Viral Hemorrhagic Septicemia Virus Infection Research Articles

RNA Aptamers Inhibit the Growth of the Fish Pathogen Viral Hemorrhagic Septicemia Virus (VHSV)

Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

Genetic Resistance to Rhabdovirus Infection in Teleost Fish Is Paralleled to the Derived Cell Resistance Status

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction - that was not observed in the susceptible cells - and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses.

Fugu double U6 promoter–driven long double-stranded RNA inhibits proliferation of viral hemorrhagic septicemia virus (VHSV) in fish cell lines

A long double-stranded RNA (dsRNA)-producing vector driven by fugu double U6 promotors, in which the two promoters were arranged in a head-to-head fashion, was newly constructed. To determine whether the DNA-vector-based long dsRNAs can induce sequence-specific RNA interference (RNAi), Epithelioma papulosum cyprini (EPC) cells and chinook salmon embryonic (CHSE-214) cells were transfected with the long dsRNA vector targeting the G gene of VHSV, and its effect on expression of the G gene and viral proliferation was investigated. The sequence-specific inhibitory effect was further confirmed by analysis of interferon (IFN)-triggered Mx1 gene expression and cross-protection against infectious hematopoietic necrosis virus (IHNV). The fugu double U6 promoter-driven vector successfully produced long dsRNAs in EPC cells, a system that allows continuous production of long dsRNAs in transfected cells. The plasmid-based long dsRNAs targeting the VHSV G gene effectively suppressed G gene expression, but control dsRNAs targeting the EGFP gene did not. Furthermore, there was no significant difference in Mx gene expression between cells transfected with the long dsRNA-producing vector and those transfected with the control empty vector. These results suggest that G gene expression was suppressed not by type-I-IFN-mediated nonspecific inhibition but in a sequence-specific manner. Both EPC and CHSE-214 cells transfected with plasmids producing long dsRNAs targeting the VHSV G gene were protected against VHSV infection but were not protected against IHNV infection, suggesting sequence-specific RNAi-mediated inhibition of viral proliferation. In conclusion, we show, for the first time, long-dsRNA-mediated RNAi in fish cells. The DNA-vector-based long dsRNAs may provide an efficient tool for analysis of gene function in fish cells without preliminary burdensome work for selection of effective siRNA clones, and it may be applied as an antiviral measure in cultured fish.

Ontogeny of anti-viral hemorrhagic septicemia virus (VHSV) immunity in developing Japanese flounder

We examined the ability of developing Japanese flounder (Paralichthys olivaceus) to acquire protective immunity after exposure to viral hemorrhagic septicemia virus (VHSV). Juveniles measuring 9.8cm average body length were not susceptible to infection with VHSV at 20°C, while the smaller fish were susceptible. Mortality was not observed after secondary infection at 15°C in the 9.8cm cohort that had previously been exposed to the virus at 20°C, while the smaller fish were susceptible to secondary infection. The expression of interferon (IFN)-related genes was shown to be better developed in larger fish upon virus infection and basal expression levels of the virus recognition proteins were higher in larger fish. Virus-specific antibody was detected in the larger fish, but not in smaller fish. These data indicate that the largest juvenile (9.8cm) acquired immunity against VHSV infection at the first virus challenge, but smaller fish did not. The anti-viral immune system in the Japanese flounder matures when juveniles reach approximately 10cm.

Predictive factors and viral genetic diversity for viral hemorrhagic septicemia virus infection in Lake Ontario and the St. Lawrence River

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of a devastating disease in fish, viral hemorrhagic septicemia (VHS), which has recently spread to the Laurentian Great Lakes. In this paper, we report the results of infection surveillance conducted during 2009 for VHSV with a focus on yellow perch (Perca flavescens) and round goby (Neogobius melanostomus). We collected 1928 fish representing eight species at nine sites in Lake Ontario and the St. Lawrence River and found fish positive for VHSV by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) at every site. Prevalence of virus-positive fish at individual sites ranged from 0.7% to 27.5%. Yellow perch were significantly less likely to test positive than round goby in this study. In logistic regression models, maturity was a significant predictor of testing positive in yellow perch, but no evaluated factor was a significant predictor of testing positive in round goby. Virus isolation in cell culture was successful in seven out of 37 high titer fish samples, and partial glycoprotein gene sequences obtained from these virus isolates showed an increase in genetic diversity relative to the previously known diversity of Laurentian Great Lakes VHSV genotype IVb. These results highlight the importance of continued movement restrictions and surveillance for VHSV in wild fish in the Laurentian Great Lakes as well as suggest future work to further elucidate the infection dynamics of this virus in wild fish populations.

