBcl-xL-VP22 was analyzed by Western blot. VP22-myc could be expressed in and spread between COS-1 cells, which indicates the validity of our VP22 expression construct. Flowcytometry analysis revealed that the isolated MSCs were CD29, CD44, and CD90 positive and were negative for the hematopoietic markers, CD34 and CD45. The co-culturing and immunofluorescence assay showed that VP22-myc, VP22-EGFP and EGFP-VP22 could traffic between COS-1 cells and MSCs, while the evidence of intercellular transportation of VP22-hBcl-xL and hBcl-xL-VP22 was not detected. Western blot analysis showed that VP22-hBcl-xL and hBcl-xL-VP22 were both insoluble in the cell lysate suggesting interactions of the fusion proteins with other cellular components. The intercellular trafficking of VP22-myc, VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs presents an intriguing prospect in the therapeutic application of VP22 as a delivery vehicle which enhances genetic modifications of MSCs. However, VP22-hBcl-xL and hBcl-xL-VP22 failed to

The Study of the Intercellular Trafficking of the Fusion Proteins of Herpes Simplex Virus Protein VP22

PubMed Central

Xue, Xiaodong; Huang, Jianhua; Wang, Huishan

2014-01-01

Background Genetic modifications can improve the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in myocardial infarction. However, so far, the efficiency of MSC modification is very low. Seeking for a more efficient way of MSC modification, we investigated the possibility of employing the intercellular trafficking capacity of the herpes simplex virus type-1 tegument protein VP22 on the enhancement of MSC modification. Methods Plasmids pVP22-myc, pVP22-EGFP, pEGFP-VP22, pVP22-hBcl-xL and phBcl-xL-VP22 were constructed for the expressions of the myc-tagged VP22 and the fusion proteins VP22-EGFP, EGFP-VP22, VP22-hBcl-xL and hBcl-xL-VP22. MSCs were isolated from rat bone marrow and the surface markers were identified by Flowcytometry. COS-1 cells were transfected with the above plasmids and co-cultured with untransfected MSCs, the intercellular transportations of the constructed proteins were studied by immunofluorescence. The solubility of VP22-hBcl-xL and hBcl-xL-VP22 was analyzed by Western blot. Results VP22-myc could be expressed in and spread between COS-1 cells, which indicates the validity of our VP22 expression construct. Flowcytometry analysis revealed that the isolated MSCs were CD29, CD44, and CD90 positive and were negative for the hematopoietic markers, CD34 and CD45. The co-culturing and immunofluorescence assay showed that VP22-myc, VP22-EGFP and EGFP-VP22 could traffic between COS-1 cells and MSCs, while the evidence of intercellular transportation of VP22-hBcl-xL and hBcl-xL-VP22 was not detected. Western blot analysis showed that VP22-hBcl-xL and hBcl-xL-VP22 were both insoluble in the cell lysate suggesting interactions of the fusion proteins with other cellular components. Conclusions The intercellular trafficking of VP22-myc, VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs presents an intriguing prospect in the therapeutic application of VP22 as a delivery vehicle which enhances genetic modifications of MSCs. However, VP

Comparative characteristics of the VP7 and VP4 antigenic epitopes of the rotaviruses circulating in Russia (Nizhny Novgorod) and the Rotarix and RotaTeq vaccines.

PubMed

Morozova, O V; Sashina, T A; Fomina, S G; Novikova, N A

2015-07-01

Two live, attenuated rotavirus A (RVA) vaccines, Rotarix and RotaTeq, have been successfully introduced into national immunization programs worldwide. The parent strains of both vaccines were obtained more than 30 years ago. Nonetheless, only very limited data are available on the molecular similarity of the vaccine strains and their genetic relationships to the wild-type strains circulating within the territory of Russian Federation. In this study, we have determined the nucleotide sequences of the genes encoding the viral proteins VP7 and VP4 (the globular domain VP8*) of vaccine strains and natural isolates of rotaviruses in Nizhny Novgorod, Russia. The VP7 and VP4 proteins contain antigenic sites that are the main targets of neutralizing antibodies. Phylogenetic analysis based on VP4 and VP7 showed that the majority of the natural RVA isolates from Nizhny Novgorod and the vaccine strains belong to different clusters. Four amino acids within the VP7 antigenic sites were common in both the wild-type and vaccine strains. The largest number of amino acid differences was found between the vaccine strain Rotarix and the Nizhny Novgorod G2 strains (19 residues out of 29). From 3 to 5 amino acid differences per strain were identified in the antigenic sites of VP4 (domain VP8*) between wild-type strains and the vaccine RotaTeq, and 6-8 substitutions were found when they were compared with the vaccine strain Rotarix. For the first time, immunodominant T-cell epitopes of VP7 were analyzed, and differences in the sequences between the vaccine and the wild-type strains were found. The accumulation of amino acid substitutions in the VP7 and VP4 antigenic sites may potentially reduce the immune protection of vaccinated children from wild-type strains of rotavirus.

Integrated Computational Approach for Virtual Hit Identification against Ebola Viral Proteins VP35 and VP40.

PubMed

Mirza, Muhammad Usman; Ikram, Nazia

2016-10-26

The Ebola virus (EBOV) has been recognised for nearly 40 years, with the most recent EBOV outbreak being in West Africa, where it created a humanitarian crisis. Mortalities reported up to 30 March 2016 totalled 11,307. However, up until now, EBOV drugs have been far from achieving regulatory (FDA) approval. It is therefore essential to identify parent compounds that have the potential to be developed into effective drugs. Studies on Ebola viral proteins have shown that some can elicit an immunological response in mice, and these are now considered essential components of a vaccine designed to protect against Ebola haemorrhagic fever. The current study focuses on chemoinformatic approaches to identify virtual hits against Ebola viral proteins (VP35 and VP40), including protein binding site prediction, drug-likeness, pharmacokinetic and pharmacodynamic properties, metabolic site prediction, and molecular docking. Retrospective validation was performed using a database of non-active compounds, and early enrichment of EBOV actives at different false positive rates was calculated. Homology modelling and subsequent superimposition of binding site residues on other strains of EBOV were carried out to check residual conformations, and hence to confirm the efficacy of potential compounds. As a mechanism for artefactual inhibition of proteins through non-specific compounds, virtual hits were assessed for their aggregator potential compared with previously reported aggregators. These systematic studies have indicated that a few compounds may be effective inhibitors of EBOV replication and therefore might have the potential to be developed as anti-EBOV drugs after subsequent testing and validation in experiments in vivo.

High-resolution Crystal Structure of Dimeric VP40 From Sudan ebolavirus.

PubMed

Clifton, Matthew C; Bruhn, Jessica F; Atkins, Kateri; Webb, Terry L; Baydo, Ruth O; Raymond, Amy; Lorimer, Donald D; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

2015-10-01

Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Identification of a T-helper cell epitope on the rotavirus VP6 protein.

PubMed Central

Baños, D M; Lopez, S; Arias, C F; Esquivel, F R

1997-01-01

In this work, we have studied the T-helper (Th)-cell response against rotavirus, in a mouse model. Adult BALB/c mice were inoculated parenterally with porcine rotavirus YM, and the Th-cell response from spleen cells against the virus and two overlapping fragments of the major capsid protein VP6 (VP6(1-192) and VP6(171-397)) were evaluated in vitro. The Th cells recognized the YM virus and the two protein fragments, suggesting that there are at least two Th-cell epitopes on the VP6 molecule. To study the specificity of Th cells against VP6 at the clonal level, we established two Th-cell hybridomas cross-reactive for the VP6 protein of rotavirus strains YM and SA11. Both hybridomas recognized the VP6(171-397) polypeptide, and a synthetic peptide comprising the amino acids 289 to 302 (RLSFQLVRPPNMTP) of YM VP6 in the context of the major histocompatibility complex class II IEd molecule. The Th-cell hybridomas recognized rotavirus VP6 in a highly cross-reactive fashion, since they could be stimulated by eight different strains of rotavirus, including the murine rotavirus EDIM, that represent five G serotypes and at least two subgroups. The amino acid sequence of the VP6 epitope is highly conserved in most group A rotavirus strains sequenced so far. On the other hand, it was found that Th cells specific for the VP6 epitope may constitute an important proportion of the total polyclonal Th-cell response against rotavirus YM in spleen cells. These results demonstrate that VP6 can be a target for highly cross-reactive Th cells. PMID:8985366

Autocatalytic Activity of the Ubiquitin-Specific Protease Domain of Herpes Simplex Virus 1 VP1-2▿†

PubMed Central

Bolstad, M.; Abaitua, F.; Crump, C. M.; O'Hare, P.

2011-01-01

The herpes simplex virus (HSV) tegument protein VP1-2 is essential for virus entry and assembly. VP1-2 also contains a highly conserved ubiquitin-specific protease (USP) domain within its N-terminal region. Despite conservation of the USP and the demonstration that it can act on artificial substrates such as polyubiquitin chains, identification of the relevance of the USP in vivo to levels or function of any substrate remains limited. Here we show that HSV VP1-2 USP can act on itself and is important for stability. VP1-2 N-terminal variants encompassing the core USP domain itself were not affected by mutation of the catalytic cysteine residue (C65). However, extending the N-terminal region resulted in protein species requiring USP activity for accumulation. In this context, C65A mutation resulted in a drastic reduction in protein levels which could be stabilized by proteosomal inhibition or by the presence of normal C65. The functional USP domain could increase abundance of unstable variants, indicating action at least in part, in trans. Interestingly, full-length variants containing the inactive USP, although unstable when expressed in isolation, were stabilized by virus infection. The catalytically inactive VP1-2 retained complementation activity of a VP1-2-negative virus. Furthermore, a recombinant virus expressing a C65A mutant VP1-2 exhibited little difference in single-step growth curves and the kinetics and abundance of VP1-2 or a number of test proteins. Despite the absence of a phenotype for these replication parameters, the USP activity of VP1-2 may be required for function, including its own stability, under certain circumstances. PMID:21715485

Benchmarking and validation activities within JEFF project

NASA Astrophysics Data System (ADS)

Cabellos, O.; Alvarez-Velarde, F.; Angelone, M.; Diez, C. J.; Dyrda, J.; Fiorito, L.; Fischer, U.; Fleming, M.; Haeck, W.; Hill, I.; Ichou, R.; Kim, D. H.; Klix, A.; Kodeli, I.; Leconte, P.; Michel-Sendis, F.; Nunnenmann, E.; Pecchia, M.; Peneliau, Y.; Plompen, A.; Rochman, D.; Romojaro, P.; Stankovskiy, A.; Sublet, J. Ch.; Tamagno, P.; Marck, S. van der

2017-09-01

The challenge for any nuclear data evaluation project is to periodically release a revised, fully consistent and complete library, with all needed data and covariances, and ensure that it is robust and reliable for a variety of applications. Within an evaluation effort, benchmarking activities play an important role in validating proposed libraries. The Joint Evaluated Fission and Fusion (JEFF) Project aims to provide such a nuclear data library, and thus, requires a coherent and efficient benchmarking process. The aim of this paper is to present the activities carried out by the new JEFF Benchmarking and Validation Working Group, and to describe the role of the NEA Data Bank in this context. The paper will also review the status of preliminary benchmarking for the next JEFF-3.3 candidate cross-section files.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

PubMed

Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

2017-09-15

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

PubMed Central

Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E.; Miorin, Lisa; Johnson, Jeffrey R.; Krogan, Nevan J.; Basler, Christopher F.; Freiberg, Alexander N.

2017-01-01

ABSTRACT Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP

Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus.

PubMed

Wang, Nian; Zhang, Lizhou; Chen, Yuming; Lu, Zhen; Gao, Li; Wang, Yongqiang; Gao, Yulong; Gao, Honglei; Cui, Hongyu; Li, Kai; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

2015-01-01

Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV.

Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

PubMed

Chung, Ivy Yeuk Wah; Paetzel, Mark

2013-05-03

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells.

PubMed

Ilinykh, Philipp A; Lubaki, Ndongala M; Widen, Steven G; Renn, Lynnsey A; Theisen, Terence C; Rabin, Ronald L; Wood, Thomas G; Bukreyev, Alexander

2015-08-01

Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC. This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal

Regional Vp, Vs, Vp/Vs, and Poisson's ratios across earthquake source zones from Memphis, Tennessee, to St. Louis, Missouri

USGS Publications Warehouse

Catchings, R.D.

1999-01-01

Models of P- and S-wave velocity, Vp/Vs ratios, Poisson's ratios, and density for the crust and upper mantle are presented along a 400-km-long profile trending from Memphis, Tennessee, to St. Louis, Missouri. The profile crosses the New Madrid seismic zone and reveals distinct regional variations in the crustal velocity structure north and south of the latitude of New Madrid. In the south near Memphis, the upper few kilometers of the crust are dominated by upper crustal sedimentary basins or graben with P-wave velocities less than 5 km/sec and S-wave velocities of about 2 km/sec. P-wave velocities of the upper and middle crust range from 6.0 to 6.5 km/sec at depths above 25 km, and corresponding S-wave velocities range from 3.5 to 3.7 km/sec. The lower crust consists of a high-velocity layer (Vp = 7.4 km/sec; Vs ~4.2 km/sec) that is up to 20-km thick at the latitude of New Madrid but thins to about 15 km near Memphis. To the north, beneath the western-most Illinois basin, low-velocity (Vp < 5 km/sec; Vs < 2.3 km/sec) sedimentary basins are less than 1-km deep. The average velocities (Vp = 6.0 km/sec; Vs = 3.5 km/sec) of the underlying, near-surface rocks argue against large thickness of unconsolidated noncarbonate sediments within 50 km of the western edge of the Illinois basin. Most of the crust beneath the Illinois basin is modeled as one layer, with velocities up to 6.8 km/sec (Vs = 3.7 km/sec) at 37-km depth. The thick, high-velocity (Vp = 7.4 km/sec; Vs ~4.2 km/sec) lower crustal layer thins from about 20 km near New Madrid to about 6 km beneath the western Illinois basin. Refractions from the Moho and upper mantle occur as first arrivals over distances as a great as 160 km and reveal upper mantle layering to 60 km depth. Upper mantle layers with P-wave velocities of 8.2 km/sec (Vs = 4.5 km/sec) and 8.4 km/sec (Vs = 4.7 km/sec) are modeled at 43 and 60 km depth, respectively. Crustal Vp/Vs ratios range between 1.74 and 1.83, and upper mantle Vp/V s ratios

The SBP-Box Gene VpSBP11 from Chinese Wild Vitis Is Involved in Floral Transition and Affects Leaf Development.

PubMed

Hou, Hongmin; Yan, Xiaoxiao; Sha, Ting; Yan, Qin; Wang, Xiping

2017-07-13

Flowering occurs in angiosperms during a major developmental transition from vegetative growth to the reproductive phase. Squamosa promoter binding protein (SBP)-box genes have been found to play critical roles in regulating flower and fruit development, but their roles in grapevine have remained unclear. To better understand the functions of the grape SBP-box genes in both vegetative and reproductive growth phases, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP11 , was obtained from Chinese wild grapevine Vitis pseudoreticulata Wen Tsai Wang (W. T. Wang) clone 'Baihe-35-1'. VpSBP11 encoded a putative polypeptide of 170 amino acids with a highly conserved SBP-domain with two zinc-binding sites of the Cx2C-x3-H-x11-C-x6-H (C2HCH) type and a nuclear localization signal. We confirmed that the VpSBP11 protein was targeted to the nucleus and possessed transcriptional activation activity by subcellular localization and trans -activation assay. Over-expression of VpSBP11 in Arabidopsis thaliana was shown to activate the FUL gene, and subsequently the AP1 and LFY genes, all of which were floral meristem identity genes, and to cause earlier flowering than in wild type (WT) plants. The pattern of vegetative growth was also different between the transgenic and WT plants. For example, in the VpSBP11 over-expressing transgenic plants, the number of rosette leaves was less than that of WT; the petiole was significantly elongated; and the rosette and cauline leaves curled upwards or downwards. These results were consistent with VpSBP11 acting as a transcription factor during the transition from the vegetative stage to the reproductive stage.

The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host.

PubMed

Calvo-Pinilla, Eva; Gubbins, Simon; Mertens, Peter; Ortego, Javier; Castillo-Olivares, Javier

2018-06-01

African horse sickness (AHS) is a lethal equine disease transmitted by Culicoides biting midges and caused by African horse sickness virus (AHSV). AHS is endemic to sub-Saharan Africa, but devastating outbreaks have been recorded periodically outside this region. The perceived risk of an AHS outbreak occurring in Europe has increased following the frequent epidemics caused in ruminants by bluetongue virus, closely related to AHSV. Attenuated vaccines for AHS are considered unsuitable for use in non-endemic countries due bio-safety concerns. Further, attenuated and inactivated vaccines are not compatible with DIVA (differentiate infected from vaccinated animals) strategies. All these factors stimulated the development of novel AHS vaccines that are safer, more efficacious and DIVA compatible. We showed previously that recombinant modified Vaccinia Ankara virus (MVA) vaccines encoding the outer capsid protein of AHSV (AHSV-VP2) induced virus neutralising antibodies (VNAb) and protection against AHSV in a mouse model and also in the horse. Passive immunisation studies demonstrated that immunity induced by MVA-VP2 was associated with pre-challenge VNAb titres in the vaccinates. Analyses of the inoculum of these MVA-VP2 experimental vaccines showed that they contained pre-formed AHSV-VP2. We continued studying the influence of pre-formed AHSV-VP2, present in the inoculum of MVA-VP2 vaccines, in the immunogenicity of MVA-VP2 vaccines. Thus, we compared correlates of immunity in challenged mice that were previously vaccinated with: a) MVA-VP2 (live); b) MVA-VP2 (live and sucrose gradient purified); c) MVA-VP2 (UV light inactivated); d) MVA-VP2 (UV light inactivated and diluted); e) MVA-VP2 (heat inactivated); f) MVA-VP2 (UV inactivated) + MVA-VP2 (purified); g) MVA-VP2 (heat inactivated) + MVA-VP2 (purified); and h) wild type-MVA (no insert). The results of these experiments showed that protection was maximal using MVA-VP2 (live) vaccine and that the protection

Climate Change Communicators: The C3E3 Project

NASA Astrophysics Data System (ADS)

Sharif, H. O.; Joseph, J.

2013-12-01

The University of Texas at San Antonio (UTSA), San Antonio College (SAC), and the University of North Dakota (UND) have partnered with NASA to provide underrepresented undergraduates from UTSA, SAC, and other community colleges climate-related research and education experiences through the Climate Change Communication: Engineer, Environmental science, and Education (C3E3) project. The program aims to develop a robust response to climate change by providing K-16 climate change education; enhance the effectiveness of K-16 education particularly in engineering and other STEM disciplines by use of new instructional technologies; increase the enrollment in engineering programs and the number of engineering degrees awarded by showing engineering's usefulness in relation to the much-discussed contemporary issue of climate change; increase persistence in STEM degrees by providing student research opportunities; and increase the ethnic diversity of those receiving engineering degrees and help ensure an ethnically diverse response to climate change. Students participated in the second summer internship funded by the project. More than 60 students participated in guided research experiences aligned with NASA Science Plan objectives for climate and Earth system science and the educational objectives of the three institutions. The students went through training in modern media technology (webcasts), and in using this technology to communicate the information on climate change to others, especially high school students, culminating in production of webcasts on investigating the aspects of climate change using NASA data. Content developed is leveraged by NASA Earth observation data and NASA Earth system models and tools. Several departments are involved in the educational program.

Differential Regulation of Interferon Responses by Ebola and Marburg Virus VP35 Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, Megan R.; Liu, Gai; Mire, Chad E.

2016-02-11

Suppression of innate immune responses during filoviral infection contributes to disease severity. Ebola (EBOV) and Marburg (MARV) viruses each encode a VP35 protein that suppresses RIG-I-like receptor signaling and interferon-α/β (IFN-α/β) production by several mechanisms, including direct binding to double stranded RNA (dsRNA). Here, we demonstrate that in cell culture, MARV infection results in a greater upregulation of IFN responses as compared to EBOV infection. This correlates with differences in the efficiencies by which EBOV and MARV VP35s antagonize RIG-I signaling. Furthermore, structural and biochemical studies suggest that differential recognition of RNA elements by the respective VP35 C-terminal IFN inhibitorymore » domain (IID) rather than affinity for RNA by the respective VP35s is critical for this observation. Our studies reveal functional differences in EBOV versus MARV VP35 RNA binding that result in unexpected differences in the host response to deadly viral pathogens.« less

Earth Science Enterprise Scientific Data Purchase Project: Verification and Validation

NASA Technical Reports Server (NTRS)

Jenner, Jeff; Policelli, Fritz; Fletcher, Rosea; Holecamp, Kara; Owen, Carolyn; Nicholson, Lamar; Dartez, Deanna

2000-01-01

This paper presents viewgraphs on the Earth Science Enterprise Scientific Data Purchase Project's verification,and validation process. The topics include: 1) What is Verification and Validation? 2) Why Verification and Validation? 3) Background; 4) ESE Data Purchas Validation Process; 5) Data Validation System and Ingest Queue; 6) Shipment Verification; 7) Tracking and Metrics; 8) Validation of Contract Specifications; 9) Earth Watch Data Validation; 10) Validation of Vertical Accuracy; and 11) Results of Vertical Accuracy Assessment.

Empirical Approach in Developing Vs/Vp Ratio for Predicting S-Wave Velocity, Study Case; Sungai Batu, Kedah

NASA Astrophysics Data System (ADS)

Sabrian, T. A.; Saad, R.; Saidin, M.; Muhammad, S. B.; Yusoh, R.

2018-04-01

In recognition of the difficulties in acquiring seismic refraction shear wave data and the ambiguities involved in its processing, Vs/Vp ratio for sedimentary areas of Sungai Batu have been developed and assessed in this study. Two seismic refraction survey line L1 and L2 were conducted using P- and S-wave were acquired and processed along the same line regarding study area. The resulting velocities were extracted from seismic tomography profile to compute specific ratios after linearity and correlation checks. It is found that Vs is linearly related to Vp, with coefficients of determination (R2) of about 0.74 and 0.52 for L1 and L2 respectively. The specific ratios were computed as 0.3 and 0.4 for L1 and L2 respectively Another data sets acquired along different lines were used to validate the ratios. The mean absolute percentage errors were calculated for both modelling and validation data sets and found that the different percentage between Vs measured and Vs calculated is 20.7% and 22% respectively.

High-yield production of the VP1 structural protein epitope from serotype O foot-and-mouth disease virus in Escherichia coli.

PubMed

Jung, Joon-Goo; Lee, Yong Jae; Velmurugan, Natarajan; Ko, Young-Joon; Lee, Hyang-Sim; Jeong, Ki Jun

2013-07-01

For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25 °C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11 g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.

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 ismore » 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.« less

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3

PubMed Central

Iverson, Eric A.; Goodman, David A.; Gorchels, Madeline E.

2017-01-01

ABSTRACT Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3, allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3.

PubMed

Iverson, Eric A; Goodman, David A; Gorchels, Madeline E; Stedman, Kenneth M

2017-05-15

Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae , where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3 , allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of

Molecular characterization of canine parvovirus variants (CPV-2a, CPV-2b, and CPV-2c) based on the VP2 gene in affected domestic dogs in Ecuador.

PubMed

la Torre, David De; Mafla, Eulalia; Puga, Byron; Erazo, Linda; Astolfi-Ferreira, Claudete; Ferreira, Antonio Piantino

2018-04-01

The objective of this study was to determine the presence of the variants of canine parvovirus (CPV)-2 in the city of Quito, Ecuador, due to the high domestic and street-type canine population, and to identify possible mutations at a genetic level that could be causing structural changes in the virus with a consequent influence on the immune response of the hosts. Thirty-five stool samples from different puppies with characteristic signs of the disease and positives for CPV through immunochromatography kits were collected from different veterinarian clinics of the city. Polymerase chain reaction and DNA sequencing were used to determine the mutations in residue 426 of the VP2 gene, which determines the variants of CPV-2; in addition, four samples were chosen for complete sequencing of the VP2 gene to identify all possible mutations in the circulating strains in this region of the country. The results revealed the presence of the three variants of CPV-2 with a prevalence of 57.1% (20/35) for CPV-2a, 8.5% (3/35) for CPV-2b, and 34.3% (12/35) for CPV-2c. In addition, complete sequencing of the VP2 gene showed amino acid substitutions in residues 87, 101, 139, 219, 297, 300, 305, 322, 324, 375, 386, 426, 440, and 514 of the three Ecuadorian variants when compared with the original CPV-2 sequence. This study describes the detection of CPV variants in the city of Quito, Ecuador. Variants of CPV-2 (2a, 2b, and 2c) have been reported in South America, and there are cases in Ecuador where CVP-2 is affecting even vaccinated puppies.

Structures of Rotavirus Reassortants Demonstrate Correlation of Altered Conformation of the VP4 Spike and Expression of Unexpected VP4-Associated Phenotypes

PubMed Central

Pesavento, Joseph B.; Billingsley, Angela M.; Roberts, Ed J.; Ramig, Robert F.; Prasad, B. V. Venkataram

2003-01-01

Numerous prior studies have indicated that viable rotavirus reassortants containing structural proteins of heterologous parental origin may express unexpected phenotypes, such as changes in infectivity and immunogenicity. To provide a structural basis for alterations in phenotypic expression, a three-dimensional structural analysis of these reassortants was conducted. The structures of the reassortants show that while VP4 generally maintains the parental structure when moved to a heterologous protein background, in certain reassortants, there are subtle alterations in the conformation of VP4. The alterations in VP4 conformation correlated with expression of unexpected VP4-associated phenotypes. Interactions between heterologous VP4 and VP7 in reassortants expressing unexpected phenotypes appeared to induce the conformational alterations seen in VP4. PMID:12584352

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

PubMed

Gc, Jeevan B; Gerstman, Bernard S; Chapagain, Prem P

2017-10-01

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP 2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP 2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

Experimental constraints on the sound velocities of cementite Fe3C to core pressures

NASA Astrophysics Data System (ADS)

Chen, Bin; Lai, Xiaojing; Li, Jie; Liu, Jiachao; Zhao, Jiyong; Bi, Wenli; Ercan Alp, E.; Hu, Michael Y.; Xiao, Yuming

2018-07-01

Sound velocities of cementite Fe3C have been measured up to 1.5 Mbar and at 300 K in a diamond anvil cell using the nuclear resonant inelastic X-ray scattering (NRIXS) technique. From the partial phonon density of states (pDOS) and equation of state (EOS) of Fe3C, we derived its elastic parameters including shear modulus, compressional (VP) and shear-wave (VS) velocities to core pressures. A pressure-induced spin-pairing transition in the powdered Fe3C sample was found to occur gradually between 10 and 50 GPa by the X-ray Emission Spectroscopy (XES) measurements. Following the completion of the spin-pairing transition, the VP and VS of low-spin Fe3C increased with pressure at a markedly lower rate than its high-spin counterpart. Our results suggest that the incorporation of carbon in solid iron to form iron carbide phases, Fe3C and Fe7C3, could effectively lower the VS but respectively raise the Poisson's ratio by 0.05 and 0.07 to approach the seismically observed values for the Earth's inner core. The comparison with the preliminary reference Earth model (PREM) implies that an inner core composition containing iron and its carbon-rich alloys can satisfactorily explain the observed seismic properties of the inner core.

