Publications by authors named "Vikram N Vakharia"

59 Publications

Evaluation of the oncolytic property of recombinant Newcastle disease virus strain R2B in 4T1 and B16-F10 cells in-vitro.

Res Vet Sci 2021 Oct 25;139:159-165. Epub 2021 Jul 25.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India. Electronic address:

Recombinant Newcastle disease virus vectors have gained a lot of interest for its oncolytic virus therapy and cancer immune therapeutic properties due to its selective replication to high titers in cancer cells. The aim of this study was to find out the oncolytic effects of mesogenic recombinant NDV strain R2B-GFP on murine mammary tumor cell line 4T1 and murine melanoma cell line B16-F10. The anti-tumor effects of R2B-GFP virus were studied via expression of virus transgene GFP in cancer cells, evaluating its cytotoxicity and cell migration efficacies by MTT and wound healing assays respectively. In addition, the underlying apoptotic mechanism of R2B-GFP virus was estimated by TUNEL assay, colorimetric estimation of Caspase-3, 8 and 9 and the estimation of Bax to Bcl-2 ratio. The results showed a significant decrease in viability of both 4T1 and B16-F10 cells infected with R2B-GFP virus at 0.1 and 1 MOI. R2B-GFP virus could significantly induce apoptosis in the 4T1 and B16-F10 cells as compared to the uninfected control. Further, a flow cytometry analysis on apoptotic cells percentage and mitochondria membrane permeability test was also studied in R2B-GFP virus treated 4T1 and B16-F10 cell lines. The R2B-GFP virus caused an increase in loss of mitochondrial membrane permeability in both 4T1 and B16-F10 cells indicating the involvement of mitochondrial regulated cell death. Thus, the recombinant virus R2B-GFP virus proved to be a valid candidate for oncolytic viral therapy in 4T1 and B16-F10 cells.
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http://dx.doi.org/10.1016/j.rvsc.2021.07.028DOI Listing
October 2021

Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo.

Viruses 2021 04 16;13(4). Epub 2021 Apr 16.

National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China.

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57-96.71% protein sequence identity) but shared only 22.65-45.85% and 23.37-43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with -C (-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 10 copies/μL.
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http://dx.doi.org/10.3390/v13040690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073260PMC
April 2021

Oral Vaccination of Grass Carp () with Baculovirus-Expressed Grass Carp Reovirus (GCRV) Proteins Induces Protective Immunity against GCRV Infection.

Vaccines (Basel) 2021 Jan 12;9(1). Epub 2021 Jan 12.

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.

The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp () industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.
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http://dx.doi.org/10.3390/vaccines9010041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827918PMC
January 2021

A Novel Subunit Vaccine Based on Outer Capsid Proteins of Grass Carp Reovirus (GCRV) Provides Protective Immunity against GCRV Infection in Rare Minnow ().

Pathogens 2020 Nov 13;9(11). Epub 2020 Nov 13.

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.

The grass carp hemorrhagic disease, caused by the grass carp reovirus (GCRV), has resulted in severe economic losses in the aquaculture industry in China. VP4 and VP35 are outer capsid proteins of GCRV and can induce an immune response in the host. Here, three recombinant baculoviruses, AcMNPV-VP35, AcMNPV-VP4, and AcMNPV-VP35-VP4, were generated to express recombinant VP4 and VP35 proteins from GCRV type II in insect cells by using the Bac-to-Bac baculovirus expression system to create a novel subunit vaccine. The expression of recombinant VP35, VP4, and VP35-VP4 proteins in Sf-9 cells were confirmed by Western blotting and immunofluorescence. Recombinant VP35, VP4, and VP35-VP4 were purified from baculovirus-infected cell lysates and injected intraperitoneally (3 μg/fish) into the model rare minnow, . After 21 days, the immunized fish were challenged with virulent GCRV. Liver, spleen, and kidney samples were collected at different time intervals to evaluate the protective efficacy of the subunit vaccines. The mRNA expression levels of some immune-related genes detected by using quantitative real-time PCR (qRT-PCR) were significantly upregulated in the liver, spleen, and kidney, with higher expression levels in the VP35-VP4 group. The nonvaccinated fish group showed 100% mortality, whereas the VP35-VP4, VP4, and VP35 groups exhibited 67%, 60%, and 33% survival, respectively. In conclusion, our results revealed that recombinant VP35 and VP4 can induce immunity and protect against GCRV infection, with their combined use providing the best effect. Therefore, VP35 and VP4 proteins can be used as a novel subunit vaccine against GCRV infection.
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http://dx.doi.org/10.3390/pathogens9110945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697209PMC
November 2020

Newcastle disease virus vectored rabies vaccine induces strong humoral and cell mediated immune responses in mice.