Induction of anti-viral genes during acute infection with Viral hemorrhagic septicemia virus (VHSV) genogroup IVa in Pacific herring ( Clupea pallasii)

Infection with the aquatic rhabdovirus Viral hemorrhagic septicemia virus (VHSV) genogroup IVa results in high mortality in Pacific herring ( Clupea pallasii) and is hypothesized to be a potential limiting factor for herring recovery. To investigate anti-viral immunity in the Pacific herring, four immune response genes were identified: the myxovirus resistance ( Clpa-Mx), a major histocompatibility complex IB (named Clpa-UAA.001), the inducible immunoproteosome subunit 9 ( Clpa-PSMB9) and the neutrophil chemotactic factor ( Clpa-LECT2). Reverse transcriptase quantitative PCR (RT-qPCR) assays were developed based on these gene sequences to investigate the host immune response to acute VHSV infection following both injection and immersion challenge. Virus levels were measured by both plaque assay and RT-qPCR and peaked at day 6 during the 10-day exposure period for both groups of fish. The interferon stimulated genes ( Clpa-Mx, −UAA.001, and −PSMB9) were significantly up-regulated in response to VHSV infection at both 6 and 10 days post-infection in both spleen and fin. Results from this study indicate that Pacific herring mount a robust, early antiviral response in both fin and spleen tissues. The immunological tools developed in this study will be useful for future studies to investigate antiviral immunity in Pacific herring.

Effect of CpG-ODNs belonging to different classes on resistance of olive flounder ( Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus (Ciliata; Scuticociliatia) infections

The objective of this study was to investigate the effects of different classes of CpG oligodeoxynucleotides (CpG-ODNs) on resistance of olive flounder ( Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus infections. Fish administered CpG-ODN 2216 (A-class ODN) showed the highest resistance against VHSV infection and the strongest expression of Mx and ISG15 genes. Fish administered CpG-ODN 2395 (C-class ODN) showed intermediate resistance against VHSV between ODN 1668 and ODN 2216. Whereas, olive flounder intraperitoneally (i.p.) injected with CpG-ODN 1668 (B-class ODN) showed no enhanced resistance against VHSV challenge and no significant increase in expression of Mx and ISG15 genes. In contrast, in the M. avidus challenge experiment, fish injected with CpG-ODN 1668 showed the highest survival rate and the strongest scuticocidal activity of serum. Similar to the results in the VHSV infection experiment, administration of CpG-ODN 2395 elicited secondly higher survival rate against M. avidus challenge. Although fish administered CpG-ODN 2216 eventually showed 100% mortality by M. avidus challenge, the cumulative mortality until 2 weeks post-challenge was similar to the group of fish injected with CpG-ODN 1668. The present results suggest that CpG-ODNs belonging to different classes have different abilities to increase resistance against different pathogens in olive flounder. Therefore, selection of a CpG-ODN appropriate to the characteristics of a certain pathogen is crucial to induce immune responses that are optimal for defense against the pathogen.

Live vaccine of viral hemorrhagic septicemia virus (VHSV) for Japanese flounder at fish rearing temperature of 21°C instead of Poly(I:C) administration

The process of "Poly(I:C) immunization" involves immunization of fish with a pathogenic live virus, followed by administration of Poly(I:C), which induces a transient, non-specific antiviral state. As a result, fish in an antiviral state survive the initial immunization with live virus. Moreover, these fish are able to mount a specific protective immune response against the injected pathogenic virus. In the present study, we investigated the optimum temperature for Poly(I:C) immunization of Japanese flounder Paralichthys olivaceus with live viral hemorrhagic septicemia virus (VHSV). It was revealed that the optimum temperature was around at 17°C for Poly(I:C) immunization in Japanese flounder. Furthermore, the protection efficacy of Poly(I:C) immunization was significantly decreased by elevation of fish rearing temperature, and no efficacy was observed at a fish rearing temperature of 25°C. Interestingly, no mortality by VHSV infection was observed in fish reared at 21°C and 25°C even when those fish were not administered Poly(I:C). All of the survivors from the first VHSV-challenge at 21°C were strongly protected from re-challenge with VHSV. However, almost all of the survivors (≥82.6%) from the first challenge at 25°C were lost by the second challenge with VHSV. It was thus concluded that by rearing fish at 21°C and challenging with live VHSV, it is possible to induce strong specific immunity in Japanese flounder without Poly(I:C) administration.

Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in Japanese flounder Paralichthys olivaceus leucocytes during Edwardsiella tarda and viral hemorrhagic septicemia virus infection

Transcriptional changes in the peripheral blood leucocytes (PBL) of Japanese flounder Paralichthys olivaceus challenged by Edwardsiella tarda and viral hemorrhagic septicemia virus (VHSV) were investigated using suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis. First, we constructed an SSH cDNA library using mRNA samples isolated from PBL of P. olivaceus that had been experimentally infected with E. tarda. We then examined the transcriptional changes occurring in the PBL due to E. tarda and VHSV infection using a cDNA microarray produced using clones produced from the SSH library. A total of 565 and 180 cDNA sequences corresponding to mRNA species that are either up- or down-regulated by E. tarda infection were isolated by SSH. While host gene expression responses in response to E. tarda and VHSV infection share several response elements, distinct patterns of gene expression were also observed. Specifically, E. tarda infection enhanced the expression of cell adhesion molecules while VHSV enhanced the expression of interferon and proteasome-related genes. In challenge trials of the two infectious agents, expression profiles of chemokines were also observed to differ. The results indicated that distinguishing between viral and bacterial infection is possible based on the RNA expression profiles of PBL from infected fish.

Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder ( Paralichthys olivaceus)

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder ( Paralichthys olivaceus) and was sampled at 3, 6, and 12 h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1β, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.

Oral immunization of olive flounder ( Paralichthys olivaceus) with recombinant live viral hemorrhagic septicemia virus (VHSV) induces protection against VHSV infection

A recombinant viral hemorrhagic septicemia virus (rVHSV-ΔNV-EGFP) that has enhanced green fluorescent protein (EGFP) gene instead of NV gene was previously generated using reverse genetics technology. In this study, potential of the rVHSV-ΔNV-EGFP to be used as a live oral vaccine candidate was assessed. The presence of the recombinant virus in internal organs of orally administered olive flounder ( Paralichthys olivaceus) was analyzed by semi-quantitative RT-PCR. Although the recombinant VHSV-specific band was detected only when the number of PCR cycle was increased to 35, the band was detected from internal organs, such as kidney, spleen, and liver of fish that were reared at either 15 °C or 20 °C till even 20 days, suggesting that a few orally administered rVHSV-ΔNV-EGFP might be transported to internal organs, and might keep weak replication ability in the organs. VHSV-neutralizing activity was induced by oral immunization of olive flounder with the NV gene knock-out recombinant VHSV not only in skin and intestinal mucus but also in serum, suggesting that mucosal and systemic adaptive immune responses were elicited by oral immunization. In challenge experiment, groups of fish immunized with 10 4, 10 5, and 2 × 10 5 PFU of rVHSV-ΔNV-EGFP/fish showed 25%, 50%, and 70% of relative percent survival (RPS), respectively. The RPSs were elevated to 60%, 75%, and 90% by a boost immunization in fish boost immunized with 10 4, 10 5, and 2 × 10 5 PFU of rVHSV-ΔNV-EGFP, respectively. The cumulative mortality of fish in the control groups was 100%. Conclusionly, the present results demonstrate that the NV gene knock-out recombinant VHSV administered orally to olive flounder can induce dose- and boosting-dependent VHSV-neutralizing antibody in mucus and serum, and can provide a high protection in olive flounder against a virulent VHSV challenge.

