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.

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.

In vivo fitness associated with high virulence in a vertebrate virus is a complex trait regulated by host entry, replication, and shedding.

The relationship between pathogen fitness and virulence is typically examined by quantifying only one or two pathogen fitness traits. More specifically, it is regularly assumed that within-host replication, as a precursor to transmission, is the driving force behind virulence. In reality, many traits contribute to pathogen fitness, and each trait could drive the evolution of virulence in different ways. Here, we independently quantified four viral infection cycle traits, namely, host entry, within-host replication, within-host coinfection fitness, and shedding, in vivo, in the vertebrate virus Infectious hematopoietic necrosis virus (IHNV). We examined how each of these stages of the viral infection cycle contributes to the fitness of IHNV genotypes that differ in virulence in rainbow trout. This enabled us to determine how infection cycle fitness traits are independently associated with virulence. We found that viral fitness was independently regulated by each of the traits examined, with the largest impact on fitness being provided by within-host replication. Furthermore, the more virulent of the two genotypes of IHNV we used had advantages in all of the traits quantified. Our results are thus congruent with the assumption that virulence and within-host replication are correlated but suggest that infection cycle fitness is complex and that replication is not the only trait associated with virulence.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 µm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 µm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic necrosis virus (IHNV).

The nature of the association between viral fitness and virulence remains elusive in vertebrate virus systems, partly due to a lack of in vivo experiments using statistically sufficient numbers of replicate hosts. We examined the relationship between virulence and fitness in Infectious hematopoietic necrosis virus (IHNV), in vivo, in intact living rainbow trout. Trout were infected with a high or low virulence genotype of M genogroup IHNV, or a mixture of the two genotypes, so as to calculate relative fitness and the effect of a competition environment on fitness. Fitness was measured as total viral load in the host at time of peak viral density, quantified by genotype-specific quantitative RT-PCR (qRT-PCR). The more virulent IHNV genotype reached higher densities in both single and mixed infections. There was no effect of competition on the performance of either genotype. Our results suggest a positive link between IHNV genotype fitness and virulence.

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout-derived cell line, RTG-2.

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout-derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

Monitoring viral-induced cell death using electric cell-substrate impedance sensing.

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.

A chlorophyll c2 analogue from the marine brown alga Eisenia bicyclis inactivates the infectious hematopoietic necrosis virus, a fish rhabdovirus.

We screened in vitro antiviral activity against a salmonid pathogenic virus, infectious hematopoietic necrosis virus (IHNV), from the extracts of a total of 342 species of marine algae collected from the Japanese coastline. The anti-IHNV activity was found primarily in MeOH extracts, and the extract from one marine brown alga in particular, Eisenia bicyclis, showed high anti-IHNV activity. The anti-IHNV compound was isolated and purified as MC15 from the E. bicyclis extract, and the chemical structure was determined by several spectrometric analyses. The antiviral compound was proved to be a chlorophyll c2 derivative lacking the metal ion Mg(2+). MC15 showed similar antiviral activity against other salmonid enveloped viruses such as Paralichthys olivaceus virus and Oncorhynchus masou virus, and stability against any pH and temperatures up to 100 degrees C. No cytotoxicity was observed at up to 5 microg/ml. The antiviral mechanism of MC15 appears to be direct inactivation of the viral particles. A time course study showed that the inactivation of IHNV was completed within 40 min when 200 PFU of IHNV was reacted with MC15 at 800 ng/ml.

Establishment of embryonic cell line from sea bass (Lates calcarifer) for virus isolation.

A continuous cell line was established from blastula stage embryos of sea bass (Lates calcarifer). The sea bass embryonic cells were maintained in Leibovitz's L-15 supplemented with 15% fetal bovine serum. The embryonic cell line was sub-cultured more than 70 passages over a period of 1.5 years and is designated as Sahul Indian sea bass embryonic (SISE) cell line. The cells were able to grow at temperatures between 25 and 32 degrees C with an optimum temperature of 28 degrees C. The growth rate of sea bass embryonic cells increased as the FBS proportion increased from 2 to 20% at 28 degrees C with optimum growth at the concentration of 15 or 20%. Polymerase chain reaction products were obtained from embryonic cells and blastula of sea bass with primer sets of microsatellite markers of sea bass. Four fish viruses were tested on this cell line to determine its susceptibility to these viruses and this cell line was found to be susceptible to IPNV VR-299 and nodavirus, and the infection was confirmed by cytopathic effect (CPE) and RT-PCR. Further, this cell line was characterized by immunocytochemistry using confocal-laser-scanning microscopy (CFLSM), transfection with pEGFP-N1, proliferate marker (BrdU).

Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens.

It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non-specific anti-viral defence mechanisms.