Viral Haemorrhagic Septicaemia Virus IVb Research Articles

Impact of feed restriction, chloroquine and deoxynivalenol on viral haemorrhagic septicaemia virus IVb in fathead minnow Pimephales promelas Rafinesque.

Autophagy can markedly alter host response to infectious disease, and several studies have demonstrated that a restricted diet or deoxynivalenol modulates autophagy and reduces mortality of fish due to bacterial disease. The picture is less clear for viral diseases of fish. Duplicate tanks of fathead minnow, Pimephales promelas Rafinesque, were fed a replete diet (control), 100µM chloroquine, 5µM deoxynivalenol, 10% (fasted) or 40% of a replete diet (pair-fed) for 2weeks and then experimentally infected by intraperitoneal injection with 2×105 viral haemorrhagic septicaemia virus IVb. Survival from highest to lowest for the different treatments was as follows: deoxynivalenol (average 43.3%); control (40.0%); pair-fed (35.0%); fasted (33.3%); and chloroquine (21.7%). No treatment significantly altered the survival rate of fathead minnow after VHSV IVb infection when compared to controls; however, the fish fed with chloroquine had significantly lower survival rate than the fish fed deoxynivalenol (p<.05).

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1.

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p<.05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.

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

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.

Regulation of endogenous antigen presentation in response to suboptimal temperatures in a walleye skin fibroblast cell line

A skin fibroblast cell line WE-skin11f from walleye (Sander vitreus) was used to study the impact of temperature (26 °C, 20 °C, 14 °C, or 4 °C) on the transcript levels of genes involved in the endogenous antigen processing and presentation pathway (EAPP), which is an important antiviral pathway of vertebrates. Partial coding sequences were found for 4 previously unidentified walleye EAPP members, calreticulin, calnexin, erp57, and tapasin, and the constitutive transcript levels of these genes in WE-skin11f was unchanged by culture incubation temperature. The viral mimic poly (I:C) and viral haemorrhagic septicaemia virus (VHSV) IVb were used to study possible induction of EAPP transcripts (b2m, mhIa, and tapasin). The walleye cells were exquisitely sensitive to poly (I:C), losing adherence and viability at concentrations greater than 100 ng/mL, particularly at suboptimal temperatures. VHSV IVb viral particles were produced from infected WE-skin11f cells at 20 °C, 14 °C, and 4 °C but with much lower production at 4 °C. Under conditions where their impact on the viability of WE-skin11f cultures was slight, poly (I:C) and VHSV IVb were shown to induce b2m, mhIa, and tapasin transcript°s at 26 °C and 20 °C respectively. However, at 4 °C, the up-regulation of EAPP transcript levels was either delayed or completely impaired when compared to the 26 °C and 20 °C control temperatures of the respective experiments. These in vitro results suggest that suboptimal temperatures may be capable of modulating the regulation of the EAPP in walleye cells during viral infection.

Effect of suboptimal temperature on the regulation of endogenous antigen presentation in a rainbow trout hypodermal fibroblast cell line

Rainbow trout (Oncorhynchus mykiss) face low environmental temperatures over winter months and during extreme low temperature events. Suboptimal temperatures are known to negatively impact the teleost immune system, although there is mixed evidence in rainbow trout as to the effect on the endogenous antigen processing and presentation pathway (EAPP). The EAPP is an important pathway for antiviral defense that involves the presentation of endogenous peptides on the cell surface for recognition by cytotoxic T cells. Using a rainbow trout hypodermal fibroblast (RTHDF) cell line as an in vitro model, we determined that constitutive EAPP transcript levels are not impaired at low temperature, but induction of up-regulation of these transcripts is delayed at the suboptimal temperature following exposure to poly(I:C) or viral haemorrhagic septicaemia virus IVb, which was still able to enter and replicate in the cell line at 4 °C, albeit with reduced efficiency. The delay in the induction of EAPP mRNA level up-regulation following poly(I:C) stimulation coincided with a delay in ifn1 transcript levels and secretion, which is important since interferon-stimulated response elements were identified in the promoter regions of the EAPP-specific members of the pathway, implying that IFN1 is involved in the regulation of these genes. Our results suggest that the ability of rainbow trout to mount an effective immune response to viral pathogens may be lessened at suboptimal temperatures.