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56–110), frg15 (65–250), frg16 (252–450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

Heavy oil exposure induces high moralities in virus carrier Japanese flounder Paralichthys olivaceus

The relationship between chemical exposure and disease outbreak in fish has not been fully defined due to the limitations of experimental systems (model fish and pathogens). Therefore, we constructed a system using the Japanese flounder, Paralichthys olivaceus, and viral haemorrhagic septicemia virus (VHSV), and evaluated it by heavy oil (HO) exposure. The fish were exposed to HO at 0.3, 0.03, 0.003, and 0g/L following VHSV infection at doses of 102.5 or 103.5 tissue culture infectious dose (TCID)50/fish. As a result, groups given the dual stressors showed more than 90% mortality. Although VHSV infection at 102.5 and 103.5 TCID50/fish without HO exposure also induced high mortality, at 68.8% and 81.3%, respectively, HO exposure induced faster and higher mortality in the virus carrier fish, indicating that chemical stressors raise the risk of disease outbreak in fish. The experimental system established in this study could be useful for chemical risk assessment.

Characterization and antiviral function of a cytosolic sensor gene, MDA5, in Japanese flounder, Paralichthys olivaceus

Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24–75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.

Molecular cloning and antiviral activity of IFN-β promoter stimulator-1 (IPS-1) gene in Japanese flounder, Paralichthys olivaceus

The mitochondrial adaptor, IFN-β promoter stimulator-1 (IPS-1), also known as MAVS/VISA/Cardif, plays a key role in the signal transduction of the RIG-1/MDA5 pathway to induce the production of interferons (IFNs) and other cytokines. In the present study, Japanese flounder ( Paralichthys olivaceus) IPS-1 cDNA was cloned from Japanese flounder spleen using PCR-based methods. The full-length cDNA has 2235 nucleotides and encodes a polypeptide of 641 amino acids. The putative Japanese flounder IPS-1 protein contains an N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to other teleost counterparts ranging from 20% to 34%. Semi-quantitative RT-PCR showed that Japanese flounder IPS-1 mRNA was expressed in all tissues examined. The expression level of flounder IPS-1 gene was unchanged in viral hemorrhagic septicemia virus (VHSV)-infected kidney as measured by quantitative real-time PCR (Q-PCR). In addition, Japanese flounder IPS-1-overexpressing cells were protected against hirame rhabdovirus (HIRRV) and VHSV infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral titers. Expression of IFN-inducible genes including Mx, ISG15 and IRF3 were also induced in the IPS-1-overexpressing cells. These results suggest that Japanese flounder IPS-1 is involved in the antiviral immunity as a one of the adaptors in fish IFN-activation pathway.

Comparative susceptibility of representative Great Lakes fish species to the North American viral hemorrhagic septicemia virus Sublineage IVb

The present study compared the susceptibility of representative Laurentian Great Lakes fish species to the emerging viral hemorrhagic septicemia virus (VHSV) Genotype IVb. The median lethal dose of infection by intraperitoneal injection (IP-LD50) was obtained from fish that were experimentally infected with the MI03 index strain of VHSV-IVb. Fish were injected at doses ranging from 7 x 10(7) to 7 x 10(-2) plaque-forming units (pfu) and maintained at 12 +/- 1 degrees C. The infection trials identified species of high, medium, and low susceptibility based on the IP-LD50 values. Pathogenicity of VHSV-IVb was highest in largemouth bass Micropterus salmoides, which resulted in an IP-LD50 of 1.5 x 10(2) pfu, while also demonstrating the clinical diathesis of VHSV-infected fish. The virus was moderately pathogenic in yellow perch Perca flavescens (IP-LD50 of 2.5 x 10(5) pfu), but also showed the classical signs of VHSV infection. Salmonids were the least susceptible to VHSV-IVb with IP-LD50 values of no less than 1.4 x 106 pfu; however, in fish that succumbed to infection, characteristic VHSV lesions were observed. Histopathologic alterations were most profound in gill, skin, muscle, gonads, and liver of largemouth bass and yellow perch, while in salmonids, hemorrhages in the swimbladder and/or degenerative changes in the liver were the most common lesions noticed. VHSV was isolated from infected fish, and its identity was confirmed by the reverse transcriptase polymerase chain reaction. These results highlight the variations among fish species susceptibility to this emerging strain of VHSV and offer insights into the potential impact of VHSV-IVb on the Laurentian Great Lakes fish community.