T helper cell-mediated interferon-gamma expression after human parvovirus B19 infection: persisting VP2-specific and transient VP1u-specific activity

PubMed Central

Franssila, R; Auramo, J; Modrow, S; Möbs, M; Oker-Blom, C; Käpylä, P; Söderlund-Venermo, M; Hedman, K

2005-01-01

Human parvovirus B19 is a small non-enveloped DNA virus with an icosahedral capsid consisting of proteins of only two species, the major protein VP2 and the minor protein VP1. VP2 is contained within VP1, which has an additional unique portion (VP1u) of 227 amino acids. We determined the ability of eukaryotically expressed parvovirus B19 virus-like particles consisting of VP1 and VP2 in the ratio recommended for vaccine use, or of VP2 alone, to stimulate, in an HLA class II restricted manner, peripheral blood mononuclear cells (PBMC) to proliferate and to secrete interferon gamma (IFN-γ) and interleukin (IL)-10 cytokines among recently and remotely B19 infected subjects. PBMC reactivity with VP1u was determined specifically with a prokaryotically expressed VP1u antigen. In general, B19-specific IFN-γ responses were stronger than IL-10 responses in both recent and remote infection; however, IL-10 responses were readily detectable among both groups, with the exception of patients with relapsed or persisting symptoms who showed strikingly low IL-10 responses. Whereas VP1u-specific IFN-γ responses were very strong among the recently infected subjects, the VP1u-specific IFN-γ and IL-10 responses were virtually absent among the remotely infected subjects. The disappearance of VP1u-specific IFN-γ expression is surprising, as B-cell immunity against VP1u is well maintained. PMID:16178856

An interference account of the missing-VP effect

PubMed Central

Häussler, Jana; Bader, Markus

2015-01-01

Sentences with doubly center-embedded relative clauses in which a verb phrase (VP) is missing are sometimes perceived as grammatical, thus giving rise to an illusion of grammaticality. In this paper, we provide a new account of why missing-VP sentences, which are both complex and ungrammatical, lead to an illusion of grammaticality, the so-called missing-VP effect. We propose that the missing-VP effect in particular, and processing difficulties with multiply center-embedded clauses more generally, are best understood as resulting from interference during cue-based retrieval. When processing a sentence with double center-embedding, a retrieval error due to interference can cause the verb of an embedded clause to be erroneously attached into a higher clause. This can lead to an illusion of grammaticality in the case of missing-VP sentences and to processing complexity in the case of complete sentences with double center-embedding. Evidence for an interference account of the missing-VP effect comes from experiments that have investigated the missing-VP effect in German using a speeded grammaticality judgments procedure. We review this evidence and then present two new experiments that show that the missing-VP effect can be found in German also with less restricting procedures. One experiment was a questionnaire study which required grammaticality judgments from participants without imposing any time constraints. The second experiment used a self-paced reading procedure and did not require any judgments. Both experiments confirm the prior findings of missing-VP effects in German and also show that the missing-VP effect is subject to a primacy effect as known from the memory literature. Based on this evidence, we argue that an account of missing-VP effects in terms of interference during cue-based retrieval is superior to accounts in terms of limited memory resources or in terms of experience with embedded structures. PMID:26136698

Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1.

PubMed

Hattori, T; Terada, T; Hamasuna, S

1995-06-01

Osem, a rice gene homologous to the wheat Em gene, which encodes one of the late-embryogenesis abundant proteins was isolated. The gene was characterized with respect to control of transcription by abscisic acid (ABA) and the transcriptional activator VP1, which is involved in the ABA-regulated gene expression during late embryo-genesis. A fusion gene (Osem-GUS) consisting of the Osem promoter and the bacterial beta-glucuronidase (GUS) gene was constructed and tested in a transient expression system, using protoplasts derived from a suspension-cultured line of rice cells, for activation by ABA and by co-transfection with an expression vector (35S-Osvp1) for the rice VP1 (OSVP1) cDNA. The expression of Osem-GUS was strongly (40- to 150-fold) activated by externally applied ABA and by over-expression of (OS)VP1. The Osem promoter has three ACGTG-containing sequences, motif A, motif B and motif A', which resemble the abscisic acid-responsive element (ABRE) that was previously identified in the wheat Em and the rice Rab16. There is also a CATGCATG sequence, which is known as the Sph box and is shown to be essential for the regulation by VP1 of the maize anthocyanin regulatory gene C1. Focusing on these sequence elements, various mutant derivatives of the Osem promoter in the transient expression system were assayed. The analysis revealed that motif A functions not only as an ABRE but also as a sequence element required for the regulation by (OS)VP1.

Pre-harvest sprouting resistance and haplotype variation of ThVp-1 gene in the collection of wheat-wheatgrass hybrids

PubMed Central

Kocheshkova, A. A.; Kroupin, P. Yu.; Bazhenov, M. S.; Karlov, G. I.; Pochtovyy, A. A.; Upelniek, V. P.; Belov, V. I.

2017-01-01

The germplasm collection of 87 wheat-wheatgrass hybrids developed in Tsitisin Main Botanical Garden (Russia, Moscow) was evaluated for resistance to pre-harvest sprouting (PHS) by spike sprouting (SS) and germination index (GI) assays as well as for spike and grain features. The PHS resistance variation and haplotype polymorphism of the wheatgrass ThVp-1 and wheat TaVp-1B genes orthologues of Vp-1 was revealed in the studied collection. Four haplotypes of ThVp-1 were revealed: ThVp-1a (41% of the entries), ThVp-1b (13%), ThVp-1c (29%), and ThVp-1d (15%). The association between the allelic state of ThVp-1 and PHS resistance in the wheat-wheatgrass hybrids was shown: haplotype ThVp-1d of the wheatgrass Vp-1 gene is significantly associated with reduced PHS in the wheat-wheatgrass hybrids (mean SS 0.33, mean GI 0.64). The resistant entries may be perspective as a source of PHS resistance in the development of commercial cultivars of perennial wheat. PMID:29131854

Comparison of C5 and C6 Aqua-MODIS Dark Target Aerosol Validation

NASA Technical Reports Server (NTRS)

Munchak, Leigh A.; Levy, Robert C.; Mattoo, Shana

2014-01-01

We compare C5 and C6 validation to compare the C6 10 km aerosol product against the well validated and trusted aerosol product on global and regional scales. Only the 10 km aerosol product is evaluated in this study, validation of the new C6 3 km aerosol product still needs to be performed. Not all of the time series has processed yet for C5 or C6, and the years processed for the 2 products is not exactly the same (this work is preliminary!). To reduce the impact of outlier observations, MODIS is spatially averaged within 27.5 km of the AERONET site, and AERONET is temporatally averaged within 30 minutes of the MODIS overpass time. Only high quality (QA = 3 over land, QA greater than 0 over ocean) pixels are included in the mean.

Enhanced and Extended Anti-Hypertensive Effect of VP5 Nanoparticles

PubMed Central

Yu, Ting; Zhao, Shengnan; Li, Ziqiang; Wang, Yi; Xu, Bei; Fang, Dailong; Wang, Fazhan; Zhang, Zhi; He, Lili; Song, Xiangrong; Yang, Jian

2016-01-01

Hypertension has become a significant global public health concern and is also one of the most common risk factors of cardiovascular disease. Recent studies have shown the promising result of peptides inhibiting angiotensin converting enzyme (ACE) in lowering the blood pressure in both animal models and humans. However, the oral bioavailability and continuous antihypertensive effectiveness require further optimization. Novel nanoparticle-based drug delivery systems are helpful to overcome these barriers. Therefore, a poly-(lactic-co-glycolic) acid nanoparticle (PLGANPs) oral delivery system, of the antihypertensive small peptides Val-Leu-Pro-Val-Pro (VLPVP, VP5) model, was developed in this study and its antihypertensive effect was investigated in spontaneously hypertensive rats (SHRs) for the first time. The obtained VP5 nanoparticles (VP5-NPs) showed a small particle size of 223.7 ± 2.3 nm and high entrapment efficiency (EE%) of 87.37% ± 0.92%. Transmission electronic microscopy (TEM) analysis showed that the nanoparticles were spherical and homogeneous. The optimal preparation of VP5-NPs exhibited sustained release of VP5 in vitro and a 96 h long-term antihypertensive effect with enhanced efficacy in vivo. This study illustrated that PLGANPs might be an optimal formulation for oral delivery of antihypertensive small peptides and VP5-NPs might be worthy of further development and use as a potential therapeutic strategy for hypertension in the future. PMID:27898022

Quercetin targets the interaction of calcineurin with LxVP-type motifs in immunosuppression

PubMed Central

Zhao, Yane; Zhang, Jin; Shi, Xiaoyu; Li, Jing; Wang, Rui; Song, Ruiwen; Wei, Qun; Cai, Huaibin; Luo, Jing

2016-01-01

Calcineurin (CN) is a unique calcium/calmodulin (CaM)-activated serine/threonine phosphatase. To perform its diverse biological functions, CN communicates with many substrates and other proteins. In the physiological activation of T cells, CN acts through transcriptional factors belonging to the NFAT family and other transcriptional effectors. The classic immunosuppressive drug cyclosporin A (CsA) can bind to cyclophilin (CyP) and compete with CN for the NFAT LxVP motif. CsA has debilitating side effects, including nephrotoxicity, hypertension and tremor. It is desirable to develop alternative immunosuppressive agents. To this end, we first tested the interactions between CN and the LxVP-type substrates, including endogenous regulators of calcineurin (RCAN1) and NFAT. Interestingly, we found that quercetin, the primary dietary flavonol, can inhibit the activity of CN and significantly disrupt the associations between CN and its LxVP-type substrates. We then validated the inhibitory effects of quercetin on the CN-NFAT interactions in cell-based assays. Further, quercetin also shows dose-dependent suppression of cytokine gene expression in mouse spleen cells. These data raise the possibility that the interactions of CN with its LxVP-type substrates are potential targets for immunosuppressive agents. PMID:27109380

Molecular characterization of canine parvovirus variants (CPV-2a, CPV-2b, and CPV-2c) based on the VP2 gene in affected domestic dogs in Ecuador

PubMed Central

la Torre, David De; Mafla, Eulalia; Puga, Byron; Erazo, Linda; Astolfi-Ferreira, Claudete; Ferreira, Antonio Piantino

2018-01-01

Aim The objective of this study was to determine the presence of the variants of canine parvovirus (CPV)-2 in the city of Quito, Ecuador, due to the high domestic and street-type canine population, and to identify possible mutations at a genetic level that could be causing structural changes in the virus with a consequent influence on the immune response of the hosts. Materials and Methods Thirty-five stool samples from different puppies with characteristic signs of the disease and positives for CPV through immunochromatography kits were collected from different veterinarian clinics of the city. Polymerase chain reaction and DNA sequencing were used to determine the mutations in residue 426 of the VP2 gene, which determines the variants of CPV-2; in addition, four samples were chosen for complete sequencing of the VP2 gene to identify all possible mutations in the circulating strains in this region of the country. Results The results revealed the presence of the three variants of CPV-2 with a prevalence of 57.1% (20/35) for CPV-2a, 8.5% (3/35) for CPV-2b, and 34.3% (12/35) for CPV-2c. In addition, complete sequencing of the VP2 gene showed amino acid substitutions in residues 87, 101, 139, 219, 297, 300, 305, 322, 324, 375, 386, 426, 440, and 514 of the three Ecuadorian variants when compared with the original CPV-2 sequence. Conclusion This study describes the detection of CPV variants in the city of Quito, Ecuador. Variants of CPV-2 (2a, 2b, and 2c) have been reported in South America, and there are cases in Ecuador where CVP-2 is affecting even vaccinated puppies. PMID:29805214

42 CFR 422.516 - Validation of Part C reporting requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 3 2014-10-01 2014-10-01 false Validation of Part C reporting requirements. 422... Procedures and Contracts for Medicare Advantage Organizations § 422.516 Validation of Part C reporting....502(f)(1) available to its enrollees upon reasonable request. (g) Data validation. Each Part C sponsor...

42 CFR 422.516 - Validation of Part C reporting requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 3 2012-10-01 2012-10-01 false Validation of Part C reporting requirements. 422... Procedures and Contracts for Medicare Advantage Organizations § 422.516 Validation of Part C reporting....502(f)(1) available to its enrollees upon reasonable request. (g) Data validation. Each Part C sponsor...

42 CFR 422.516 - Validation of Part C reporting requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 3 2013-10-01 2013-10-01 false Validation of Part C reporting requirements. 422... Procedures and Contracts for Medicare Advantage Organizations § 422.516 Validation of Part C reporting....502(f)(1) available to its enrollees upon reasonable request. (g) Data validation. Each Part C sponsor...

Crystal Structures of Major Envelope Proteins VP26 and VP28 from White Spot Syndrome Virus Shed Light on Their Evolutionary Relationship

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang,X.; Wu, J.; Sivaraman, J.

2007-01-01

White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 {angstrom}, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelopemore » proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt {beta}-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core {beta}-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.« less

Phylogenetic characterization of Canine Parvovirus VP2 partial sequences from symptomatic dogs samples.

PubMed

Zienius, D; Lelešius, R; Kavaliauskis, H; Stankevičius, A; Šalomskas, A

2016-01-01

The aim of the present study was to detect canine parvovirus (CPV) from faecal samples of clinically ill domestic dogs by polymerase chain reaction (PCR) followed by VP2 gene partial sequencing and molecular characterization of circulating strains in Lithuania. Eleven clinically and antigen-tested positive dog faecal samples, collected during the period of 2014-2015, were investigated by using PCR. The phylogenetic investigations indicated that the Lithuanian CPV VP2 partial sequences (3025-3706 cds) were closely related and showed 99.0-99.9% identity. All Lithuanian sequences were associated with one phylogroup, but grouped in different clusters. Ten of investigated Lithuanian CPV VP2 sequences were closely associated with CPV 2a antigenic variant (99.4% nt identity). Five CPV VP2 sequences from Lithuania were related to CPV-2a, but were rather divergent (6.8 nt differences). Only one CPV VP2 sequence from Lithuania was associated (99.3% nt identity) with CPV-2b VP2 sequences from France, Italy, USA and Korea. The four of eleven investigated Lithuanian dogs with CPV infection symptoms were vaccinated with CPV-2 vaccine, but their VP2 sequences were phylogenetically distantly associated with CPV vaccine strains VP2 sequences (11.5-15.8 nt differences). Ten Lithuanian CPV VP2 sequences had monophyletic relations among the close geographically associated samples, but five of them were rather divergent (1.0% less sequence similarity). The one Lithuanian CPV VP2 sequence was closely related with CPV-2b antigenic variant. All the Lithuanian CPV VP2 partial sequences were conservative and phylogenetically low associated with most commonly used CPV vaccine strains.

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Recombinant VP1, an Akt Inhibitor, Suppresses Progression of Hepatocellular Carcinoma by Inducing Apoptosis and Modulation of CCL2 Production

PubMed Central

Chen, Tai-An; Wang, Jui-Ling; Hung, Shao-Wen; Chu, Chiao-Li; Cheng, Yung-Chih; Liang, Shu-Mei

2011-01-01

Background The application of viral elements in tumor therapy is one facet of cancer research. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus has previously been demonstrated to induce apoptosis in cancer cell lines. Here, we aim to further investigate its apoptotic mechanism and possible anti-metastatic effect in murine models of hepatocellular carcinoma (HCC), one of the most common human cancers worldwide. Methodology/Principal Findings Treatment with rVP1 inhibited cell proliferation in two murine HCC cell lines, BNL and Hepa1-6, with IC50 values in the range of 0.1–0.2 µM. rVP1 also induced apoptosis in these cells, which was mediated by Akt deactivation and dissociation of Ku70-Bax, and resulted in conformational changes and mitochondrial translocation of Bax, leading to the activation of caspases-9, -3 and -7. Treatment with 0.025 µM rVP1, which did not affect the viability of normal hepatocytes, suppressed cell migration and invasion via attenuating CCL2 production. The production of CCL2 was modulated by Akt-dependent NF-κB activation that was decreased after rVP1 treatment. The in vivo antitumor effects of rVP1 were assessed in both subcutaneous and orthotopic mouse models of HCC in immune-competent BALB/c mice. Intratumoral delivery of rVP1 inhibited subcutaneous tumor growth as a result of increased apoptosis. Intravenous administration of rVP1 in an orthotopic HCC model suppressed tumor growth, inhibited intra-hepatic metastasis, and prolonged survival. Furthermore, a decrease in the serum level of CCL2 was observed in rVP1-treated mice. Conclusions/Significance The data presented herein suggest that, via inhibiting Akt phosphorylation, rVP1 suppresses the growth, migration, and invasion of murine HCC cells by inducing apoptosis and attenuating CCL2 production both in vitro and in vivo. Recombinant protein VP1 thus has the potential to be developed as a new therapeutic agent for HCC. PMID:21826248

Localization of the herpes simplex virus type 1 major capsid protein VP5 to the cell nucleus requires the abundant scaffolding protein VP22a.

PubMed

Nicholson, P; Addison, C; Cross, A M; Kennard, J; Preston, V G; Rixon, F J

1994-05-01

The intracellular distributions of three herpes simplex virus type 1 (HSV-1) capsid proteins, VP23, VP5 and VP22a, were examined using vaccinia virus and plasmid expression systems. During infection of cells with HSV-1 wild-type virus, all three proteins were predominantly located in the nucleus, which is the site of capsid assembly. However, when expressed in the absence of any other HSV-1 proteins, although VP22a was found exclusively in the nucleus as expected, VP5 and VP23 were distributed throughout the cell. Thus nuclear localization is not an intrinsic property of these proteins but must be mediated by one or more HSV-1-induced proteins. Co-expression experiments demonstrated that VP5 was efficiently transported to the nucleus in the presence of VP22a, but the distribution of VP23 was unaffected by the presence of either or both of the other two proteins.

Reservoir Structure and Wastewater-Induced Seismicity at the Val d'Agri Oilfield (Italy) Shown by Three-Dimensional Vp and Vp/Vs Local Earthquake Tomography

NASA Astrophysics Data System (ADS)

Improta, L.; Bagh, S.; De Gori, P.; Valoroso, L.; Pastori, M.; Piccinini, D.; Chiarabba, C.; Anselmi, M.; Buttinelli, M.

2017-11-01

Wastewater injection into a high-rate well in the Val d'Agri oilfield, the largest in onshore Europe, has induced swarm microseismicity since the initiation of disposal in 2006. To investigate the reservoir structure and to track seismicity, we performed a high-spatial resolution local earthquake tomography using 1,281 natural and induced earthquakes recorded by local networks. The properties of the carbonate reservoir (rock fracturing, pore fluid pressure) and inherited faults control the occurrence and spatiotemporal distribution of seismicity. A low-Vp, high-Vp/Vs region under the well represents a fluid saturated fault zone ruptured by induced seismicity. High-Vp, high-Vp/Vs bumps match reservoir culminations indicating saturated liquid-bearing zones, whereas a very low Vp, low Vp/Vs anomaly might represent a strongly fractured and depleted zone of the hydrocarbon reservoir characterized by significant fluid withdrawal. The comprehensive picture of the injection-linked seismicity obtained by integrating reservoir-scale tomography, high-precision earthquake locations, and geophysical and injection data suggests that the driving mechanism is the channeling of pore pressure perturbations through a high permeable fault damage zone within the reservoir. The damage zone surrounds a Pliocene reverse fault optimally oriented in the current extensional stress field. The ruptured damage zone measures 2 km along strike and 3 km along dip and is confined between low permeability ductile formations. Injection pressure is the primary parameter controlling seismicity rate. Our study underlines that local earthquake tomography also using wastewater-induced seismicity can give useful insights into the physical mechanism leading to these earthquakes.

Immunogenicity and protective efficacy of rotavirus VP8* fused to cholera toxin B subunit in a mouse model.

PubMed

Xue, Miaoge; Yu, Linqi; Jia, Lianzhi; Li, Yijian; Zeng, Yuanjun; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

2016-11-01

In attempts to develop recombinant subunit vaccines against rotavirus disease, it was previously shown that the N-terminal truncated VP8* protein, VP8-1 (aa26-231), is a good vaccine candidate when used for immunization in combination with Freund's adjuvant. However, this protein stimulated only weak immune response when aluminum hydroxide was used as an adjuvant. In this study, the nontoxic B subunit of cholera toxin (CTB) was employed as intra-molecular adjuvant to improve the immunogenicity of VP8-1. Both, the N-terminal and C-terminal fusion proteins, were purified to homogeneity, at which stage they formed pentamers, and showed significantly higher immunogenicity and protective efficacy than a VP8-1/aluminum hydroxide mixture in a mouse model. Compared to VP8-1-CTB, CTB-VP8-1 showed higher binding activity to both, GM1 and the conformation sensitive neutralizing monoclonal antibodies specific to VP8. More importantly, CTB-VP8-1 elicited higher titers of neutralizing antibodies and conferred higher protective efficacy than VP8-1-CTB. Therefore, the protein CTB-VP8-1, with enhanced immunogenicity and immunoprotectivity, could be considered as a viable candidate for further development of an alternative, replication-incompetent, parenterally administered vaccine against rotavirus disease.

Image quality and radiation dose of brain computed tomography in children: effects of decreasing tube voltage from 120 kVp to 80 kVp.

PubMed

Park, Ji Eun; Choi, Young Hun; Cheon, Jung-Eun; Kim, Woo Sun; Kim, In-One; Cho, Hyun Suk; Ryu, Young Jin; Kim, Yu Jin

2017-05-01

Computed tomography (CT) has generated public concern associated with radiation exposure, especially for children. Lowering the tube voltage is one strategy to reduce radiation dose. To assess the image quality and radiation dose of non-enhanced brain CT scans acquired at 80 kilo-voltage peak (kVp) compared to those at 120 kVp in children. Thirty children who had undergone both 80- and 120-kVp non-enhanced brain CT were enrolled. For quantitative analysis, the mean attenuation of white and gray matter, attenuation difference, noise, signal-to-noise ratio, contrast-to-noise ratio and posterior fossa artifact index were measured. For qualitative analysis, noise, gray-white matter differentiation, artifact and overall image quality were scored. Radiation doses were evaluated by CT dose index, dose-length product and effective dose. The mean attenuations of gray and white matter and contrast-to-noise ratio were significantly increased at 80 kVp, while parameters related to image noise, i.e. noise, signal-to-noise ratio and posterior fossa artifact index were higher at 80 kVp than at 120 kVp. In qualitative analysis, 80-kVp images showed improved gray-white differentiation but more artifacts compared to 120-kVp images. Subjective image noise and overall image quality scores were similar between the two scans. Radiation dose parameters were significantly lower at 80 kVp than at 120 kVp. In pediatric non-enhanced brain CT scans, a decrease in tube voltage from 120 kVp to 80 kVp resulted in improved gray-white matter contrast, comparable image quality and decreased radiation dose.

A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35–NP Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Gai; Nash, Peter J.; Johnson, Britney

The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarizationmore » assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP–VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.« less

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.

The VP35 and VP40 proteins of filoviruses. Homology between Marburg and Ebola viruses.

PubMed

Bukreyev, A A; Volchkov, V E; Blinov, V M; Netesov, S V

1993-05-03

The fragments of genomic RNA sequences of Marburg (MBG) and Ebola (EBO) viruses are reported. These fragments were found to encode the VP35 and VP40 proteins. The canonic sequences were revealed before and after each open reading frame. It is suggested that these sequences are mRNA extremities and at the same time the regulatory elements for mRNA transcription. Homology between the MBG and EBO proteins was discovered.

Sequence analysis of VP7 and VP4 genes of G1P[8] rotaviruses circulating among diarrhoeic children in Pune, India: a comparison with Rotarix and RotaTeq vaccine strains.

PubMed

Kulkarni, Ruta; Arora, Ritu; Arora, Rashmi; Chitambar, Shobha D

2014-08-11

The G1P[8] rotaviruses are a common cause of rotavirus diarrhoea among children in India. Two rotavirus vaccines licensed in India, Rotarix and RotaTeq, contain strains with G1 and P[8] genotypes. A comparative analysis of these genotypes in the live rotavirus vaccines with circulating rotavirus strains is essential for assessment of rotavirus diversity. G1P[8] strains detected during rotavirus surveillance among diarrhoeic children hospitalized in Pune in 1992-1993 and 2006-2008, were included in the study. Amplification, sequencing and phylogenetic analysis of the VP7 and VP4 genes were carried out for identification of the G1 and P[8] lineages, respectively. Antigenic epitopes of VP7 and VP4 encoded proteins were compared to determine the differences between the G1P[8] strains from Pune and the vaccine strains. G1-Lineage 1, P[8]-Lineage 3 strains were predominant in Pune during 1992-1993 and 2006-2008. Strains of G1-Lineage 2, P[8]-Lineage 3 and G1-Lineage 1, P[8]-Lineage 4 were detected at low levels during 2006-2008. The G1-Lineage 1, P[8]-Lineage 3 strains showed up to eight amino acid changes, each in the VP7 and VP4 epitopes, with respect to the Rotarix vaccine strain (G1-Lineage 2, P[8]-Lineage 1) and the G1 (Lineage-3) and P[8] (Lineage 2) components of the RotaTeq vaccine. The G1-Lineage 2 strains were closer to both vaccine strains with no or only two amino acid substitutions in the VP7 epitopes. The divergent P[8]-Lineage 4 (OP354-like) strains showed fourteen and fifteen amino acid differences, with Rotarix and RotaTeq vaccine strains, respectively, in the VP4 epitopes. The differences between the G1P[8] strains in Pune and the G1 and P[8] components of the vaccine strains need to be described for appropriate evaluation of vaccine shedding. Continuous monitoring of the G1P[8] subgenotypic lineages would be necessary to study any long term impact of vaccine use on G1P[8] strain evolution. Copyright © 2014. Published by Elsevier Ltd.

