Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins.

Exon-Intron Circular RNA circRNF217 Promotes Innate Immunity and Antibacterial Activity in Teleost Fish by Reducing miR-130-3p Function.

Circular RNA (circRNA) is produced by splicing head to tail and is widely distributed in multicellular organisms, and circRNA reportedly can participate in various cell biological processes. In this study, we discovered a novel exon-intron circRNA derived from probable E3 ubiquitin-protein ligase RNF217 (RNF217) gene, namely, circRNF217, which was related to the antibacterial responses in teleost fish. Results indicated that circRNF217 played essential roles in host antibacterial immunity and inhibited the Vibrio anguillarum invasion into cells. Our study also found a microRNA miR-130-3p, which could inhibit antibacterial immune response and promote V. anguillarum invasion into cells by targeting NOD1. Moreover, we also found that the antibacterial effect inhibited by miR-130-3p could be reversed with circRNF217. In mechanism, our data revealed that circRNF217 was a competing endogenous RNA of NOD1 by sponging miR-130-3p, leading to activation of the NF-κB pathway and then enhancing the innate antibacterial responses. In addition, we also found that circRNF217 can promote the antiviral response caused by Siniperca chuatsi rhabdovirus through targeting NOD1. Our study provides new insights for understanding the impact of circRNA on host-pathogen interactions and formulating fish disease prevention to resist the severely harmful V. anguillarum infection.

N Protein of Viral Hemorrhagic Septicemia Virus Suppresses STAT1-Mediated MHC Class II Transcription to Impair Antigen Presentation in Sea Perch, Lateolabrax japonicus.

Upon virus invasion of the host, APCs process Ags to short peptides for presentation by MHC class II (MHC-II). The recognition of virus-derived peptides in the context of MHC-II by CD4+ T cells initiates the adaptive immune response for virus clearance. As a survival instinct, viruses have evolved mechanisms to evade Ag processing and presentation. In this study, we discovered that IFN-γ induced endogenous MHC-II expression by a sea perch brain cell line through the STAT1/IFN regulatory factor 1 (IRF1)/CIITA signaling pathway. Furthermore, viral hemorrhagic septicemia virus infection significantly inhibited the IFN-γ-induced expression of IRF1, CIITA, MHC-II-α, and MHC-II-ß genes. By contrast, although STAT1 transcript was upregulated, paradoxically, the STAT1 protein level was attenuated. Moreover, overexpression analysis revealed that viral hemorrhagic septicemia virus N protein blocked the IFN-γ-induced expression of IRF1, CIITA, MHC-II-α, and MHC-II-ß genes, but not the STAT1 gene. We also found out that N protein interacted with STAT1 and enhanced the overall ubiquitination level of proteins, including STAT1 in Lateolabrax japonicus brain cells. Enhanced ubiquitination of STAT1 through K48-linked ubiquitination led to its degradation through the ubiquitin-proteasome pathway, thereby inhibiting the biological function of STAT1. Our study suggests that aquatic viruses target Ag presentation in lower vertebrates for immune evasion as do mammalian viruses.

The mTOR/PGC-1α/SIRT3 Pathway Drives Reductive Glutamine Metabolism to Reduce Oxidative Stress Caused by ISKNV in CPB Cells.