Investigation of round goby viral haemorrhagic septicaemia outbreak in New York

Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT-qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.

Pathogenesis of experimental viral hemorrhagic septicemia virus IVb infection in adult sea lamprey (Petromyzon marinus)

Abstract In 2008, viral hemorrhagic septicemia virus (VHSV) genotype IVb, the causative agent of viral hemorrhagic septicemia (VHS) in a wide range of fish, was isolated from sea lamprey (Petromyzon marinus) in the Great Lakes Basin. To better understand the implications of the reported isolation, we utilized four virus detection methods to follow the pathogenesis on an experimental infection of VHSV IVb in 60 wild, adult lamprey. Thirty lamprey were infected by intracoelomic cavity injections with 106 plaque forming units (pfu) of the VHSV IVb MI03 isolate, and 30 control lamprey were injected with sterile culture media. All media-injected controls tested negative by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and cell culture. Twenty-six of the 30 infected lamprey were positive for VHSV by qRT-PCR, and nine were positive by cell culture. In-vivo replication was demonstrated by a significant temporal increase in the mean concentration of VHS viral RNA copy number equivalents in pooled viscera measured by qRT-PCR from day 7 to day 28 and an increase in cell culture positive pools at day 28. Microscopically, infected lamprey did not develop lesions typical of virulent VHSV. Despite this, the virus was identified by immunohistochemistry in multiple organs, most readily in the kidney, where localization progressed from the interstitium to the tubular lumens. The combined results from qRT-PCR, cell culture, histopathology, and immunohistochemistry suggest that VHSV causes a sublethal infection in lamprey characterized by depletion of renal interstitial cells and in-vivo viral replication with maintenance in the brain at later stages of infection.

Complete sequences of 4 viral hemorrhagic septicemia virus IVb isolates and their virulence in northern pike fry.

Four viral hemorrhagic septicemia virus (VHSV) genotype IVb isolates were sequenced, their genetic variation explored, and comparative virulence assayed with experimental infections of northern pike Esox lucius fry. In addition to the type strain MI03, the complete 11183 bp genome of the first round goby Neogobius melanostomus isolate from the St. Lawrence River, and the 2013 and 2014 isolates from gizzard shad Dorosoma cepedianum die-offs in Irondequoit Bay, Lake Ontario and Dunkirk Harbor, Lake Erie were all deep sequenced on an Illumina platform. Mutations documented in the 11 yr since the MI03 index case from Lake St. Clair muskellunge Esox masquinongy showed 87 polymorphisms among the 4 isolates. Twenty-six mutations were non-synonymous and located at 18 different positions within the matrix protein, glycoprotein, non-virion protein, and RNA polymerase genes. The same 4 isolates were used to infect northern pike fry by a single 1 h bath exposure. Cumulative percent mortality varied from 42.5 to 62.5%. VHSV was detected in 57% (41/72) of the survivors at the end of the 21-d trial, suggesting that the virus was not rapidly cleared. Lesions were observed in many of the moribund and dead northern pike, such as hemorrhaging in the skin and fins, as well as hydrocephalus. Mean viral load measured from the trunk and visceral tissues of MI03-infected pike was significantly higher than the quantities detected in fish infected with the most recent isolates of genotype IVb, but there were no differences in cumulative mortality observed.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD+poly I:C, lipopolysaccharide (LPS), LPS+poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P<0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

Viral hemorrhagic septicemia IVb status in the United States: Inferences from surveillance activities and regional context