Ebolavirus VP35 is a multifunctional virulence factor.

PubMed

Leung, Daisy W; Prins, Kathleen C; Basler, Christopher F; Amarasinghe, Gaya K

2010-01-01

Ebola virus (EBOV) is a member of the filoviridae family that causes severe hemorrhagic fever during sporadic outbreaks, and no approved treatments are currently available. The multifunctional EBOV VP35 protein facilitates immune evasion by antagonizing antiviral signaling pathways and is important for viral RNA synthesis. In order to elucidate regulatory mechanisms and to develop countermeasures, we recently solved the structures of the Zaire and Reston EBOV VP35 interferon inhibitory domain (IID) in the free form and of the Zaire EBOV VP35 IID bound to dsRNA. Together with biochemical, cell biological, and virological studies, our structural work revealed that distinct regions within EBOV VP35 IID contribute to virulence through host immune evasion and viral RNA synthesis. Here we summarize our recent structural and functional studies and discuss the potential of multifunctional Ebola VP35 as a therapeutic target.

Small interfering RNA against the 2C genomic region of coxsackievirus B3 exerts potential antiviral effects in permissive HeLa cells.

PubMed

Luan, Ying; Dai, Hai-Li; Yang, Dan; Zhu, Lin; Gao, Tie-Lei; Shao, Hong-Jiang; Peng, Xue; Jin, Zhan-Feng

2012-01-01

Coxsackievirus B3 (CVB3) is the most important causal agent of viral heart muscle disease, but no specific antiviral drug is currently available. Small interfering RNA (siRNA) has been used as an antiviral therapeutic strategy via posttranscriptional gene silencing. In this study, eleven siRNAs were designed to target seven distinct regions of the CVB3 genome including VP1, VP2, VP3, 2A, 2C, 3C, and 3D. All of the siRNAs were individually transfected into HeLa cells, which were subsequently infected with CVB3. The impacts of RNA interference (RNAi) on viral replication were evaluated using five measures: cytopathic effect (CPE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 50% tissue culture infectious dose (TCID(50)), real-time RT-PCR, and Western blot. Five of the eleven siRNAs were highly efficient at inhibiting viral replication. This was especially true for siRNA-5, which targeted the ATPase 2C. However, antiviral activity varied significantly among siRNA-9, -10, and -11 even though that they all targeted the 3D region. Our results revealed several effective targets for CVB3 silencing, and provided evidence that sequences except CRE within the 2C region may also be potential targets for CVB3-specific siRNAs design. These data supported a potential role of RNA interference in future antiviral intervention therapies. Copyright © 2011 Elsevier B.V. All rights reserved.

Confirmation of the genome position and mass of the viral protien, VP3, of Solenopsis Invicta Virus 1 (Picornavirales: Dicistroviridae) infecting the Red Imported Fire Ant (Hymenoptera: Formicidae)

USDA-ARS?s Scientific Manuscript database

A predicted peptide sequence corresponding to the capsid protein VP3 of SINV-1 was used to prepare a polyclonal antibody preparation and probe SDS-PAGE separated proteins from SINV-1 particles and S. invicta ants. The SINV-1 VP3 antibody preparation recognized a 24 kDa protein from denatured SINV-1...

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

PubMed Central

Wijesinghe, Kaveesha J.; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E.; Gerstman, Bernard S.; Chapagain, Prem P.; Li, Sheng; Stahelin, Robert V.

2017-01-01

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. PMID:28167534

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

PubMed

Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E; Gerstman, Bernard S; Chapagain, Prem P; Li, Sheng; Stahelin, Robert V

2017-04-14

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Ebolavirus VP35 is a multifunctional virulence factor

PubMed Central

Leung, Daisy W; Prins, Kathleen C; Basler, Christopher F

2010-01-01

Ebola virus (EBOV) is a member of the filoviridae family that causes severe hemorrhagic fever during sporadic outbreaks, and no approved treatments are currently available. The multifunctional EBOV VP35 protein facilitates immune evasion by antagonizing antiviral signaling pathways and is important for viral RNA synthesis. In order to elucidate regulatory mechanisms and to develop countermeasures, we recently solved the structures of the Zaire and Reston EBOV VP35 interferon inhibitory domain (IID) in the free form and of the Zaire EBOV VP35 IID bound to dsRNA. Together with biochemical, cell biological and virological studies, our structural work revealed that distinct regions within EBOV VP35 IID contribute to virulence through host immune evasion and viral RNA synthesis. Here we summarize our recent structural and functional studies and discuss the potential of multifunctional Ebola VP35 as a therapeutic target. PMID:21178490

Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina

2010-06-21

VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6{sub 1}22, P2{sub 1}2{sub 1}2{sub 1} and P2{sub 1}, respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Lightmore » Source and the Advanced Photon Source.« less

A novel parallel pipeline structure of VP9 decoder

NASA Astrophysics Data System (ADS)

Qin, Huabiao; Chen, Wu; Yi, Sijun; Tan, Yunfei; Yi, Huan

2018-04-01

To improve the efficiency of VP9 decoder, a novel parallel pipeline structure of VP9 decoder is presented in this paper. According to the decoding workflow, VP9 decoder can be divided into sub-modules which include entropy decoding, inverse quantization, inverse transform, intra prediction, inter prediction, deblocking and pixel adaptive compensation. By analyzing the computing time of each module, hotspot modules are located and the causes of low efficiency of VP9 decoder can be found. Then, a novel pipeline decoder structure is designed by using mixed parallel decoding methods of data division and function division. The experimental results show that this structure can greatly improve the decoding efficiency of VP9.

Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport.

PubMed

Takamatsu, Yuki; Kolesnikova, Larissa; Becker, Stephan

2018-01-30

The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different protein compositions. Only three of the five EBOV nucleocapsid proteins-nucleoprotein, VP35, and VP24-were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.

Controlled conformational transitions in the MVM virion expose the VP1 N-terminus and viral genome without particle disassembly.

PubMed

Cotmore, S F; D'abramo, A M; Ticknor, C M; Tattersall, P

1999-02-01

Antisera were raised against peptides corresponding to the N-termini of capsid proteins VP1 and VP2 from the parvovirus minute virus of mice. Epitopes in the 142-amino-acid VP1-specific region were not accessible in the great majority of newly released viral particles, and sera directed against them failed to neutralize virus directly or deplete stocks of infectious virions. However, brief exposure to temperatures of 45 degreesC or more induced a conformational transition in a population of full virions, but not in empty viral particles, in which VP1-specific sequences became externally accessible. In contrast, the VP2 N-terminus was antibody-accessible in all full, but not empty, particles without prior treatment. An electrophoretic mobility shift assay, in which particles were heat-treated and/or preincubated with antibodies prior to electrophoresis, confirmed this pattern of epitope accessibility, showing that the heat-induced conformational transition produces a retarded form of virion that can be supershifted by incubation with VP1-specific sera. The proportion of virions undergoing transition increased with temperature, but at all temperatures up to 70 degreesC viral particles retained structure-specific antigenic determinants and remained essentially intact, without shedding individual polypeptide species or subunits. However, despite the apparent integrity of its protective coat, the genome became accessible to externally applied enzymes in an increasing proportion of virions through this temperature range, suggesting that the conformational transitions that expose VP1 likely also allow access to the genome. Heating particles to 80 degreesC or above finally induced disassembly to polypeptide monomers. Copyright 1999 Academic Press.

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.

Detection of Rare G3P[19] Porcine Rotavirus Strains in Chiang Mai, Thailand, Provides Evidence for Origin of the VP4 Genes of Mc323 and Mc345 Human Rotaviruses▿

PubMed Central

Maneekarn, Niwat; Khamrin, Pattara; Chan-it, Wisoot; Peerakome, Supatra; Sukchai, Sujin; Pringprao, Kidsadagon; Ushijima, Hiroshi

2006-01-01

Among 175 fecal specimens collected from diarrheic piglets during a surveillance of porcine rotavirus (PoRV) strains in Chiang Mai, Thailand, 39 (22.3%) were positive for group A rotaviruses. Of these, 33.3% (13 of 39) belonged to G3P[19], which was a rare P genotype seldom reported. Interestingly, their VP4 nucleotide sequences were most closely related to human P[19] strains (Mc323 and Mc345) isolated in 1989 from the same geographical area where these PoRV strains were isolated. These P[19] PoRV strains were also closely related to another human P[19] strain (RMC321), isolated from India in 1990. The VP4 sequence identities with human P[19] were 95.4% to 97.4%, while those to a porcine P[19] strain (4F) were only 87.6 to 89.1%. Phylogenetic analysis of the VP4 gene revealed that PoRV P[19] strains clustered with human P[19] strains in a monophyletic branch separated from strain 4F. Analysis of the VP7 gene confirmed that these strains belonged to the G3 genotype and shared 97.7% to 98.3% nucleotide identities with other G3 PoRV strains circulating in the regions. This close genetic relationship was also reflected in the phylogenetic analysis of their VP7 genes. Altogether, the findings provided peculiar evidence that supported the porcine origin of VP4 genes of Mc323 and Mc345 human rotaviruses. PMID:16988014

Genetic Analyses Reveal Differences in the VP7 and VP4 Antigenic Epitopes between Human Rotaviruses Circulating in Belgium and Rotaviruses in Rotarix and RotaTeq

PubMed Central

Zeller, Mark; Patton, John T.; Heylen, Elisabeth; De Coster, Sarah; Ciarlet, Max; Van Ranst, Marc

2012-01-01

Two live-attenuated rotavirus group A (RVA) vaccines, Rotarix (G1P[8]) and RotaTeq (G1-G4, P[8]), have been successfully introduced in many countries worldwide, including Belgium. The parental RVA strains used to generate the vaccines were isolated more than 20 years ago in France (G4 parental strain in RotaTeq) and the United States (all other parental strains). At present, little is known about the relationship between currently circulating human RVAs and the vaccine strains. In this study, we determined sequences for the VP7 and VP4 outer capsid proteins of representative G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] RVAs circulating in Belgium during 2007 to 2009. The analyses showed that multiple amino acid differences existed between the VP7 and VP4 antigenic epitopes of the vaccine viruses and the Belgian isolates, regardless of their G and P genotypes. However, the highest variability was observed among the circulating G1P[8] RVA strains and the G1 and P[8] components of both RVA vaccines. In particular, RVA strains of the P[8] lineage 4 (OP354-like) showed a significant number of amino acid differences with the P[8] VP4 of both vaccines. In addition, the circulating Belgian G3 RVA strains were found to possibly possess an extra N-linked glycosylation site compared to the G3 RVA vaccine strain of RotaTeq. These results indicate that the antigenic epitopes of RVA strains contained in the vaccines differ substantially from those of the currently circulating RVA strains in Belgium. Over time, these differences might result in selection for strains that escape the RVA neutralizing-antibody pressure induced by vaccines. PMID:22189107

Genetic analyses reveal differences in the VP7 and VP4 antigenic epitopes between human rotaviruses circulating in Belgium and rotaviruses in Rotarix and RotaTeq.

PubMed

Zeller, Mark; Patton, John T; Heylen, Elisabeth; De Coster, Sarah; Ciarlet, Max; Van Ranst, Marc; Matthijnssens, Jelle

2012-03-01

Two live-attenuated rotavirus group A (RVA) vaccines, Rotarix (G1P[8]) and RotaTeq (G1-G4, P[8]), have been successfully introduced in many countries worldwide, including Belgium. The parental RVA strains used to generate the vaccines were isolated more than 20 years ago in France (G4 parental strain in RotaTeq) and the United States (all other parental strains). At present, little is known about the relationship between currently circulating human RVAs and the vaccine strains. In this study, we determined sequences for the VP7 and VP4 outer capsid proteins of representative G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] RVAs circulating in Belgium during 2007 to 2009. The analyses showed that multiple amino acid differences existed between the VP7 and VP4 antigenic epitopes of the vaccine viruses and the Belgian isolates, regardless of their G and P genotypes. However, the highest variability was observed among the circulating G1P[8] RVA strains and the G1 and P[8] components of both RVA vaccines. In particular, RVA strains of the P[8] lineage 4 (OP354-like) showed a significant number of amino acid differences with the P[8] VP4 of both vaccines. In addition, the circulating Belgian G3 RVA strains were found to possibly possess an extra N-linked glycosylation site compared to the G3 RVA vaccine strain of RotaTeq. These results indicate that the antigenic epitopes of RVA strains contained in the vaccines differ substantially from those of the currently circulating RVA strains in Belgium. Over time, these differences might result in selection for strains that escape the RVA neutralizing-antibody pressure induced by vaccines.

Isolation and characterization of two VpYABBY genes from wild Chinese Vitis pseudoreticulata.

PubMed

Xiang, J; Liu, R Q; Li, T M; Han, L J; Zou, Y; Xu, T F; Wei, J Y; Wang, Y J; Xu, Y

2013-12-01

The establishment of abaxial-adaxial polarity is an important feature of the development of lateral organs in plants. Members of the YABBY gene family may be specific to seed-plant-specific transcriptional regulators that play critical roles in promoting abaxial cell fate in the model eudicot, Arabidopsis thaliana. However, recent study has shown that the roles of YABBY genes are not conserved in the development of angiosperms. The establishment of abaxial-adaxial polarity has not been studied in perennial fruit crops. Grapes are an important fruit crop in many regions of the world. Investigating YABBY genes in grapevines should help us to discover more about the key genetic and molecular pathways in grapevine development. To understand the characterization of YABBY genes in grapevines, two YABBY genes, VpYABBY1 (GenBank accession No. KC139089) and VpYABBY2 (GenBank accession No. KC139090), were isolated from the wild Chinese species Vitis pseudoreticulata. Both of these encode YABBY proteins. Sequence characterization and phylogenetic analyses show that VpYABBY1 is group classified into the FIL subfamily while VpYABBY2 is a member of the YAB2 subfamily of Arabidopsis thaliana. Subcellular localization analysis indicates that VpYABBY1 and VpYABBY2 proteins are localized in the nucleus. Tissue specific expressional analysis reveals that VpYABBY1 is expressed strongly in young leaves of grape but only weakly in the mature leaves. Meanwhile, VpYABBY2 is expressed in grape stems, flowers, tendrils, and leaves. Transgenic Arabidopsis plants ectopically expressing VpYABBY1 caused the partial abaxialization of the adaxial epidermises of leaves, behaving similarly to those over-expressing FIL or YAB3 with abaxialized lateral organs. By contrast, ectopic expression of VpYABBY2 in Arabidopsis did not cause any alteration in the adaxial-abaxial polarity. Sequence characterization and phylogenetic analysis revealed that VpYABBY1 and VpYABBY2 are group-classified into two

Identification of Continuous Human B-Cell Epitopes in the VP35, VP40, Nucleoprotein and Glycoprotein of Ebola Virus

PubMed Central

Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M.

2014-01-01

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7–12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods. PMID:24914933

Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

PubMed

Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M

2014-01-01

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

Renal Cyst Pseudoenhancement: Intraindividual Comparison Between Virtual Monochromatic Spectral Images and Conventional Polychromatic 120-kVp Images Obtained During the Same CT Examination and Comparisons Among Images Reconstructed Using Filtered Back Projection, Adaptive Statistical Iterative Reconstruction, and Model-Based Iterative Reconstruction

PubMed Central

Yamada, Yoshitake; Yamada, Minoru; Sugisawa, Koichi; Akita, Hirotaka; Shiomi, Eisuke; Abe, Takayuki; Okuda, Shigeo; Jinzaki, Masahiro

2015-01-01

Abstract The purpose of this study was to compare renal cyst pseudoenhancement between virtual monochromatic spectral (VMS) and conventional polychromatic 120-kVp images obtained during the same abdominal computed tomography (CT) examination and among images reconstructed using filtered back projection (FBP), adaptive statistical iterative reconstruction (ASIR), and model-based iterative reconstruction (MBIR). Our institutional review board approved this prospective study; each participant provided written informed consent. Thirty-one patients (19 men, 12 women; age range, 59–85 years; mean age, 73.2 ± 5.5 years) with renal cysts underwent unenhanced 120-kVp CT followed by sequential fast kVp-switching dual-energy (80/140 kVp) and 120-kVp abdominal enhanced CT in the nephrographic phase over a 10-cm scan length with a random acquisition order and 4.5-second intervals. Fifty-one renal cysts (maximal diameter, 18.0 ± 14.7 mm [range, 4–61 mm]) were identified. The CT attenuation values of the cysts as well as of the kidneys were measured on the unenhanced images, enhanced VMS images (at 70 keV) reconstructed using FBP and ASIR from dual-energy data, and enhanced 120-kVp images reconstructed using FBP, ASIR, and MBIR. The results were analyzed using the mixed-effects model and paired t test with Bonferroni correction. The attenuation increases (pseudoenhancement) of the renal cysts on the VMS images reconstructed using FBP/ASIR (least square mean, 5.0/6.0 Hounsfield units [HU]; 95% confidence interval, 2.6–7.4/3.6–8.4 HU) were significantly lower than those on the conventional 120-kVp images reconstructed using FBP/ASIR/MBIR (least square mean, 12.1/12.8/11.8 HU; 95% confidence interval, 9.8–14.5/10.4–15.1/9.4–14.2 HU) (all P < .001); on the other hand, the CT attenuation values of the kidneys on the VMS images were comparable to those on the 120-kVp images. Regardless of the reconstruction algorithm, 70-keV VMS images showed

Radiation dose and image conspicuity comparison between conventional 120 kVp and 150 kVp with spectral beam shaping for temporal bone CT.

PubMed

Kim, Chang Rae; Jeon, Ji Young

2018-05-01

The purpose of this article is to compare radiation doses and conspicuity of anatomic landmarks of the temporal bone between the CT technique using spectral beam shaping at 150 kVp with a dedicated tin filter (150 kVp-Sn) and the conventional protocol at 120 kVp. 25 patients (mean age, 46.8 ± 21.2 years) were examined using the 150-kVp Sn protocol (200 reference mAs using automated tube current modulation, 64 × 0.6 mm collimation, 0.6 mm slice thickness, pitch 0.8), whereas 30 patients (mean age, 54.5 ± 17.8 years) underwent the 120-kVp protocol (180 mAs, 128 × 0.6 mm collimation, 0.6 mm slice thickness, pitch 0.8). Radiation doses were compared between the two acquisition techniques, and dosimetric data from the literature were reviewed for comparison of radiation dose reduction. Subjective conspicuity of 23 anatomic landmarks of the temporal bone, expressed by 5-point rating scale and objective conspicuity by signal-to-noise ratio (SNR) which measured in 4 different regions of interest (ROI), were compared between 150-kVp Sn and 120-kVp acquisitions. The mean dose-length-product (DLP) and effective dose were significantly lower for the 150-kVp Sn scans (0.26 ± 0.26 mSv) compared with the 120-kVp scans (0.92 ± 0.10 mSv, p < 0.001). The lowest effective dose from the literature-based protocols was 0.31 ± 0.12 mSv, which proposed as a low-dose protocol in the setting of spiral multislice temporal bone CT. SNR was slightly superior for 120-kVp images, however analyzability of the 23 anatomic structures did not differ significantly between 150-kVp Sn and 120-kVp scans. Temporal bone CT performed at 150 kVp with an additional tin filter for spectral shaping markedly reduced radiation exposure when compared with conventional temporal bone CT at 120 kVp while maintaining anatomic conspicuity. The decreased radiation dose of the 150-kVp Sn was also lower in comparison to the previous literature-based low

Live Attenuated Vaccine Based on Duck Enteritis Virus against Duck Hepatitis A Virus Types 1 and 3

PubMed Central

Zou, Zhong; Ma, Ji; Huang, Kun; Chen, Huanchun; Liu, Ziduo; Jin, Meilin

2016-01-01

As causative agents of duck viral hepatitis, duck hepatitis A virus type 1 (DHAV-1) and type 3 (DHAV-3) causes significant economic losses in the duck industry. However, a licensed commercial vaccine that simultaneously controls both pathogens is currently unavailable. Here, we generated duck enteritis virus recombinants (rC-KCE-2VP1) containing both VP1 from DHAV-1 (VP1/DHAV-1) and VP1 from DHAV-3 (VP1/DHAV-3) between UL27 and UL26. A self-cleaving 2A-element of FMDV was inserted between the two different types of VP1, allowing production of both proteins from a single open reading frame. Immunofluorescence and Western blot analysis results demonstrated that both VP1 proteins were robustly expressed in rC-KCE-2VP1-infected chicken embryo fibroblasts. Ducks that received a single dose of rC-KCE-2VP1 showed potent humoral and cellular immune responses and were completely protected against challenges of both pathogenic DHAV-1 and DHAV-3 strains. The protection was rapid, achieved as early as 3 days after vaccination. Moreover, viral replication was fully blocked in vaccinated ducks as early as 1 week post-vaccination. These results demonstrated, for the first time, that recombinant rC-KCE-2VP1 is potential fast-acting vaccine against DHAV-1 and DHAV-3. PMID:27777571

First study conducted in Northern India that identifies group C rotavirus as the etiological agent of severe diarrhea in children in Delhi.

PubMed

Tiku, Vasundhara Razdan; Jiang, Baoming; Kumar, Praveen; Aneja, Satender; Bagga, Arvind; Bhan, Maharaj Kishen; Ray, Pratima

2017-05-30

Group C Rotavirus (RVC) is an enteric pathogen responsible for acute gastroenteritis in children and adults globally. At present there are no surveillance studies on group C Rotaviruses in India and therefore their prevalence in India remains unknown. The present study aimed to evaluate group C rotavirus infection among <5 years old children hospitalized with acute gastroenteritis in New Delhi. A total of 350 fecal specimens were collected during September 2013 to November 2014 from <5 years old diarrheal patients admitted at KSCH hospital, Delhi. The samples found negative for group A rotavirus (N = 180) by Enzyme immunoassay were screened for group C rotavirus by RT-PCR with VP6, VP7 and VP4 gene specific primers. The PCR products were further sequenced (VP6, VP7, VP4) and analyzed to ascertain their origin and G and P genotypes. Six out of 180 (group A rotavirus negative) samples were found positive for group C rotavirus by VP6 gene specific RT-PCR, of which 3 were also found positive for VP7 and VP4 genes. Phylogenetic analysis of VP7 and VP4 genes of these showed them to be G4 and P[2] genotypes. Overall, the nucleotide sequence data (VP6, VP7 and VP4) revealed a close relationship with the human group C rotavirus with no evidence of animal ancestry. Interestingly, the nucleotide sequence analysis of various genes also indicated differences in their origin. While the identity matrix of VP4 gene (n = 3) showed high amino acid sequence identity (97.60 to 98.20%) with Korean strain, the VP6 gene (n = 6) showed maximum identity with Nigerian strain (96.40 to 97.60%) and VP7 gene (n = 3) with Bangladeshi and USA strains. This is true for all analyzed samples. Our study demonstrated the group C rotavirus as the cause of severe diarrhea in young children in Delhi and provides insights on the origin of group C rotavirus genes among the local strains indicating their source of transmission. Our study also highlights the need for a simple and reliable

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain.

PubMed

Bruhn, Jessica F; Kirchdoerfer, Robert N; Urata, Sarah M; Li, Sheng; Tickle, Ian J; Bricogne, Gérard; Saphire, Erica Ollmann

2017-01-15

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics. Copyright © 2017 American Society for Microbiology.

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruhn, Jessica F.; Kirchdoerfer, Robert N.; Urata, Sarah M.

ABSTRACT Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (generaMarburgvirusandEbolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOVmore » VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. IMPORTANCEMarburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.« less

WEC3: Wave Energy Converter Code Comparison Project: Preprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Combourieu, Adrien; Lawson, Michael; Babarit, Aurelien

This paper describes the recently launched Wave Energy Converter Code Comparison (WEC3) project and present preliminary results from this effort. The objectives of WEC3 are to verify and validate numerical modelling tools that have been developed specifically to simulate wave energy conversion devices and to inform the upcoming IEA OES Annex VI Ocean Energy Modelling Verification and Validation project. WEC3 is divided into two phases. Phase 1 consists of a code-to-code verification and Phase II entails code-to-experiment validation. WEC3 focuses on mid-fidelity codes that simulate WECs using time-domain multibody dynamics methods to model device motions and hydrodynamic coefficients to modelmore » hydrodynamic forces. Consequently, high-fidelity numerical modelling tools, such as Navier-Stokes computational fluid dynamics simulation, and simple frequency domain modelling tools were not included in the WEC3 project.« less

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. © 2014 Poultry Science Association Inc.

Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein.

PubMed

Sun, Jianhui; Huang, Liping; Wei, Yanwu; Wang, Yiping; Chen, Dongjie; Du, Wenjuan; Wu, Hongli; Feng, Li; Liu, Changming

2015-11-01

Porcine parvovirus type 1 (PPV1) is a major causative agent of embryonic and fetal death in swine. The PPV1 VP2 protein is closely associated with viral immunogenicity for eliciting neutralizing antibodies, but its antigenic structures have been largely unknown. We generated three monoclonal antibodies (MAbs) against baculovirus-expressed recombinant PPV1 VP2 protein. A PEPSCAN analysis identified the minimal B cell linear epitopes of PPV1 VP2 based on these MAbs. Three core epitopes, (228)QQITDA(233), (284)RSLGLPPK(291), and (344)FEYSNGGPFLTPI(356), were defined and mapped onto three-dimensional models of the PPV1 virion and VP2 monomer. The epitope (228)QQITDA(233) is exposed on the virion surface, and the other two are located inside the protein. An alignment of the PPV1 VP2 amino acid sequences showed that (284)RSLGLPPK(291) and (344)FEYSNGGPFLTPI(356) are absolutely conserved, whereas (228)QQITDA(233) has a single substitution at residue 233 in some (S → A or T). We developed a VP2 epitope-based indirect enzyme-linked immunosorbent assay (iELISA) to test for anti-PPV1 antibodies. In a comparative analysis with an immunoperoxidase monolayer assay using 135 guinea pig sera, the VP2-epitope-based iELISA had a concordance rate of 85.19 %, sensitivity of 83.33 %, and specificity of 85.47 %. MAb 8H6 was used to monitor VP2 during the PPV1 replication cycle in vitro with an indirect immunofluorescence assay, which indicated that newly encapsulated virions are released from the nucleus at 24 h postinfection and the PPV1 replication cycle takes less than 24 h. This study provides valuable information clarifying the antigenic structure of PPV1 VP2 and lays the foundations for PPV1 serodiagnosis and antigen detection.