Under oxidative stress, viruses prefer glycolysis as an ATP source, and glutamine is required as an anaplerotic substrate to replenish the TCA cycle. Infectious spleen and kidney necrosis virus (ISKNV) induces reductive glutamine metabolism in the host cells. Here we report that ISKNV infection the increased NAD+/NADH ratio and the gene expression of glutaminase 1 (GLS1), glutamate dehydrogenase (GDH), and isocitrate dehydrogenase (IDH2) resulted in the phosphorylation and activation of mammalian target of rapamycin (mTOR) in CPB cells. Inhibition of mTOR signaling attenuates ISKNV-induced the upregulation of GLS1, GDH, and IDH2 genes expression, and exhibits significant antiviral activity. Moreover, the expression of silent information regulation 2 homolog 3 (SIRT3) in mRNA level is increased to enhance the reductive glutamine metabolism in ISKNV-infected cells. And those were verified by the expression levels of metabolic genes and the activities of metabolic enzymes in SIRT3-overexpressed or SIRT3-knocked down cells. Remarkably, activation of mTOR signaling upregulates the expression of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene, leading to increased expression of SIRT3 and metabolic genes. These results indicate that mTOR signaling manipulates reductive glutamine metabolism in ISKNV-infected cells through PGC-1α-dependent regulation of SIRT3. Our findings reveal new insights on ISKNV-host interactions and will contribute new cellular targets to antiviral therapy. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of farmed fish disease that has caused huge economic losses in fresh and marine fish aquaculture. The redox state of cells is shaped by virus into a favorable microenvironment for virus replication and proliferation. Our previous study demonstrated that ISKNV replication induced glutamine metabolism reprogramming, and it is necessary for the ISKNV multiplication. In this study, the mechanistic link between the mTOR/PGC-1α/SIRT3 pathway and reductive glutamine metabolism in the ISKNV-infected cells was provided, which will contribute new insights into the pathogenesis of ISKNV and antiviral treatment strategies.

Novel picornavirus (family Picornaviridae) from freshwater fishes (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) in Hungary.

In this study, a novel picornavirus (perchPV/M9/2015/HUN, GenBank accession no. MW590713) was detected in eight (12.9%) out of 62 faecal samples collected from three (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) out of 13 freshwater fish species tested and genetically characterized using viral metagenomics and RT-PCR methods. The complete genome of perchPV/M9/2015/HUN is 7,741 nt long, excluding the poly(A) tail, and has the genome organization 5'UTRIRES-?/P1(VP0-VP3-VP1)/P2(2A1NPG↓P-2A2H-box/NC-2B-2C)/P3(3A-3BVPg-3CPro-3DPol)/3'UTR-poly(A). The P1, 2C, and 3CD proteins had 41.4%, 38.1%, and 47.3% amino acid sequence identity to the corresponding proteins of Wenling lepidotrigla picornavirus (MG600079), eel picornavirus (NC_022332), and Wenling pleuronectiformes picornavirus (MG600098), respectively, as the closest relatives in the genus Potamipivirus. PerchPV/M9/2015/HUN represents a potential novel fish-origin species in an unassigned genus in the family Picornaviridae.

Comparative transcriptome analysis reveals the role of p53 signalling pathway during red-spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells.

Nervous necrosis virus (NNV) is one of the fish pathogens that have caused mass mortalities of many marine and freshwater fishes in the world. To better comprehend the molecular immune mechanism of sea perch (Lateolabrax japonicus) against NNV infection, the comparative transcriptome analysis of red-spotted grouper nervous necrosis virus (RGNNV)-infected or mock-infected L. japonicus brain (LJB) cells was performed via RNA sequencing technology. Here, 1,969 up-regulated genes and 9,858 down-regulated genes, which were widely implicated in immune response pathways, were identified. Furthermore, we confirmed that p53 signalling pathway was repressed at 48 hr post-RGNNV infection, as indicated by up-regulation of Mdm2 and down-regulation of p53 and its downstream target genes, including Bax, Casp8 and CytC. Overexpression of L. japonicus p53 (Ljp53) significantly inhibited RGNNV replication and up-regulated the expression of apoptosis-related genes, whereas the down-regulation caused by pifithrin-α led to the opposite effect, suggesting Ljp53 might promote cell apoptosis to repress virus replication. Luciferase assay indicated that Ljp53 could enhance the promoter activities of zebrafish interferon (IFN)1, indicating that Ljp53 could exert its anti-RGNNV activities by enforcing the type I IFN response. This study revealed the potential antiviral role of p53 during NNV infection.