Vet Microbiol 2020 Dec 12;251:108890. Epub 2020 Oct 12.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243 122, India. Electronic address:

Rabies is a devastating disease affecting almost all mammalian animal species including humans. Vaccines are available to combat the disease. Protection against the disease is rendered by assessing the humoral immune response. Recent reports suggest the role of cell mediated immune response (CMI) in assessing vaccine efficacy. In the present study, two live vectored vaccine candidates containing glycoprotein G of rabies virus were generated using the mesogenic Newcastle disease virus (NDV) strain R2B and another with NDV with an altered fusion protein cleavage site as backbones. The efficacy of these vaccine candidates on testing in experimental mouse model indicated generation of robust humoral and CMI responses. The recombinant NDV containing the altered fusion protein cleavage site with glycoprotein G showed the highest CMI response in mice indicating its usage as a potential live vectored vaccine candidate against the disease.
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http://dx.doi.org/10.1016/j.vetmic.2020.108890DOI Listing
December 2020

Effect of the Viral Hemorrhagic Septicemia Virus Nonvirion Protein on Translation via PERK-eIF2α Pathway.

Viruses 2020 04 30;12(5). Epub 2020 Apr 30.

Department of Biological Sciences, University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606, USA.

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.
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http://dx.doi.org/10.3390/v12050499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290495PMC
April 2020

Genotype-matched Newcastle disease virus vaccine confers improved protection against genotype XII challenge: The importance of cytoplasmic tails in viral replication and vaccine design.

PLoS One 2019 14;14(11):e0209539. Epub 2019 Nov 14.

Institute of Marine & Environmental Technology, University of Maryland-Baltimore County, Baltimore, MD, United States of America.

Although typical Newcastle disease virus (NDV) vaccines can prevent mortality, they are not effective in preventing viral shedding. To overcome this, genotype-matched vaccines have been proposed. To date, this approach has never been tested against genotype XII strains. In this study, we generated and assessed the protection against genotype XII challenge of two chimeric NDV vaccine strains (rLS1-XII-1 and rLS1-XII-2). The rLS1-XII-1 virus has the complete fusion protein (F) and the hemagglutinin-neuraminidase (HN) open reading frames replaced with those from genotype XII strain NDV/peacock/Peru/2011 (PP2011) in a recombinant LaSota (rLS1) backbone. In rLS1-XII-2 virus, cytoplasmic tails of F and HN proteins were restored to those of rLS1. In vitro evaluation showed that rLS1-XII-2 and the parental rLS1 strains replicate at higher efficiencies than rLS1-XII-1. In the first vaccine/challenge experiment, SPF chickens vaccinated with rLS1-XII-1 virus showed only 71.3% protection, whereas, rLS1 and rLS1-XII-2 vaccinated chickens were fully protected. In a second experiment, both rLS1-XII-2 and the commercial vaccine strain LaSota induced 100% protection. However, rLS1-XII-2 virus significantly reduced viral shedding, both in the number of shedding birds and in quantity of shed virus. In conclusion, we have developed a vaccine candidate capable of fully protecting chickens against genotype XII challenges. Furthermore, we have shown the importance of cytoplasmic tails in virus replication and vaccine competence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209539PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855454PMC
March 2020

Recombinant Newcastle Disease Virus (NDV) Expressing Sigma C Protein of Avian Reovirus (ARV) Protects against Both ARV and NDV in Chickens.

Pathogens 2019 Sep 10;8(3). Epub 2019 Sep 10.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India.

Newcastle disease (ND) and avian reovirus (ARV) infections are a serious threat to the poultry industry, which causes heavy economic losses. The mesogenic NDV strain R2B is commonly used as a booster vaccine in many Asian countries to control the disease. In this seminal work, a recombinant NDV strain R2B expressing the sigma C (σC) gene of ARV (rNDV-R2B-σC) was generated by reverse genetics, characterized in vitro and tested as a bivalent vaccine candidate in chickens. The recombinant rNDV-R2B-σC virus was attenuated as compared to the parent rNDV-R2B virus as revealed by standard pathogenicity assays. The generated vaccine candidate, rNDV-R2B-σC, could induce both humoral and cell mediated immune responses in birds and gave complete protection against virulent NDV and ARV challenges. Post-challenge virus shedding analysis revealed a drastic reduction in NDV shed, as compared to unvaccinated birds.
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http://dx.doi.org/10.3390/pathogens8030145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789743PMC
September 2019

The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout.