Unusual structural transition of antimicrobial VP1 peptide.

PubMed

Shanmugam, Ganesh; Phambu, Nsoki; Polavarapu, Prasad L

2011-05-01

VP1 peptide, an active domain of m-calpain enzyme with antimicrobial activity is found to undergo an unusual conformational transition in trifluoroethanol (TFE) solvent. The nature of, and time dependent variations in, circular dichroism associated with the amide I vibrations, suggest that VP1 undergoes self-aggregation forming anti-parallel β-sheet structure in TFE. Transmission electron micrograph (TEM) images revealed that β-sheet aggregates formed by VP1 possess fibril-like assemblies. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

PubMed

Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

2010-11-01

In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

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.

Genotyping of polyomavirus BK by Real Time PCR for VP1 gene.

PubMed

Gambarino, Stefano; Costa, Cristina; Astegiano, Sara; Piasentin, Elsa Alessio; Segoloni, Giuseppe P; Cavallo, Rossana; Bergallo, Massimiliano

2011-10-01

Polyomavirus BK latently persist in different sites, including the renourinary tract, and may reactivate causing nephropathy in renal transplant recipients or hemorrhagic cystitis in bone marrow recipients. Based on the sequence of the VP1 gene, four genotypes have been described, corresponding to the four serologically differentiated subtypes I-IV, with different prevalence and geographic distribution. In this study, the development and clinical validation of four different Real-Time PCR assays for the detection and discrimination of BKV genotypes as a substitute of DNA sequencing are described. 379 BK VP1 sequences, belonging to the main four genotypes, were aligned and "hot spots" of mutation specific for all the strains or isolates were identified. Specific primers and probes for the detection and discrimination of each genotype by four Real-Time PCR assays were designed and technically validated. Subsequently, the four Real-Time PCR assays were used to test 20 BK-positive urine specimens from renal transplant patients, and evidenced a prevalence of BK genotype I, as previously reported in Europe. Results were confirmed by sequencing. The availability of a rapid and simple genotyping method could be useful for the evaluation of BK genotypes prevalence and studies on the impact of the infecting genotype on viral biological behavior, pathogenic role, and immune evasion strategies.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

PubMed Central

Ren, Shoufeng; Wei, Qimei; Cai, Liya; Yang, Xuejing; Xing, Cuicui; Tan, Feng; Leavenworth, Jianmei W.; Liang, Shaohui; Liu, Wenquan

2018-01-01

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention. PMID:29375526

The ebolavirus VP24 interferon antagonist: know your enemy.

PubMed

Zhang, Adrianna P P; Abelson, Dafna M; Bornholdt, Zachary A; Liu, Tong; Woods, Virgil L; Saphire, Erica Ollmann

2012-08-15

Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways. The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation. A more recent discovery is that VP24 also binds STAT1 directly, suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses.

Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle.

PubMed

Biedenkopf, Nadine; Lier, Clemens; Becker, Stephan

2016-05-15

Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle. The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that

Genetic relatedness among human rotavirus genes coding for VP7, a major neutralization protein, and its application to serotype identification.

PubMed Central

Midthun, K; Flores, J; Taniguchi, K; Urasawa, S; Kapikian, A Z; Chanock, R M

1987-01-01

Antigenic characterization of human rotaviruses by plaque reduction neutralization assay has revealed four distinct serotypes. The outer capsid protein VP7, coded for by gene 8 or 9, is a major neutralization protein; however, studies of rotaviruses derived from genetic reassortment between two strains have confirmed that another outer capsid protein, VP3, is in some cases equally important in neutralization. In this study, the genetic relatedness of the genes coding for VP7 of human rotaviruses belonging to serotypes 1 through 4 was examined by hybridization of their denatured double-stranded genomic RNAs to labeled single-stranded mRNA probes derived from human-animal rotavirus reassortants containing only the VP7 gene of their human rotavirus parent. A high degree of homology was demonstrated between the VP7 genes of strain D and other serotype 1 human rotaviruses, strain DS-1 and other serotype 2 human rotaviruses, strain P and other serotype 3 human rotaviruses, and strain ST3 and other serotype 4 human rotaviruses. Hybrid bands could not be demonstrated between the VP7 gene of D, DS-1, P, or ST3 and the corresponding gene of human rotaviruses belonging to a different serotype. RNA specimens extracted from the stools of 15 Venezuelan children hospitalized with rotavirus diarrhea were hybridized to each of the reassortant probes representing the four human serotypes. All five viruses with short RNA patterns showed homology with the DS-1 strain VP7 gene; two of these were previously adapted to tissue culture and shown to be serotype 2 strains by tissue culture neutralization. Of the remaining 10 viruses with long RNA patterns, 2 hybridized only to the D strain VP7 gene, 6 hybridized only to the P strain VP7 gene, and 2 hybridized only to the ST3 strain VP7 gene. Hybridization using single human rotavirus gene substitution reassortants as probes may provide an alternative method for identifying the VP7 serotype of field isolates that would circumvent the need for

Evaluation of imaging quality for flat-panel detector based low dose C-arm CT system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Chang-Woo; Cha, Bo Kyung; Jeon, Sungchae

The image quality associated with the extent of the angle of gantry rotation, the number of projection views, and the dose of X-ray radiation was investigated in flat-panel detector (FPD) based C-arm cone-beam computed tomography (CBCT) system for medical applications. A prototype CBCT system for the projection acquisition used the X-ray tube (A-132, Varian inc.) having rhenium-tungsten molybdenum target and flat panel a-Si X-ray detector (PaxScan 4030CB, Varian inc.) having a 397 x 298 mm active area with 388 μm pixel pitch and 1024 x 768 pixels in 2 by 2 binning mode. The performance comparison of X-ray imaging qualitymore » was carried out using the Feldkamp, Davis, and Kress (FDK) reconstruction algorithm between different conditions of projection acquisition. In this work, head-and-dental (75 kVp/20 mA) and chest (90 kVp/25 mA) phantoms were used to evaluate the image quality. The 361 (30 fps x 12 s) projection data during 360 deg. gantry rotation with 1 deg. interval for the 3D reconstruction were acquired. Parke weighting function were applied to handle redundant data and improve the reconstructed image quality in a mobile C-arm system with limited rotation angles. The reconstructed 3D images were investigated for comparison of qualitative image quality in terms of scan protocols (projection views, rotation angles and exposure dose). Furthermore, the performance evaluation in image quality will be investigated regarding X-ray dose and limited projection data for a FPD based mobile C-arm CBCT system. (authors)« less

High-kVp Assisted Metal Artifact Reduction for X-ray Computed Tomography

PubMed Central

Xi, Yan; Jin, Yannan; De Man, Bruno; Wang, Ge

2016-01-01

In X-ray computed tomography (CT), the presence of metallic parts in patients causes serious artifacts and degrades image quality. Many algorithms were published for metal artifact reduction (MAR) over the past decades with various degrees of success but without a perfect solution. Some MAR algorithms are based on the assumption that metal artifacts are due only to strong beam hardening and may fail in the case of serious photon starvation. Iterative methods handle photon starvation by discarding or underweighting corrupted data, but the results are not always stable and they come with high computational cost. In this paper, we propose a high-kVp-assisted CT scan mode combining a standard CT scan with a few projection views at a high-kVp value to obtain critical projection information near the metal parts. This method only requires minor hardware modifications on a modern CT scanner. Two MAR algorithms are proposed: dual-energy normalized MAR (DNMAR) and high-energy embedded MAR (HEMAR), aiming at situations without and with photon starvation respectively. Simulation results obtained with the CT simulator CatSim demonstrate that the proposed DNMAR and HEMAR methods can eliminate metal artifacts effectively. PMID:27891293

Metric analysis and data validation across FORTRAN projects

NASA Technical Reports Server (NTRS)

Basili, Victor R.; Selby, Richard W., Jr.; Phillips, Tsai-Yun

1983-01-01

The desire to predict the effort in developing or explaining the quality of software has led to the proposal of several metrics. As a step toward validating these metrics, the Software Engineering Laboratory (SEL) has analyzed the software science metrics, cyclomatic complexity, and various standard program measures for their relation to effort (including design through acceptance testing), development errors (both discrete and weighted according to the amount of time to locate and fix), and one another. The data investigated are collected from a project FORTRAN environment and examined across several projects at once, within individual projects and by reporting accuracy checks demonstrating the need to validate a database. When the data comes from individual programmers or certain validated projects, the metrics' correlations with actual effort seem to be strongest. For modules developed entirely by individual programmers, the validity ratios induce a statistically significant ordering of several of the metrics' correlations. When comparing the strongest correlations, neither software science's E metric cyclomatic complexity not source lines of code appears to relate convincingly better with effort than the others.

Differential expression of VGLUT1 or VGLUT2 in the trigeminothalamic or trigeminocerebellar projection neurons in the rat.

PubMed

Ge, Shun-Nan; Li, Zhi-Hong; Tang, Jun; Ma, Yunfei; Hioki, Hiroyuki; Zhang, Ting; Lu, Ya-Cheng; Zhang, Fu-Xing; Mizuno, Noboru; Kaneko, Takeshi; Liu, Ying-Ying; Lung, Mandy Siu Yu; Gao, Guo-Dong; Li, Jin-Lian

2014-01-01

The vesicular glutamate transporters, VGLUT1 and VGLUT2, reportedly display complementary distribution in the rat brain. However, co-expression of them in single neurons has been reported in some brain areas. We previously found co-expression of VGLUT1 and VGLUT2 mRNAs in a number of single neurons in the principal sensory trigeminal nucleus (Vp) of the adult rat; the majority of these neurons sent their axons to the thalamic regions around the posteromedial ventral nucleus (VPM) and the posterior nuclei (Po). It is well known that trigeminothalamic (T-T) projection fibers arise not only from the Vp but also from the spinal trigeminal nucleus (Vsp), and that trigeminocerebellar (T-C) projection fibers take their origins from both of the Vp and Vsp. Thus, in the present study, we examined the expression of VGLUT1 and VGLUT2 in Vp and Vsp neurons that sent their axons to the VPM/Po regions or the cortical regions of the cerebellum. For this purpose, we combined fluorescence in situ hybridization (FISH) histochemistry with retrograde tract-tracing; immunofluorescence histochemistry was also combined with anterograde tract-tracing. The results indicate that glutamatergic Vsp neurons sending their axons to the cerebellar cortical regions mainly express VGLUT1, whereas glutamatergic Vsp neurons sending their axons to the thalamic regions express VGLUT2. The present data, in combination with those of our previous study, indicate that glutamatergic Vp neurons projecting to the cerebellar cortical regions express mainly VGLUT1, whereas the majority of glutamatergic Vp neurons projecting to the thalamus co-express VGLUT1 and VGLUT2.

The Predictive Validity of Projective Measures.

ERIC Educational Resources Information Center

Suinn, Richard M.; Oskamp, Stuart

Written for use by clinical practitioners as well as psychological researchers, this book surveys recent literature (1950-1965) on projective test validity by reviewing and critically evaluating studies which shed light on what may reliably be predicted from projective test results. Two major instruments are covered: the Rorschach and the Thematic…

Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress.

PubMed

Loughran, H Marie; Han, Ziying; Wrobel, Jay E; Decker, Sarah E; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N; Reitz, Allen B

2016-08-01

We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome.

PubMed

Lin, Chun-Yu; Chiu, Chun-Ching; Cheng, Ju; Lin, Chia-Yun; Shi, Ya-Fang; Tsai, Chun-Chou; Tzang, Bor-Show; Hsu, Tsai-Ching

2018-01-01

Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61-227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21-227 to 121-227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61-227 and 101-227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u. The mice that received amino acid residues 31-227 or 61-227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21-227, 31-227, 61-227, 71-227, 82-227, 91-227, 101-227 or 114-227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These

CD8+ T Lymphocyte Epitopes From The Herpes Simplex Virus Type 2 ICP27, VP22 and VP13/14 Proteins To Facilitate Vaccine Design And Characterization

PubMed Central

Platt, Rebecca J.; Khodai, Tansi; Townend, Tim J.; Bright, Helen H.; Cockle, Paul; Perez-Tosar, Luis; Webster, Rob; Champion, Brian; Hickling, Timothy P.; Mirza, Fareed

2013-01-01

CD8+ T cells have the potential to control HSV-2 infection. However, limited information has been available on CD8+ T cell epitopes or the functionality of antigen specific T cells during infection or following immunization with experimental vaccines. Peptide panels from HSV-2 proteins ICP27, VP22 and VP13/14 were selected from in silico predictions of binding to human HLA-A*0201 and mouse H-2Kd, Ld and Dd molecules. Nine previously uncharacterized CD8+ T cell epitopes were identified from HSV-2 infected BALB/c mice. HSV-2 specific peptide sequences stabilized HLA-A*02 surface expression with intermediate or high affinity binding. Peptide specific CD8+ human T cell lines from peripheral blood lymphocytes were generated from a HLA-A*02+ donor. High frequencies of peptide specific CD8+ T cell responses were elicited in mice by DNA vaccination with ICP27, VP22 and VP13/14, as demonstrated by CD107a mobilization. Vaccine driven T cell responses displayed a more focused immune response than those induced by viral infection. Furthermore, vaccination with ICP27 reduced viral shedding and reduced the clinical impact of disease. In conclusion, this study describes novel HSV-2 epitopes eliciting strong CD8+ T cell responses that may facilitate epitope based vaccine design and aid immunomonitoring of antigen specific T cell frequencies in preclinical and clinical settings. PMID:24709642

Immunogenicity of virus-like particles containing modified goose parvovirus VP2 protein.

PubMed

Chen, Zongyan; Li, Chuanfeng; Zhu, Yingqi; Wang, Binbin; Meng, Chunchun; Liu, Guangqing

2012-10-01

The major capsid protein VP2 of goose parvovirus (GPV) expressed using a baculovirus expression system (BES) assembles into virus-like particles (VLPs). To optimize VP2 gene expression in Sf9 cells, we converted wild-type VP2 (VP2) codons into codons that are more common in insect genes. This change greatly increased VP2 protein production in Sf9 cells. The protein generated from the codon-optimized VP2 (optVP2) was detected by immunoblotting and an indirect immunofluorescence assay (IFA). Transmission electron microscopy analysis revealed the formation of VLPs. These findings indicate that optVP2 yielded stable and high-quality VLPs. Immunogenicity assays revealed that the VLPs are highly immunogenic, elicit a high level of neutralizing antibodies and provide protection against lethal challenge. The antibody levels appeared to be directly related to the number of GP-Ag-positive hepatocytes. The variation trends for GP-Ag-positive hepatocytes were similar in the vaccine groups. In comparison with the control group, the optVP2 VLPs groups exhibited obviously better responses. These data indicate that the VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Thus, GPV optVP2 appears to be a good candidate for the vaccination of goslings. Copyright © 2012 Elsevier B.V. All rights reserved.

NASA Countermeasures Evaluation and Validation Project

NASA Technical Reports Server (NTRS)

Lundquist, Charlie M.; Paloski, William H. (Technical Monitor)

2000-01-01

To support its ISS and exploration class mission objectives, NASA has developed a Countermeasure Evaluation and Validation Project (CEVP). The goal of this project is to evaluate and validate the optimal complement of countermeasures required to maintain astronaut health, safety, and functional ability during and after short- and long-duration space flight missions. The CEVP is the final element of the process in which ideas and concepts emerging from basic research evolve into operational countermeasures. The CEVP is accomplishing these objectives by conducting operational/clinical research to evaluate and validate countermeasures to mitigate these maladaptive responses. Evaluation is accomplished by testing in space flight analog facilities, and validation is accomplished by space flight testing. Both will utilize a standardized complement of integrated physiological and psychological tests, termed the Integrated Testing Regimen (ITR) to examine candidate countermeasure efficacy and intersystem effects. The CEVP emphasis is currently placed on validating the initial complement of ISS countermeasures targeting bone, muscle, and aerobic fitness; followed by countermeasures for neurological, psychological, immunological, nutrition and metabolism, and radiation risks associated with space flight. This presentation will review the processes, plans, and procedures that will enable CEVP to play a vital role in transitioning promising research results into operational countermeasures necessary to maintain crew health and performance during long duration space flight.

Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

PubMed

Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

2017-12-01

Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

Supramolecular Assembly of Gold Nanoparticles in PS-b-P2VP Diblock Copolymers via Hydrogen Bonding

NASA Astrophysics Data System (ADS)

Jang, Se Gyu; Hawker, Craig J.; Kramer, Edward J.

2011-03-01

We report a simple route to control the spatial distribution of Au nanoparticles (Au-NPs) in PS- b -P2VP diblock copolymers using hydrogen bonding between P2VP and the hydroxyl-containing (PI-OH) units in PS- b -PIOH thiol-terminated ligands on Au-NP. End-functional thiol ligands of poly(styrene- b -1,2&3,4-isoprene-SH) are synthesized by anionic polymerization. After synthesis of Au-NPs, the inner PI block is hydroxylated by hydroboration and the resulting micelle-like Au-NPs consist of a hydrophobic PS outer brush and a hydrophilic inner PI-OH block. The influence of the hydroxyl groups is significant with strong segregation being observed to the PS/P2VP interface and then to the P2VP domain of lamellar-forming PS-b-P2VP diblock copolymers as the length of the PI-OH block is increased. The strong hydrogen bonding between nanoparticle block copolymer ligands and the P2VP block allows the Au-NPs to be incorporated within the P2VP domain to high Au--NP volume fractions ϕp without macrophase separation, driving transitions from lamellar to bicontinuous morphologies as ϕp increases.

Antigenic and Genomic Diversity of Human Rotavirus VP4 in Two Consecutive Epidemic Seasons in Mexico

PubMed Central

Padilla-Noriega, Luis; Méndez-Toss, Martha; Menchaca, Griselda; Contreras, Juan F.; Romero-Guido, Pedro; Puerto, Fernando I.; Guiscafré, Héctor; Mota, Felipe; Herrera, Ismael; Cedillo, Roberto; Muñoz, Onofre; Calva, Juan; Guerrero, María de Lourdes; Coulson, Barbara S.; Greenberg, Harry B.; López, Susana; Arias, Carlos F.

1998-01-01

In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods. PMID:9620401

Radiation dose reduction using 100-kVp and a sinogram-affirmed iterative reconstruction algorithm in adolescent head CT: Impact on grey-white matter contrast and image noise.

PubMed

Nagayama, Yasunori; Nakaura, Takeshi; Tsuji, Akinori; Urata, Joji; Furusawa, Mitsuhiro; Yuki, Hideaki; Hirarta, Kenichiro; Kidoh, Masafumi; Oda, Seitaro; Utsunomiya, Daisuke; Yamashita, Yasuyuki

2017-07-01

To retrospectively evaluate the image quality and radiation dose of 100-kVp scans with sinogram-affirmed iterative reconstruction (IR) for unenhanced head CT in adolescents. Sixty-nine patients aged 12-17 years underwent head CT under 120- (n = 34) or 100-kVp (n = 35) protocols. The 120-kVp images were reconstructed with filtered back-projection (FBP), 100-kVp images with FBP (100-kVp-F) and sinogram-affirmed IR (100-kVp-S). We compared the effective dose (ED), grey-white matter (GM-WM) contrast, image noise, and contrast-to-noise ratio (CNR) between protocols in supratentorial (ST) and posterior fossa (PS). We also assessed GM-WM contrast, image noise, sharpness, artifacts, and overall image quality on a four-point scale. ED was 46% lower with 100- than 120-kVp (p < 0.001). GM-WM contrast was higher, and image noise was lower, on 100-kVp-S than 120-kVp at ST (p < 0.001). CNR of 100-kVp-S was higher than of 120-kVp (p < 0.001). GM-WM contrast of 100-kVp-S was subjectively rated as better than of 120-kVp (p < 0.001). There were no significant differences in the other criteria between 100-kVp-S and 120-kVp (p = 0.072-0.966). The 100-kVp with sinogram-affirmed IR facilitated dramatic radiation reduction and better GM-WM contrast without increasing image noise in adolescent head CT. • 100-kVp head CT provides 46% radiation dose reduction compared with 120-kVp. • 100-kVp scanning improves subjective and objective GM-WM contrast. • Sinogram-affirmed IR decreases head CT image noise, especially in supratentorial region. • 100-kVp protocol with sinogram-affirmed IR is suited for adolescent head CT.

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.

Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen.

PubMed

Nasiri, K; Nassiri, M R; Tahmoorespur, M; Haghparast, A; Zibaee, S

2016-01-01

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. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus.

Constraining the Dynamic Rupture Properties with Moment Tensor Derived Vp/Vs Ratios.

NASA Astrophysics Data System (ADS)

Smith-Boughner, L.; Baig, A. M.; Urbancic, T.; Viegas, G. F.

2014-12-01

The goal of hydraulic fracturing is to increase the permeability of rocks to extract hydrocarbons from "tight" formations. This process stimulates fluid-driven fractures which induce microseismic events. Successfully treating the formations, stimulating large volumes of the reservoir, depends on targeting parts of the formation with more "brittleness", a property which is frequently characterized from the mechanical properties of the rock. Typically, these properties are constrained using well-logs, vertical seismic profiles and 3-D seismic surveys. Such tools provide a static view of the reservoir on very large or very small scales. While lithology controls the average rock strength within a unit, the content (gas or fluid filled), the shape of the pore space and the concentration of micro-fractures alters the mechanical properties of the reservoir. Seismic moment tensor inversion of the events generated during these stimulations reveals that they are significantly non-double-couple, and are described by a tensile angle and a Poisson's ratio (or, equivalently, ratio of shear to compressional velocities, Vp/Vs) of the rock-fracture system. Following Vavryčuk (2011), the mechanical properties of the reservoir (i.e. Vp/Vs ratio) are estimated as the hydraulic fracture progresses from an extensive catalog of microseismic events spanning magnitudes of -1.5 to 0.8 in the Horn-River Basin, Canada. Studying several fracture stages in the reservoir reveals temporal and spatial variations in the rock strength within a unit as hydraulic fracturing proceeds. Initially, the estimated values of Vp/Vs are quite close to those determined from 3-D seismic surveys. As the stage progresses, previously fractured regions have lower Vp/Vs values. At the onset of maximum treating pressure, regions have anomalously high Vp/Vs values, which could reflect short-term local concentrations of high pore pressures or other interactions of the treatment with the formation. The relationship

Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes.

PubMed

Wagner, Melany J; Smiley, James R

2009-12-01

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus.

Neonatal treatment philosophy in Dutch and German NICUs: health-related quality of life in adulthood of VP/VLBW infants.

PubMed

Breeman, Linda D; van der Pal, Sylvia; Verrips, Gijsbert H W; Baumann, Nicole; Bartmann, Peter; Wolke, Dieter

2017-04-01

Although survival after very preterm birth (VP)/very low birth weight (VLBW) has improved, a significant number of VP/VLBW individuals develop physical and cognitive problems during their life course that may affect their health-related quality of life (HRQoL). We compared HRQoL in VP/VLBW cohorts from two countries: The Netherlands (n = 314) versus Germany (n = 260) and examined whether different neonatal treatment and rates of disability affect HRQoL in adulthood. To analyse whether cohorts differed in adult HRQoL, linear regression analyses were performed for three HRQoL outcomes assessed with the Health Utilities Index 3 (HUI3), the London Handicap Scale (LHS), and the WHO Quality of Life instrument (WHOQOL-BREF). Stepwise hierarchical linear regression was used to test whether neonatal physical health and treatment, social environment, and intelligence (IQ) were related to VP/VLBW adults' HRQoL and cohort differences. Dutch VP/VLBW adults reported a significantly higher HRQoL on all three general HRQoL measures than German VP/VLBW adults (HUI3: .86 vs .83, p = .036; LHS: .93 vs. .90, p = .018; WHOQOL-BREF: 82.8 vs. 78.3, p < .001). Main predictor of cohort differences in all three HRQoL measures was adult IQ (p < .001). Lower HRQoL in German versus Dutch adults was related to more cognitive impairment in German adults. Due to different policies, German VP/VLBW infants received more intensive treatment that may have affected their cognitive development. Our findings stress the importance of examining effects of different neonatal treatment policies for VP/VLBW adults' life.

Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

PubMed Central

Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei

2014-01-01

ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661

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

Image quality and radiation dose of lower extremity CT angiography at 70 kVp on an integrated circuit detector dual-source computed tomography.

PubMed

Qi, Li; Zhao, Yan'E; Zhou, Chang Sheng; Spearman, James V; Renker, Matthias; Schoepf, U Joseph; Zhang, Long Jiang; Lu, Guang Ming

2015-06-01

Despite the well-established requirement for radiation dose reduction there are few studies examining the potential for lower extremity CT angiography (CTA) at 70 kVp. To compare the image quality and radiation dose of lower extremity CTA at 70 kVp using a dual-source CT system with an integrated circuit detector to similar studies at 120 kVp. A total of 62 patients underwent lower extremity CTA. Thirty-one patients were examined at 70 kVp using a second generation dual-source CT with an integrated circuit detector (70 kVp group) and 31 patients were evaluated at 120 kVp using a first generation dual-source CT (120 kVp group). The attenuation and image noise were measured and signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. Two radiologists assessed image quality. Radiation dose was compared. The mean attenuation of the 70 kVp group was higher than the 120 kVp group (575 ± 149 Hounsfield units [HU] vs. 258 ± 38 HU, respectively, P < 0.001) as was SNR (44.0 ± 22.0 vs 32.7 ± 13.3, respectively, P = 0.017), CNR (39.7 ± 20.6 vs 26.6 ± 11.7, respectively, P = 0.003) and the mean image quality score (3.7 ± 0.1 vs. 3.2 ± 0.3, respectively, P < 0.001). The inter-observer agreement was good for the 70 kVp group and moderate for the 120 kVp group. The dose-length product was lower in the 70 kVp group (264.5 ± 63.1 mGy × cm vs. 412.4 ± 81.5 mGy × cm, P < 0.001). Lower extremity CTA at 70 kVp allows for lower radiation dose with higher SNR, CNR, and image quality when compared with standard 120 kVp. © The Foundation Acta Radiologica 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Effect of bladder augmentation on VP shunt failure rates in spina bifida.

