Histological, serological and virulence studies on rainbow trout experimentally infected with recombinant infectious hematopoietic necrosis viruses

Several recombinant infectious hematopoietic necrosis viruses (IHNV) were produced by reverse genetics and their pathogenicity in trout was evaluated and compared to that of the wild type (wt) viruses IHNV and viral haemorrhagic septicemia virus (VHSV). Recombinant IHNVs used in this study were: rIHNV, identical to the wtIHNV; rIHNV-Gvhsv, a recombinant virus expressing the VHSV G gene instead of the IHNV G gene; rIHNV-Gmut, which possesses 2 targeted mutations in the glycoprotein; and rIHNVmut-Gmut, which is similar to the rIHNV-Gmut, but exhibits additional mutations along the genome. Results obtained in experimental infections showed that the rIHNV and rIHNV-Gmut were the most virulent recombinant viruses. Severity of the lesions induced by the different recombinant viruses was in agreement with mortality data. The kidney and the liver were the organs most affected by the most pathogenic viruses, and the lesions observed resembled those produced by wtIHNV. The introduction of mutations did not alter the tissue tropism of the virus. The recombinant viruses were able to replicate in fish, as shown by immunoperoxidase assay and RT-PCR. Antibodies against IHNV were detected in the fish inoculated with IHNV, rIHNV, rIHNV-Gmut and rIHNVmut-Gmut, and antibodies against VHSV were also found in fish infected with rIHNV-Gvhsv. Finally, antibody production was highest in fish infected with the rIHNVmut-Gmut even though this virus was the least virulent.     

Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) are rhabdoviruses that infect salmonids, producing serious economic losses. Two recombinant IHN viruses were generated by reverse genetics. For one (rIHNV GFP) the IHNV NV gene was replaced with the green fluorescent protein (GFP) gene. In the other (rIHNV-Gvhsv GFP) the G gene was also exchanged for that of VHSV. No mortalities, external signs or histological lesions were observed in experimental infections conducted with the recombinant viruses. Neither the rIHNV GFP nor rIHNV-Gvhsv GFP was detected by RT-PCR in any of the examined tissues from experimentally infected fish. In order to assess their potential as vaccines against the wild type viruses, rainbow trout were vaccinated with the recombinant viruses by intraperitoneal injection and challenged 30 d later with virulent IHNV or VHSV. The GFP viruses provided protection against both wild type viruses. None of the recombinant viruses induced antibody production, and the expression of interferon (IFNalpha4) and interferon induced genes such as Mx protein and ISG-15 was not different to that of controls. The rIHNV-Gvhsv GFP did not inhibit cellular apoptosis as it was observed in an IHNV inoculated fish cell line. These studies suggest that the recombinant rIHNV-Gvhsv GFP is a promising candidate as a live recombinant vaccine and also provides a good model to further study viral pathogenicity and the molecular basis of protection against these viral infections.     

Heterologous exchanges of the glycoprotein and the matrix protein in a Novirhabdovirus

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are two salmonid rhabdoviruses replicating at low temperatures (14 to 20 degrees C). Both viruses belong to the Novirhabdovirus genus, but they are only distantly related and do not cross antigenically. By using a recently developed reverse-genetic system based on IHNV (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000), we investigated the ability to exchange IHNV glycoprotein G with that of VHSV. Thus, the IHNV genome was modified so that the VHSV G gene replaced the complete IHNV G gene. A recombinant virus expressing VHSV G instead of IHNV G, rIHNV-Gvhsv, was generated and was shown to replicate as well as the wild-type rIHNV in cell culture. This study was extended by exchanging IHNV G with that of a fish vesiculovirus able to replicate at high temperatures (up to 28 degrees C), the spring viremia of carp virus (SVCV). rIHNV-Gsvcv was successfully recovered; however, its growth was restricted to 14 to 20 degrees C. These results show the nonspecific sequence requirement for the insertion of heterologous glycoproteins into IHNV virions and also demonstrate that an IHNV protein other than the G protein is responsible for the low-temperature restriction on growth. To determine to what extent the matrix (M) protein interacts with G, a series of chimeric pIHNV constructs in which all or part of the M gene was replaced with the VHSV counterpart was engineered and used to recover the respective recombinant viruses. Despite the very low percentage (38%) of amino acid identity between the IHNV and VHSV matrix proteins, viable chimeric IHNVs, harboring either the matrix protein or both the glycoprotein and the matrix protein from VHSV, were recovered and propagated. Altogether, these data show the extreme flexibility of IHNV to accommodate heterologous structural proteins.     

Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.     

Bacterially expressed nucleoprotein of infectious hematopoietic necrosis virus augments protective immunity induced by the glycoprotein vaccine in fish.

The ribonucleoprotein gene of infectious hematopoietic necrosis virus (IHNV) has been expressed in Escherichia coli as a trpE fusion protein. This viral protein does not induce protective immunity to lethal IHNV infection in fish, and virus-neutralizing antibodies do not react with this viral protein. However, when it was administered with a bacterial lysate containing a region of the IHNV glycoprotein, there was enhanced resistance in immunized fish to lethal virus infection.     

Wide range of susceptibility to rhabdoviruses in homozygous clones of rainbow trout

Inbred lines differentially susceptible to diseases are a powerful tool to get insights into the mechanisms of genetic resistance to pathogens. In fish, chromosome manipulation techniques allow a quick production of such homozygous lines. Using gynogenesis, we produced nine homozygous clones of rainbow trout from a domestic population (INRA Sy strain). We examined the variability between clones for resistance to two rhabdoviruses, the viral haemorrhagic septicaemia virus (VHSV) and the infectious haematopoietic necrosis virus (IHNV). Intraperitoneal injections and waterborne infections were performed in parallel for both viruses. No survival was recorded after intraperitoneal injection of VHSV or IHNV, indicating that fish from all clones were fully susceptible to both viruses by this route of infection. In contrast, the different clones showed a wide range of survival frequency after waterborne infection. The resistance levels to VHSV ranged from 0 to 99% and resistance was not abrogated when resistant and sensitive animals were mixed and subjected to waterborne infection. VHSV was recovered from 10% of resistant fish after waterborne infection, confirming that virus replication was possible in this context but effective only in a low proportion of the population. The different clones also exhibited a wide range of survival (0-68%) after a waterborne infection with IHNV. Although VHSV-resistant clones were not fully resistant to IHNV, the susceptibility to IHNV and VHSV tended to be correlated, suggesting that non-specific mechanisms common to both viruses were involved.     

Study of the viral interference between infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

The resistance of rainbow trout (Oncorhynchus mykiss) to an infectious haematopoietic necrosis virus (IHNV) challenge following a preceding non-lethal infection with infectious pancreatic necrosis virus (IPNV) was investigated through experimental dual infections. Trout initially infected with IPNV were inoculated 14 days later with IHNV. Single infections of trout with 1 of the 2 viruses or with cell culture supernatant were also carried out and constituted control groups. No mortality was noted in fish after a single infection with IPNV. This virus had no influence on the head kidney leucocyte phagocytic activity and plasma haemolytic complement activity. IHNV induced a high mortality (72%) and reduced the macrophage phagocytic activity and complement haemolytic activity. It also induced a late production of anti-IHNV antibodies which occurred after clearance of the virus in the fish. In trout co-infected with both viruses, a mortality rate of 2% occurred and the immune parameters were similar to those observed in the fish infected with IPNV only, demonstrating that in co-infected trout IPNV inhibits the effects of IHNV. The studied parameters did not allow us to define the mechanism of interference occurring between these 2 viruses, but some hypothesis are put forward to explain the interference between the 2 viruses.     

Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.     

Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout

Novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) are fish rhabdoviruses that, in comparison to the other rhabdoviruses, contain an additional gene coding for a small nonvirion (NV) protein of unassigned function. A recombinant IHNV with the NV gene deleted but expressing the green fluorescent protein (rIHNV-Delta NV) has previously been shown to be efficiently recovered by reverse genetics (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000). However, preliminary experiments suggested that the growth in cell culture of rIHNV-Delta NV was affected by the NV deletion. In the present study, we show that the growth in cell culture of rIHNV-Delta NV is indeed severely impaired but that a normal growth of rIHNV-Delta NV can be restored when NV is provided in trans by using fish cell clones constitutively expressing the NV protein. These results indicate that NV is a protein that has a crucial biological role for optimal replication of IHNV in cell culture. Although IHNV and VHSV NV proteins do not share any significant identity, we show here that both NV proteins play a similar role since a recombinant IHNV virus, rIHNV-NV(VHSV), in which the IHNV NV open reading frame has been replaced by that of VHSV, was shown to replicate as well as the wild-type (wt) IHNV into fish cells. Finally, data provided by experimental fish infections with the various recombinant viruses strongly suggest an essential role of the NV protein for the pathogenicity of IHNV. Furthermore, we show that juvenile trout immunized with NV-knockout IHNV were protected against challenge with wt IHNV. That opens a new perspective for the development of IHNV attenuated live vaccines.     