The United States (U.S.) response to viral hemorrhagic septicemia virus (VHSV) IVb emergence in the Laurentian Great Lakes (GL) included risk-based surveillance for cost-effective decision support regarding the health of fish populations in open systems. All U.S. VHSV IVb isolations to date derive from free-ranging fish from GL States. Most originate in the region designated by US Geological Survey hydrologic unit code (HUC) 04, with the exception of two detections in neighboring Upper Mississippi (HUC 05) and Ohio (HUC 07) regions. For States outside the GL system, disease probability was assessed using multiple evidence sources. None substantiated VHSV IVb absence using surveillance alone, in part due to the limited temporal relevance of data in open systems. However, Bayesian odds risk-based analysis of surveillance and population context, coupled with exclusions where water temperatures likely preclude viral replication, achieved VHSV IVb freedom assurance for 14 non-GL States by the end of 2012, with partial evidence obtained for another 17 States. The non-GL region (defined as the aggregate of 4-digit HUCs located outside of GL States) met disease freedom targets for 2012 and is projected to maintain this status through 2016 without additional active surveillance. Projections hinge on continued basic biosecurity conditions such as movement restrictions and passive surveillance. Areas with navigable waterway connections to VHSV IVb-affected HUCs (and conducive water temperatures) should receive priority for resources in future surveillance or capacity building efforts. However, 6 years of absence of detections in non-GL States suggests that existing controls limit pathogen spread, and that even spread via natural pathways (e.g., water movement or migratory fish) appears contained to the Great Lakes system. This report exemplifies the cost-effective use of risk-based surveillance in decision support to assess and manage aquatic animal population health in open systems.

Susceptibility of various Japanese freshwater fish species to an isolate of viral haemorrhagic septicaemia virus (VHSV) genotype IVb

Genotype IVb of viral haemorrhagic septicaemia virus (VHSV) was isolated for the first time in the Great Lakes basin in 2003, where it spread and caused mass mortalities in several wild fish species throughout the basin. In order to prevent further spreading of the disease and to assess risks of new genotypes invading new watersheds, basic microbiological information such as pathogenicity studies are essential. In this study, experimental infections were conducted on 7 indigenous freshwater fish species from Japan by immersion with a VHSV genotype IVb isolate. In Expt 1, cumulative mortalities in bluegill Lepomis macrochirus used as positive controls, Japanese fluvial sculpin Cottus pollux, and iwana Salvelinus leucomaenis pluvius were 50, 80 and 0%, respectively. In Expt 2, cumulative mortalities of 100, 100 and 10% were observed in Japanese fluvial sculpin C. pollux, Japanese rice fish Oryzias latipes and yoshinobori Rhinogobius sp., respectively. No mortality was observed in honmoroko Gnathopogon caerulescens, akaza Liobagrus reini or Japanese striped loach Cobitis biwae. VHSV was detected by RT-PCR from samples of kidney, spleen, and brain from all dead fish, and virus re-isolation by cell culture was successful from all dead fish. We detected the virus in the brain from a few surviving bluegill 50 d post exposure by both cell culture and RT-PCR. These results revealed that VHSV IVb could become a serious threat to wild freshwater fish species in Japan, and that some surviving fish might become healthy carriers of the virus.

Development of a walleye cell line and use to study the effects of temperature on infection by viral haemorrhagic septicaemia virus group IVb.

A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a walleye caudal fin and used to study the intersection of thermobiology of walleye, Sander vitreus (Mitchill), with the thermal requirements for replication of viral haemorrhagic septicaemia virus (VHSV) IVb. WE-cfin11f proliferated from 10 to 32°C and endured as a monolayer for at least a week at 1-34°C. WE-cfin11f adopted an epithelial shape and did not proliferate at 4°C. Adding VHSV IVb to cultures at 4 and 14°C but not 26°C led to cytopathic effects (CPE) and virus production. At 4°C, virus production developed more slowly, but Western blotting showed more N protein accumulation. Infecting monolayer cultures at 4°C for 7days and then shifting them to 26°C resulted in the monolayers being broken in small areas by CPE, but with time at 26°C, the monolayers were restored. These results suggest that at 26°C, the VHSV IVb life cycle stages responsible for CPE can be completed, but the production of virus and the initiation of infections cannot be accomplished.