J Virol 2019 09 28;93(18). Epub 2019 Aug 28.

Western Fisheries Research Center, U.S. Geological Survey, Seattle, Washington, USA

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout (). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral nucleoprotein (N), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric recombinant VHSV (rVHSV) by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout virulence phenotype of either parental strain. However, reciprocal exchanges of the viral N gene resulted in a partial gain of function in the chimeric trout-avirulent strain (22% mortality) and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and P genes together resulted in complete gain of function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout. Rainbow trout farming is a major food source industry worldwide that has suffered great economic losses due to host jumps of fish rhabdovirus pathogens, followed by evolution of dramatic increases in trout-specific virulence. However, the genetic determinants of host jumps and increased virulence in rainbow trout are unknown for any fish rhabdovirus. Previous attempts to identify the viral genes containing trout virulence determinants of viral hemorrhagic septicemia virus (VHSV) have not been successful. We show here that, somewhat surprisingly, the viral nucleocapsid (N) and phosphoprotein (P) genes together contain the determinants responsible for trout virulence in VHSV. This suggests a novel host-specific virulence mechanism involving the viral polymerase and a host component. This differs from the known virulence mechanisms of mammalian rhabdoviruses based on the viral P or M (matrix) protein.
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http://dx.doi.org/10.1128/JVI.00382-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714817PMC
September 2019

The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout.

Virol J 2019 03 7;16(1):31. Epub 2019 Mar 7.

Institute of Marine & Environmental Technology, University of Maryland Baltimore County, 701 E. Pratt Street, Baltimore, MD, 21202, USA.

Background: Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence).

Methods: Overlapping cDNA fragments of the DK-3592B genome were cloned after RT-PCR amplification, and their DNA sequenced by the di-deoxy chain termination method. A full-length cDNA copy (pVHSVdk) of the DK-3592B strain genome was constructed by assembling six overlapping cDNA fragments by using natural or artificially created unique restriction sites in the overlapping regions of the clones. Using an existing clone of the trout-avirulent VHSV-IVb strain MI03 (pVHSVmi), eight chimeric VHSV clones were constructed in which the coding region(s) of the glycoprotein (G), non-virion protein (NV), G and NV, or G, NV and L (polymerase) genes together, were exchanged between the two clones. Ten recombinant VHSVs (rVHSVs) were generated, including two parental rVHSVs, by transfecting fish cells with ten individual full-length plasmid constructs along with supporting plasmids using the established protocol. Recovered rVHSVs were characterized for viability and growth in vitro and used to challenge groups of juvenile rainbow trout by intraperitoneal injection.

Results: Complete sequence of the VHSV DK-3592B genome was determined from the cloned cDNA and deposited in GenBank under the accession no. KC778774. The trout-virulent DK-3592B genome (genotype Ia) is 11,159 nt in length and differs from the trout-avirulent MI03 genome (pVHSVmi) by 13% at the nucleotide level. When the rVHSVs were assessed for the trout-virulence phenotype in vivo, the parental rVHSVdk and rVHSVmi were virulent and avirulent, respectively, as expected. Four chimeric rVHSVdk viruses with the substitutions of the G, NV, G and NV, or G, NV and L genes from the avirulent pVHSVmi constructs were still highly virulent (100% mortality), while the reciprocal four chimeric rVHSVmi viruses with genes from pVHSVdk remained avirulent (0-10% mortality).

Conclusions: When chimeric rVHSVs, containing all the G, NV, and L gene substitutions, were tested in vivo, they did not exhibit any change in trout-virulence relative to the background clones. These results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.
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http://dx.doi.org/10.1186/s12985-019-1139-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407216PMC
March 2019

Oral vaccination of Macrobrachium rosenbergii with baculovirus-expressed M. rosenbergii nodavirus (MrNV) capsid protein induces protective immunity against MrNV challenge.

Fish Shellfish Immunol 2019 Mar 27;86:1123-1129. Epub 2018 Dec 27.