Isolation, characterization and molecular identification of a novel aquareovirus that infects endangered fountain darter Etheostoma fonticola.

The fountain darter Etheostoma fonticola (FOD) is a federally endangered fish listed under the US Endangered Species Act. Here, we identified and characterized a novel aquareovirus isolated from wild fountain darters inhabiting the San Marcos River. This virus was propagated in Chinook salmon embryo (CHSE)-214, rainbow trout gonad-2 and fathead minnow cells at 15°C. The epithelioma papulosum cyprini cell line was refractory at all temperatures evaluated. High throughput sequencing technologies facilitated the complete genome sequencing of this virus utilizing ribosomal RNA-depleted RNA extracted from infected CHSE-214 cells. Conventional PCR primer sets were developed for the detection and confirmation of this virus to assist diagnostic screening methods. Phylogenetic analysis suggests this virus belongs to the Aquareovirus A genus. This research provides requisite initial data critical to support hatchery and refugia biosecurity measures for this endangered species.

Opportunistic disease in yellow perch in response to decadal changes in the chemistry of oil sands-affected waters.

Oil sands-affected water from mining must eventually be incorporated into the reclaimed landscape or treated and released. However, this material contains petrogenic organic compounds, such as naphthenic acids and traces of polycyclic aromatic hydrocarbons. This has raised concerns for impacts of oil sands process-affected waters on the heath of wildlife and humans downstream of receiving environments. The objective of this study was to evaluate the temporal association of disease states in fish with water chemistry of oil sands-affected waters over more than a decade and determine the pathogens associated with disease pathologies. Yellow perch (Perca flavescens) captured from nearby lakes were stocked into two experimental ponds during 1995-1997 and 2008-2010. South Bison Pond is a drainage basin that has received unextracted oil sands-contaminated material. Demonstration Pond is a constructed pond containing mature fine tailings capped with fresh water. Two disease pathologies, fin erosion for which a suspected bacterial pathogen (Acinetobacter Iwoffi) is identified, and lymphocystis (confirmed using a real-time PCR) were associated with oil sands-affected water exposure. From 1995 to 1997 pathologies were most prevalent in the South Bison Pond; however, from 2008 to 2009, disease was more frequently observed in the Demonstration Pond. CYP1A activity was 3-16 fold higher in fish from experimental ponds as compared to reference populations and this pattern was consistent across all sampling years. Bile fluorescence displayed a gradient of exposure with experimental ponds being elevated over local perch populations. Naphthenic acids decreased in the Bison Pond from approximately 12 mg/L to <4 mg/L while naphthenic acids increased in the Demonstration Pond from 6 mg/L to 12 mg/L due to tailings densification. Temporal changes in naphthenic acid levels, CYP1A activity and bile fluorescent metabolites correlate positively with incidence of disease pathologies whereas all inorganic water quality changes (major ions, pH, metals) were not associated with disease responses.

The pathogenicity and biological features of Santee-Cooper Ranaviruses isolated from Chinese perch and snakehead fish.

Ranavirus has become a noticeable threat to both farmed and natural populations of fish and amphibians. Herein, we reported that 3 strains of novel viruses, designated as ScRIV-GM-20150902, CmRIV-XT-20150917 and ScRIV-ZS-20151201, were isolated from diseased Chinese perch and snakehead fish in China. Efficient propagation of these isolates were determined in Chinese perch brain (CPB) cell line by the means of cytopathic effect observation, PCR amplification and electron microscopy observation. And their viral titers in CPB cells reached 108.13 TCID50 ml-1, 107.71 TCID50 ml-1 and 107.94 TCID50 ml-1, respectively. While the challenge experiment results showed that 3 isolates resulted in 100% mortality of Chinese perch after virus infection. Electron microscopy analysis showed that two kinds of viral inclusion bodies (intracytoplasmic and intranuclear inclusion body) were observed in infected CPB cells. Sequence alignment and phylogenetic analysis of major capsid protein gene sequences of isolates revealed that these isolates belonged to the species Santee-Cooper Ranavirus.