Pepscan Mapping of Viral Hemorrhagic Septicemia Virus Glycoprotein G Major Lineal Determinants Implicated in Triggering Host Cell Antiviral Responses Mediated by Type I Interferon

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.

Monthly occurrence and prevalence of viral haemorrhagic septicaemia virus (VHSV) in whiting Merlangius merlangus

A field survey was carried out to determine the occurrence and spread of viral haemorrhagic septicaemia virus (VHSV) in whiting Merlangius merlangus (L.), a key prey species in the southeastern Black Sea region. In March, April, September and December 2007 and monthly in 2008, 70 to 105 M. merlangus captured by gill nets were examined in pools of 7 fish per pool (total number of fish = 1603). After determining the seasonal occurrence of VHSV in whiting, 43, 80 and 88 whiting were sampled individually in January, February and March 2009, respectively, to determine age and size of whiting commonly infected with VHSV. The samples were tested in duplicate using BF-2 and EPC cell lines for virus isolation. Positive cultures (those demonstrating cytopathic effect, [CPE]), were tested further with a commercial ELISA kit to confirm that the observed CPE was caused by VHSV. VHSV was detected in M. merlangus only between February and April following a drop in the surface water temperature to 9 degrees C. All infected fish from the sampled whiting were asymptomatic carriers with low levels of virus. The estimated prevalence from pooled samples ranged between 1.35% (2.5 to 97.5 percentiles; 0.04 to 7.32%) to 3.14% (0.36 to 10.95%), whereas prevalence estimated from individual sampling was 7, 5 and 1% in January, February and March, respectively. Individual sampling also revealed that prevalence was higher among young fish (1 to 1.5 yr old) and decreased with increasing age. This is the first report of VHSV infection in whiting in the Black Sea.

Chronic and persistent viral hemorrhagic septicemia virus infections in Pacific herring

Chronic viral hemorrhagic septicemia virus (VHSV) infections were established in a laboratory stock of Pacific herring Clupea pallasii held in a large-volume tank supplied with pathogen-free seawater at temperatures ranging from 6.8 to 11.6 degrees C. The infections were characterized by viral persistence for extended periods and near-background levels of host mortality. Infectious virus was recovered from mortalities occurring up to 167 d post-exposure and was detected in normal-appearing herring for as long as 224 d following initial challenge. Geometric mean viral titers were generally as high as or higher in brain tissues than in pools of kidney and spleen tissues, with overall prevalence of infection being higher in the brain. Upon re-exposure to VHSV in a standard laboratory challenge, negligible mortality occurred among groups of herring that were either chronically infected or fully recovered, indicating that survival from chronic manifestations conferred protection against future disease. However, some survivors of chronic VHS infections were capable of replicating virus upon re-exposure. Demonstration of a chronic manifestation of VHSV infection among Pacific herring maintained at ambient seawater temperatures provides insights into the mechanisms by which the virus is maintained among populations of endemic hosts.

Combining surveillance and expert evidence of viral hemorrhagic septicemia freedom: A decision science approach

The ability to combine evidence streams to establish disease freedom or prioritize surveillance is important for the evaluation of emerging diseases, such as viral hemorrhagic septicemia virus (VHSV) IVb in freshwater systems of the United States and Canada. Waterways provide a relatively unconstrained pathway for the spread of VHSV; and structured surveillance for emerging disease in open systems has many challenges. We introduce a decision framework for estimating VHSV infection probability that draws from multiple evidence streams and addresses challenges associated with the assessment of emerging disease. Using this approach, historical and risk-based evidence, whether empirical or expert-derived, supplement surveillance data to estimate disease probability. Surveillance-based estimates of VHSV prevalence were described using beta distributions. Subjective likelihood ratios (LRs), representing contextual risk, were elicited by asking experts to estimate the predicted occurrence of risk factors among VHSV-affected vs. VHSV-unaffected watersheds. We used the odds form of Bayes’ theorem to aggregate expert and surveillance evidence to predict the risk-adjusted posterior probability of VHSV-infection for given watersheds. We also used LRs representing contextual risk to quantify the time value of past surveillance data. This evidence aggregation model predicts disease probability from the combined assessment of multiple sources of information. The method also provides a flexible framework for iterative revision of disease freedom status as knowledge and data evolve.