Institute of Marine and Environmental Technology (IMET), University of Maryland Baltimore Country (UMBC), Baltimore, MD, 21202, USA. Electronic address:

White Tail Disease (WTD) is one of the important viral diseases of fresh water giant prawn Macrobrachium rosenbergii, which is caused by Macrobrachium rosenbergii nodavirus (MrNV). In the present study, the capsid protein gene of MrNV containing a His-tag was cloned into a baculovirus vector pVL1393 and expressed the recombinant MrNV protein in insect cells, using a baculovirus expression system. A band corresponding to the MrNV protein of 43 kDa was characterized after fractionating the proteins of baculovirus-infected cell lysates by SDS-polyacrylamide gel, and immunostaining with His-tag monoclonal antibody. Furthermore, purified MrNV capsid protein assembled into virus-like particles (VLPs) of ∼30 nm in diameter, when examined by transmission electron microscopy (TEM). To vaccinate the larvae by oral route, the recombinant MrNV (r-MrNV) protein was coated with artificial prawn feed and fed to M. rosenbergii larvae (90 ± 10 mg) for 60 days. After 30 and 60 days of vaccine treatment, group of prawns were challenged with virulent MrNV orally. Samples were collected at different time intervals to evaluate the survival of larvae and to analyze the presence of MrNV by double-step PCR and expression of immune/ toll-like receptor (TLR) genes. Non-vaccinated group of M. rosenbergii larvae succumbed to death and had 90% mortality, whereas the r-MrNV protein treated groups exhibited 65 and 80% survival (P  ≤  0.001) for 30 and 60 days post-vaccination (dpv), respectively. Double-step PCR diagnosis revealed that there was 100% positive signals observed in non-vaccinated prawn group, whereas the infection was reduced significantly (P < 0.001) to 32 and 17% respectively in 30 and 60 dpv. Among the four different immune/ TLR genes such as antimicrobial peptide (Mramp), lysozyme (MrLY), proPhenol Oxidase (MrPPO) and Toll-Like Receptor (MrToll) expression screening, Mramp was successfully expressed in the MrNV subunit protein vaccinated prawns, whereas the non-vaccinated prawn had no immune/TLR gene expression. Taken together, our results demonstrate that oral vaccination of M. rosenbergii larvae with baculovirus-expressed MrNV capsid protein confer up to 78% protection against MrNV infection.
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http://dx.doi.org/10.1016/j.fsi.2018.12.010DOI Listing
March 2019

ICTV virus taxonomy profile: Picobirnaviridae.

J Gen Virol 2019 02 28;100(2):133-134. Epub 2018 Nov 28.

6​Department of Marine Biotechnology, University of Maryland, Baltimore County, 701, EastPratt Street, Baltimore, MD 21202, USA.

Picobirnaviridae is a family of viruses with bi-segmented (rarely unsegmented) dsRNA genomes comprising about 4.4 kbp in total, with small, non-enveloped spherical virions. The family includes one genus (Picobirnavirus) grouping three genetic clusters with high sequence variability, two defined by viruses infecting vertebrates and a third with viruses found in invertebrates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Picobirnaviridae, which is available at www.ictv.global/report/picobirnaviridae.
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http://dx.doi.org/10.1099/jgv.0.001186DOI Listing
February 2019

ICTV virus taxonomy profile: Birnaviridae.

J Gen Virol 2019 01 28;100(1):5-6. Epub 2018 Nov 28.

6​Department of Marine Biotechnology, University of Maryland, Baltimore County, 701, East Pratt Street, Baltimore, MD 21202, USA.

Birnaviridae is a family of viruses with bi-segmented dsRNA genomes totalling about 6 kbp forming icosahedral, non-enveloped virions. The family includes four genera, members of three of which (Aquabirnavirus, Avibirnavirus and Blosnavirus) infect vertebrates (excluding mammals), whereas members of the fourth genus (Entomobirnavirus) infect insects. Each genus includes 1-3 species. Infectious pancreatic necrosis virus of salmonids and infectious bursal disease virus of poultry are two economically important birnaviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Birnaviridae, which is available at www.ictv.global/report/birnaviridae.
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http://dx.doi.org/10.1099/jgv.0.001185DOI Listing
January 2019

Macrobrachium rosenbergii nodavirus (MrNV)-CP-RNA-2 DNA vaccine confers protective immunity in giant freshwater prawn Macrobrachium rosenbergii against MrNV infection.

Fish Shellfish Immunol 2019 Mar 22;86:319-326. Epub 2018 Nov 22.

Institute of Marine and Environmental Technology (IMET), University of Maryland Baltimore Country (UMBC), Baltimore, MD, 21202, USA. Electronic address:

Macrobrachium rosenbergii Nodavirus (MrNV) causes white tail disease (WTD) in Giant freshwater prawn Macrobrachium rosenbergii which leads to immense economic losses in hatcheries and farms. In the present study, we cloned the capsid protein gene of MrNV-CP-RNA-2 (1146 bp) into a DNA vaccine vector pVAX1 to form MrNV-CP-RNA-2- pVAX1. The bacterial transformant, containing the MrNV-CP gene, was coated on the fish diet pellets and fed to juvenile M. rosenbergii for 40 days. After the vaccine delivery, group of M. rosenbergii were challenged with virulent MrNV on 20 and 40 days post-vaccination (dpv) respectively and monitored for the survival. The non-vaccinated M. rosenbergii succumbed to death (100%) within 5 days, whereas the MrNV-CP-RNA-2- pVAX1 treated groups had the survivals of 60 and 80% in 20 and 40 dpv respectively (P ≤ 0.001). To study the MrNV infection level, double step PCR was performed at different dpv. The results revealed that in 20 dpv group, the infection was decreased to 65% and in 40 dpv group the infection decreased to 69% from control diet fed prawns (P < 0.001). Haematological parameters like coagulation time, total haemocyte count (THC) and oxyhaemocyanin levels were performed for the control and vaccinated prawns. The vaccination helped to decrease the time of coagulation, improved THC and oxyhaemocyanin levels at a significant level (p < 0.001) when compared to the non-vaccinated group. The immunological parameters like prophenol oxidase (ProPO), superoxide anion and intra-agar lysozyme activity were also performed and the results revealed that the level of proPO, superoxide anion and lysozyme activities were significantly (P ≤ 0.05) increased in 20 and 40 dpv groups respectively, when compared with the non-vaccinated groups. Based on the vaccination trials, the DNA vaccine construct MrNV-CP-RNA-2-pVAX1 effectively improved the survival against MrNV challenge, helped to decrease viral load and enhanced the immune system to protect the prawn from MrNV infection. This vaccine construct is highly useful to protect the M. rosenbergii from MrNV infection.
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http://dx.doi.org/10.1016/j.fsi.2018.11.049DOI Listing
March 2019

Development of a novel Newcastle disease virus (NDV) neutralization test based on recombinant NDV expressing enhanced green fluorescent protein.

Virol J 2017 11 23;14(1):232. Epub 2017 Nov 23.

Universidad Nacional Mayor de San Marcos, School of Veterinary Medicine, San Borja, Lima, Peru.

Background: Newcastle disease is one of the most important infectious diseases of poultry, caused by Newcastle disease virus (NDV). This virus is distributed worldwide and it can cause severe economic losses in the poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. Protection against NDV in chickens has been associated with development of humoral response. Although hemagglutination inhibition (HI) assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV.

Methods: In this study, we established a system to recover a recombinant NDV (rLS1) from a cloned cDNA, which is able to accept exogenous genes in desired positions. An enhanced green fluorescent protein (eGFP) gene was engineered in the first position of the NDV genome and we generated a recombinant NDV carrying eGFP. This NDV- eGFP reporter virus was used to develop an eGFP-based neutralization test (eGFP-NT), in which nAbs titers were expressed as the reciprocal of the highest dilution that expressed the eGFP.

Results: The eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were assayed by conventional neutralization (NT) and eGFP-NT. Additionally, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R  = 0.816) and ELISA (R  = 0.791) showed substantial correlation with conventional NT, eGFP-NT showed higher correlation (R  = 0.994), indicating that eGFP-NT is more accurate method to quantify nAbs.

Conclusions: Overall, the neutralization test developed here is a simple, rapid and reliable method for quantitation of NDV specific nAbs. It is suitable for vaccine studies and diagnostics.
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http://dx.doi.org/10.1186/s12985-017-0900-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701357PMC
November 2017

Newcastle Disease Virus Vectored Bivalent Vaccine against Virulent Infectious Bursal Disease and Newcastle Disease of Chickens.

Vaccines (Basel) 2017 Sep 26;5(4). Epub 2017 Sep 26.

Institute of Marine and Environmental Technology, University of Maryland Baltimore County, Baltimore, MD 21202, USA.

Newcastle disease virus (NDV) strain F is a lentogenic vaccine strain used for primary vaccination in day-old chickens against Newcastle disease (ND) in India and Southeast Asian countries. Recombinant NDV-F virus and another recombinant NDV harboring the major capsid protein VP2 gene of a very virulent infectious bursal disease virus (IBDV); namely rNDV-F and rNDV-F/VP2, respectively, were generated using the NDV F strain. The rNDV-F/VP2 virus was slightly attenuated, as compared to the rNDV-F virus, as evidenced from the mean death time and intracerebral pathogenicity index analysis. This result indicates that rNDV-F/VP2 behaves as a lentogenic virus and it is stable even after 10 serial passages in embryonated chicken eggs. When chickens were vaccinated with the rNDV F/VP2, it induced both humoral and cell mediated immunity, and was able to confer complete protection against very virulent IBDV challenge and 80% protection against virulent NDV challenge. These results suggest that rNDV-F could be an effective and inherently safe vaccine vector. Here, we demonstrate that a bivalent NDV-IBDV vaccine candidate generated by reverse genetics method is safe, efficacious and cost-effective, which will greatly aid the poultry industry in developing countries.
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http://dx.doi.org/10.3390/vaccines5040031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748598PMC
September 2017

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

J Virol 2017 10 12;91(19). Epub 2017 Sep 12.