Experimental Infection of Rainbow Trout, Oncorhynchus mykiss , and Hybrid Striped Bass, Morone chrysops ♂ × Morone saxatilis ♀, with Viral Hemorrhagic Septicemia Virus Genotype IVb

The emergence of a new sublineage of viral hemorrhagic septicemia virus (VHSV) within the Laurentian Great Lakes has caused concern for aquaculture in the United States. Because of the occurrence of VHSV in a new geographic location, new host species have been identified and the complete host range has not been determined. This study confirmed the high resistance of rainbow trout, Oncorhynchus mykiss, to VHSV type IVb infection. In addition, the experimental susceptibility of hybrid striped bass, Morone chrysops ♂ × Morone saxatilis ♀, to VHSV type IVb infection was examined but determined to be highly dependent on age of fish and exposure temperature. No mortality was observed in adult fish infected via intraperitoneal (IP) injection at 15 C, whereas yearling fish infected via IP injection under the same conditions experienced 20.8% mortality. Among yearling fish infected via IP injection, mortality increased to 100% when exposure to VHSV occurred at 10 C. An LD50 for yearling hybrid striped bass exposed to VHSV at 10 C by IP injection was determined to be 1.4 × 104 pfu (SE = 2.1). Thus, at 10 C, yearling hybrid striped bass experience a high mortality when exposed to VHSV IVb by IP injection.

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-1β 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.

Comparison of treatments to inactivate viral hemorrhagic septicemia virus (VHSV-IVb) in frozen baitfish

Current US state and federal fish health regulations target the spread of viral hemorrhagic septicemia virus-IVb (VHSV-IVb) through movement restrictions of live fish; however, they largely ignore the potential for the virus to be spread through commercial distribution and use of frozen baitfish from VHSV-IVb-positive regions. Some state laws do require treatment of frozen baitfish to inactivate VHSV, and additional methods have been proposed, but few scientific studies have examined the efficacy of these treatments. In this study, bluegills Lepomis macrochirus were challenged with VHSV-IVb and frozen to represent standard industry methods, disinfected by various treatments, and tested for infectious VHSV-IVb using virus isolation. The virus was isolated from 70% of fish subjected to 3 freeze/thaw cycles. All other treatment methods were effective in inactivating the virus, including treatment with isopropyl alcohol, mineral oil, salt and borax, and dehydration. Dehydration followed by rehydration is rapid and effective, and therefore, seems to be the best option for inactivating VHSV-IVb present in frozen baitfish while maintaining their usefulness as bait.

Comparative susceptibility among three stocks of yellow perch, Perca flavescens (Mitchill), to viral haemorrhagic septicaemia virus strain IVb from the Great Lakes

The Great Lakes strain of viral haemorrhagic septicaemia virus IVb (VHSV-IVb) is capable of infecting a wide number of naive species and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. The yellow perch, Perca flavescens (Mitchill), a freshwater species commonly found throughout inland waters of the United States and prized for its high value in sport and commercial fisheries, is a species documented in several fish kills affiliated with VHS. In the present study, differences in survival after infection with VHSV IVb were observed among juvenile fish from three yellow perch broodstocks that were originally derived from distinct wild populations, suggesting innate differences in susceptibility due to genetic variance. While all three stocks were susceptible upon waterborne exposure to VHS virus infection, fish derived from the Midwest (Lake Winnebago, WI) showed significantly lower cumulative % survival compared with two perch stocks derived from the East Coast (Perquimans River, NC and Choptank River, MD) of the United States. However, despite differences in apparent susceptibility, clinical signs did not vary between stocks and included moderate-to-severe haemorrhages at the pelvic and pectoral fin bases and exophthalmia. After the 28-day challenge was complete, VHS virus was analysed in subsets of whole fish that had either survived or succumbed to the infection using both plaque assay and quantitative PCR methodologies. A direct correlation was identified between the two methods, suggesting the potential for both methods to be used to detect virus in a research setting.

Experimental Infection of Koi Carp with Viral Hemorrhagic Septicemia Virus Type IVb

Viral hemorrhagic septicemia virus (VHSV) type IVb has a wide host range that includes at least three cyprinid species: Fathead Minnow Pimephales promelas, Emerald Shiner Notropis atherinoides, and Bluntnose Minnow P. notatus. To date, VHSV IVb has only been found in wild fish. However, the possibility of infection in culture facilities remains. Koi Carp Cyprinus carpio are a major ornamental aquaculture species in the United States; however, their potential to become infected with VHSV IVb has not yet been examined. In this study, we exposed Koi to 3 × 10(6) PFU VHSV Great Lakes isolate MI03 by intraperitoneal injection. While we observed low mortality (0-5%), VHSV was isolated in cell culture from the majority of fish up to 28 d postexposure (DPE) and was detected by a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay up to 90 DPE, when the trial was terminated. The results of this study strongly suggest that Koi are at risk for VHSV infection, although their susceptibility by intraperitoneal injection appears to be low. This study also provides more evidence of the sensitivity of qRT-PCR for detection of VHSV IVb.