PubMed

Gonzalez, Dani O; Cooper, Jennifer N; McLeod, Daryl J

2017-12-11

Most patients with spina bifida require ventriculoperitoneal (VP) shunt placement. Some also require bladder augmentation, which may increase the risk of VP shunt malfunction and/or failure. The aim of this study was to assess whether bladder augmentation affects the rate of VP shunt failure in this population. Using the Pediatric Health Information System, we studied patients with spina bifida born between 1992 and 2014 who underwent VP shunt placement. Using conditional logistic regression, we compared age- and hospital-matched patients who did and did not undergo a bladder augmentation to determine their difference in rates of VP shunt failure. There were 4192 patients with spina bifida who underwent both surgical closure and VP shunt placement. Of these, 203 patients with bladder augmentation could be matched to 593 patients without bladder augmentation. VP shunt failure occurred within 2 years in 7.7% of patients, the majority of whom were in the group who underwent bladder augmentation (87%). After adjusting for confounders, undergoing bladder augmentation was independently associated with VP shunt failure (HR: 33.5, 95% CI: 13.15-85.44, p< 0.001). Bladder augmentation appears to be associated with VP shunt failure. Additional studies are necessary to better define this relationship and identify risk-reduction techniques.

Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer.

PubMed

Bhattarai, Nisha; Gc, Jeevan B; Gerstman, Bernard S; Stahelin, Robert V; Chapagain, Prem P

2017-04-26

Filovirus infections cause hemorrhagic fever in humans and non-human primates that often results in high fatality rates. The Marburg virus is a lipid-enveloped virus from the Filoviridae family and is closely related to the Ebola virus. The viral matrix layer underneath the lipid envelope is formed by the matrix protein VP40 (VP40), which is also involved in other functions during the viral life-cycle. As in the Ebola virus VP40 (eVP40), the recently determined X-ray crystal structure of the Marburg virus VP40 (mVP40) features loops containing cationic residues that form a lipid binding basic patch. However, the mVP40 basic patch is significantly flatter with a more extended surface than in eVP40, suggesting the possibility of differences in the plasma membrane interactions and phospholipid specificity between the VP40 dimers. In this paper, we report on molecular dynamics simulations that investigate the roles of various residues and lipid types in PM association as well as the conformational changes of the mVP40 dimer facilitated by membrane association. We compared the structural changes of the mVP40 dimer with the mVP40 dimer in both lipid free and membrane associated conditions. Despite the significant structural differences in the crystal structure, the Marburg VP40 dimer is found to adopt a configuration very similar to the Ebola VP40 dimer after associating with the membrane. This conformational rearrangement upon lipid binding allows Marburg VP40 to localize and stabilize at the membrane surface in a manner similar to the Ebola VP40 dimer. Consideration of the structural information in its lipid-interacting condition may be important in targeting mVP40 for novel drugs to inhibit viral budding from the plasma membrane.

Hydrometer in the mantle: dln(Vs)/dln(Vp)

NASA Astrophysics Data System (ADS)

Li, L.; Weidner, D. J.

2010-12-01

The absorption of water into nominally non-hydrous phases is the probable storage mechanism of hydrogen throughout most of the mantle. Thus the water capacity in the mantle is greatest in the transition zone owing to the large water-solubility of ringwoodite and wadsleyite. However, the actual amount of water that is stored there is highly uncertain. Since water is probably brought down by subduction activity, it’s abundance is probably laterally variable. Thus, a metric that is sensitive to variations of water content are good candidates for hydrometers. Here we evaluate the parameter, dln(Vs)/dln(Vp), as such a parameter. It is useful to detect lateral variations of water if the effects of hydration on the parameter are different than those of temperature or composition. We compare the value of dln(Vs)/dln(Vp) due to the temperature with that due to the water content as a function of depth for the upper mantle. We have calculated dln(Vs)/dln(Vp) due to both water and temperature using a density functional theory approach, and available experimental data. Our results indicate that dln(Vs)/dln(Vp) due to water is distinguishable from dln(Vs)/dln(Vp) due to temperature or variations in iron content, particularly in ringwoodite. The difference increases with depth and making the lower part of the transition zone most identifiable as a water reservoir.

MicroCT imaging dose to mouse organs using a validated Monte Carlo model of the small animal radiation research platform (SARRP)

NASA Astrophysics Data System (ADS)

Johnstone, Christopher Daniel; Bazalova-Carter, Magdalena

2018-06-01

The goal of this work was to establish imaging dose to mouse organs with a validated Monte Carlo (MC) model of the image-guided Small Animal Radiation Research Platform (SARRP) and to investigate the effect of scatter from the internal walls on animal therapy dose determination. A MC model of the SARRP was built in the BEAMnrc code and validated with a series of homogeneous and heterogeneous phantom measurements. A segmented microCT scan of a mouse was used in DOSXYZnrc to determine mouse organ microCT imaging doses to 15–35 g mice for the SARRP pancake (mouse lying on couch) and standard (mouse standing on couch) imaging geometries for 40–80 kVp tube voltages. Imaging dose for off-center positioning shifts and maintaining image noise across tube voltages were also calculated. Half-value layer (HVL) measurements for the 220 kVp therapy beam in the presence of the SARRP shielding cabinet were modeled in BEAMnrc and compared to the 100 cm source-to-detector distance (SDD) in the scatter free, narrow-beam geometry recommended by the American Association of Physicists in Medicine Task Group 61 (AAPM TG-61). For a 60 kVp, 0.8 mA, and 60 s scan protocol, maximum mean organ imaging doses to boney and non-boney structures were 10.5 cGy and 3.5 cGy, respectively, for an average size 20 g mouse. Current-exposure combinations above 323, 203, 147, 116, and 95 mAs for 40–80 kVp tube voltages, respectively, will increase body doses above 10 cGy. MicroCT mean body dose was 18% lower in pancake compared to standard imaging geometry. An 11% difference in measured HVL at a 50 cm SDD was found compared to MC simulated HVL for the AAPM TG-61 recommended scatter free geometry at a 100 cm SDD. This change in HVL resulted in a 0.5% change in absorbed dose to water calculations for the treatment beam.

A porcine G9 rotavirus strain shares neutralization and VP7 phylogenetic sequence lineage 3 characteristics with contemporary human G9 rotavirus strains.

PubMed

Hoshino, Yasutaka; Honma, Shinjiro; Jones, Ronald W; Ross, Jerri; Santos, Norma; Gentsch, Jon R; Kapikian, Albert Z; Hesse, Richard A

2005-02-05

Of five globally important VP7 (G) serotypes (G1-4 and 9) of group A rotaviruses (the single most important etiologic agents of infantile diarrhea worldwide), G9 continues to attract considerable attention because of its unique natural history. Serotype G9 rotavirus was isolated from a child with diarrhea first in the United States in 1983 and subsequently in Japan in 1985. Curiously, soon after their detection, G9 rotaviruses were not detected for about a decade in both countries and then reemerged in both countries in the mid-1990s. Unexpectedly, however, such reemerged G9 strains were distinct genetically and molecularly from those isolated in the 1980s. Thus, the origin of the reemerged G9 viruses remains an enigma. Sequence analysis has demonstrated that the G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages: lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Currently, lineage 3 G9 viruses are the most frequently detected G9 strains globally. We characterized a porcine rotavirus (A2 strain) isolated in the United States that was known to belong to the P[7] genotype but had not been serotyped by neutralization. The A2 strain was found to bear serotype G9 and P9 specificities as well as NSP4 [B] and subgroup I characteristics. By VP7-specific neutralization, the porcine G9 strain was more closely related to lineage 3 viruses than to lineage 1 or 2 viruses. Furthermore, by sequence analysis, the A2 VP7 was shown to belong to lineage 3 G9. These findings raise intriguing questions regarding possible explanations for the emergence of variations among the G9 strains.

Cerebral bone subtraction CT angiography using 80 kVp and sinogram-affirmed iterative reconstruction: contrast medium and radiation dose reduction with improvement of image quality.

PubMed

Nagayama, Yasunori; Nakaura, Takeshi; Tsuji, Akinori; Urata, Joji; Furusawa, Mitsuhiro; Yuki, Hideaki; Hirarta, Kenichiro; Oda, Seitaro; Kidoh, Masafumi; Utsunomiya, Daisuke; Yamashita, Yasuyuki

2017-02-01

The purpose of this study was to evaluate the feasibility of a contrast medium (CM), radiation dose reduction protocol for cerebral bone-subtraction CT angiography (BSCTA) using 80-kVp and sinogram-affirmed iterative reconstruction (SAFIRE). Seventy-five patients who had undergone BSCTA under the 120- (n = 37) or the 80-kVp protocol (n = 38) were included. CM was 370 mgI/kg for the 120-kVp and 296 mgI/kg for the 80-kVp protocol; the 120- and the 80-kVp images were reconstructed with filtered back-projection (FBP) and SAFIRE, respectively. We compared effective dose (ED), CT attenuation, image noise, and contrast-to-noise ratio (CNR) of two protocols. We also scored arterial contrast, sharpness, depiction of small arteries, visibility near skull base/clip, and overall image quality on a four-point scale. ED was 62% lower at 80- than 120-kVp (0.59 ± 0.06 vs 1.56 ± 0.13 mSv, p < 0.01). CT attenuation of the internal carotid artery (ICA) and middle cerebral artery (MCA) was significantly higher on 80- than 120-kVp (ICA: 557.4 ± 105.7 vs 370.0 ± 59.3 Hounsfield units (HU), p < 0.01; MCA: 551.9 ± 107.9 vs 364.6 ± 62.2 HU, p < 0.01). The CNR was also significantly higher on 80- than 120-kVp (ICA: 46.2 ± 10.2 vs 36.9 ± 7.6, p < 0.01; MCA: 45.7 ± 10.0 vs 35.7 ± 9.0, p < 0.01). Visibility near skull base and clip was not significantly different (p = 0.45). The other subjective scores were higher with the 80- than the 120-kVp protocol (p < 0.05). The 80-kVp acquisition with SAFIRE yields better image quality for BSCTA and substantial reduction in the radiation and CM dose compared to the 120-kVp with FBP protocol.

Herpes Simplex Virus Requires VP11/12 To Induce Phosphorylation of the Activation Loop Tyrosine (Y394) of the Src Family Kinase Lck in T Lymphocytes ▿

PubMed Central

Wagner, Melany J.; Smiley, James R.

2009-01-01

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. PMID:19776125

Low-tube voltage 100 kVp MDCT in screening of cocaine body packing: image quality and radiation dose compared to 120 kVp MDCT.

PubMed

Aissa, Joel; Rubbert, Christian; Boos, Johannes; Schleich, Christoph; Thomas, Christoph; Kröpil, Patric; Antoch, Gerald; Miese, Falk

2015-10-01

The aim of this study was to evaluate the impact of a reduced tube potential (100 kVp) for non-enhanced abdominal low-dose CT on radiation dose and image quality (IQ) in the detection of body packing. This retrospective study was approved by the local research ethics committee of our clinic. From March 2012 to July 2014, 99 subjects were referred to our institute with suspected body packing. 50 CT scans were performed using a 120 kVp protocol (group A), and 49 CTs were performed using a low-dose protocol with a tube voltage of 100 kVp (group B). Subjective and objective IQ were assessed. DLP and CTDIvol were analyzed. All examinations were of diagnostic IQ. Objective IQ was not significantly different between the 120 kVp and 100 kVp protocol. Mean density of solid and liquid body packets was 210 ± 60.2 HU at 120 kVp and 250.6 ± 29.7 HU at 100 kVp. Radiation dose was significantly lower in group B as compared to group A (p < 0.05). In group A, body packs were detected in 16 (32%) of the 50 patients. In group B, packets were observed in 15 (31%) of 49 patients. Laboratory analysis detected cocaine in all smuggled body packs. Low-tube voltage 100 kVp MDCT with automated tube current modulation in screening of illegal drugs leads to a diagnostic IQ and significant dose reduction compared to 120 kVp low-tube voltage protocols. Despite lower radiation dose, liquid and solid cocaine containers retain high attenuation and are easily detected.

Overexpression of VP, a vacuolar H+-pyrophosphatase gene in wheat (Triticum aestivum L.), improves tobacco plant growth under Pi and N deprivation, high salinity, and drought.

PubMed

Li, Xiaojuan; Guo, Chengjin; Gu, Juntao; Duan, Weiwei; Zhao, Miao; Ma, Chunying; Du, Xiaoming; Lu, Wenjing; Xiao, Kai

2014-02-01

Establishing crop cultivars with strong tolerance to P and N deprivation, high salinity, and drought is an effective way to improve crop yield and promote sustainable agriculture worldwide. A vacuolar H+-pyrophosphatase (V-H+-PPase) gene in wheat (TaVP) was functionally characterized in this study. TaVP cDNA is 2586-bp long and encodes a 775-amino-acid polypeptide that contains 10 conserved membrane-spanning domains. Transcription of TaVP was upregulated by inorganic phosphate (Pi) and N deprivation, high salinity, and drought. Transgene analysis revealed that TaVP overexpression improved plant growth under normal conditions and specifically under Pi and N deprivation stresses, high salinity, and drought. The improvement of growth of the transgenic plants was found to be closely related to elevated V-H+-PPase activities in their tonoplasts and enlarged root systems, which possibly resulted from elevated expression of auxin transport-associated genes. TaVP-overexpressing plants showed high dry mass, photosynthetic efficiencies, antioxidant enzyme activities, and P, N, and soluble carbohydrate concentrations under various growth conditions, particularly under the stress conditions. The transcription of phosphate and nitrate transporter genes was not altered in TaVP-overexpressing plants compared with the wild type, suggesting that high P and N concentrations regulated by TaVP were caused by increased root absorption area instead of alteration of Pi and NO3- acquisition kinetics. TaVP is important in the tolerance of multiple stresses and can serve as a useful genetic resource to improve plant P- and N-use efficiencies and to increase tolerance to high salinity and drought.

Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

PubMed

Marín-López, Alejandro; Ortego, Javier

2016-01-01

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Juan; Department of Microbiology and Immunology, Nanjing Medical University; Wang, Shixia

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 71more » (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.« less

Expression of the VP40 antigen from the Zaire ebolavirus in tobacco plants.

PubMed

Monreal-Escalante, Elizabeth; Ramos-Vega, Abel A; Salazar-González, Jorge A; Bañuelos-Hernández, Bernardo; Angulo, Carlos; Rosales-Mendoza, Sergio

2017-07-01

The plant cell is able to produce the VP40 antigen from the Zaire ebolavirus , retaining the antigenicity and the ability to induce immune responses in BALB/c mice. The recent Ebola outbreak evidenced the need for having vaccines approved for human use. Herein we report the expression of the VP40 antigen from the Ebola virus as an initial effort in the development of a plant-made vaccine that could offer the advantages of being cheap and scalable, which is proposed to overcome the rapid need for having vaccines to deal with future outbreaks. Tobacco plants were transformed by stable DNA integration into the nuclear genome using the CaMV35S promoter and a signal peptide to access the endoplasmic reticulum, reaching accumulation levels up to 2.6 µg g -1 FW leaf tissues. The antigenicity of the plant-made VP40 antigen was evidenced by Western blot and an initial immunogenicity assessment in test animals that revealed the induction of immune responses in BALB/c mice following three weekly oral or subcutaneous immunizations at very low doses (125 and 25 ng, respectively) without accessory adjuvants. Therefore, this plant-based vaccination prototype is proposed as an attractive platform for the production of vaccines in the fight against Ebola virus disease outbreaks.

Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells.

PubMed

Zhu, Yali; Cherukuri, Nil Celebi; Jackel, Jamie N; Wu, Zetang; Crary, Monica; Buckley, Kenneth J; Bisaro, David M; Parris, Deborah S

2012-03-01

Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

Long-term archives of land surface albedo products through the EUMETSAT/LSA-SAF and ECMWF/C3S projects: status and project development plan

NASA Astrophysics Data System (ADS)

Carrer, D.; Pinault, F.; Ceamanos, X.; Meurey, C.; Moparthy, S.; Swinnen, E.; Trigo, I.

2017-12-01

The two space programs of EUMETSAT (project CDOP3, LSA-SAF) and ECMWF (the Copernicus Climate Change Service; C3S_312a Lot9) provide (or will provide) added-value satellite products for the meteorological and environmental science communities, especially in the fields of climate modeling, environmental management, natural hazards management, and climate change detection. The EUMETSAT/LSA-SAF project started in 1999 with research and development activities. The Third Continuous Development and Operations Phase (CDOP-3) starts in March 2017 and will end in 2022. This project uses instruments on board European satellites that were, or will be, launched between 2004 and 2022. Unlike the LSA-SAF, the COPERNICUS/C3S_312a project has no NRT constraint. Its first phase started in november 2016. One of the major objective of the COPERNICUS/C3S_312a project is to harmonize datasets from various sensors in order to provide consistent and continuous ECV products from the 80's until now.Presently, the delivered operational products comprise several surface albedo products using data from various space missions (METEOSAT, NOAA, METOP, …). We present here the portfolio of the surface albedo products that are disseminated with an operational status. Their characteristics and accuracy are detailed here after. Also we will present the development plan to produce long-term re-analysis and to prepare the arrival of the next generation of satellite (MTG, EPS-SG, ...). This work will lead in 2018 to 40 years of products characterizing the albedo properties of the surface. These programs provide a great opportunity to monitor and identify human-induced climate change since consistent production of data sets is guaranteed until at least 2022.

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

PubMed

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.

The C3PO project: a laser communication system concept for small satellites

NASA Astrophysics Data System (ADS)

d'Humières, Benoît; Esmiller, Bruno; Gouy, Yann; Steck, Emilie; Quintana, Crisanto; Faulkner, Graham; O'Brien, Dominic; Sproll, Fabian; Wagner, Paul; Hampf, Daniel; Riede, Wolfgang; Salter, Michael; Wang, Qin; Platt, Duncan; Jakonis, Darius; Piao, Xiaoyu; Karlsson, Mikael; Oberg, Olof; Petermann, Ingemar; Michalkiewicz, Aneta; Krezel, Jerzy; Debowska, Anna; Thueux, Yoann

2017-02-01

The satellite market is shifting towards smaller (micro and nanosatellites), lowered mass and increased performance platforms. Nanosatellites and picosatellites have been used for a number of new, innovative and unique payloads and missions. This trend requires new concepts for a reduced size, a better performance/weight ratio and a reduction of onboard power consumption. In this context, disruptive technologies, such as laser-optical communication systems, are opening new possibilities. This paper presents the C3PO1 system, "advanced Concept for laser uplink/ downlink CommuniCation with sPace Objects", and the first results of the development of its key technologies. This project targets the design of a communications system that uses a ground-based laser to illuminate a satellite, and a Modulating Retro-Reflector (MRR) to return a beam of light modulated by data to the ground. This enables a downlink, without a laser source on the satellite. This architecture suits well to small satellite applications so as high data rates are potentially provided with very low board mass. C3PO project aims to achieve data rates of 1Gbit/s between LEO satellites and Earth with a communication payload mass of less than 1kilogram. In this paper, results of the initial experiments and demonstration of the key technologies will be shown.

Sequence analysis of porcine kobuvirus VP1 region detected in pigs in Japan and Thailand.

PubMed

Okitsu, Shoko; Khamrin, Pattara; Thongprachum, Aksara; Hidaka, Satoshi; Kongkaew, Sompreeya; Kongkaew, Apisek; Maneekarn, Niwat; Mizuguchi, Masashi; Hayakawa, Satoshi; Ushijima, Hiroshi

2012-04-01

Porcine kobuvirus is a new candidate species of the genus Kobuvirus in the family Picornaviridae, and information is still limited. The identification of porcine kobuvirus has been performed by the sequence analyses of the 3D region of the viruses. Therefore, the purpose of this study was to characterize the molecular properties of VP1 nucleotide sequences of the porcine kobuviruses isolated from porcine stool samples in Japan during 2009 and Thailand between 2006 and 2008. In addition, previous identification of a unique porcine kobuvirus; Japanese H023/2009/JP, which is a bovine kobuvirus-like strain based on sequence analysis of the 3D region, was also included in this study. All of the strains were amplified by the VP1-specific primer pair: the amplicons were subjected to direct sequencing and compared with the VP1 nucleotide sequences of reference strains. The VP1 sequences of strains from the GenBank database revealed high nucleotide sequence identity at 84.3-100%. On the other hand, the nucleotide identities among the 15 porcine kobuvirus strains analyzed in this study ranged from 78.8 to 99.8%. The results revealed that diversity of the strains in this study were higher than those of the strains in previous studies. Furthermore, it was found that the VP1 region of the bovine kobuvirus-like strain, H023/2009/JP, clustered with nine porcine kobuvirus strains that were isolated in Thailand and Japan. Since this strain was previously found to be closely related to bovine kobuviruses in the 3D gene region, it may be a natural recombinant.

Insights into the homo-oligomerization properties of N-terminal coiled-coil domain of Ebola virus VP35 protein.

PubMed

Ramaswamy, Venkata Krishnan; Di Palma, Francesco; Vargiu, Attilio V; Corona, Angela; Piano, Dario; Ruggerone, Paolo; Zinzula, Luca; Tramontano, Enzo

2018-03-02

The multifunctional Ebola virus (EBOV) VP35 protein is a key determinant of virulence. VP35 is essential for EBOV replication, is a component of the viral RNA polymerase and participates in nucleocapsid formation. Furthermore, VP35 contributes to EBOV evasion of the host innate immune response by suppressing RNA silencing and blocking RIG-I like receptors' pathways that lead to type I interferon (IFN) production. VP35 homo-oligomerization has been reported to be critical for its replicative function and to increase its IFN-antagonism properties. Moreover, homo-oligomerization is mediated by a predicted coiled-coil (CC) domain located within its N-terminal region. Here we report the homo-oligomerization profile of full-length recombinant EBOV VP35 (rVP35) assessed by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Based on our biochemical results and in agreement with previous experimental observations, we have built an in silico 3D model of the so-far structurally unsolved EBOV VP35 CC domain and performed self-assembly homo-oligomerization simulations by means of molecular dynamics. Our model advances the understanding of how VP35 may associate in different homo-oligomeric species, a crucial process for EBOV replication and pathogenicity. Copyright © 2018 Elsevier B.V. All rights reserved.

Unenhanced third-generation dual-source chest CT using a tin filter for spectral shaping at 100kVp.

PubMed

Haubenreisser, Holger; Meyer, Mathias; Sudarski, Sonja; Allmendinger, Thomas; Schoenberg, Stefan O; Henzler, Thomas

2015-08-01

To prospectively investigate image quality and radiation dose of 100kVp spectral shaping chest CT using a dedicated tin filter on a 3rd generation dual-source CT (DSCT) in comparison to standard 100kVp chest CT. Sixty patients referred for a non-contrast chest on a 3rd generation DSCT were prospectively included and examined at 100kVp with a dedicated tin filter. These patients were retrospectively matched with patients that were examined on a 2nd generation DSCT at 100kVp without tin filter. Objective and subjective image quality was assessed in various anatomic regions and radiation dose was compared. Radiation dose was decreased by 90% using the tin filter (3.0 vs 0.32mSv). Soft tissue attenuation and image noise was not statistically different for both examination techniques (p>0.05), however image noise was found to be significantly higher in the trachea when using the additional tin filter (p=0.002). SNR was found to be statistically similar in pulmonary tissue, significantly lower when measured in air and significantly higher in the aorta for the scans on the 3rd generation DSCT. Subjective image quality with regard to overall quality and image noise and sharpness was not statistically significantly different (p>0.05). 100kVp spectral shaping chest CT by means of a tube-based tin-filter on a 3rd generation DSCT allows 90% dose reduction when compared to 100kVp chest CT on a 2nd generation DSCT without spectral shaping. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

VP7 and VP8* genetic characterization of group A rotavirus genotype G12P[8]: Emergence and spreading in the Eastern Brazilian coast in 2014.

PubMed

da Silva, Marcelle Figueira Marques; Fumian, Tulio Machado; de Assis, Rosane Maria Santos; Fialho, Alexandre Madi; Carvalho-Costa, Filipe Anibal; da Silva Ribeiro de Andrade, Juliana; Leite, José Paulo Gagliardi

2017-01-01

Group A rotavirus (RVA) genotype G12 is habitually associated with diarrhea disease (DD) in African children and recently its detection has increased worldwide. A total of 970 stool samples collected from individuals with DD in the Northeastern, Southeastern, and Southern Brazilian regions, Eastern coast, were analyzed and 321 (33%) were positive for RVA and of these, 241 (75%) genotyped as G12P[8]. The rate of RVA positivity was higher among children aged 5-10 years old (60%). All RVA infections observed in adults aged >21 years were G12P[8] (n = 27) showing that this genotype affected older age groups during the year of 2014 in Brazil. Phylogenetic analysis of VP7 and VP8* G12P[8] strains demonstrated an elevated similarity among Brazilian and G12-III prototypes strains circulating worldwide recently, suggesting that this lineage is associated with the global spread of the G12 genotype, considered as the 6th most prevalent human RVA genotype nowadays; while other G12 lineages remain sporadically detected and usually detected in association with other P genotypes. VP8* analysis revealed that Brazilian strains belong to P[8]-3 lineage, the single P[8] lineage presently detected in the country. No major nucleotide/amino acid disparities were observed among strains recovered from children and adults for VP7 and VP8* genes. These data are essential to support the surveillance studies, particularly in countries where the RVA vaccine was introduced in their National Immunization Program enabling identification of potential alterations in the epidemiological profile that can impact its efficacy in vaccination programs. J. Med. Virol. 89:64-70, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

PubMed

Panjwani, Anusha; Strauss, Mike; Gold, Sarah; Wenham, Hannah; Jackson, Terry; Chou, James J; Rowlands, David J; Stonehouse, Nicola J; Hogle, James M; Tuthill, Tobias J

2014-08-01

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

The Role of Exosomal VP40 in Ebola Virus Disease.