Co-infections of infectious spleen and kidney necrosis virus and Siniperca chuatsi rhabdovirus in Chinese perch (Siniperca chuatsi).

In spite of the quite common co-infections of viruses in the cultured fish, most of the previous studies have just simply focused on the infection of a single pathogen. In this report, we observed that about 13% of cultured Chinese perch have been co-infected by infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Furthermore, Chinese perch could co-infected by ISKNV and SCRV by intraperitoneally injection with the two viruses. Interestingly, we revealed that the two viruses could even co-infect a single cell of Chinese perch in vivo and a single Chinese perch brain cells (CPB) cell in vitro. The dynamic co-infected viruses loads in the different tissues of Chinese perch showed dependent. When CPB cells were infected with the same 10 MOI of SCRV and ISKNV, the replication of SCRV overwhelmed the replication of ISKNV. When the MOI of ISKNV (10 MOI) was 10,000 times of MOI of SCRV (0.001 MOI), the dynamic virus loads of the two viruses in CPB cells indicated that co-infections could synergistically stimulate both viruses replication at the late time points but not at early time points. The co-infections of ISKNV and SCRV in the cultured Chinese perch will shed a new light on the prevention of the viral diseases of Chinese perch. The development of multivalent vaccine which could be effective for preventing against the co-infections of the viruses is highly needed.

Establishment and characterization of a brain cell line from sea perch, Lateolabrax japonicus.

A continuous cell line, designated LJB, derived from the brain of sea perch (Lateolabrax japonicus) was established. LJB cells have been subcultured for more than 60 times in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS) since the initial primary culture. LJB cells exhibited maximum growth rate at 28°C in DMEM supplemented with 20% FBS. Cytogenetic analysis indicated that the modal chromosome number was 48, which was identical with the chromosome number of embryonic stem-like cells of sea perch. Comparison of the 18S ribosomal RNA gene sequences of LJB cells and sea perch confirmed that LJB cells originated from sea perch. After transfected with pEGFP-N3 plasmid, LJB cells showed a transfection efficiency of about 40% which was indicated by the percentage of cells expressing green fluorescence protein, indicating the potential application of LJB cells in gene expression studies. Cytopathic effect was clearly observed, and RNA-dependent RNA polymerase gene was also detected in LJB cells post red-spotted grouper nervous necrosis virus (RGNNV) infection. Furthermore, virus replication was confirmed by quantitative RT-PCR, virus titer, and transmission electron microscopy assay in RGNNV-infected LJB cells. The LJB cell line might be used as an ideal in vitro tool for analyzing and understanding the mechanisms of nervous necrosis virus-host interaction.

Glutamine and glutaminolysis are required for efficient replication of infectious spleen and kidney necrosis virus in Chinese perch brain cells.

Viruses rely on host cellular metabolism for energy and macromolecule synthesis during their replication. Infectious spleen and kidney necrosis virus (ISKNV) causes significant economic losses in the Chinese perch (Siniperca chuatsi) industry worldwide. However, little is known about the relationship between ISKNV replication and cellular metabolism. Using transcriptomic analysis, we observed that glutamine metabolism in Chinese perch brain (CPB) cells is altered during ISKNV infection. Moreover, ISKNV replication was decreased in CPB cells cultured in the glutamine-depleted medium. ISKNV replication was also inhibited in CPB cells cultured in the presence of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (an inhibitor of glutaminase), (-)-epigallocatechinmo nogallate (an inhibitor of glutamate dehydrogenase) or L-buthionine sulfoximine (an inhibitor of glutathione synthesis). However, virus replication was rescued by the addition of multiple tricarboxylic acid cycle intermediates, ATP, or glutathione reduced ethyl ester. ATP and reduced glutathione/oxidized glutathione levels were increased in CPB cells infected with ISKNV, but were decreased in CPB cells cultured in glutamine-depleted medium. These results indicate ISKNV infection induces glutaminolysis to accommodate the biosynthetic and energy needs for its efficient virus replication.

