Generation of microRNA-30e-producing recombinant viral hemorrhagic septicemia virus (VHSV) and its effect on in vitro immune responses

ABSTRACT Surface glycoproteins of enveloped virus are potent elicitors of type I interferon (IFN)-mediated antiviral responses in a way that may be independent of the well-studied genome-mediated route. However, the viral glycoprotein determinants responsible for initiating the IFN response remain unidentified. In this study, we have used a collection of 60 synthetic 20-mer overlapping peptides (pepscan) spanning the full length of glycoprotein G (gpG) of viral hemorrhagic septicemia virus (VHSV) to investigate what regions of this protein are implicated in triggering the type I IFN-associated immune responses. Briefly, two regions with ability to increase severalfold the basal expression level of the IFN-stimulated mx gene and to restrict the spread of virus among responder cells were mapped to amino acid residues 280 to 310 and 340 to 370 of the gpG protein of VHSV. In addition, the results obtained suggest that an interaction between VHSV gpG and integrins might trigger the host IFN-mediated antiviral response after VHSV infection. Since it is known that type I IFN plays an important role in determining/modulating the protective-antigen-specific immune responses, the identification of viral glycoprotein determinants directly implicated in the type I IFN induction might be of special interest for designing new adjuvants and/or more-efficient and cost-effective viral vaccines as well as for improving our knowledge on how to stimulate the innate immune system.

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Genomic and immunogenic changes over the history of the Viral Hemorrhagic Septicemia (VHS-IVb) fish virus (=Piscine novirhabdovirus) in the Laurentian Great Lakes

10.1101/2020.04.27.063842 ◽

2020 ◽

Author(s):

Megan D. Niner ◽

Carol A. Stepien ◽

Bartolomeo Gorgoglione ◽

Douglas W. Leaman

Keyword(s):

Great Lakes ◽

Fish Host ◽

Laurentian Great Lakes ◽

Viral Hemorrhagic Septicemia Virus ◽

Coding Region ◽

Nucleotide Substitutions ◽

Whole Genome Analysis ◽

Viral Hemorrhagic Septicemia ◽

History Of

AbstractViral Hemorrhagic Septicemia Virus (VHSV) (=Piscine novirhabdovirus) appeared in the Laurentian Great Lakes in 2005, constituting a unique and highly virulent genogroup (IVb), which killed >32 fish species in large 2005 and 2006. Periods of apparent dormancy punctuated smaller outbreaks in 2007, 2008, and 2017. We conducted the first whole genome analysis of IVb, evaluating its evolutionary changes using 46 isolates, in reference to immunogenicity in cell culture, and the genomes of other VHS genogroups (I–IVa) and other Novirhabdoviruses. IVb isolates had 253 genomic nucleotide substitutions (2.3% of the total 11,158 nucleotides), with 85 (16.6%) being non-synonymous. The greatest number of substitutions occurred in the non-coding region (NCDS; 4.3%) followed by the Nv- (3.8%), and M- (2.8%) genes. The M-gene possessed the greatest proportions of amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHS genogroups, IVa from the northeastern Pacific exhibited the fastest substitution rate (2.01×10-3), followed by Ivb (6.64×10−5), and I/III from Europe (4.09×10−5). A 2016 gizzard shad isolate from Lake Erie was the most divergent IVb isolate (38 NT, 15.0%, 15 AA), yet exhibited reduced virulence with in vitro immunogenicity analyses, as did other 2016 isolates, in comparison to the first IVb isolate (2003). The 2016 isolates exhibited lower impact on innate antiviral responses, suggesting phenotypic effects. Results suggest continued sequence change and lower virulence over the history of IVb, which may facilitate its long-term persistence in fish host populations.

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Genomic and immunogenic changes of Piscine novirhabdovirus (Viral Hemorrhagic Septicemia Virus) over its evolutionary history in the Laurentian Great Lakes

PLoS ONE ◽

10.1371/journal.pone.0232923 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0232923

Author(s):

Megan D. Niner ◽

Carol A. Stepien ◽

Bartolomeo Gorgoglione ◽

Douglas W. Leaman

Keyword(s):