PubMed

Pleet, Michelle L; DeMarino, Catherine; Lepene, Benjamin; Aman, M Javad; Kashanchi, Fatah

2017-04-01

Ebola virus (EBOV) can cause a devastating hemorrhagic disease, leading to death in a short period of time. After infection, the resulting EBOV disease results in high levels of circulating cytokines, endothelial dysfunction, coagulopathy, and bystander lymphocyte apoptosis in humans and nonhuman primates. The VP40 matrix protein of EBOV is essential for viral assembly and budding from the host cell. Recent data have shown that VP40 exists in the extracellular environment, including in exosomes, and exosomal VP40 can impact the viability of recipient immune cells, including myeloid and T cells, through the regulation of the RNAi and endosomal sorting complexes required for transport pathways. In this study, we discuss the latest findings of the impact of exosomal VP40 on immune cells in vitro and its potential implications for pathogenesis in vivo.

Development and Validation of a Project Package for Junior Secondary School Basic Science

ERIC Educational Resources Information Center

Udofia, Nsikak-Abasi

2014-01-01

This was a Research and Developmental study designed to develop and validate projects for Junior Secondary School Basic Science instruction and evaluation. The projects were developed using the project blueprint and sent for validation by experts in science education and measurement and evaluation; using a project validation scale. They were to…

Interaction of monomeric Ebola VP40 protein with a plasma membrane: A coarse-grained molecular dynamics (CGMD) simulation study.

PubMed

Mohamad Yusoff, Mohamad Ariff; Abdul Hamid, Azzmer Azzar; Mohammad Bunori, Noraslinda; Abd Halim, Khairul Bariyyah

2018-06-01

Ebola virus is a lipid-enveloped filamentous virus that affects human and non-human primates and consists of several types of protein: nucleoprotein, VP30, VP35, L protein, VP40, VP24, and transmembrane glycoprotein. Among the Ebola virus proteins, its matrix protein VP40 is abundantly expressed during infection and plays a number of critical roles in oligomerization, budding and egress from the host cell. VP40 exists predominantly as a monomer at the inner leaflet of the plasma membrane, and has been suggested to interact with negatively charged lipids such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylserine (PS) via its cationic patch. The hydrophobic loop at the C-terminal domain has also been shown to be important in the interaction between the VP40 and the membrane. However, details of the molecular mechanisms underpinning their interactions are not fully understood. This study aimed at investigating the effects of mutation in the cationic patch and hydrophobic loop on the interaction between the VP40 monomer and the plasma membrane using coarse-grained molecular dynamics simulation (CGMD). Our simulations revealed that the interaction between VP40 and the plasma membrane is mediated by the cationic patch residues. This led to the clustering of PIP 2 around the protein in the inner leaflet as a result of interactions between some cationic residues including R52, K127, K221, K224, K225, K256, K270, K274, K275 and K279 and PIP 2 lipids via electrostatic interactions. Mutation of the cationic patch or hydrophobic loop amino acids caused the protein to bind at the inner leaflet of the plasma membrane in a different orientation, where no significant clustering of PIP 2 was observed around the mutated protein. This study provides basic understanding of the interaction of the VP40 monomer and its mutants with the plasma membrane. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural and Functional Characterization of Reston Ebola Virus VP35 Interferon Inhibitory Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Daisy W.; Shabman, Reed S.; Farahbakhsh, Mina

2010-09-21

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address thismore » question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-{angstrom} crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 {angstrom}, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for

Structural and functional characterization of Reston Ebola virus VP35 interferon inhibitory domain.

PubMed

Leung, Daisy W; Shabman, Reed S; Farahbakhsh, Mina; Prins, Kathleen C; Borek, Dominika M; Wang, Tianjiao; Mühlberger, Elke; Basler, Christopher F; Amarasinghe, Gaya K

2010-06-11

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-A crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled

Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression.

PubMed

Oda, Shun-Ichiro; Noda, Takeshi; Wijesinghe, Kaveesha J; Halfmann, Peter; Bornholdt, Zachary A; Abelson, Dafna M; Armbrust, Tammy; Stahelin, Robert V; Kawaoka, Yoshihiro; Saphire, Erica Ollmann

2016-02-15

Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

PubMed

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

2011-12-01

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)→Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

Complete Statistical Survey Results of 1982 Texas Competency Validation Project.

ERIC Educational Resources Information Center

Rogers, Sandra K.; Dahlberg, Maurine F.

This report documents a project to develop current statewide validated competencies for auto mechanics, diesel mechanics, welding, office occupations, and printing. Section 1 describes the four steps used in the current competency validation project and provides a standardized process for conducting future studies at the local or statewide level.…

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro

PubMed Central

van de Water, Sandra G. P.; Potgieter, Christiaan A.; van Rijn, Piet A.

2016-01-01

been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro. Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses. PMID:27903804

Vaccination with multimeric recombinant VP28 induces high protection against white spot syndrome virus in shrimp.

PubMed

Taengchaiyaphum, Suparat; Nakayama, Hideki; Srisala, Jiraporn; Khiev, Ratny; Aldama-Cano, Diva January; Thitamadee, Siripong; Sritunyalucksana, Kallaya

2017-11-01

To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28. Copyright © 2017 Elsevier Ltd. All rights reserved.

Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

NASA Astrophysics Data System (ADS)

Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

2016-11-01

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

Rotavirus diarrhea severity is related to the VP4 type in Mexican children.

PubMed

Mota-Hernández, Felipe; Calva, Juan José; Gutiérrez-Camacho, Claudia; Villa-Contreras, Sofía; Arias, Carlos F; Padilla-Noriega, Luis; Guiscafré-Gallardo, Héctor; de Lourdes Guerrero, María; López, Susana; Muñoz, Onofre; Contreras, Juan F; Cedillo, Roberto; Herrera, Ismael; Puerto, Fernando I

2003-07-01

This report is of a community-based case control study to assess whether the severity of acute diarrhea by rotavirus (RV) in young children is associated with a particular VP7 (G) or VP4 (P) RV serotype. Five hundred twenty children younger than 2 years of age with diarrhea lasting less than 3 days were age and gender matched with 520 children with no diarrhea. The G and P serotypes were determined with specific monoclonal antibodies, and the VP4 serotype specificity in a subgroup was confirmed by genotyping. Infection with a G3 serotype led to a higher risk of diarrhea than infection with a G1 serotype. Infection with a G3-nontypeable-P serotype was associated with more severe gastroenteritis than infection with a G3 (or G1) P1A[8] serotype. A child with diarrhea-associated dehydration was almost five times more likely to be infected with a G3-nontypeable-P serotype than a child without dehydration (P < 0.001). Moreover, the two predominant monotypes within serotype P1A[8] had significantly different clinical manifestations. In this study, the severity of RV-associated diarrhea was related to different P serotypes rather than to G serotypes. The relationship between serotype and clinical outcomes seems to be complex and to vary among different geographic areas.

Rotavirus Diarrhea Severity Is Related to the VP4 Type in Mexican Children

PubMed Central

Mota-Hernández, Felipe; José Calva, Juan; Gutiérrez-Camacho, Claudia; Villa-Contreras, Sofía; Arias, Carlos F.; Padilla-Noriega, Luis; Guiscafré-Gallardo, Héctor; Guerrero, María de Lourdes; López, Susana; Muñoz, Onofre; Contreras, Juan F.; Cedillo, Roberto; Herrera, Ismael; Puerto, Fernando I.

2003-01-01

This report is of a community-based case control study to assess whether the severity of acute diarrhea by rotavirus (RV) in young children is associated with a particular VP7 (G) or VP4 (P) RV serotype. Five hundred twenty children younger than 2 years of age with diarrhea lasting less than 3 days were age and gender matched with 520 children with no diarrhea. The G and P serotypes were determined with specific monoclonal antibodies, and the VP4 serotype specificity in a subgroup was confirmed by genotyping. Infection with a G3 serotype led to a higher risk of diarrhea than infection with a G1 serotype. Infection with a G3-nontypeable-P serotype was associated with more severe gastroenteritis than infection with a G3 (or G1) P1A[8] serotype. A child with diarrhea-associated dehydration was almost five times more likely to be infected with a G3-nontypeable-P serotype than a child without dehydration (P < 0.001). Moreover, the two predominant monotypes within serotype P1A[8] had significantly different clinical manifestations. In this study, the severity of RV-associated diarrhea was related to different P serotypes rather than to G serotypes. The relationship between serotype and clinical outcomes seems to be complex and to vary among different geographic areas. PMID:12843057

Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393.

PubMed

Yin, Ji-Yuan; Guo, Chao-Qun; Wang, Zi; Yu, Mei-Ling; Gao, Shuai; Bukhari, Syed M; Tang, Li-Jie; Xu, Yi-Gang; Li, Yi-Jing

2016-11-01

Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.

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.

LANL C10.2 Projects in FY13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Batha, Steven H.; Fincke, James R.; Schmitt, Mark J.

2012-06-07

LANL has two projects in C10.2: Defect-Induced Mix Experiment (DIME) (ongoing, several runs at Omega; NIF shots this summer); and Shock/Shear (tested at Omega for two years; NIF shots in second half of FY13). Each project is jointly funded by C10.2, other C10 MTEs, and Science Campaigns. DIME is investigating 4{pi} and feature-induced mix in spherically convergent ICF implosions by using imaging of the mix layer. DIME prepared for NIF by demonstrating its PDD mix platform on Omega including imaging mid-Z doped layers and defects. DIME in FY13 will focus on PDD symmetry-dependent mix and moving burn into the mixmore » region for validation of mix/burn models. Re-Shock and Shear are two laser-driven experiments designed to study the turbulent mixing of materials. In FY-2012 43 shear and re-shock experimental shots were executed on the OMEGA laser and a complete time history obtained for both. The FY-2013 goal is to transition the experiment to NIF where the larger scale will provide a longer time period for mix layer growth.« less

A conserved carboxy-terminal domain in the major tegument structural protein VP22 facilitates virion packaging of a chimeric protein during productive herpes simplex virus 1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlegel, Elisabeth F.M.; Blaho, John A., E-mail: john.blaho@mssm.ed

2009-05-10

Recombinant virus HSV-1(RF177) was previously generated to examine tegument protein VP22 function by inserting the GFP gene into the gene encoding VP22. During a detailed analysis of this virus, we discovered that RF177 produces a novel fusion protein between the last 15 amino acids of VP22 and GFP, termed GCT-VP22. Thus, the VP22 carboxy-terminal specific antibody 22-3 and two anti-GFP antibodies reacted with an approximately 28 kDa protein from RF177-infected Vero cells. GCT-VP22 was detected at 1 and 3 hpi. Examination of purified virions indicated that GCT-VP22 was incorporated into RF177 virus particles. These observations imply that at least amore » portion of the information required for virion targeting is located in this domain of VP22. Indirect immunofluorescence analyses showed that GCT-VP22 also localized to areas of marginalized chromatin during RF177 infection. These results indicate that the last fifteen amino acids of VP22 participate in virion targeting during HSV-1 infection.« less

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

PubMed Central

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. PMID:26173145

Type-specific and cross-reactive antibodies and T cell responses in norovirus VLP immunized mice are targeted both to conserved and variable domains of capsid VP1 protein.

PubMed

Malm, Maria; Tamminen, Kirsi; Vesikari, Timo; Blazevic, Vesna

2016-10-01

Norovirus (NoV)-specific antibodies, which block binding of the virus-like particles (VLPs) to the cell receptors are conformation dependent and directed towards the most exposed domain of the NoV capsid VP1 protein, the P2 domain. Limited data are available on the antibodies directed to other domains of the VP1, and even less on the NoV VP1-specific T cell epitopes. In here, BALB/c mice were immunized with six VLPs derived from NoV GII.4-1999, GII.4-2009 (New Orleans), GII.4-2012 (Sydney), GII.12, GI.1, and G1.3. Serum immunoglobulin G binding antibodies, histo-blood group antigen blocking antibodies and T cell responses using type-specific and heterologous NoV VLPs, P-dimers and 76 overlapping synthetic peptides, spanning the entire 539 amino acid sequence of GII.4 VP1, were determined. The results showed that at least half of the total antibody content is directed towards conserved S domain of the VP1. Only a small fraction (<1%) of the VP1 binding antibodies were blocking/neutralizing. With the use of matrix peptide pools and individual peptides, seven CD4 + and CD8 + T cell restricted epitopes were mapped, two located in S domain, four in P2 domain and one in P1 domain of NoV VP1. The epitopes were GII.4 strain-specific but also common GII.4 genotype-specific T cell epitopes were identified. More importantly, the results suggest a 9-amino acids long sequence ( 318 PAPLGTPDF 326 ) in P2 domain of VP1 as a universal NoV genogroup II-specific CD8 + T cell epitope. Distribution of the T cell epitopes alongside the capsid VP1 indicates the need of the complete protein for high immunogenicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rhesus rotavirus VP6 regulates ERK-dependent calcium influx in cholangiocytes.

PubMed

Lobeck, Inna; Donnelly, Bryan; Dupree, Phylicia; Mahe, Maxime M; McNeal, Monica; Mohanty, Sujit K; Tiao, Greg

2016-12-01

The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical composition of the continental crust: Insights from a quantitative interpretation of the Vp/Vs ratio

NASA Astrophysics Data System (ADS)

Guerri, M.; Youssof, M.; Fullea, J.

2017-12-01

The processes driving continental crust formation are not yet fully understood. One of the fundamental keys necessary to investigate the enigma is represented by crustal composition. The Vp/Vs ratio from seismic receiver functions or tomography studies is a powerful tool to constrain the crustal composition. However, to date only qualitative relationships between Vp/Vs and composition have been proposed. We present a quantitative interpretation of the Vp/Vs in terms of major oxide components, based on thermo-elastic constrained modelling of rock phase equilibria and physical properties. The geophysical-petrological approach is implemented in the new release of the software package LitMod, which now allows for integrated and self-consistent modeling of the entire lithosphere (crust + lithospheric mantle) and upper mantle. Forward modelling of the Vp/Vs, based on petrology and thermodynamics, reveals that, as expected, mafic compositions have higher Vp/Vs than felsic ones. However, in high temperature settings (surface heat flow > 75 mW/m2), the quartz alpha / quartz beta transition strongly increases Vp, leaving Vs almost unaltered, leading to SiO2-rich compositions displaying Vp/Vs values higher than those associated with mafic compositions. Additionally, we highlight the importance of H2O, the presence of which stabilizes amphibole (in place of pyroxene), characterized by a relatively low Vp/Vs. If H2O is present, mafic compositions show Vp/Vs ratios that are comparable to those produced by anhydrous SiO2-rich compositions. The destabilization of amphibole (in favour of pyroxene) generates a sharp seismic discontinuity, potentially detectable by, for example, seismic refraction and receiver function investigations. We invert the Vp/Vs ratio for composition and hydrous state of the crust in the Southern African cratons. Our results show that the Kaapvaal craton, Archean in age, has an intermediate (SiO2 60 wt%) composition. The finding has implications on our

System and method for investigating sub-surface features and 3D imaging of non-linear property, compressional velocity VP, shear velocity VS and velocity ratio VP/VS of a rock formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vu, Cung Khac; Skelt, Christopher; Nihei, Kurt

A system and a method for generating a three-dimensional image of a rock formation, compressional velocity VP, shear velocity VS and velocity ratio VP/VS of a rock formation are provided. A first acoustic signal includes a first plurality of pulses. A second acoustic signal from a second source includes a second plurality of pulses. A detected signal returning to the borehole includes a signal generated by a non-linear mixing process from the first and second acoustic signals in a non-linear mixing zone within an intersection volume. The received signal is processed to extract the signal over noise and/or signals resultingmore » from linear interaction and the three dimensional image of is generated.« less

Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pleet, Michelle L.; Mathiesen, Allison; DeMarino, Catherine

Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80–90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, wemore » examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. In addition, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the

Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

DOE PAGES

Pleet, Michelle L.; Mathiesen, Allison; DeMarino, Catherine; ...

2016-11-07

Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80–90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, wemore » examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. In addition, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the

Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction.

PubMed

Pleet, Michelle L; Mathiesen, Allison; DeMarino, Catherine; Akpamagbo, Yao A; Barclay, Robert A; Schwab, Angela; Iordanskiy, Sergey; Sampey, Gavin C; Lepene, Benjamin; Nekhai, Sergei; Aman, M J; Kashanchi, Fatah

2016-01-01

Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80-90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, we examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. Additionally, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the deregulation

Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.

2009-09-01

White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 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 thatmore » rVP28 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 rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.« less

The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

PubMed

Adu-Gyamfi, Emmanuel; Soni, Smita P; Xue, Yi; Digman, Michelle A; Gratton, Enrico; Stahelin, Robert V

2013-02-22

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

Phylogenetic analysis of VP2 gene of canine parvovirus and comparison with Indian and world isolates.

PubMed

Kaur, G; Chandra, M; Dwivedi, P N

2016-03-01

Canine parvovirus (CPV) causes hemorrhagic enteritis, especially in young dogs, leading to high morbidity and mortality. It has four main antigenic types CPV-2, CPV-2a, CPV-2b and CPV-2c. Virus protein 2 (VP2) is the main capsid protein and mutations affecting VP2 gene are responsible for the evolution of various antigenic types of CPV. Full length VP2 gene from field isolates was amplified and cloned for sequence analysis. The sequences were submitted to the GenBank and were assigned Acc. Nos., viz. KP406928.1 for P12, KP406927.1 for P15, KP406930.1 for P32, KP406926.1 for Megavac-6 and KP406929.1 for NobivacDHPPi. Phylogenetic analysis indicated that the samples were forming a separate clad with vaccine strains. When the samples were compared with the world and Indian isolates, it was observed that samples formed a separate node indicating regional genetic variation in CPV.

Characterization and protective efficacy in an animal model of a novel truncated rotavirus VP8 subunit parenteral vaccine candidate.

PubMed

Xue, Miaoge; Yu, Linqi; Che, Yaojian; Lin, Haijun; Zeng, Yuanjun; Fang, Mujin; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

2015-05-21

The cell-attachment protein VP8* of rotavirus is a potential candidate parenteral vaccine. However, the yield of full-length VP8 protein (VP8*, residues 1-231) expressed in Escherichia coli was low, and a truncated VP8 protein (ΔVP8*, residues 65-231) cannot elicit efficient protective immunity in a mouse model. In this study, tow novel truncated VP8 proteins, VP8-1 (residues 26-231) and VP8-2 (residues 51-231), were expressed in E. coli and evaluated for immunogenicity and protective efficacy, compared with VP8* and ΔVP8*. As well as ΔVP8*, the protein VP8-1 and VP8-2 were successfully expressed in high yield and purified in homogeneous dimeric forms, while the protein VP8* was expressed with lower yield and prone to aggregation and degradation in solution. Although the immunogenicity of the protein VP8*, VP8-1, VP8-2 and ΔVP8* was comparable, immunization of VP8* and VP8-1 elicited significantly higher neutralizing antibody titers than that of VP8-2 and ΔVP8* in mice. Furthermore, when assessed using a mouse maternal antibody model, the efficacy of VP8-1 to protect against rotavirus-induced diarrhea in pups was comparable to that of VP8*, both were dramatically higher than that of VP8-2 and ΔVP8*. Taken together, the novel truncated protein VP8-1, with increased yield, improved homogeneity and high protective efficacy, is a viable candidate for further development of a parenterally administrated prophylactic vaccine against rotavirus infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Daisy W.; Prins, Kathleen C.; Borek, Dominika M.

2010-03-12

Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also importantmore » for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.« less

Liver fat quantification using fast kVp-switching dual energy CT

NASA Astrophysics Data System (ADS)

Kriston, Andras; Mendonça, Paulo; Silva, Alvin; Paden, Robert G.; Pavlicek, William; Sahani, Dushyant; Janos Kis, Benedek; Rusko, Laszlo; Okerlund, Darin; Bhotika, Rahul

2011-03-01

Nonalcoholic steatohepatitis (NASH) is a liver disease that occurs in patients that lack a history of the well-proven association of alcohol use. A major symptom of NASH is increased fat deposition in the liver. Gemstone Spectral Imaging (GSI) with fast kVp-switching enables projection-based material decomposition, offering the opportunity to accurately characterize tissue types, e.g., fat and healthy liver tissue, based on their energy-sensitive material attenuation and density. We describe our pilot efforts to apply GSI to locate and quantify the amount of fat deposition in the liver. Two approaches are presented, one that computes percentage fat from the difference in HU values at high and low energies and the second based on directly computing fat volume fraction at each voxel using multi-material decomposition. Simulation software was used to create a phantom with a set of concentric rings, each composed of fat and soft tissue in different relative amounts with attenuation values obtained from the National Institute of Standards and Technology. Monte Carlo 80 and 140 kVp X-ray projections were acquired and CT images of the phantom were reconstructed. Results demonstrated the sensitivity of dual energy CT to the presence of fat and its ability to distinguish fat from soft tissue. Additionally, actual patient (liver) datasets were acquired using GSI and monochromatic images at 70 and 140 keV were reconstructed. Preliminary results demonstrate a tissue sensitivity that appears sufficient to quantify fat content with a degree of accuracy as may be needed for non-invasive clinical assessment of NASH.

Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

PubMed

Biswal, Jitendra K; Das, Biswajit; Sharma, Gaurav K; Khulape, Sagar A; Pattnaik, Bramhadev

2016-04-01

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142 → R or H142 → F or H142 → A substitutions resulted in non-infectious FMDV, H142 → D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A.

Flight Research and Validation Formerly Experimental Capabilities Supersonic Project

NASA Technical Reports Server (NTRS)

Banks, Daniel

2009-01-01

This slide presentation reviews the work of the Experimental Capabilities Supersonic project, that is being reorganized into Flight Research and Validation. The work of Experimental Capabilities Project in FY '09 is reviewed, and the specific centers that is assigned to do the work is given. The portfolio of the newly formed Flight Research and Validation (FRV) group is also reviewed. The various projects for FY '10 for the FRV are detailed. These projects include: Eagle Probe, Channeled Centerbody Inlet Experiment (CCIE), Supersonic Boundary layer Transition test (SBLT), Aero-elastic Test Wing-2 (ATW-2), G-V External Vision Systems (G5 XVS), Air-to-Air Schlieren (A2A), In Flight Background Oriented Schlieren (BOS), Dynamic Inertia Measurement Technique (DIM), and Advanced In-Flight IR Thermography (AIR-T).

Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luthra, Priya; Jordan, David S.; Leung, Daisy W.

2015-03-04

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

The AgMIP Coordinated Climate-Crop Modeling Project (C3MP): Methods and Protocols

NASA Technical Reports Server (NTRS)

Shukla, Sonali P.; Ruane, Alexander Clark

2014-01-01

Climate change is expected to alter a multitude of factors important to agricultural systems, including pests, diseases, weeds, extreme climate events, water resources, soil degradation, and socio-economic pressures. Changes to carbon dioxide concentration ([CO2]), temperature, and water (CTW) will be the primary drivers of change in crop growth and agricultural systems. Therefore, establishing the CTW-change sensitivity of crop yields is an urgent research need and warrants diverse methods of investigation. Crop models provide a biophysical, process-based tool to investigate crop responses across varying environmental conditions and farm management techniques, and have been applied in climate impact assessment by using a variety of methods (White et al., 2011, and references therein). However, there is a significant amount of divergence between various crop models' responses to CTW changes (Rotter et al., 2011). While the application of a site-based crop model is relatively simple, the coordination of such agricultural impact assessments on larger scales requires consistent and timely contributions from a large number of crop modelers, each time a new global climate model (GCM) scenario or downscaling technique is created. A coordinated, global effort to rapidly examine CTW sensitivity across multiple crops, crop models, and sites is needed to aid model development and enhance the assessment of climate impacts (Deser et al., 2012). To fulfill this need, the Coordinated Climate-Crop Modeling Project (C3MP) (Ruane et al., 2014) was initiated within the Agricultural Model Intercomparison and Improvement Project (AgMIP; Rosenzweig et al., 2013). The submitted results from C3MP Phase 1 (February 15, 2013-December 31, 2013) are currently being analyzed. This chapter serves to present and update the C3MP protocols, discuss the initial participation and general findings, comment on needed adjustments, and describe continued and future development. AgMIP aims to improve

Seismotectonics of the Loma Prieta, California, region determined from three-dimensional Vp, Vp/Vs, and seismicity

USGS Publications Warehouse

Eberhart-Phillips, D.; Michael, A.J.

1998-01-01

Three-dimensional Vp and Vp/Vs velocity models for the Loma Prieta region were developed from the inversion of local travel time data (21,925 P arrivals and 1,116 S arrivals) from earthquakes, refraction shots, and blasts recorded on 1700 stations from the Northern California Seismic Network and numerous portable seismograph deployments. The velocity and density models and microearthquake hypocenters reveal a complex structure that includes a San Andreas fault extending to the base of the seismogenic layer. A body with high Vp extends the length of the rupture and fills the 5 km wide volume between the Loma Prieta mainshock rupture and the San Andreas and Sargent faults. We suggest that this body controls both the pattern of background seismicity on the San Andreas and Sargent faults and the extent of rupture during the mainshock, thus explaining how the background seismicity outlined the along-strike and depth extent of the mainshock rupture on a different fault plane 5 km away. New aftershock focal mechanisms, based on three-dimensional ray tracing through the velocity model, support a heterogeneous postseismic stress field and can not resolve a uniform fault normal compression. The subvertical (or steeply dipping) San Andreas fault and the fault surfaces that ruptured in the 1989 Loma Prieta earthquake are both parts of the San Andreas fault zone and this section of the fault zone does not have a single type of characteristic event.

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

PubMed

Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

1995-11-01

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.

ICH guidance in practice: validated reversed-phase HPLC method for the determination of active mangiferin from extracts of Mangifera indica Linn.

PubMed

Gowda, Nagaraj; Kumar, Pradeep; Panghal, Surender; Rajshree, Mashru

2010-02-01

This study presents the development and validation of a reversed-phase liquid chromatographic method for the determination of mangiferin (MGN) in alcoholic extracts of mangifera indica. A Lichrospher 100 C(18)-ODS (250 x 4.6 mm, 5 microm size) (Merck, Whitehouse Station, NJ) prepacked column and a mobile phase of potassium dihydrogen orthophosphate (0.01M) pH 2.7 +/- 0.2-acetonitrile (15:85, v/v) with the flow rate of 1 mL/min was used. MGN detection was achieved at a wavelength monitored at 254 nm with SPD-M 10A vp PDA detector or SPD 10AD vp UV detector in combination with class LC 10A software. The proposed method was validated as prescribed by International Conference on Harmonization (ICH) with respect to linearity, specificity, accuracy, precision, stability, and quantification. The method validation was realized using alcoholic extracts and raw materials of leaves and barks. All the validation parameters were within the acceptable limits, and the developed analytical method can successfully be applied for MGN determination.

Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA.

PubMed Central

Kirkegaard, K; Nelsen, B

1990-01-01

Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report. Images PMID:2152811

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia.

PubMed

Khaw, Yam Sim; Chan, Yoke Fun; Jafar, Faizatul Lela; Othman, Norlijah; Chee, Hui Yee

2016-01-01

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5' and 3' non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63-81% among themselves and 63-96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection.

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia

PubMed Central

Khaw, Yam Sim; Chan, Yoke Fun; Jafar, Faizatul Lela; Othman, Norlijah; Chee, Hui Yee

2016-01-01

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5′ and 3′ non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63–81% among themselves and 63–96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection. PMID:27199901

Phylogenetic analysis of G1P[8] and G12P[8] rotavirus A samples obtained in the pre- and post-vaccine periods, and molecular modeling of VP4 and VP7 proteins.

PubMed

Almeida, Tâmera Nunes Vieira; de Sousa, Teresinha Teixeira; da Silva, Roosevelt Alves; Fiaccadori, Fabíola Souza; Souza, Menira; Badr, Kareem Rady; de Paula Cardoso, Divina das Dôres

2017-09-01

Reduction in morbimortality rates for acute gastroenteritis (AGE) by Rotavirus A (RVA) has been observed after the introduction of vaccines, however the agent continues to circulate. The present study described the genomic characterization of the 11 dsRNA segments of two RVA samples G1P[8] obtained in the pre- and post-vaccination periods and one of G12P[8] sample (post-vaccine), compared to Rotarix™ vaccine. Analysis by molecular sequencing of the samples showed that the three samples belonged to genogroup I. In addition, the analysis of VP7 gene revealed that the samples G1 (pre-vaccine), G1 (post-vaccine) and G12 were characterized as lineages II, I and III, respectively. Regarding to VP4 and NSP4 gene it was observed that all samples belonged to lineage III, whereas for VP6 gene, the sample of the pre- and post-vaccine belonged to the lineage IV and I, respectively. Considering the VP7 gene, it was observed high nucleotide and amino acid identity for the two G1 samples when compared to Rotarix™ vaccine and lesser identity for the G12 sample. In relation to antigenic epitope of VP7 greater modifications were observed for the G12 sample in the 7-2 epitope that was confirmed by molecular modeling. On the other hand, for VP4, some changes in the 8-1 and 8-3 antigenic epitopes was observed for the three samples. This data could be interpreted as a low selective pressure exerted by vaccination in relation to G1P[8] samples and lesser protection in relation to G12P[8]. Thus, the continuous monitoring of RVA circulating samples remains important. Copyright © 2017 Elsevier B.V. All rights reserved.

Vp/Vs Ratio of Juan de Fuca Plate Sediments Along the Cascadia Deformation Front From Analysis of Controlled-Source Multi-Component OBS Records

NASA Astrophysics Data System (ADS)

Zhu, J.; Canales, J. P.; Han, S.; Carbotte, S. M.; Arnulf, A. F.; Nedimovic, M. R.

2017-12-01

The seismic characteristics (Vp, Vs, Vp/Vs, anisotropy) and derived physical properties (porosity, effective stress, pore fluid pressure, and crack density/orientation) of sediments entering a subduction zone are key parameters to understand subduction plate interface properties and seismogenic behavior. Here we present preliminary results of the average Vp/Vs within the 2-3-km-thick sediment section along the Cascadia deformation front between 44°-48°N offshore Oregon and Washington. We use data acquired in 2012 during the Juan de Fuca Ridge to Trench (R2T) controlled-source seismic experiment. We use P-waves and PPS converted-wave modes (i.e., P-to-S conversions of up-going waves at the crust-sediment interface) observed in 25 multi-component, short-period ocean bottom seismometers (OBS) deployed along an 400-km-long profile located 10-15 km seaward from the trench. The Vp/Vs was calculated following the method of Tsuji et al. (2011), which uses the seafloor-to-basement two-way traveltime determined from a coincident multichannel seismic reflection profile, and the time lag DT between the crustal P-refracted and PPS-converted waves. Processing of the OBS data included rotation into radial and traverse components, bandpass filtering, and predictive deconvolution. Our preliminary results show that the average sediment Vp/Vs along the profile varies from 2.8 to 3.35. Along the central-northern Oregon margin, Vp/Vs ranges between 2.8 and 2.95, while along the northern Washington margin Vp/Vs are slightly higher (2.95-3.05). We find the largest Vp/Vs values (≥3.1) offshore southern WA (between 46°-46.7°N). These preliminary results imply along-margin variations in subducting sediments along Cascadia. Implications for the physical properties of the sediments entering Cascadia are currently being explored and will be presented at the meeting.

Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus.

PubMed

He, Lei; Hu, Xiaolong; Zhu, Min; Liang, Zi; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2017-09-05

The genome of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) contains 10 double stranded RNA segments (S1-S10). The segment 7 (S7) encodes 50kDa protein which is considered as a structural protein. The expression pattern and function of p50 in the virus life cycle are still unclear. In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The expression pattern of vp7 gene was investigated by its overexpression in BmN cells. In addition to VP7, supplementary band was identified with western blotting technique. The virion, BmCPV infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. We concluded that the novel viral product was generated with a leaky scanning mechanism and the VDAC may be an interacted protein with VP7. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of a novel scoring and grading model for VP-based exams in postgraduate nurse education.

PubMed

Forsberg, Elenita; Ziegert, Kristina; Hult, Håkan; Fors, Uno

2015-12-01

For Virtual Patient-based exams, several scoring and grading methods have been proposed, but none have yet been validated. The aim of this study was to evaluate a new scoring and grading model for VP-based exams in postgraduate paediatric nurse education. The same student group of 19 students performed a VP-based exam in three consecutive courses. When using the scoring and grading assessment model, which contains a deduction system for unnecessary or unwanted actions, a progression was found in the three courses: 53% of the students passed the first exam, 63% the second and 84% passed the final exam. The most common reason for deduction of points was due to students asking too many interview questions or ordering too many laboratory tests. The results showed that the new scoring model made it possible to judge the students' clinical reasoning process as well as their progress. Copyright © 2015 Elsevier Ltd. All rights reserved.

Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40.

PubMed

Wijesinghe, Kaveesha J; Stahelin, Robert V

2015-12-30

Marburg virus (MARV), which belongs to the virus family Filoviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface. Marburg virus (MARV) is a lipid-enveloped filamentous virus from the family Filoviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane. In vitro and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Clinical application and validation of an iterative forward projection matching algorithm for permanent brachytherapy seed localization from conebeam-CT x-ray projections.

PubMed

Pokhrel, Damodar; Murphy, Martin J; Todor, Dorin A; Weiss, Elisabeth; Williamson, Jeffrey F

2010-09-01

To experimentally validate a new algorithm for reconstructing the 3D positions of implanted brachytherapy seeds from postoperatively acquired 2D conebeam-CT (CBCT) projection images. The iterative forward projection matching (IFPM) algorithm finds the 3D seed geometry that minimizes the sum of the squared intensity differences between computed projections of an initial estimate of the seed configuration and radiographic projections of the implant. In-house machined phantoms, containing arrays of 12 and 72 seeds, respectively, are used to validate this method. Also, four 103Pd postimplant patients are scanned using an ACUITY digital simulator. Three to ten x-ray images are selected from the CBCT projection set and processed to create binary seed-only images. To quantify IFPM accuracy, the reconstructed seed positions are forward projected and overlaid on the measured seed images to find the nearest-neighbor distance between measured and computed seed positions for each image pair. Also, the estimated 3D seed coordinates are compared to known seed positions in the phantom and clinically obtained VariSeed planning coordinates for the patient data. For the phantom study, seed localization error is (0.58 +/- 0.33) mm. For all four patient cases, the mean registration error is better than 1 mm while compared against the measured seed projections. IFPM converges in 20-28 iterations, with a computation time of about 1.9-2.8 min/ iteration on a 1 GHz processor. The IFPM algorithm avoids the need to match corresponding seeds in each projection as required by standard back-projection methods. The authors' results demonstrate approximately 1 mm accuracy in reconstructing the 3D positions of brachytherapy seeds from the measured 2D projections. This algorithm also successfully localizes overlapping clustered and highly migrated seeds in the implant.

Clinical application and validation of an iterative forward projection matching algorithm for permanent brachytherapy seed localization from conebeam-CT x-ray projections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pokhrel, Damodar; Murphy, Martin J.; Todor, Dorin A.

2010-09-15

Purpose: To experimentally validate a new algorithm for reconstructing the 3D positions of implanted brachytherapy seeds from postoperatively acquired 2D conebeam-CT (CBCT) projection images. Methods: The iterative forward projection matching (IFPM) algorithm finds the 3D seed geometry that minimizes the sum of the squared intensity differences between computed projections of an initial estimate of the seed configuration and radiographic projections of the implant. In-house machined phantoms, containing arrays of 12 and 72 seeds, respectively, are used to validate this method. Also, four {sup 103}Pd postimplant patients are scanned using an ACUITY digital simulator. Three to ten x-ray images are selectedmore » from the CBCT projection set and processed to create binary seed-only images. To quantify IFPM accuracy, the reconstructed seed positions are forward projected and overlaid on the measured seed images to find the nearest-neighbor distance between measured and computed seed positions for each image pair. Also, the estimated 3D seed coordinates are compared to known seed positions in the phantom and clinically obtained VariSeed planning coordinates for the patient data. Results: For the phantom study, seed localization error is (0.58{+-}0.33) mm. For all four patient cases, the mean registration error is better than 1 mm while compared against the measured seed projections. IFPM converges in 20-28 iterations, with a computation time of about 1.9-2.8 min/iteration on a 1 GHz processor. Conclusions: The IFPM algorithm avoids the need to match corresponding seeds in each projection as required by standard back-projection methods. The authors' results demonstrate {approx}1 mm accuracy in reconstructing the 3D positions of brachytherapy seeds from the measured 2D projections. This algorithm also successfully localizes overlapping clustered and highly migrated seeds in the implant.« less

[Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

PubMed

Niu, Xue-Feng; Liu, Xiao-Hui; Sun, Zhao-Jin; Shi, He-He; Chen, Jing; Jiang, Bido; Sun, Jing-Chen; Guo, Xiao-Feng

2009-09-01

To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

VP1 amino acid residue 145 of enterovirus 71 is a key residue for its receptor attachment and resistance to neutralizing antibody during cynomolgus monkey infection.

PubMed

Fujii, Ken; Sudaka, Yui; Takashino, Ayako; Kobayashi, Kyousuke; Kataoka, Chikako; Suzuki, Tadaki; Iwata-Yoshikawa, Naoko; Kotani, Osamu; Ami, Yasushi; Shimizu, Hiroyuki; Nagata, Noriyo; Mizuta, Katsumi; Matsuzaki, Yoko; Koike, Satoshi

2018-05-30

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G to E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses in vitro A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence. IMPORTANCE The contribution of VP1-145 variants in humans is not fully understood. In some reports, VP1-145G/Q viruses were more frequently isolated from severely affected than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper, we showed that VP1-145E viruses are more virulent than VP1-145G viruses in

Rotavirus A strains obtained from children with acute gastroenteritis in Mozambique, 2012-2013: G and P genotypes and phylogenetic analysis of VP7 and partial VP4 genes.

PubMed

João, Eva Dora; Strydom, Amy; O'Neill, Hester G; Cuamba, Assa; Cassocera, Marta; Acácio, Sozinho; Mandomando, Inácio; Motanyane, Lithabiso; Page, Nicola; de Deus, Nilsa

2018-01-01

In Mozambique rotavirus (RV) was shown to be the greatest cause of acute diarrhoea in infants from 0 to 11 months, and in 2015, national rotavirus vaccination was introduced. As with other developing countries, there is very limited active strain characterisation. Rotavirus positive clinical specimens, collected between 2012 and 2013, have now provided information on the genotypes circulating in southern Mozambique prior to vaccine introduction. Genotypes G2 (32.4%), G12 (28.0%), P[4] (41.4%) and P[6] (22.9%) (n = 157) strains were commonly detected with G2P[4] (42.3%) RVs being predominant, specifically during 2013. Phylogenetic evaluation of the VP7 and VP8* encoding genes showed, for the majority of the Mozambican strains, that they clustered with other African strains based on genotype. RVA/Human-wt/MOZ/0153/2013/G2P[4], RVA/Human-wt/MOZ/0308/2012/G2P[4] and RVA/Human-wt/MOZ/0288/2012/G12P[8] formed separate clusters from the other Mozambican strains with similar genotypes, suggesting possible reassortment. Amino acid substitutions in selected epitope regions also supported phylogenetic clustering. As expected, the VP7 and VP8* genes from the Mozambican strains differed from both the RotaTeq ® (SC2-9) G2P[5] and Rotarix ® (A41CB052A) G1P[8] genes. This study provides information on the genetic diversity of rotavirus strains prior to vaccine introduction and generates baseline data for future monitoring of any changes in rotavirus strains in response to vaccine pressure.

Genetic diversity of G1P[8] rotavirus VP7 and VP8* antigens in Finland over a 20-year period: No evidence for selection pressure by universal mass vaccination with RotaTeq® vaccine.

PubMed

Hemming, Maria; Vesikari, Timo

2013-10-01

Two live-attenuated oral vaccines (Rotarix™ and Rotateq®) against rotavirus gastroenteritis were licensed in 2006 and have been introduced into National Immunization Programs (NIPs) of several countries. Large scale use of rotavirus vaccines might cause antigenic pressure on circulating rotavirus types or lead to selection of new rotaviruses thus decreasing vaccine efficacy. We examined the nucleotide and amino acid sequences of the surface proteins VP7 and VP4 (cleaved to VP8(*) and VP5(*)) of a total of 108 G1P[8] rotavirus strains collected over a 20-year period from 1992, including the years 2006-2009 when rotavirus vaccine (mainly Rotarix™) was available, and the years 2009-2012 after implementation of RotaTeq® vaccine into the NIP of Finland. In G1 VP7 no changes at amino acid level were observed. In VP8(*) periodical fluctuation of the sublineage over the study period was found with multiple changes both at nucleotide and amino acid levels. Most amino acid changes were in the dominant antigenic epitopes of VP8(*). A change in VP8(*) sublineage occurred between 2008 and 2009, with a temporal correlation to the use of Rotarix™ up to 30% coverage in the period. In contrast, no antigenic changes in the VP8(*) protein appeared to be correlated to the exclusive use of RotaTeq® vaccine after 2009. Nevertheless, long-term surveillance of antigenic changes in VP4 and also VP7 proteins in wild-type rotavirus strains is warranted in countries with large scale use of the currently licensed live oral rotavirus vaccines. Copyright © 2013 Elsevier B.V. All rights reserved.

Genetic characterization of type 2a canine parvoviruses from Taiwan reveals the emergence of an Ile324 mutation in VP2

PubMed Central

2014-01-01

Background Canine parvovirus 2 (CPV 2) is a major infectious cause of mortality in puppies. The characteristic symptom of CPV 2 disease is intestinal hemorrhage with severe bloody diarrhea. Soon after CPV was first recognized in the late 1970s, the original virus, CPV 2, was replaced in the canine population by strains carrying minor antigenic variants (termed 2a, 2b, and 2c) of the VP2 gene that could be distinguished using monoclonal antibodies and molecular analyses. Here, we provide an updated molecular characterization of the CPV 2 circulating in Taiwan. Methods In this study, 28 isolates of CPV 2 from 144 dogs with suspected CPV infection were obtained from northern, central, and southern Taiwan from 2008 to 2012 and screened by PCR. The 28 isolates were sequenced, and a phylogenetic analysis of the VP2 gene was performed. Results Of the 28 Taiwanese CPV 2 isolates, 15 were identified as new CPV 2a, and 13 were identified as new CPV 2b. Compared to the reference CPV 2a, all 15 of the CPV 2a sequences collected in this study contain an Ile324 mutation caused by a TAT to ATT mutation at nucleotides 970–972 of the VP2 gene. Conclusion Our VP2 sequence data revealed that both types are currently prevalent CPV 2 field strains circulating in Taiwan, and a unique Ile324 VP2 mutation was found in our Taiwanese CPV 2a isolates and recent Asian isolates. CPV 2c was not observed in this study. PMID:24568207

A pragmatic approach to determine the optimal kVp in cone beam CT: balancing contrast-to-noise ratio and radiation dose

PubMed Central

Silkosessak, O; Jacobs, R; Bogaerts, R; Bosmans, H; Panmekiate, S

2014-01-01

Objectives: To determine the optimal kVp setting for a particular cone beam CT (CBCT) device by maximizing technical image quality at a fixed radiation dose. Methods: The 3D Accuitomo 170 (J. Morita Mfg. Corp., Kyoto, Japan) CBCT was used. The radiation dose as a function of kVp was measured in a cylindrical polymethyl methacrylate (PMMA) phantom using a small-volume ion chamber. Contrast-to-noise ratio (CNR) was measured using a PMMA phantom containing four materials (air, aluminium, polytetrafluoroethylene and low-density polyethylene), which was scanned using 180 combinations of kVp/mA, ranging from 60/1 to 90/8. The CNR was measured for each material using PMMA as background material. The pure effect of kVp and mAs on the CNR values was analysed. Using a polynomial fit for CNR as a function of mA for each kVp value, the optimal kVp was determined at five dose levels. Results: Absorbed doses ranged between 0.034 mGy mAs−1 (14 × 10 cm, 60 kVp) and 0.108 mGy mAs−1 (14 × 10 cm, 90 kVp). The relation between kVp and dose was quasilinear (R2 > 0.99). The effect of mA and kVp on CNR could be modelled using a second-degree polynomial. At a fixed dose, there was a tendency for higher CNR values at increasing kVp values, especially at low dose levels. A dose reduction through mA was more efficient than an equivalent reduction through kVp in terms of image quality deterioration. Conclusions: For the investigated CBCT model, the most optimal contrast at a fixed dose was found at the highest available kVp setting. There is great potential for dose reduction through mA with a minimal loss in image quality. PMID:24708447

42 CFR 51c.203 - Project elements.

Code of Federal Regulations, 2010 CFR

2010-10-01

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42 CFR 51c.403 - Project elements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Project elements. 51c.403 Section 51c.403 Public... SERVICES Grants for Operating Community Health Projects § 51c.403 Project elements. A project for the operation of a community health project supported under this subpart must: (a) Meet all of the requirements...

42 CFR 51c.403 - Project elements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Project elements. 51c.403 Section 51c.403 Public... SERVICES Grants for Operating Community Health Projects § 51c.403 Project elements. A project for the operation of a community health project supported under this subpart must: (a) Meet all of the requirements...

42 CFR 51c.403 - Project elements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Project elements. 51c.403 Section 51c.403 Public... SERVICES Grants for Operating Community Health Projects § 51c.403 Project elements. A project for the operation of a community health project supported under this subpart must: (a) Meet all of the requirements...

42 CFR 51c.203 - Project elements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Project elements. 51c.203 Section 51c.203 Public... SERVICES Grants for Planning and Developing Community Health Centers § 51c.203 Project elements. A project... community health center and the gradual assumption of operational status of the project so that the project...

42 CFR 51c.203 - Project elements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Project elements. 51c.203 Section 51c.203 Public... SERVICES Grants for Planning and Developing Community Health Centers § 51c.203 Project elements. A project... community health center and the gradual assumption of operational status of the project so that the project...

42 CFR 51c.403 - Project elements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Project elements. 51c.403 Section 51c.403 Public... SERVICES Grants for Operating Community Health Projects § 51c.403 Project elements. A project for the operation of a community health project supported under this subpart must: (a) Meet all of the requirements...

42 CFR 51c.403 - Project elements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Project elements. 51c.403 Section 51c.403 Public... SERVICES Grants for Operating Community Health Projects § 51c.403 Project elements. A project for the operation of a community health project supported under this subpart must: (a) Meet all of the requirements...

42 CFR 51c.203 - Project elements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Project elements. 51c.203 Section 51c.203 Public... SERVICES Grants for Planning and Developing Community Health Centers § 51c.203 Project elements. A project... community health center and the gradual assumption of operational status of the project so that the project...

42 CFR 51c.203 - Project elements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Project elements. 51c.203 Section 51c.203 Public... SERVICES Grants for Planning and Developing Community Health Centers § 51c.203 Project elements. A project... community health center and the gradual assumption of operational status of the project so that the project...

Poliovirus serotype-specific VP1 sequencing primers.

PubMed

Kilpatrick, David R; Iber, Jane C; Chen, Qi; Ching, Karen; Yang, Su-Ju; De, Lina; Mandelbaum, Mark D; Emery, Brian; Campagnoli, Ray; Burns, Cara C; Kew, Olen

2011-06-01

The Global Polio Laboratory Network routinely uses poliovirus-specific PCR primers and probes to determine the serotype and genotype of poliovirus isolates obtained as part of global poliovirus surveillance. To provide detailed molecular epidemiologic information, poliovirus isolates are further characterized by sequencing the ~900-nucleotide region encoding the major capsid protein, VP1. It is difficult to obtain quality sequence information when clinical or environmental samples contain poliovirus mixtures. As an alternative to conventional methods for resolving poliovirus mixtures, sets of serotype-specific primers were developed for amplifying and sequencing the VP1 regions of individual components of mixed populations of vaccine-vaccine, vaccine-wild, and wild-wild polioviruses. Published by Elsevier B.V.

Expression and characterization of a novel truncated rotavirus VP4 for the development of a recombinant rotavirus vaccine.

PubMed

Li, Yijian; Xue, Miaoge; Yu, Linqi; Luo, Guoxing; Yang, Han; Jia, Lianzhi; Zeng, Yuanjun; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

2018-04-12

The outer capsid protein VP4 is an important target for the development of a recombinant rotavirus vaccine because it mediates the attachment and penetration of rotavirus. Due to the poor solubility of full-length VP4, VP8 was explored as candidate rotavirus vaccines in the past years. In previous studies, it has been found that the N-terminal truncated VP8 protein, VP8-1 (aa26-231), could be expressed in soluble form with improved immunogenicity compared to the core of VP8 (aa65-223). However, this protein stimulated only a weak immune response when aluminum hydroxide was used as an adjuvant. In addition, it should be noted that the protective efficacy of VP4 was higher than that of VP8 and VP5. In this study, it was found that when the N-terminal 25 amino acids were deleted, the truncated VP4 ∗ (aa26-476) containing VP8 and the stalk domain of VP5 could be expressed in soluble form in E. coli and purified to homogeneous trimers. Furthermore, the truncated VP4 could induce high titers of neutralizing antibodies when aluminum adjuvant was used and conferred high protective efficacy in reducing the severity of diarrhea and rotavirus shedding in stools in animal models. The immunogenicity of the truncated VP4 was significantly higher than that of VP8 ∗ and VP5 ∗ alone. Taken together, the truncated VP4 ∗ (aa26-476), with enhanced immunogenicity and immunoprotectivity, could be considered as a viable candidate for further development and has the potential to become a parenterally administered rotavirus vaccine. Copyright © 2018 Elsevier Ltd. All rights reserved.

Validity of palpation of the C1 transverse process: comparison with a radiographic reference standard

PubMed Central

Cooperstein, Robert; Young, Morgan; Lew, Makani

2015-01-01

Objectives: Primary goal: to determine the validity of C1 transverse process (TVP) palpation compared to an imaging reference standard. Methods: Radiopaque markers were affixed to the skin at the putative location of the C1 TVPs in 21 participants receiving APOM radiographs. The radiographic vertical distances from the marker to the C1 TVP, mastoid process, and C2 TVP were evaluated to determine palpatory accuracy. Results: Interexaminer agreement for radiometric analysis was “excellent.” Stringent accuracy (marker placed ±4mm from the most lateral projection of the C1 TVP) = 57.1%; expansive accuracy (marker placed closer to contiguous structures) = 90.5%. Mean Absolute Deviation (MAD) = 4.34 (3.65, 5.03) mm; root-mean-squared error = 5.40mm. Conclusions: Manual palpation of the C1 TVP can be very accurate and likely to direct a manual therapist or other health professional to the intended diagnostic or therapeutic target. This work is relevant to manual therapists, anesthetists, surgeons, and other health professionals. PMID:26136601

First genetic characterization of rotavirus C in Russia.

PubMed

Zhirakovskaia, Elena; Tikunov, Artem; Klemesheva, Vera; Loginovskikh, Natalia; Netesov, Sergey; Tikunova, Nina

2016-04-01

Rotaviruses C (RVC) cause sporadic cases and outbreaks of diarrhea in humans and animals worldwide. The aim of this study was to monitor RVC during a surveillance study of sporadic cases of viral gastroenteritis in the Novosibirsk and Omsk regions of Russia from 2006 to 2011. A total of 2144 stool samples from children and adults hospitalized with acute gastroenteritis were tested for RVC by RT-PCR. Sixteen RVC-positive stool samples were detected at a rate of 0.6% (13/2037) in children and 2.8% (3/107) in adults. The low detection rate suggested that RVC infection was an uncommon cause of hospitalization in Russia. The complete VP7, VP4, VP6, and NSP4 gene sequences were determined. It was found that RVCs with at least two different genome backgrounds circulated in Siberia. VP4, VP6, and NSP4 gene sequences of most Russian RVC strains clustered with South Asian strains, while the VP7 gene showed a closer relationship to European strains. Meanwhile, only VP4 and NSP4 sequences of the strain Omsk08-386 clustered with South Asian strains, while its VP6 and VP7 sequences clustered with European strains. This is the first genetic characterization of Russian RVC strains and the first report on the prevalence of RVC in the Asian part of Russia. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural Insight into Nucleoprotein Conformation Change Chaperoned by VP35 Peptide in Marburg Virus.