Department of Biological Sciences, The University of Toledo, Toledo, Ohio, USA

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression. Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.
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http://dx.doi.org/10.1128/JVI.00279-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599748PMC
October 2017

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

Proc Natl Acad Sci U S A 2017 08 24;114(32):8550-8555. Epub 2017 Jul 24.

Department of BioSciences, Rice University, Houston, TX 77251;

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.
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http://dx.doi.org/10.1073/pnas.1701747114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559005PMC
August 2017

Rescue of a recombinant Newcastle disease virus strain R2B expressing green fluorescent protein.

Virus Genes 2017 Jun 9;53(3):410-417. Epub 2017 Feb 9.

Institute of Marine and Environmental Technology, University of Maryland, Baltimore County, Baltimore, MD, USA.

Newcastle disease virus (NDV), strain R2B is a mesogenic vaccine strain used for booster vaccination in chickens against Newcastle disease in India and many south East Asian countries. A full-length cDNA clone of the virus was generated by ligating eight overlapping fragments generated by reverse transcription polymerase chain reaction having unique restriction enzyme sites within them. This full-length cDNA clone was flanked by hammerhead ribozyme and hepatitis delta virus ribozyme sequences. Defined genetic markers were introduced into the NDV genome to differentiate the rescued virus from the parent virus. A gene cassette containing the reporter gene, green fluorescent protein flanked by NDV gene-start and gene-end signals was generated by PCR and introduced into the full-length clone of NDV between the P and M genes. Recombinant NDV encoding the GFP gene was rescued having precise termini when transfected into permissive Vero cells along with support plasmids harbouring the nucleoprotein, phosphoprotein and polymerase genes. The recombinant virus had similar growth kinetics as that of the parent virus with a moderate reduction in the virulence. The generation of reverse genetics system for NDV strain R2B will help in the development of multivalent vaccines against viral diseases of livestock and poultry.
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http://dx.doi.org/10.1007/s11262-017-1433-3DOI Listing
June 2017

Molecular characterization of infectious pancreatic necrosis virus strains isolated from the three types of salmonids farmed in Chile.

Virol J 2017 01 31;14(1):17. Epub 2017 Jan 31.

Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.

Background: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level.

Methods: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S).

Results: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates.

Conclusions: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.
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http://dx.doi.org/10.1186/s12985-017-0684-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282824PMC
January 2017

Phylogenetic analysis of the glycoprotein gene of viral hemorrhagic septicemia virus from Iranian trout farms points towards a common European origin.

Vet Microbiol 2016 Apr 26;186:97-101. Epub 2016 Feb 26.

Qiagen-Aarhus, Aarhus, Denmark.

Viral haemorrhagic septicaemia virus (VHSV), a member of family Rhabdoviridae and genus Novirhabdoviridae, causes mortality in numerous marine and freshwater hosts located in northern hemisphere. To evaluate the genetic diversity of VHSV from the North and South West of Iran, the sequences of a 1483bp nt region of the glycoprotein gene were determined for four Iranian isolates. These sequences were analysed to evaluate their genetic relatedness with 86 worldwide isolates representing the four known genogroups of VHSV. Phylogenetic analysis by nucleotide sequences showed that all the VHSV isolates studied were closest related to the 19 fresh water strains from Germany grouped within the European genogroup Ia-2. This finding indicates that Iranian VHSV most likely was introduced to Iran by the movement of contaminated fish fry from a source in Europe.
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http://dx.doi.org/10.1016/j.vetmic.2016.02.019DOI Listing
April 2016

Identification and Characterization of MicroRNAs in Snakehead Fish Cell Line upon Snakehead Fish Vesiculovirus Infection.

Int J Mol Sci 2016 Jan 26;17(2). Epub 2016 Jan 26.

Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China.

