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

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

Virulence and Infectivity of UC, MD, and L Strains of Infectious Hematopoietic Necrosis Virus (IHNV) in Four Populations of Columbia River Basin Chinook Salmon.

Infectious Hematopoietic Necrosis Virus (IHNV) infects juvenile salmonid fish in conservation hatcheries and aquaculture facilities, and in some cases, causes lethal disease. This study assesses intra-specific variation in the IHNV susceptibility of Chinook salmon (Oncorhynchus tshawytscha) in the Columbia River Basin (CRB), in the northwestern United States. The virulence and infectivity of IHNV strains from three divergent virus genogroups are measured in four Chinook salmon populations, including spring-run and fall-run fish from the lower or upper regions of the CRB. Following controlled laboratory exposures, our results show that the positive control L strain had significantly higher virulence, and the UC and MD strains that predominate in the CRB had equivalently low virulence, consistent with field observations. By several experimental measures, there was little variation in host susceptibility to infection or disease. However, a small number of exceptions suggested that the lower CRB spring-run Chinook salmon population may be less susceptible than other populations tested. The UC and MD viruses did not differ in infectivity, indicating that the observed asymmetric field prevalence in which IHNV detected in CRB Chinook salmon is 83% UC and 17% MD is not due to the UC virus being more infectious. Overall, we report little intra-species variation in CRB Chinook salmon susceptibility to UC or MD IHNV infection or disease, and suggest that other factors may instead influence the ecology of IHNV in the CRB.

The L-domains in M and G proteins of infectious hematopoietic necrosis virus (IHNV) affect viral budding and pathogenicity.

RNA viruses including many retroviruses encode "late-domain" motifs that can interact with host proteins to mediate viral assembly and affect viral budding and pathogenicity. For IHNV, our previous studies demonstrated that the respective interactions of the L domains of IHNV with host proteins could mediate viral assembly and budding. To our knowledge, the role of L domains of the IHNV in the budding and pathogenicity has not investigated yet. In this study, we generated two recombinant IHNV strains rIHNV-M(PH>A4) and rIHNV-G(PS>A4) with mutations in the L domains (PPPH to AAAA or PSAP to AARA) of IHNV by reverse genetics and explored the effect of the mutations on budding and pathogenicity of the two recombinant viruses. The RT-qPCR results showed that the production levels of the extracellular particles of rIHNV-M(PH>A4) or rIHNV-G(PS>A4) declined significantly, compared with those of wild-type (wt) IHNV HLJ-09. Furthermore, the challenge test showed that the survival rates of juvenile rainbow trout challenged with rIHNV-M(PH>A4) or rIHNV-G(PS>A4) were 90% or 87%, respectively; however, the survivability was zero in groups challenged with wtIHNV HLJ-09 or rIHNV HLJ-09 (recombinant IHNV). Additionally, the RT-qPCR results showed that the recombinant viruses induced higher expression levels of IFN1, IL-1ß, and IL-8 compared with those induced by wtIHNV HLJ-09 as well as the ELISA results showed that fish vaccinated with recombinant viruses produced high levels of specific IgM antibodies, demonstrating that the two recombinant viruses may induce immune responses to resist infection by IHNV. Also, these results demonstrated for the first time that the L domains of the M and G proteins of IHNV could affect the budding and pathogenicity of IHNV, which may be beneficial in the prevention and control of IHNV infections in fish. Taken together, our study as the first research provides the foundation for effect of rhabdovirus L domains on viral budding and pathogenicity.

Co-infection of rainbow trout (Oncorhynchus mykiss) with infectious hematopoietic necrosis virus and Flavobacterium psychrophilum.

Co-infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co-infection has not been well characterized or documented. In this study, it was hypothesized that fish co-infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co-infected with low doses of either IHNV or F. psychrophilum, and at 2 days post-initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%-100%), while mortality from a single pathogen infection with the same respective dose was low (5%-20%). The onset of mortality was earlier in the co-infected group (3-4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co-infection led to a significant increase in the number of F. psychrophilum colony-forming units and IHNV plaque-forming units within tissues. This finding confirms that when present together in co-infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co-infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co-infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host-pathogen interactions related to co-infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.