Integrated spatial health assessment of yellow perch (Perca flavescens) populations from the St. Lawrence River, Quebec, Canada) part A: physiological parameters and pathogen assessment.

A multi-disciplinary approach was used to evaluate the health of yellow perch (Perca flavescens) in the St. Lawrence River (Quebec, Canada), which is experiencing a severe population decline in the downstream portion of the river. Physiological parameters, liver alterations, trace metal concentrations, parasite prevalence and abundance, stable isotope composition, and the presence/absence of the viral hemorragic septicemia virus (VHSV) were evaluated in perch collected at six sites along the river: Lake St. François, Lake St. Louis (north and south), Beauregard Island, and Lake St. Pierre (north and south). Trace metal concentrations in surface water were higher in Lake St. Louis and downstream of a major urban wastewater treatment plant discharge, indicating that this effluent was a significant source of Cu, As, Ag, Zn, and Cd. Levels of Pb in surface water exceeded thresholds for the protection of aquatic life in Lake St. Louis and were negatively correlated with body condition index in this lake. In Lake St. Pierre, Cu, Ag, and Cd bioaccumulated significantly in perch liver and lower body condition index and greater liver damage were observed compared to upstream sites. Parasite analyses indicated a higher abundance of metacercariae of the trematodes Apophallus brevis and Diplostomum spp. in Lake St. Louis, and VHSV was not detected in the liver of yellow perch for all studied sites. Overall, results suggested that the global health of yellow perch from Lake St. Pierre is lower compared to upstream studied sites, which could contribute to the documented population collapse at this site.

Susceptibility of Chinese Perch Brain (CPB) Cell and Mandarin Fish to Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) Infection.

Nervous necrosis virus (NNV) is the causative agent of viral encephalopathy and retinopathy (VER), a neurological disease responsible for high mortality of fish species worldwide. Taking advantage of our established Chinese perch brain (CPB) cell line derived from brain tissues of Mandarin fish (Siniperca chuatsi), the susceptibility of CPB cell to Red-Spotted Grouper nervous necrosis virus (RGNNV) was evaluated. The results showed that RGNNV replicated well in CPB cells, resulting in cellular apoptosis. Moreover, the susceptibility of Mandarin fish to RGNNV was also evaluated. Abnormal swimming was observed in RGNNV-infected Mandarin fish. In addition, the cellular vacuolation and viral particles were also observed in brain tissues of RGNNV-infected Mandarin fish by Hematoxylin-eosin staining or electronic microscopy. The established RGNNV susceptible brain cell line from freshwater fish will pave a new way for the study of the pathogenicity and replication of NNV in the future.

Endogenous grouper and barramundi Mx proteins facilitated the clearance of betanodavirus RNA-dependent RNA polymerase.

This study confirmed that the infection of nervous necrosis virus (NNV), belonging to the betanodavirus, can induce the expression of endogenous Mx in grouper fin-3 (GF-3), grouper brain (cGB), and barramundi brain (cBB) cells, but not in grouper fin-1 (GF-1) cells. In a co-sedimentation assay, RdRp appeared in the mitochondrial pellet of GF-1 cells without endogenous Mx expression. However, in GF-3, cGB, and cBB cells, RdRp was detected in the nuclear pellet accompanied by endogenous Mx. By immunostaining, RdRp was found to colocalize with not only endogenous Mx but also lysosomes and monodansylcadaverine (MDC)-labeled autophagic vacuoles. In GF-1 cells, the RdRp level continuously increased during 24-72 h post infection (hpi). When endogenous Mx expressed during 24-72 hpi in virus-infected GF-3, cGB, and cBB cells, the RdRp level peaked at 24 hpi but decreased at 48-72 hpi. The degradation of RdRp could be suppressed by treatment with 3-methyladenine (3MA), NH4Cl, and Mx-specific siRNA respectively. After poly I:C transfection, the endogenous Mx level peaked at 3 days post transfection (dpt) and then spontaneously decreased at 5-7 dpt. The poly I:C-indued Mx also colocalized with MDC-labeled autophagic vacuoles at 3 dpt, and its degradation could be inhibited by 3MA or NH4Cl treatments. Therefore, the anti-NNV mechanism of endogenous grouper and barramundi Mx is suggested to sequester RdRp for degradation through autophagy and lysosomes.