MicroRNAs (miRNAs) play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection. Snakehead fish Vesiculovirus (SHVV) has caused mass mortality in snakehead fish during the past few years. To identify specific miRNAs involved in SHVV infection, we performed microRNA deep sequencing on a snakehead fish cell line (SSN-1) with or without SHVV infection. A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs, and nine novel miRNAs were identified using MiRDeep2 software. Eighteen and 143 of the 205 known miRNAs were differentially expressed at three and 24 h post-infection (poi), respectively. From the differentially-expressed miRNAs, five were randomly selected to validate their expression profiles using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and their expression profiles were consistent with the microRNA sequencing results. In addition, the target gene prediction of the SHVV genome was performed for the differentially-expressed host miRNAs, and a total of 10 and 58 differentially-expressed miRNAs were predicted to bind to the SHVV genome at three and 24 h poi, respectively. The effects of three selected miRNAs (miR-130-5p, miR-214 and miR-216b) on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting. The results showed that all three miRNAs were able to inhibit the multiplication of SHVV; whereas the mechanisms underlying the SHVV multiplication inhibited by the specific miRNAs need to be further characterized in the future.
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http://dx.doi.org/10.3390/ijms17020154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783888PMC
January 2016

The zebrafish galectins Drgal1-L2 and Drgal3-L1 bind in vitro to the infectious hematopoietic necrosis virus (IHNV) glycoprotein and reduce viral adhesion to fish epithelial cells.

Dev Comp Immunol 2016 Feb 30;55:241-252. Epub 2015 Sep 30.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Institute of Marine and Environmental Technology, Baltimore, Maryland, USA.

The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells. Our results suggest that the extracellular Drgal1-L2 and Drgal3-L1 interact directly and in a carbohydrate-dependent manner with the IHNV glycosylated envelope and glycans on the epithelial cell surface, significantly reducing viral adhesion.
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http://dx.doi.org/10.1016/j.dci.2015.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684960PMC
February 2016

Protective and immunogenic effects of Escherichia coli-expressed infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in rainbow trout.

Fish Shellfish Immunol 2015 Nov 8;47(1):390-6. Epub 2015 Sep 8.

Iranian Fisheries Research Organization, Inland Water Aquaculture Research Center, Bandar Anzali, Iran.

Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae which causes significant losses in the aquaculture industry. To develop a recombinant vaccine for IPNV, a cDNA construct of IPNV VP2-VP3 fusion gene was prepared and cloned into an Escherichia coli (E. coli) expression vector (pET-26b) to obtain recombinant protein products. A study was conducted to determine the antibody responses and protective capacity of this recombinant vaccine expressing VP2-VP3 fusion protein. Subsequently, juvenile rainbow trout were inoculated by injecting purified recombinant IPNV VP2-VP3 proteins, followed by challenge with virulent IPNV in rainbow trout. Our results demonstrate that recombinant E. coli derived VP2-VP3 fusion protein induced a strong and significantly (P < 0.05) higher IgM antibody response in serum samples compared to control groups. Following intraperitoneal challenge, the relative percent survival (RPS) rate of survivors was 83% for the vaccinated group. Statistical analysis of IgM levels indicated that immunogenicity of recombinant VP2-VP3 protein, combined with adjuvant, was much higher than any other groups of rainbow trout challenged with virulent IPNV. This result was confirmed by measuring the viral loads of IPNV in immunized rainbow trout which was drastically reduced, as analyzed by real-time RT-PCR. In summary, we demonstrate that E. coli-expressed IPNV VP2-VP3 injectable vaccine is highly immunogenic and protective against IPNV infection.
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http://dx.doi.org/10.1016/j.fsi.2015.09.007DOI Listing
November 2015

Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens.

Vaccine 2015 Feb 14;33(8):1033-9. Epub 2015 Jan 14.

Department of Marine Biotechnology, University of Maryland, Baltimore County, 701, East Pratt Street, Baltimore, MD 21202, USA.

Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a 'DNA prime-protein boost' approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and 'DNA prime-protein boost' elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and 'DNA prime-protein boost' groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p<0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to 'DNA prime-protein boost' group. Additionally, chickens immunized with pCIVP2-cHSP70 and 'DNA prime-protein boost' vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens.
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http://dx.doi.org/10.1016/j.vaccine.2015.01.006DOI Listing
February 2015

Specific nucleotides at the 3'-terminal promoter of viral hemorrhagic septicemia virus are important for virulence in vitro and in vivo.

Virology 2015 Feb 30;476:226-232. Epub 2014 Dec 30.

Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Bioproduction, P.O. Box 8146 Dep, N-0033 Oslo, Norway.