PubMed

Liu, Baocheng; Dong, Shishang; Li, Guobang; Wang, Wenming; Liu, Xiang; Wang, Yantong; Yang, Cheng; Rao, Zihe; Guo, Yu

2017-08-15

Marburg virus (MARV) encodes a nucleoprotein (NP) to encapsidate its genome by oligomerization and form a ribonucleoprotein complex (RNP). According to previous investigation on nonsegmented negative-sense RNA viruses (nsNSV), the newly synthesized NPs must be prevented from indiscriminately binding to noncognate RNAs. During the viral RNA synthesis process, the RNPs undergo a transition from an RNA-bound form to a template-free form, to open access for the interaction between the viral polymerase and the RNA template. In filoviruses, this transition is regulated by VP35 peptide and other viral components. To further understand the dynamic process of filovirus RNP formation, we report here the structure of MARV NP core , both in the apo form and in the VP35 peptide-chaperoned form. These structures reveal a typical bilobed structure, with a positive-charged RNA binding groove between two lobes. In the apo form, the MARV NP exists in an interesting hexameric state formed by the hydrophobic interaction within the long helix of the NP core C-terminal region, which shows high structural flexibility among filoviruses and may imply critical function during RNP formation. Moreover, the VP35 peptide-chaperoned NP core remains in a monomeric state and completely loses its affinity for single-stranded RNA (ssRNA). The structural comparison reveals that the RNA binding groove undergoes a transition from closed state to open state, chaperoned by VP35 peptide, thus preventing the interaction for viral RNA. Our investigation provides considerable structural insight into the filovirus RNP working mechanism and may support the development of antiviral therapies targeting the RNP formation of filovirus. IMPORTANCE Marburg virus is one of the most dangerous viruses, with high morbidity and mortality. A recent outbreak in Angola in 2005 caused the deaths of 272 persons. NP is one of the most essential proteins, as it encapsidates and protects the whole virus genome simultaneously

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

PubMed

Dilley, Kari A; Voorhies, Alexander A; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B; Lorenzi, Hernan; Basler, Christopher F; Shabman, Reed S

2017-01-01

Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing

PubMed Central

Voorhies, Alexander A.; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B.; Lorenzi, Hernan; Basler, Christopher F.; Shabman, Reed S.

2017-01-01

Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation. PMID:28636653

Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geiss, Brian J.; Cano, Gina L.; Tavis, John E.

2004-12-05

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22.more » Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.« less

Structural properties of the promiscuous VP16 activation domain.

PubMed

Jonker, Hendrik R A; Wechselberger, Rainer W; Boelens, Rolf; Folkers, Gert E; Kaptein, Rob

2005-01-25

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

PubMed Central

Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

1995-01-01

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine. PMID:7474152

Multiplicity distributions of charged hadrons in vp and charged current interactions

NASA Astrophysics Data System (ADS)

Jones, G. T.; Jones, R. W. L.; Kennedy, B. W.; Morrison, D. R. O.; Mobayyen, M. M.; Wainstein, S.; Aderholz, M.; Hantke, D.; Katz, U. F.; Kern, J.; Schmitz, N.; Wittek, W.; Borner, H. P.; Myatt, G.; Radojicic, D.; Burke, S.

1992-03-01

Using data on vp andbar vp charged current interactions from a bubble chamber experiment with BEBC at CERN, the multiplicity distributions of charged hadrons are investigated. The analysis is based on ˜20000 events with incident v and ˜10000 events with incidentbar v. The invariant mass W of the total hadronic system ranges from 3 GeV to ˜14 GeV. The experimental multiplicity distributions are fitted by the binomial function (for different intervals of W and in different intervals of the rapidity y), by the Levy function and the lognormal function. All three parametrizations give acceptable values for X 2. For fixed W, forward and backward multiplicities are found to be uncorrelated. The normalized moments of the charged multiplicity distributions are measured as a function of W. They show a violation of KNO scaling.

[Genetic variation analysis of canine parvovirus VP2 gene in China].

PubMed

Yi, Li; Cheng, Shi-Peng; Yan, Xi-Jun; Wang, Jian-Ke; Luo, Bin

2009-11-01

To recognize the molecular biology character, phylogenetic relationship and the state quo prevalent of Canine parvovirus (CPV), Faecal samnples from pet dogs with acute enteritis in the cities of Beijing, Wuhan, and Nanjing were collected and tested for CPV by PCR and other assay between 2006 and 2008. There was no CPV to FPV (MEV) variation by PCR-RFLP analysis in all samples. The complete ORFs of VP2 genes were obtained by PCR from 15 clinical CPVs and 2 CPV vaccine strains. All amplicons were cloned and sequenced. Analysis of the VP2 sequences showed that clinical CPVs both belong to CPV-2a subtype, and could be classified into a new cluster by amino acids contrasting which contains Tyr-->Ile (324) mutation. Besides the 2 CPV vaccine strains belong to CPV-2 subtype, and both of them have scattered variation in amino acids residues of VP2 protein. Construction of the phylogenetic tree based on CPV VP2 sequence showed these 15 CPV clinical strains were in close relationship with Korea strain K001 than CPV-2a isolates in other countries at early time, It is indicated that the canine parvovirus genetic variation was associated with location and time in some degree. The survey of CPV capsid protein VP2 gene provided the useful information for the identification of CPV types and understanding of their genetic relationship.

[The cause of polyurethane catheter cracking during constant infusion of etoposide (VP-16) injection].

PubMed

Yokoyama, H; Aoyama, T; Matsuyama, T; Yamamura, Y; Nakajima, K; Nakamura, K; Sato, H; Kotaki, H; Chiba, S; Hirai, H; Yazaki, Y; Iga, T

1998-12-01

We studied the cause of cracking of a clinically used polyurethane (PU) catheter during the constant infusion of etoposide (VP-16) injection (Lastet), administered without dilution to patients as a part of combination high-dose chemotherapy. After VP-16 injection was infused into the PU catheter at a constant infusion rate (30 ml/h) for 24 h, a decrease in the elasticity (36% of untreated) and on increase in the length of the catheter (3.7%) were observed. These changes were significantly higher than those treated with the control saline. The similar changes of the PU catheter were observed after treatment with a basal solution containing polyethylene glycol 400 (PEG 400), polysorbate 80 and ethanol, which is the vehicle of the VP-16 injection, and with ethanol alone. Moreover, obvious degeneration of the internal wall (occurrence of spots like melting) and cutting face (micro-cracking) of the catheter was observed with an electron microscope after treatment with the vehicle. On the other hand, the elasticity or extension of the PU catheter were not changed after treatment with saline or PEG 400. From these findings, it was suggested that the degeneration and subsequent cracking of the PU catheter during the infusion of VP-16 injection was caused by ethanol contained in its injection solution. No cracking or morphological changes of polyvinyl chloride (PVC) and silicone catheters were found after treatment with the vehicle solution. However, since it has been reported in previous reports that di(2-ethylhexyl)phthalate was leached from PVC bags, the high dose chemotherapy with the dilution-free VP-16 injection should be achieved safely and effectively using a silicon catheter, rather than the PU catheter.

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.

2010-10-11

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that lossmore » of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.« less

Rapid detection of EBOLA VP40 in microchip immunofiltration assay

NASA Astrophysics Data System (ADS)

Miethe, Peter; Gary, Dominik; Hlawatsch, Nadine; Gad, Anne-Marie

2015-05-01

In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.

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. Published by Elsevier Ltd.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bucks, Michelle A.; O'Regan, Kevin J.; Murphy, Michael A.

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 associatedmore » 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.« less

The Importance of the C3 Framework

ERIC Educational Resources Information Center

Social Education, 2013

2013-01-01

"The C3 Framework for Social Studies State Standards will soon be released under the title "The College, Career, and Civic Life (C3) Framework for Social Studies State Standards: State Guidance for Enhancing the Rigor of K-12 Civics, Economics, Geography, and History." The C3 Project Director and Lead Writer was NCSS member Kathy…

Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging.

PubMed

Banadyga, Logan; Hoenen, Thomas; Ambroggio, Xavier; Dunham, Eric; Groseth, Allison; Ebihara, Hideki

2017-08-09

Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.

Value of 100 kVp scan with sinogram-affirmed iterative reconstruction algorithm on a single-source CT system during whole-body CT for radiation and contrast medium dose reduction: an intra-individual feasibility study.

PubMed

Nagayama, Y; Nakaura, T; Oda, S; Tsuji, A; Urata, J; Furusawa, M; Tanoue, S; Utsunomiya, D; Yamashita, Y

2018-02-01

To perform an intra-individual investigation of the usefulness of a contrast medium (CM) and radiation dose-reduction protocol using single-source computed tomography (CT) combined with 100 kVp and sinogram-affirmed iterative reconstruction (SAFIRE) for whole-body CT (WBCT; chest-abdomen-pelvis CT) in oncology patients. Forty-three oncology patients who had undergone WBCT under both 120 and 100 kVp protocols at different time points (mean interscan intervals: 98 days) were included retrospectively. The CM doses for the 120 and 100 kVp protocols were 600 and 480 mg iodine/kg, respectively; 120 kVp images were reconstructed with filtered back-projection (FBP), whereas 100 kVp images were reconstructed with FBP (100 kVp-F) and the SAFIRE (100 kVp-S). The size-specific dose estimate (SSDE), iodine load and image quality of each protocol were compared. The SSDE and iodine load of 100 kVp protocol were 34% and 21%, respectively, lower than of 120 kVp protocol (SSDE: 10.6±1.1 versus 16.1±1.8 mGy; iodine load: 24.8±4versus 31.5±5.5 g iodine, p<0.01). Contrast enhancement, objective image noise, contrast-to-noise-ratio, and visual score of 100 kVp-S were similar to or better than of 120 kVp protocol. Compared with the 120 kVp protocol, the combined use of 100 kVp and SAFIRE in WBCT for oncology assessment with an SSCT facilitated substantial reduction in the CM and radiation dose while maintaining image quality. Copyright © 2017 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

The Ebola virus matrix protein VP40 selectively induces vesiculation from phosphatidylserine-enriched membranes.

PubMed

Soni, Smita P; Stahelin, Robert V

2014-11-28

Ebola virus is from the Filoviridae family of viruses and is one of the most virulent pathogens known with ∼ 60% clinical fatality. The Ebola virus negative sense RNA genome encodes seven proteins including viral matrix protein 40 (VP40), which is the most abundant protein found in the virions. Within infected cells VP40 localizes at the inner leaflet of the plasma membrane (PM), binds lipids, and regulates formation of new virus particles. Expression of VP40 in mammalian cells is sufficient to form virus-like particles that are nearly indistinguishable from the authentic virions. However, how VP40 interacts with the PM and forms virus-like particles is for the most part unknown. To investigate VP40 lipid specificity in a model of viral egress we employed giant unilamellar vesicles with different lipid compositions. The results demonstrate VP40 selectively induces vesiculation from membranes containing phosphatidylserine (PS) at concentrations of PS that are representative of the PM inner leaflet content. The formation of intraluminal vesicles was not significantly detected in the presence of other important PM lipids including cholesterol and polyvalent phosphoinositides, further demonstrating PS selectivity. Taken together, these studies suggest that PM phosphatidylserine may be an important component of Ebola virus budding and that VP40 may be able to mediate PM scission. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Concentration of acrylamide in a polyacrylamide gel affects VP4 gene coding assignment of group A equine rotavirus strains with P[12] specificity

PubMed Central

2010-01-01

Background It is universally acknowledged that genome segment 4 of group A rotavirus, the major etiologic agent of severe diarrhea in infants and neonatal farm animals, encodes outer capsid neutralization and protective antigen VP4. Results To determine which genome segment of three group A equine rotavirus strains (H-2, FI-14 and FI-23) with P[12] specificity encodes the VP4, we analyzed dsRNAs of strains H-2, FI-14 and FI-23 as well as their reassortants by polyacrylamide gel electrophoresis (PAGE) at varying concentrations of acrylamide. The relative position of the VP4 gene of the three equine P[12] strains varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P[3], P[4], P[6], P[7], P[8] or P[18] specificity did not exhibit this phenomenon when the PAGE running conditions were varied. Conclusions The concentration of acrylamide in a PAGE gel affected VP4 gene coding assignment of equine rotavirus strains bearing P[12] specificity. PMID:20573245

[Immune Response of Recombinant Pseudorabies Virus rPRV-VP2 Expressing VP2 Gene of Porcine Parvovirus in Mice].

PubMed

Fu, Pengfei; Pan, Xinlong; Han, Qiao; Yang, Xingwu; Zhu, Qianlei; Guo, Xiaoqing; Zhang, Yu; Chen, Hongying

2016-03-01

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.

The C8 health project: design, methods, and participants.

PubMed

Frisbee, Stephanie J; Brooks, A Paul; Maher, Arthur; Flensborg, Patsy; Arnold, Susan; Fletcher, Tony; Steenland, Kyle; Shankar, Anoop; Knox, Sarah S; Pollard, Cecil; Halverson, Joel A; Vieira, Verónica M; Jin, Chuanfang; Leyden, Kevin M; Ducatman, Alan M

2009-12-01

The C8 Health Project was created, authorized, and funded as part of the settlement agreement reached in the case of Jack W. Leach, et al. v. E.I. du Pont de Nemours & Company (no. 01-C-608 W.Va., Wood County Circuit Court, filed 10 April 2002). The settlement stemmed from the perfluorooctanoic acid (PFOA, or C8) contamination of drinking water in six water districts in two states near the DuPont Washington Works facility near Parkersburg, West Virginia. This study reports on the methods and results from the C8 Health Project, a population study created to gather data that would allow class members to know their own PFOA levels and permit subsequent epidemiologic investigations. Final study participation was 69,030, enrolled over a 13-month period in 2005-2006. Extensive data were collected, including demographic data, medical diagnoses (both self-report and medical records review), clinical laboratory testing, and determination of serum concentrations of 10 perfluorocarbons (PFCs). Here we describe the processes used to collect, validate, and store these health data. We also describe survey participants and their serum PFC levels. The population geometric mean for serum PFOA was 32.91 ng/mL, 500% higher than previously reported for a representative American population. Serum concentrations for perfluorohexane sulfonate and perfluorononanoic acid were elevated 39% and 73% respectively, whereas perfluorooctanesulfonate was present at levels similar to those in the U.S. population. This largest known population study of community PFC exposure permits new evaluations of associations between PFOA, in particular, and a range of health parameters. These will contribute to understanding of the biology of PFC exposure. The C8 Health Project also represents an unprecedented effort to gather basic data on an exposed population; its achievements and limitations can inform future legal settlements for populations exposed to environmental contaminants.

SERT C project study

NASA Technical Reports Server (NTRS)

1974-01-01

The SERT C (Space Electric Rocket Test - C) project study defines a spacecraft mission that would demonstrate the technology readiness of ion thruster systems for primary propulsion and station keeping applications. As a low cost precursor, SERT C develops the components and systems required for subsequent Solar Electric Propulsion (SEP) applications. The SERT C mission requirements and preliminary spacecraft and subsystem design are described.

Expression and characterization of human group C rotavirus virus-like particles in insect cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, Kristina B.; Lin, S.-C.; Humphrey, Charles

2009-05-10

Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpCmore » RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.« less

Functional Investigation of NCI-H460-Inducible Myofibroblasts on the Chemoresistance to VP-16 with a Microfluidic 3D Co-Culture Device

PubMed Central

He, Jiarui; Guo, Zhe; Ying, Li; Xu, Zhiyun; Zhang, Jianing; Lu, Jianxin; Wang, Qi

2013-01-01

Fibroblasts, the major cell type in tumor stroma, are essential for tumor growth and survival, and represent an important therapeutic target for cancers. Here we presented a microfluidic co-culture device in which the three-dimensional (3D) matrix was employed to reconstruct an in vivo-like fibroblast-tumor cell microenvironment for investigation of the role of myofibroblasts induced by lung cancer cells in the chemoresistance to VP-16. Composed of a double-layer chip and an injection pump, the device houses fibroblasts and lung cancer cells co-cultured in 3D matrix and 2D mode to induce fibroblasts to become myofibroblasts with the supplement of the medium continuously. With this device, we verified that the cytokines secreted by lung cancer cells could effectively transform the fibroblasts into myofibroblasts. Moreover, compared to fibroblasts, the myofibroblasts showed higher resistance to anticancer drug VP-16. We also demonstrated that this kind of acquired resistance in myofibroblasts was associated with the expression of Glucose-regulated protein 78 (GP78). We concluded that this device allows for the assay to characterize various cellular events in a single device sequentially, facilitating a better understanding of the interactions among heterotypic cells in a sophisticated microenvironment. PMID:23613925

Comparison of Validated Assessment Scales and 3D digital fringe projection method to assess lifetime development of wrinkles in men.

PubMed

Luebberding, Stefanie; Krueger, Nils; Kerscher, Martina

2014-02-01

The assessment of wrinkle severity is an important evaluation criterion to determine the efficacy of aesthetic treatments. Aim of the present study was to compare Validated Assessment Scales (VAS) and 3D fringe projection (PRIMOS(®) ) for the evaluation of facial wrinkles in men and to determine standard values for each level of the VAS. 150 male subjects (20 to 70 years) were selected following strict criteria. Wrinkle severity at periorbital, glabella and forehead lines was evaluated using the 3D fringe projection and 5-point photonumeric VAS. The results of both methods were matched by determining quantitative values for each level of the clinical rating scale. High average correlation with age was found for VAS, Wd, maxWd, lWd, Wv, aWa and pWa. With a Wd of 60 to 70μm crow's feet and forehead lines are pronounced first, whereas glabella lines develop in subject's mid-forties, by an Wd of 180μm. Wrinkle severity increases at all locations every 10 years of age by one level of the VAS. This increase corresponds to an increase of Wd about 100 μm at glabella and forehead lines, and about 50 μm at crow's feet. The presented reference values for the Validated Assessment Scale are an important step towards an optimized assessment of skin aging and aesthetic dermatological treatments. The data helps to combine the precession of a biophysical measurement with the practical relevance of a clinical rating. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Recombinant Vp2 protein of infectious bursal disease virus AH1 strain expressed in insect cells: a vaccine candidate].

PubMed

Ouyang, Wei; Wang, Yongshan; Zhou, Yu; Zhang, Haibin; Tang, Yude

2010-05-01

Protective immune response of the available IBD vaccine is insufficient to fully protect against the prevailing strain of the infectious bursal disease virus (IBDV). Such a vaccination escape IBDV field isolate idenfied from Anhui province of China in December 2007, where IBD broke out at 2 weeks post vaccination. The IBDV vp2 gene was cloned into pFastBacHTA donor plasmid, followed by generation of the recombinant bacmid DNA pBac-VP2. The latter was used to transfect insect cell Sf9 with Lipofectamine to produce recombinant baculovirus vBac-VP2. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay (IFA), which was also confirmed by the detection of IBDV Vp2 protein in the infected Sf9 cells by IBDV sandwich ELISA. Western blotting revealed that the calculated protein of approximately 53 kDa was in the expressed in the insect cells. Moreover, virus-like particles (VLPs) and "inclusion body-like"structure in the infected Sf9 cells were observed under electron microscopy. We further developed an indirect ELISA for the detection of the IBDV antibodies, which was specific and sensitive. In addition, the lysates of vBac-VP2 infected cells was used to immunize 2-week-old SPF chickens, followed by challenging with the virulent IBDV, the survival rate was 30% at 14 days post primary immunization, however, the survival rate was 100% at 14 d after the booster vaccination. The ELISA antibody titers was up to 3.2 x 10(3) and neutralization antibody titer was 2536, significantly higher than those of one-shot vaccination, 8 x 10(2) and 1106, respectively. The immunized chickens did not show any clinical signs and histopathological changes of infection in 7-days trial time. The bursa/body-weight ratios were higher than those of the unimmunized control (P < 0.05). The virus-like-particle recombinant Vp2 protein expressed in insect cells promises to be a novel subunit vaccine and diagnostic reagent candidate

Study of canine parvovirus evolution: comparative analysis of full-length VP2 gene sequences from Argentina and international field strains.

PubMed

Gallo Calderón, Marina; Wilda, Maximiliano; Boado, Lorena; Keller, Leticia; Malirat, Viviana; Iglesias, Marcela; Mattion, Nora; La Torre, Jose

2012-02-01

The continuous emergence of new strains of canine parvovirus (CPV), poorly protected by current vaccination, is a concern among breeders, veterinarians, and dog owners around the world. Therefore, the understanding of the genetic variation in emerging CPV strains is crucial for the design of disease control strategies, including vaccines. In this paper, we obtained the sequences of the full-length gene encoding for the main capsid protein (VP2) of 11 canine parvovirus type 2 (CPV-2) Argentine representative field strains, selected from a total of 75 positive samples studied in our laboratory in the last 9 years. A comparative sequence analysis was performed on 9 CPV-2c, one CPV-2a, and one CPV-2b Argentine strains with respect to international strains reported in the GenBank database. In agreement with previous reports, a high degree of identity was found among CPV-2c Argentine strains (99.6-100% and 99.7-100% at nucleotide and amino acid levels, respectively). However, the appearance of a new substitution in the 440 position (T440A) in four CPV-2c Argentine strains obtained after the year 2009 gives support to the variability observed for this position located within the VP2, three-fold spike. This is the first report on the genetic characterization of the full-length VP2 gene of emerging CPV strains in South America and shows that all the Argentine CPV-2c isolates cluster together with European and North American CPV-2c strains.

42 CFR 51c.504 - Project elements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Project elements. 51c.504 Section 51c.504 Public... SERVICES Acquisition and Modernization of Existing Buildings § 51c.504 Project elements. (a) General requirements. A grantee which has received a grant under section 330 of the Act for a project which includes...

42 CFR 51c.504 - Project elements.

Code of Federal Regulations, 2011 CFR

2011-10-01

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42 CFR 51c.504 - Project elements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Project elements. 51c.504 Section 51c.504 Public... SERVICES Acquisition and Modernization of Existing Buildings § 51c.504 Project elements. (a) General requirements. A grantee which has received a grant under section 330 of the Act for a project which includes...

42 CFR 51c.504 - Project elements.

Code of Federal Regulations, 2013 CFR

2013-10-01

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42 CFR 51c.504 - Project elements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Project elements. 51c.504 Section 51c.504 Public... SERVICES Acquisition and Modernization of Existing Buildings § 51c.504 Project elements. (a) General requirements. A grantee which has received a grant under section 330 of the Act for a project which includes...

Genetics, recombination and clinical features of human rhinovirus species C (HRV-C) infections; interactions of HRV-C with other respiratory viruses.

PubMed

Wisdom, Anne; Kutkowska, Aldona E; McWilliam Leitch, E Carol; Gaunt, Eleanor; Templeton, Kate; Harvala, Heli; Simmonds, Peter

2009-12-30

To estimate the frequency, molecular epidemiological and clinical associations of infection with the newly described species C variants of human rhinoviruses (HRV), 3243 diagnostic respiratory samples referred for diagnostic testing in Edinburgh were screened using a VP4-encoding region-based selective polymerase chain reaction (PCR) for HRV-C along with parallel PCR testing for 13 other respiratory viruses. HRV-C was the third most frequently detected behind respiratory syncytial virus (RSV) and adenovirus, with 141 infection episodes detected among 1885 subjects over 13 months (7.5%). Infections predominantly targeted the very young (median age 6-12 months; 80% of infections in those <2 years), occurred throughout the year but with peak incidence in early winter months. HRV-C was detected significantly more frequently among subjects with lower (LRT) and upper respiratory tract (URT) disease than controls without respiratory symptoms; HRV-C mono-infections were the second most frequently detected virus (behind RSV) in both disease presentations (6.9% and 7.8% of all cases respectively). HRV variants were classified by VP4/VP2 sequencing into 39 genotypically defined types, increasing the current total worldwide to 60. Through sequence comparisons of the 5'untranslated region (5'UTR), the majority grouped with species A (n = 96; 68%, described as HRV-Ca), the remainder forming a phylogenetically distinct 5'UTR group (HRV-Cc). Multiple and bidirectional recombination events between HRV-Ca and HRV-Cc variants and with HRV species A represents the most parsimonious explanation for their interspersed phylogeny relationships in the VP4/VP2-encoding region. No difference in age distribution, seasonality or disease associations was identified between HRV-Ca and HRV-Cc variants. HRV-C-infected subjects showed markedly reduced detection frequencies of RSV and other respiratory viruses, providing evidence for a major interfering effect of HRV-C on susceptibility to other

Testing & Validating: 3D Seismic Travel Time Tomography (Detailed Shallow Subsurface Imaging)

NASA Astrophysics Data System (ADS)

Marti, David; Marzan, Ignacio; Alvarez-Marron, Joaquina; Carbonell, Ramon

2016-04-01

A detailed full 3 dimensional P wave seismic velocity model was constrained by a high-resolution seismic tomography experiment. A regular and dense grid of shots and receivers was use to image a 500x500x200 m volume of the shallow subsurface. 10 GEODE's resulting in a 240 channels recording system and a 250 kg weight drop were used for the acquisition. The recording geometry consisted in 10x20m geophone grid spacing, and a 20x20 m stagered source spacing. A total of 1200 receivers and 676 source points. The study area is located within the Iberian Meseta, in Villar de Cañas (Cuenca, Spain). The lithological/geological target consisted in a Neogen sedimentary sequence formed from bottom to top by a transition from gyspum to silstones. The main objectives consisted in resolving the underground structure: contacts/discontinuities; constrain the 3D geometry of the lithology (possible cavities, faults/fractures). These targets were achieved by mapping the 3D distribution of the physical properties (P-wave velocity). The regularly space dense acquisition grid forced to acquire the survey in different stages and with a variety of weather conditions. Therefore, a careful quality control was required. More than a half million first arrivals were inverted to provide a 3D Vp velocity model that reached depths of 120 m in the areas with the highest ray coverage. An extended borehole campaign, that included borehole geophysical measurements in some wells provided unique tight constraints on the lithology an a validation scheme for the tomographic results. The final image reveals a laterally variable structure consisting of four different lithological units. In this methodological validation test travel-time tomography features a high capacity of imaging in detail the lithological contrasts for complex structures located at very shallow depths.

Mathematically Gifted High School Students' Approaches to Developing Visual Proofs (VP) and Preliminary Ideas about VP

ERIC Educational Resources Information Center

Ugurel, Isikhan; Morali, H. Sevgi; Karahan, Ozge; Boz, Burcak

2016-01-01

The purpose of this study is to describe the procedure and examples of visual proofs (VP-or proof without words) developed by gifted mathematics secondary school students after their experiences. The participants of this study are three male 9th grade students enrolled in a private science high school. In the first stage of the research a briefing…

42 CFR 51c.303 - Project elements.