Age- and weight-dependent susceptibility of rainbow trout Oncorhynchus mykiss to isolates of infectious haematopoietic necrosis virus (IHNV) of varying virulence

The virulence of 5 European and 1 North American isolate of infectious haematopoietic necrosis virus (IHNV) was compared by infecting female sibling rainbow trout ('Isle of Man' strain) of different weights and ages (2, 20 and 50 g). The fish were exposed to 10(4) TCID50 IHNV per ml of water by immersion, and the mortality was recorded for 28 d. Two new IHNV isolates from Germany were included in the investigation. One was isolated from European eels kept at 23 degrees C (+/- 2 degrees C) and the other was not detectable by immunofluorescence with commercially available monoclonal antibodies recognising the viral G protein. The results showed that IHNV isolates of high or low virulence persisted in rainbow trout of all ages/weights for 28 d, with the exception of fish over 15 g in the eel IHNV (DF [diagnostic fish] 13/98)-infected groups from which the virus could not be reisolated on Day 28. The smallest fish were most susceptible to an infection with any of the IHNV isolates. The lowest cumulative mortality (18%) was observed in fingerlings infected with the North American isolate HAG (obtained from Hagerman Valley), and the highest mortality (100%) in DF 04/99 infected fish. The DF 04/99 and O-13/95 viruses caused mortality in fish independent of their weight or age. The isolates FR-32/87 and I-4008 were virulent in fish up to a weight of 20 g and caused no mortality in larger fish. In the IHNV HAG- and DF 13/98 (eel)-infected rainbow trout, no signs of disease were observed in fish weighing between 15 and 50 g. An age/weight related susceptibility of rainbow trout was demonstrated under the defined conditions for all IHNV isolates tested.     

Novel Form of Fibronectin from Zebrafish Mediates Infectious Hematopoietic Necrosis Virus Infection

The presence of a novel form of zebrafish fibronectin (FN2) on the cell surface increased the cell's susceptibility to infection by infectious hematopoietic necrosis virus (IHNV). Unlike other fibronectins, FN2 possesses a truncated structure and accumulates on the cell surface instead of in the extracellular matrix. Fish embryo cells expressing recombinant FN2 were more susceptible to IHNV infection, with a greater percentage of cells exhibiting cytopathic effect (CPE) compared to nontransfected control cells. Incubation of nontransfected cells with soluble recombinant FN2 increased IHNV infection, as measured by plaque assay. The number of plaques increased in correlation with the amount of protein added and the length of time that cells were incubated with the protein. Incubation of IHNV with soluble FN2 before addition to cells also increased infection. FN2 immobilized on the culture surface inhibited IHNV infection. The results indicate that FN2 present on the cell surface is able to mediate IHNV attachment and cell entry.     

Effects of salmonid fish viruses on Mx gene expression and resistance to single or dual viral infections

We examined the ability of several fish viruses to induce protection against homologous or heterologous viruses in single or double infections, and assessed whether such protection is correlated with innate immunity or expression of the Mx gene. Monolayers of BF2 cells pre-treated with supernatants of brown trout (Salmo trutta L.) macrophage cultures that had been stimulated with either polyinosinic polycytidylic acid (poly I:C) or viruses, such as infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) or a mixture of the two, showed varying degrees of protection against viral infections. The virus showing the strongest induction was IPNV, and the antiviral activity against IHNV was also high: around 6 log(10) reduction of virus yield. Consequently, the IPNV-IHNV co-infection yield was also reduced by varying amounts. In vivo, the cumulative mortality observed in the IPNV-IHNV co-infected fish was always less than that in those with a single infection. Stimulation with poly I:C for 7 days significantly reduced cumulative mortality in single-infected fish, but not in the double-infected, in which the IPNV was the only virus isolated from moribund animals. By RT-PCR, Mx was expressed in all the organ samples tested (kidney, liver and spleen) from virus-stimulated fish at 1, 2 and 3 days. By qRT-PCR the extent and timing of Mx expression was shown to differ in the poly I:C and the single or dual viral infections. The highest increase in Mx expression (21.6-fold above basal levels) occurred (after 24 h) in fish infected with the IHNV, and expression remained high until day 7. Mx expression in fish infected with IPNV peaked later, at 2 days post infection, and also remained high until day 7. The dual infection with IPNV-IHNV induced high Mx expression on day 1, which peaked on day 2 and remained high until day 7. These results indicate that activation of the immune system could explain the interference and loss of IHNV in the IPNV-IHNV co-infections.     