Synergistic osmoregulatory dysfunction during salmon lice (Lepeophtheirus salmonis) and infectious hematopoietic necrosis virus co-infection in sockeye salmon (Oncorhynchus nerka) smolts.

While co-infections are common in both wild and cultured fish, knowledge of the interactive effects of multiple pathogens on host physiology, gene expression and immune response is limited. To evaluate the impact of co-infection on host survival, physiology and gene expression, sockeye salmon Oncorhynchus nerka smolts were infected with the salmon louse Lepeophtheirus salmonis (V-/SL+), infectious hematopoietic necrosis virus (IHNV; V+/SL-), both (V+/SL+), or neither (V-/SL-). Survival in the V+/SL+ group was significantly lower than the V-/SL- and V-/SL+ groups (p = 0.024). Co-infected salmon had elevated osmoregulatory indicators and lowered haematocrit values as compared to the uninfected control. Expression of 12 genes associated with the host immune response was analysed in anterior kidney and skin. The only evidence of L. salmonis-induced modulation of the host antiviral response was down-regulation of mhc I although the possibility of modulation cannot be ruled out for mx-1 and rsad2. Co-infection did not influence the expression of genes associated with the host response to L. salmonis. Therefore, we conclude that the reduced survival in co-infected sockeye salmon resulted from the osmoregulatory consequences of the sea lice infections which were amplified due to infection with IHNV.

N-linked glycosylation sites in G protein of infectious hematopoietic necrosis virus (IHNV) affect its virulence and immunogenicity in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis in salmonid fish, resulting in substantial economic losses to the aquaculture industry worldwide. The G protein, which harbors the major antigenic determinants of IHNV, is an envelope glycoprotein that plays an important role in both pathogenicity and immunogenicity of IHNV. Previous studies have demonstrated that changes to viral glycosylation sites may affect replication and immunogenicity, but little is known about the specific contributions of G protein glycosylation to IHNV replication and pathogenicity. In this study, we predicted four N-linked glycosylation sites at position 56, 379, 401, and 438 Asp (N) in G protein, and using a reverse genetics system developed in our laboratory, constructed nine recombinant viruses with single, triple, or quadruple glycosylation site disruptions using alanine substitutions in the following combinations: rIHNV-N56A, rIHNV-N379A, rIHNV-N401A, rIHNV-N438A, rIHNV-N56A-N379A-N401A, rIHNV-N56A-N379A-N438A, rIHNV-N56A-N401A-N438A, rIHNV-N379A-N401A-N438A, and rIHNV-N56A-N379A-N401A-N438A. Our results confirmed that all four asparagines are sites of N-linked glycosylation, and Western blot confirmed that mutation of each predicted N-glycosylation sited impaired glycosylation. Among the nine recombinant IHNVs, replication levels decreased significantly in vitro and in vivo in the triple and quadruple mutants that combined mutation of asparagines 401 and 438, indicating the importance of glycosylation at these sites for efficient replication. Moreover, juvenile rainbow trout mortality after challenge by each of the nine mutants showed that, while eight mutants suffered almost 100% cumulative mortality over 30 days, the mutant with a single alanine substitution at position 438 resulted in cumulative mortality of less than 50% over 30 days. This mutant also elicited specific anti-IHNV IgM production earlier than other mutants, suggesting that glycosylation of asparagine 438 may be important for viral immune escape. In conclusion, our study reveals the effect of G protein glycosylation on the pathogenicity and immunogenicity of IHNV and provides a foundation for developing a live-attenuated vaccine.

Recovery of recombinant infectious hematopoietic necrosis virus strain Sn1203 using the mammalian cell line BHK-21.