Experimental Infection of Four Aquacultured Species with Viral Hemorrhagic Septicemia Virus Type IVb

Viral hemorrhagic septicemia virus (VHSV) is the etiologic agent of a disease of great concern to aquaculture. We performed experimental infections with intraperitoneal VHSV IVb injections at 106 pfu/fish in four species: tiger muskellunge, ♂Esox lucius×♀Esox masquinongy; Atlantic salmon, Salmo salar; channel catfish, Ictalurus punctatus; and walleye, Sander vitreus. The fish were thermal shocked concurrent with infection to cause immunosuppression and increase the probability of eliciting disease. Each species was observed for morbidity and mortality and periodic samples taken. Virus was detected using virus isolation and real‐time quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR). Histopathology confirmed lesions in tiger muskellunge and Atlantic salmon, and immunohistochemistry showed virus associated with some lesions. Tiger muskellunge were highly susceptible to VHSV IVb, and high viral levels were detected in exposed fish. A single mortality was observed in exposed Atlantic salmon with a short viral replication period. Channel catfish and walleye were resistant to developing clinical signs of VHS. The results show that these species vary in the development of VHS under these conditions, and qRT‐PCR testing enabled virus detection in subclinically affected fish. Basic concordance statistics between the qRT‐PCR test results and the virus isolation results were evaluated.

Detection of VHSV IVb within the gonads of Great Lakes fish using in situ hybridization

Viral haemorrhagic septicaemia virus (VHSV) genotype IVb was recently detected as the cause of numerous mortality events in Great Lakes fish. In situ hybridization was used to examine the gonads from 13 fish, including freshwater drum Aplodinotus grunniens and muskellunge Esox masquinongy that were infected naturally, as well as rainbow trout Oncorhynchus mykiss and fathead minnows Pimphales promelas, which were experimentally infected. Although the ovaries and testes of fish infected by VHSV IVb had few lesions, viral RNA was present in the ovaries of the rainbow trout and fathead minnow and was abundant in the gonads of muskellunge and in the ovaries of freshwater drum. Viral RNA was present mainly surrounding yolk vacuoles/granules or adjacent to the germinal vesicle, with lesser amounts found within the germinal vesicle, in the mesovarium and/or tunica albuginea and blood vessels of the ovary. Viral RNA was also found in and surrounding primary and secondary spermatocytes of the muskellunge.

Replication and persistence of VHSV IVb in freshwater turtles

With the emergence of viral hemorrhagic septicemia virus (VHSV) strain IVb in the Great Lakes of North America, hatchery managers have become concerned that this important pathogen could be transmitted by animals other than fish. Turtles are likely candidates because they are poikilotherms that feed on dead fish, but there are very few reports of rhabdovirus infections in reptiles and no reports of the fish rhabdoviruses in animals other than teleosts. We injected common snapping turtles Chelydra serpentine and red-eared sliders Trachemys scripta elegans intraperitoneally with 10(4) median tissue culture infectious dose (TCID50) of VHSV-IVb and 21 d later were able to detect the virus by quantitative real-time reverse transcriptase PCR (qrt-RTPCR) in pools of kidney, liver, and spleen. In a second experiment, snapping turtles, red-eared sliders, yellow-bellied sliders T. scripta scripta, and northern map turtles Grapetemys geographica at 14 degrees C were allowed to feed on tissues from bluegill dying of VHSV IVb disease. Turtle kidney, spleen, and brain pools were not positive by qrt-RTPCR on Day 3 post feeding, but were positive on Days 10 and 20. Map turtles on Day 20 post-feeding were positive by both qrt-RTPCR and by cell culture. Our work shows that turtles that consume infected fish are a possible vector for VHSV IVb, and that the fish rhabdoviruses may have a broader host range than previously suspected.