Viral hemorrhagic septicemia virus (VHSV), a member of the Novirhabdovirus genus, contains an 11-nucleotide conserved sequence at the terminal 3'- and 5'-untranslated regions (UTRs) that are complementary. To study the importance of nucleotides in the 3'-UTR of VHSV for replication of novirhabdoviruses, we performed site-directed mutagenesis of selected residues at the 3'-terminus and generated mutant viruses using a reverse genetics approach. Assessment of growth kinetics and in vitro real-time cytopathogenicity studies showed that the order of two nucleotides (A4G5) of the 3'-terminus of VHSV directly affects growth kinetics in vitro. The mutant A4G-G5A virus has reduced total positive-strand RNA synthesis efficiency (51% of wild-type) at 48h post-transfection and 70h delay in causing complete cytopathic effect in susceptible fish cells, as compared to the WT-VHSV. Furthermore, when the A4G-G5A virus was used to challenge zebrafish, it exhibited reduced pathogenicity (54% lower end-point mortality) compared to the WT-VHSV. From these studies, we infer that specific residues in the 3'-UTR of VHSV have a promoter function and are essential to modulate the virulence in cells and pathogenicity in fish.
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http://dx.doi.org/10.1016/j.virol.2014.12.003DOI Listing
February 2015

Interchange of L polymerase protein between two strains of viral hemorrhagic septicemia virus (VHSV) genotype IV alters temperature sensitivities in vitro.

Virus Res 2015 Jan 18;195:203-6. Epub 2014 Oct 18.

Norwegian University of Life Sciences, PO Box 8146 Dep, N-0033 Oslo, Norway. Electronic address:

Viral hemorrhagic septicemia virus (VHSV) has four genotypes (I-IV) and sub-lineages within genotype I and IV. Using a reverse genetics approach, we explored the importance of the L gene for growth characteristics at different temperatures following interchange of the L gene within genotype IV (IVa and IVb) strains. VHSV strains harboring heterologous L gene were recovered and we show that the L gene determines growth characteristics at different temperatures in permissive cell lines.
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http://dx.doi.org/10.1016/j.virusres.2014.10.013DOI Listing
January 2015

Genotype characterization of commonly used Newcastle disease virus vaccine strains of India.

PLoS One 2014 4;9(6):e98869. Epub 2014 Jun 4.

Institute of Marine and Environmental Technology, University of Maryland, Baltimore County, Baltimore, Maryland, United States of America.

Newcastle disease is an avian pathogen causing severe economic losses to the Indian poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. India being an endemic country, advocates vaccination against the virus using lentogenic and mesogenic strains. Two virus strains which are commonly used for vaccination are strain F (a lentogenic virus) and strain R2B (a mesogenic virus). Strain F is given to 0-7 days old chicks and R2B is given to older birds which are around 6-8 weeks old. To understand the genetic makeup of these two strains, a complete genome study and phylogenetic analysis of the F, HN genes of these vaccine strains were carried out. Both the viral strains had a genome length of 15,186 nucleotides and consisted of six genes with conserved complimentary 3' leader and 5' trailer regions. The fusion protein cleavage site of strain F is GGRQGRL and strain R2B is RRQKRF. Although both the viral strains had different virulence attributes, the length of the HN protein was similar with 577 amino acids. Phylogenetic analysis of F, HN and complete genome sequences grouped these two strains in genotype II category which are considered as early genotypes and corroborated with their years of isolation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098869PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045777PMC
August 2015

A single amino acid in VP2 is critical for the attachment of infectious bursal disease subviral particles to immobilized metal ions and DF-1 cells.

Biochim Biophys Acta 2014 Jul 13;1844(7):1173-82. Epub 2014 Apr 13.

Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan. Electronic address:

VP2 protein is the primary host-protective immunogen of infectious bursal disease virus (IBDV). His249 and His253 are two surface histidine residues in IBDV subviral particles (SVP), which is formed by twenty VP2 trimers when the VP2 protein of a local isolate is expressed. Here, a systemic study was performed to investigate His249 or/and His253 on self-assembly, cell attachment and immunogenicity of SVP. Point-mutagenesis of either or both histidine residues to alanine did not affect self-assembly of the SVP, but the SVP lost its Ni-NTA binding affinity when the His253 was mutated. Indirect immunofluorescence assays and inhibitory experiments also showed that His253 is essential for SVP to attach onto the DF-1 cells and to inhibit IBDV infection of DF-1 cells. Finally, enzyme-linked immunosorbent assays and chicken protection assays demonstrated that SVP with a mutation of His253 to alanine induced comparable neutralizing antibody titers in chickens as the wild-type SVP did. It was concluded that VP2's His253, a site not significant for the overall immunogenicity induced by SVP, is crucial for the binding affinity of SVP to Ni-NTA and the attachment of an IBDV host cell line. This is the first paper to decipher the role of His253 played in receptor interaction and immunogenicity.
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http://dx.doi.org/10.1016/j.bbapap.2014.04.004DOI Listing
July 2014
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