Reverse genetics systems are powerful tools for understanding the virulence mechanisms and gene functions of negative-sense RNA viruses. The reverse genetics systems commonly used for recombinant infectious hematopoietic necrosis virus (IHNV) are based on vaccinia virus infection. To avoid the potential biological safety risks associated with vaccinia virus, a recombinant IHNV virus strain Sn1203 (rIHNV-Sn1203) was rescued in this study using a mammalian cell line, BHK-21. The genome sequence authenticity of rIHNV-Sn1203 was confirmed using two silent genetic tags introduced by site-directed mutagenesis. Indirect immunofluorescence assays and transmission electron microscopy revealed that rIHNV-Sn1203 and wild-type IHNV-Sn1203 (wtIHNV-Sn1203) had identical immunogenicity and virion morphology. The virulence and pathogenicity of rIHNV-Sn1203 were assessed in vitro and in vivo. Although rIHNV-Sn1203 displayed trends toward delayed intracellular viral replication and lower virion yields compared with wtIHNV-Sn1203, statistical analyses revealed no significant differences between these two viruses. Moreover, rainbow trout challenged with rIHNV-Sn1203 and wtIHNV-Sn1203 showed indistinguishable mortality. Together, these results show that IHNV was successfully rescued using BHK-21 cells. This method is very convenient and may also be suitable for use in the recovery of other Novirhabdoviruses.

Transchem project - Part II: Transgenerational effects of long-term exposure to pendimethalin at environmental concentrations on the early development and viral pathogen susceptibility of rainbow trout (Oncorhynchus mykiss).

In the Transchem project, rainbow trout genitors were exposed to environmental concentrations of pendimethalin over a period of 18 months and two new first generations of offspring, F1_2013 and F1_2014, were obtained. We investigated the impact of direct chemical exposure on juveniles as well as the potential cumulative transgenerational and direct effects on the larval development and on the pathogen susceptibility of offspring. Depending on the chemical treatment or not of the adults, their offspring were distributed in the tanks of our experimental system, in two batches i.e. juveniles from the control genitors (G-) and others from the contaminated ones (G+), and then, half of the tanks were exposed daily to pendimethalin (Off+) while the others were used as controls (Off-). Viral challenges were performed on the offspring, before and after three months of direct chemical exposure, with strains of infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and sleeping disease alphavirus (SDV). Direct and transgenerational macroscopic effects were observed on offspring, with a percentage of abnormalities in offspring derived from the genitors exposed to pendimethalin (G+) significantly higher compared to those from the genitors from non-exposed group (G-). Before the direct chemical exposure, similar kinetics of mortality was observed between the offspring from the contaminated or control genitors after VHSV infection. With IHNV, the G+ group died in a slightly larger proportion compared to the G- group and seroconversion was greater for the G- group. For the SDV challenge, the mortality was delayed for the G+ offspring compared to the G- and seroconversion reached 65% in the G+ group compared to 45% in the G-, with similar antibody titres. After three months of direct chemical exposure, kinetics of mortality induced by IHNV infection were similar for all groups studied. Infection with SDV resulted in a cumulative mortality of 40% for the G- groups (Off- and Off+), significantly higher than those observed from the contaminated genitors G+. Proportion of seropositivity for SDV varied from 24 to 47% depending on the group, with very low quantities of secreted antibodies. Lastly, the direct exposure of offspring could impact the capacity of fish to adapt their haematological parameters to environmental and physiological changes, and underlines the potential toxic effects on the next generations.

Identification of amino acid residues in infectious hematopoietic necrosis virus (IHNV) NV protein necessary for viral replication and pathogenicity.

Our previous studies demonstrated that the nonstructural NV protein of infectious hematopoietic necrosis virus (IHNV) was essential for efficient viral replication and pathogenicity, and that the amino acid residues 32EGDL35 of the NV protein were responsible for nuclear localization, and played important roles in suppressing IFN and inhibiting NF-κB activity. However, little is known about the influence of 32EGDL35 on IHNV replication and pathogenicity. In the present study, two recombinant IHNV strains with deletions of NV 32EGDL35 were generated and the effect on IHNV replication and pathogenicity was explored. Our results showed that both mutants stably replicated in Chinook salmon embryo cells for 15 consecutive passages, and had similar host-tropism as wild-type (wt) IHNV; however, titers of the mutants were lower than those of wt IHNV in CHSE-214 cells. Infection of rainbow trout showed wt IHNV produced 90% cumulative mortality, while the mutants produced 55% and 60% cumulative mortality, respectively. Histopathological evaluation showed that tissues from the liver, brain, kidney, and heart of fish infected with wt IHNV exhibited pathological changes, but significant lesions were found only in the liver and heart of fish infected with the recombinant viruses. In addition, the recombinant viruses induced higher expression levels of IFN1, Mx-1, and IL-6 compared with those induced by wt IHNV. These results indicated that the 32EGDL35 residues were essential for the efficient anti-IFN and NF-κB-inhibiting activity of NV. Our results provide a basis for understanding the roles of 32EGDL35 in IHNV replication and pathogenicity, and may prove beneficial in the prevention and control of IHNV infections of fish.

Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish.

Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full-length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G-gene to make the rIHNV-Gsvcv. The chimeric rIHNV-Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild-type SVCV infection was tested. The rIHNV-Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV-Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV-Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV-Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.

Infectious hematopoietic necrosis virus (IHNV) outbreak in farmed rainbow trout in Iran: Viral isolation, pathological findings, molecular confirmation, and genetic analysis.

Infectious hematopoietic necrosis virus (IHNV) is the etiological agent of a contagious disease (IHN) mainly in salmonid fish. In the present study, we isolated and identified IHNV in trout fry from Iranian trout farms with unexplained high mortality in 2016. The affected fry showed cumulative mortality of 90% with the gross pathological signs including exophthalmia and hemorrhage of the eye, skin darkening, abdominal distension, ulceration of the snout, and the visceral pallor and yellowish fluid in the intestine. Histopathological examination revealed marked necrosis in the anterior kidney, liver and spleen with the intracytoplasmic inclusion bodies in the liver sections. Also, intranuclear inclusion body and marginated chromatin were observable in the hematopoietic cells of the kidney. The homogenates tissues of infected fry induced IHNV-positive cytopathic effects (CPE) in EPC cells and confirmed by RT-PCR reactions and sequencing. Phylogenetic analysis revealed the Iranian IHNV isolates belonged to the European (E) genogroup with 100% identity to some Italian isolates. This is the first report of IHNV infection in farmed trout fry in Iran describing the viral isolation, clinical symptoms, histopathological findings, molecular confirmation, and genetic analysis suggestion of the specific country of origin.

Virulence and serological studies of recombinant infectious hematopoietic necrosis virus (IHNV) in rainbow trout.

Infectious hematopoietic necrosis virus is a highly contagious disease of juvenile salmonid species. From the IHNV HLJ-09 isolated in China, two recombinant viruses were generated by reverse genetics using the RNA polymerase II transcription system. The recombinant viruses were confirmed by RT-PCR, indirect immunofluorescence assay and electron microscopy. They were referred to as rIHNV HLJ-09 and rIHNV-EGFP. rIHNV HLJ-09 and rIHNV-EGFP could stably replicate in EPC cell lines and had the same cellular tropism as wtIHNV HLJ-09. But the titer of rIHNV-EGFP was significantly lower than rIHNV HLJ-09 and wtIHNV HLJ-09. rIHNV-EGFP strain could express EGFP stably at least in 20 passages, and the fluorescence could be observed clearly. To assess the virulence and pathogenicity of the recombinant viruses in vivo, juvenile rainbow trout were challenged by intraperitoneal injection with 20µl of rIHNV HLJ-09, rIHNV-EGFP or wtIHNV HLJ-09 (1×10(6)pfuml(-1)). Fish challenged with rIHNV HLJ-09 and wtIHNV HLJ-09 exhibited clinical signs typical of IHN disease and both produced 90% cumulative percent mortality, whlie rIHNV-EGFP produced only 5%. Pathological sectioning results showed that the tissues (liver, kidney, heart muscle, back muscle) of the fish infected with rIHNV HLJ-09 exhibited pathological changes, with the exception of cerebral neurons and the cheek. However, no lesions of liver, kidney, heart, muscle, brain in rainbow trout of rIHNV-EGFP or the control group were observed. Indirect ELISA results showed that a high level of serum antibody was detected in the experimental fish challenged with rIHNV HLJ-09, just as the same as wtIHNV HLJ-09, while a lower titer was detecred in the fish infected with rIHNV-EGFP. This indicated that the recombinant viruses could induce humoral immune response in the experimental fish. The recombinant viruses had unique genetic tags and could be used for genetic engineering, laying new ground for further investigation of IHNV pathopoiesis molecular mechanism, host tropism and the development of novel vaccines against IHN.