Inactivation of Infectious Hematopoietic Necrosis Virus by Low Levels of Iodine

The fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) was rapidly inactivated by extremely low concentrations of iodine in water. A 99.9% virus reduction was obtained in 7.5 s when virus (10⁵ PFU/ml) and iodine (0.1 mg/liter, final concentration) were combined in distilled-deionized or hatchery water. Iodine efficacy decreased at pHs greater than 7.5 or when proteinaceous material was added to the water. Bovine serum albumin blocked iodine inactivation of the virus more effectively than did equal concentrations of fetal bovine serum or river sediment. Sodium thiosulfate effectively neutralized free iodine. Powder, iodophor, and crystalline iodine solutions inactivated IHNV equally. Iodine rapidly inactivated IHNV isolates representing each of the five electropherotypes. Under the conditions used in this study, inactivation was not affected by temperature, salinity, or water hardness. When Dworshak National Fish Hatchery water was continuously treated to provide a free iodine concentration of 0.14 mg/liter, a 7.5-s exposure to iodine was sufficient to inactivate 99.9% of the IHNV. Iodine added to water that contained IHNV prevented infection of rainbow trout (Oncorhynchus mykiss) fry. These results suggest that the waterborne route of IHNV transmission can be blocked by adding low iodine concentrations to the water supplies of hatcheries.     

Comparison of Representative Strains of Infectious Hematopoietic Necrosis Virus by Serological Neutralization and Cross-Protection Assays

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of young salmon and trout. Viral epizootics among these fish in private and public rearing facilities have been a problem in the northwestern United States from California to Alaska, and an IHNV vaccine has been sought by the aquaculture experts. Since an IHNV vaccine must be designed to immunize against all viral serotypes, an analysis of IHNV serotypes was made. A large number of viruses from widely separated geographic locations and different fish species had already been placed in one of five electropherotypes by the migration of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Also, there was evidence that some of these virus isolates had differences in virulence for chinook salmon, rainbow trout, or kokanee salmon. Previous serological studies with polyclonal rabbit antisera and three IHNV isolates indicated that there was only one serotype (B. B. McCain, J. L. Fryer, and K. S. Pilcher, Proc. Soc. Exp. Biol. Med. 137:1042-1046, 1971). A substantial number of new IHNV isolations have been made since that study, and thus a more extensive comparison was made of 10 different IHNV isolates representing the five electropherotypes. This report shows that the glycoprotein from a single isolate of IHNV can induce a protective immune response in vivo to the five IHNV electropherotypes. Plaque reduction neutralization assays indicated that there was only one serotype. Thus, despite the differences observed in the migration of the structural proteins for IHNV isolated from separate geographic locations and different fish species, only one neutralizing virus type was identified.     