First isolation of a rhabdovirus from perch Perca fluviatilis in Switzerland.

Perca fluviatilis is a fish species of increasing interest to the Swiss fish farming industry. In recent years, recirculation systems have been specifically set up to increase production. In one of these farms, abnormal spiral swimming associated with elevated mortalities occurred in repeated batches of imported perch shortly after stocking on several occasions. No bacterial or parasitic etiology was detected, but a virus grown in bluegill fry (BF-2) cells was identified as perch rhabdovirus. Subsequent investigations of other samples suggested a viral tropism for the central nervous system (CNS). Phylogenetic analysis of the partial N and entire G gene sequences positioned this isolate in genogroup C of the species Perch rhabdovirus, with high nucleotide and amino acid (aa) sequence identities with the DK5533 strain isolated in Denmark in 1989. Comparative studies using other closely related isolates allowed the distinction of 2 serological patterns among perch rhabdoviruses and the identification of a proline substitution by a serine in position 147 of the glycoprotein potentially involved in antigenic differentiation. Even if perch imported onto the farm tested negative by virus isolation prior to transport, they may have been the origin of this outbreak since CNS tissue was not included in the samples that were analyzed. Another possibility might be a sub-clinical infection with a viral load in resident fish too low to be detected. This study reports the first isolation of a perch rhabdovirus in Switzerland, and emphasizes the necessity of optimizing diagnostic tools that facilitate better control of the risks associated with fish translocation.

Characterisation of the genomes of four putative vesiculoviruses: tench rhabdovirus, grass carp rhabdovirus, perch rhabdovirus and eel rhabdovirus European X.

The complete coding sequences were determined for four putative vesiculoviruses isolated from fish. Sequence alignment and phylogenetic analysis based on the predicted amino acid sequences of the five main proteins assigned tench rhabdovirus and grass carp rhabdovirus together with spring viraemia of carp and pike fry rhabdovirus to a lineage that was distinct from the mammalian vesiculoviruses. Perch rhabdovirus, eel virus European X, lake trout rhabdovirus 903/87 and sea trout virus were placed in a second lineage that was also distinct from the recognised genera in the family Rhabdoviridae. Establishment of two new rhabdovirus genera, "Perhabdovirus" and "Sprivivirus", is discussed.

Expression kinetics of key genes in the early innate immune response to Great Lakes viral hemorrhagic septicemia virus IVb infection in yellow perch (Perca flavescens).

The recently discovered strain of viral hemorrhagic septicemia virus, VHSV-IVb, represents an example of the introduction of an extremely pathogenic rhabdovirus capable of infecting a wide variety of new fish species in a new host-environment. The goal of the present study was to delineate the expression kinetics of key genes in the innate immune response relative to the very early stages of VHSV-IVb infection using the yellow perch (Perca flavescens) as a model. Administration of VHSV-IVb by IP-injection into juvenile yellow perch resulted in 84% cumulative mortality, indicating their high susceptibility to this disease. In fish sampled in the very early stages of infection, a significant up-regulation of Mx gene expression in the liver, as well as IL-1ß and SAA activation in the head kidney, spleen, and liver was directly correlated to viral load. The potential down-regulation of Mx in the hematopoietic tissues, head kidney and spleen, may represent a strategy utilized by the virus to increase replication.

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.