Gene Diversification of an Emerging Pathogen: A Decade of Mutation in a Novel Fish Viral Hemorrhagic Septicemia (VHS) Substrain since Its First Appearance in the Laurentian Great Lakes.

Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus, which causes one of the world's most serious fish diseases, infecting >80 freshwater and marine species across the Northern Hemisphere. A new, novel, and especially virulent substrain-VHSv-IVb-first appeared in the Laurentian Great Lakes about a decade ago, resulting in massive fish kills. It rapidly spread and has genetically diversified. This study analyzes temporal and spatial mutational patterns of VHSv-IVb across the Great Lakes for the novel non-virion (Nv) gene that is unique to this group of novirhabdoviruses, in relation to its glycoprotein (G), phosphoprotein (P), and matrix (M) genes. Results show that the Nv-gene has been evolving the fastest (k = 2.0 x 10-3 substitutions/site/year), with the G-gene at ~1/7 that rate (k = 2.8 x 10-4). Most (all but one) of the 12 unique Nv- haplotypes identified encode different amino acids, totaling 26 changes. Among the 12 corresponding G-gene haplotypes, seven vary in amino acids with eight total changes. The P- and M- genes are more evolutionarily conserved, evolving at just ~1/15 (k = 1.2 x 10-4) of the Nv-gene's rate. The 12 isolates contained four P-gene haplotypes with two amino acid changes, and six M-gene haplotypes with three amino acid differences. Patterns of evolutionary changes coincided among the genes for some of the isolates, but appeared independent in others. New viral variants were discovered following the large 2006 outbreak; such differentiation may have been in response to fish populations developing resistance, meriting further investigation. Two 2012 variants were isolated by us from central Lake Erie fish that lacked classic VHSv symptoms, having genetically distinctive Nv-, G-, and M-gene sequences (with one of them also differing in its P-gene); they differ from each other by a G-gene amino acid change and also differ from all other isolates by a shared Nv-gene amino acid change. Such rapid evolutionary differentiation may allow new viral variants to evade fish host recognition and immune responses, facilitating long-time persistence along with expansion to new geographic areas.

Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): enhanced quality control with internal standards.

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.

Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv).

Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus that causes one of the most important finfish diseases, affecting over 70 marine and freshwater species. It was discovered in European cultured fish in 1938 and since has been described across the Northern Hemisphere. Four strains and several substrains have been hypothesized, whose phylogenetic relationships and evolutionary radiation are evaluated here in light of a quasispecies model, including an in-depth analysis of the novel and especially virulent new substrain (IVb) that first appeared in the North American Laurentian Great Lakes in 2003. We analyze the evolutionary patterns, genetic diversity, and biogeography of VHSv using all available RNA sequences from the glycoprotein (G), nucleoprotein (N), and non-virion (Nv) genes, with Maximum Likelihood and bayesian approaches. Results indicate that the G gene evolves at an estimated rate of µ=2.58×10(-4) nucleotide substitutions per site per year, the N gene at µ=4.26×10(-4), and Nv fastest at µ=1.25×10(-3). Phylogenetic trees from the three genes largely are congruent, distinguishing strains I-IV as reciprocally monophyletic with high bootstrap and posterior probability support. VHSv appears to have originated from a marine ancestor in the North Atlantic Ocean, diverging into two primary clades: strain IV in North America (the Northwestern Atlantic Ocean), and strains I-III in the Northeastern Atlantic region (Europe). Strain II may comprise the basal group of the latter clade and diverged in Baltic Sea estuarine waters; strains I and III appear to be sister groups (according to the G and Nv genes), with the former mostly in European freshwaters and the latter in North Sea marine/estuarine waters. Strain IV is differentiated into three monophyletic substrains, with IVa infecting Northeastern Pacific salmonids and many marine fishes (with 44 unique G gene haplotypes), IVb endemic to the freshwater Great Lakes (11 haplotypes), and a newly-designated IVc in marine/estuarine North Atlantic waters (five haplotypes). Two separate substrains independently appeared in the Northwestern Pacific region (Asia) in 1996, with Ib originating from the west and IVa from the east. Our results depict an evolutionary history of relatively rapid population diversifications in star-like patterns, following a quasispecies model. This study provides a baseline for future tracking of VHSv spread and interpreting its evolutionary diversification pathways.