Immunogenicity of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein), produced in Spodoptera frugiperda (Sf9) cells following infection with a baculovirus vector containing the full-length (1.6 kb) glycoprotein gene, provided very limited protection in rainbow trout Oncorhynchus mykiss challenged with IHNV. Fish were injected intraperitoneally (i.p.) with Sf9 cells grown at 20 degrees C (RecGlow) or 27 degrees C (RecGhigh) expressing the glycoprotein gene. Various antigen (Ag) preparations were administered to adult rainbow trout or rainbow trout fry. Sera collected from adult fish were evaluated for IHNV neutralization activity by a complement-dependent neutralization assay. Anti-IHNV neutralizing activity was observed in sera, but the percent of fish responding was significantly lower (p < 0.05) in comparison to fish immunized with a low virulence strain of IHNV (LV-IHNV). A small number of fish immunized with RecGlow or RecGhigh possessed IHNV G protein specific antibodies (Abs) in their serum. Cumulative mortality (CM) of rainbow trout fry (mean weight, 1 g) vaccinated by i.p. injection of freeze/thawed Sf9 cells producing RecGlow was 18% in initial trials following IHNV challenge. This level of protection was significant (p < 0.05) but was not long lasting, and neutralizing Abs were not detected in pooled serum samples. When trout fry (mean weight, 0.6 g) were vaccinated with supernatant collected from sonicated Sf9 cells, Sf9 cells producing RecGlow, or Sf9 cells producing RecGhigh, CM averaged 46%. Protection was enhanced over negative controls, but not the positive controls (2% CM), suggesting that in the first trial soluble cellular proteins may have provided some level of non-specific protection, regardless of recombinant protein expression. Although some immunity was elicited in fish, and RecGlow provided short-term protection from IHNV, Ab-mediated protection could not be demonstrated. The results suggest that recombinant G proteins produced in insect cells lack the immunogenicity associated with vaccination of fish with an attenuated strain of IHNV.     

Occurrence and genetic typing of infectious hematopoietic necrosis virus in Kamchatka, Russia

Infectious hematopoietic necrosis virus (IHNV) is a well known rhabdoviral pathogen of salmonid fish in North America that has become established in Asia and Europe. On the Pacific coast of Russia, IHNV was first detected in hatchery sockeye from the Kamchatka Peninsula in 2001. Results of virological examinations of over 10,000 wild and cultured salmonid fish from Kamchatka during 1996 to 2005 revealed IHNV in several sockeye salmon Oncorhynchus nerka populations. The virus was isolated from spawning adults and from juveniles undergoing epidemics in both hatchery and wild sockeye populations from the Bolshaya watershed. No virus was detected in 2 other watersheds, or in species other than sockeye salmon. Genetic typing of 8 virus isolates by sequence analysis of partial glycoprotein and nucleocapsid genes revealed that they were genetically homogeneous and fell within the U genogroup of IHNV. In phylogenetic analyses, the Russian IHNV sequences were indistinguishable from the sequences of North American U genogroup isolates that occur throughout Alaska, British Columbia, Washington, and Oregon. The high similarity, and in some cases identity, between Russian and North American IHNV isolates suggests virus transmission or exposure to a common viral reservoir in the North Pacific Ocean.     

A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the (32)EGDL(35) residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the (32)EGDL(35) was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of (32)EGDL(35) responsible for nuclear localization are important for the inhibitory activity of NV.     

Transmission routes maintaining a viral pathogen of steelhead trout within a complex multi‐host assemblage

This is the first comprehensive region wide, spatially explicit epidemiologic analysis of surveillance data of the aquatic viral pathogen infectious hematopoietic necrosis virus (IHNV) infecting native salmonid fish. The pathogen has been documented in the freshwater ecosystem of the Pacific Northwest of North America since the 1950s, and the current report describes the disease ecology of IHNV during 2000–2012. Prevalence of IHNV infection in monitored salmonid host cohorts ranged from 8% to 30%, with the highest levels observed in juvenile steelhead trout. The spatial distribution of all IHNV‐infected cohorts was concentrated in two sub‐regions of the study area, where historic burden of the viral disease has been high. During the study period, prevalence levels fluctuated with a temporal peak in 2002. Virologic and genetic surveillance data were analyzed for evidence of three separate but not mutually exclusive transmission routes hypothesized to be maintaining IHNV in the freshwater ecosystem. Transmission between year classes of juvenile fish at individual sites (route 1) was supported at varying levels of certainty in 10%–55% of candidate cases, transmission between neighboring juvenile cohorts (route 2) was supported in 31%–78% of candidate cases, and transmission from adult fish returning to the same site as an infected juvenile cohort was supported in 26%–74% of candidate cases. The results of this study indicate that multiple specific transmission routes are acting to maintain IHNV in juvenile fish, providing concrete evidence that can be used to improve resource management. Furthermore, these results demonstrate that more sophisticated analysis of available spatio‐temporal and genetic data is likely to yield greater insight in future studies.