Great Lakes ◽

Evolutionary History ◽

Fish Host ◽

Laurentian Great Lakes ◽

Viral Hemorrhagic Septicemia Virus ◽

Sequencing Analysis ◽

Dorosoma Cepedianum ◽

Coding Region ◽

Viral Hemorrhagic Septicemia

A unique and highly virulent subgenogroup (-IVb) of Piscine novirhabdovirus, also known as Viral Hemorrhagic Septicemia Virus (VHSV), suddenly appeared in the Laurentian Great Lakes, causing large mortality outbreaks in 2005 and 2006, and affecting >32 freshwater fish species. Periods of apparent dormancy have punctuated smaller and more geographically-restricted outbreaks in 2007, 2008, and 2017. In this study, we conduct the largest whole genome sequencing analysis of VHSV-IVb to date, evaluating its evolutionary changes from 48 isolates in relation to immunogenicity in cell culture. Our investigation compares genomic and genetic variation, selection, and rates of sequence changes in VHSV-IVb, in relation to other VHSV genogroups (VHSV-I, VHSV-II, VHSV-III, and VHSV-IVa) and with other Novirhabdoviruses. Results show that the VHSV-IVb isolates we sequenced contain 253 SNPs (2.3% of the total 11,158 nucleotides) across their entire genomes, with 85 (33.6%) of them being non-synonymous. The most substitutions occurred in the non-coding region (NCDS; 4.3%), followed by the Nv- (3.8%), and M- (2.8%) genes. Proportionally more M-gene substitutions encoded amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHSV genogroups and subgenogroups, VHSV-IVa from the northeastern Pacific Ocean has shown the fastest substitution rate (2.01x10-3), followed by VHSV-IVb (6.64x10-5) and by the VHSV-I, -II and-III genogroups from Europe (4.09x10-5). A 2016 gizzard shad (Dorosoma cepedianum) from Lake Erie possessed the most divergent VHSV-IVb sequence. The in vitro immunogenicity analysis of that sample displayed reduced virulence (as did the other samples from 2016), in comparison to the original VHSV-IVb isolate (which had been traced back to 2003, as an origin date). The 2016 isolates that we tested induced milder impacts on fish host cell innate antiviral responses, suggesting altered phenotypic effects. In conclusion, our overall findings indicate that VHSV-IVb has undergone continued sequence change and a trend to lower virulence over its evolutionary history (2003 through present-day), which may facilitate its long-term persistence in fish host populations.

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In vitro transcribed dsRNA limits viral hemorrhagic septicemia virus (VHSV)-IVb infection in a novel fathead minnow (Pimephales promelas) skin cell line

Fish & Shellfish Immunology ◽

10.1016/j.fsi.2018.11.053 ◽

2019 ◽

Vol 86 ◽

pp. 403-409 ◽

Cited By ~ 3

Author(s):

Sarah J. Poynter ◽

Eric M. Leis ◽

Stephanie J. DeWitte-Orr

Keyword(s):

Cell Line ◽

Fathead Minnow ◽

Pimephales Promelas ◽

Viral Hemorrhagic Septicemia Virus ◽

Viral Hemorrhagic Septicemia ◽

Skin Cell

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Differences in Virulence of Marine and Freshwater Isolates of Viral Hemorrhagic Septicemia Virus In Vivo Correlate with In Vitro Ability To Infect Gill Epithelial Cells and Macrophages of Rainbow Trout (Oncorhynchus mykiss)

Journal of Virology ◽

10.1128/jvi.01009-08 ◽

2008 ◽

Vol 82 (21) ◽

pp. 10359-10365 ◽

Cited By ~ 31

Author(s):

Bjørn E. Brudeseth ◽

Helle F. Skall ◽

Øystein Evensen

Keyword(s):

Rainbow Trout ◽

Epithelial Cells ◽

Oncorhynchus Mykiss ◽

Time Course ◽

Primary Cultures ◽

Head Kidney ◽

Viral Hemorrhagic Septicemia Virus ◽

Viral Hemorrhagic Septicemia

ABSTRACT Two strains of viral hemorrhagic septicemia virus (VHSV) with known different virulence characteristics in vivo were studied (by a time course approach) for their abilities to infect and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout (RBT; Oncorhynchus mykiss). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim being to determine any correlation between in vivo virulence and in vitro infectivity. The in vitro studies showed that the fw-VHSV isolate infected and caused a cytotoxic effect in monolayers of GEC (demonstrating virulence) at an early time point (2 h postinoculation) and that the same virus strain had translocated over a confluent, polarized GEC layer by 2 h postinoculation. The marine isolate did not infect monolayers of GEC, and delayed translocation across polarized GEC was seen by 48 h postinoculation. Primary cultures of head kidney macrophages were also infected with fw-VHSV, with a maximum of 9.5% virus-positive cells by 3 days postinfection, while for the ma-VHSV strain, only 0.5% of the macrophages were positive after 3 days of culture. In vivo studies showed that the fw-VHSV strain was highly virulent for RBT fry and caused high mortality, with classical features of viral hemorrhagic septicemia. The ma-VHSV showed a very low level of virulence (only one pool of samples from the dead fish was VHSV positive). This study has shown that the differences in virulence between marine and freshwater strains of VHSV following the in vivo infection of RBT correlate with in vitro abilities to infect primary cultures of GEC and head kidney macrophages of the same species.

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