Characterization of infectious dose and lethal dose of two strains of infectious hematopoietic necrosis virus (IHNV).

The ability to infect a host is a key trait of a virus, and differences in infectivity could put one virus at an evolutionary advantage over another. In this study we have quantified the infectivity of two strains of infectious hematopoietic necrosis virus (IHNV) that are known to differ in fitness and virulence. By exposing juvenile rainbow trout (Oncorhynchus mykiss) hosts to a wide range of virus doses, we were able to calculate the infectious dose in terms of ID50 values for the two genotypes. Lethal dose experiments were also conducted to confirm the virulence difference between the two virus genotypes, using a range of virus doses and holding fish either in isolation or in batch so as to calculate LD50 values. We found that infectivity is positively correlated with virulence, with the more virulent genotype having higher infectivity. Additionally, infectivity increases more steeply over a short range of doses compared to virulence, which has a shallower increase. We also examined the data using models of virion interaction and found no evidence to suggest that virions have either an antagonistic or a synergistic effect on each other, supporting the independent action hypothesis in the process of IHNV infection of rainbow trout.

The zebrafish galectins Drgal1-L2 and Drgal3-L1 bind in vitro to the infectious hematopoietic necrosis virus (IHNV) glycoprotein and reduce viral adhesion to fish epithelial cells.

The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells. Our results suggest that the extracellular Drgal1-L2 and Drgal3-L1 interact directly and in a carbohydrate-dependent manner with the IHNV glycosylated envelope and glycans on the epithelial cell surface, significantly reducing viral adhesion.

Atlantic salmon, Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus.

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U-group strains ranged from 20 to 100%, four M-group strains ranged 30-63% and two L-group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.

The role of virulence in in vivo superinfection fitness of the vertebrate RNA virus infectious hematopoietic necrosis virus.

We have developed a novel in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a coevolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 h to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal-virulence and unequal-virulence genotype pairs, the frequency of superinfection in most cases was the same regardless of which genotype was used in the primary exposure. The ability to replicate in the context of superinfection also did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural vertebrate host.

Lack of correlation between the resistances to two rhabdovirus infections in rainbow trout.

The Viral Hemorrhagic Septicemia Virus (VHSV) and the Infectious Hematopoietic Necrosis Virus (IHNV) are two rhabdoviruses responsible for serious outbreaks in salmonid farms. To date, little is known about the variability of host response to these viruses. Using gynogenetic clonal lines of rainbow trout exhibiting a wide range of resistance to viral infections, we showed that there was no correlation between the resistance to VHSV and IHNV. We also confirmed the importance of fish weight for its susceptibility to IHNV infection. Finally, using a chimeric recombinant IHNV expressing the VHSV glycoprotein, we showed that the glycoprotein plays a key role in the virulence and in the level of resistance observed in different genetic backgrounds. Taken together, our results provide new prospects for a better understanding of host responses to rhabdovirus infections in salmonids.

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.

In vivo fitness correlates with host-specific virulence of Infectious hematopoietic necrosis virus (IHNV) in sockeye salmon and rainbow trout.

The relationship between virulence and overall within-host fitness of the fish rhabdovirus Infectious hematopoietic necrosis virus (IHNV) was empirically investigated in vivo for two virus isolates belonging to different IHNV genogroups that exhibit opposing host-specific virulence. U group isolates are more virulent in sockeye salmon and M group isolates are more virulent in rainbow trout. In both single and mixed infections in the two fish hosts, the more virulent IHNV type exhibited higher prevalence and higher viral load than the less virulent type. Thus, a positive correlation was observed between higher in vivo fitness and higher host-specific virulence in sockeye salmon and rainbow trout. Comparisons of mean viral loads in single and mixed infections revealed no evidence for limitation due to competition effects between U and M viruses in either rainbow trout or sockeye salmon co-infections.

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.