Anti-viral mechanism of barramundi Mx against betanodavirus involves the inhibition of viral RNA synthesis through the interference of RdRp

Nervous necrosis virus (NNV) belongs to the betanodavirus of the Nodaviridae family. It is the causative agent of viral nervous necrosis (VNN) disease, and has inflicted devastating damage on the world-wide aquaculture industry. The fish that survived after the outbreak of VNN become persistently NNV-infected carriers. NNV-persistent infection has been demonstrated in a barramundi brain (BB) cell line, and it involves the type I interferon (IFN) response with the expression of Mx gene. However, little of the defense mechanism in fish cells against NNV infection is understood. In this study, the anti-NNV mechanism of barramundi Mx protein (BMx) was elucidated in cBB cells which were derived from BB cell line after serial treatments by NNV-specific antiserum and then became an NNV-free cell line. After NNV infection of cBB cells, the level of viral RNA-dependent RNA polymerase (RdRp) increased with time over a period of 24 h post-infection (hpi), but decreased when the BMx expression increased 48 and 72 hpi. When the expression of BMx was down-regulated by BMx-specific siRNA, the expression levels of viral RNA, proteins and progeny viral titers were restored. The BMx was found to colocalize with viral RdRp at the perinuclear area 24 hpi and coprecipitate with viral RdRp, indicating that they could bind with each other. Viral RdRp was also revealed to colocalize with lysosomes 48 hpi as the NNV RdRp level started to decline. Therefore, it is suggested that BMx inhibited the viral RNA synthesis by interaction with viral RdRp, and redistributed RdRp to perinuclear area for degradation.     

Murine gammaherpesvirus targets type I IFN receptor but not type III IFN receptor early in infection

The innate immune response represents a primary line of defense against invading viral pathogens. Since epithelial cells are the primary site of gammaherpesvirus replication during infection in vivo and there are no information on activity of IFN-III signaling against gammaherpesviruses in this cell type, in present study, we evaluated the expression profile and virus-host interactions in mouse mammary epithelial cell (NMuMG) infected with three strains of murine gammaherpesvirus, MHV-68, MHV-72 and MHV-4556. Studying three strains of murine gammaherpesvirus, which differ in nucleotide sequence of some structural and non-structural genes, allowed us to compare the strain-dependent interactions with host organism. Our results clearly demonstrate that: (i) MHV-68, MHV-72 and MHV-4556 differentially interact with intracellular signaling and dysregulate IFN signal transduction; (ii) MHV-68, MHV-72 and MHV-4556 degrade type I IFN receptor in very early stages of infection (2–4 hpi), but not type III IFN receptor; (iii) type III IFN signaling might play a key role in antiviral defense of epithelial cells in early stages of murine gammaherpesvirus replication; (iv) NMuMG cells are an appropriate model for study of not only type I IFN signaling, but also type III IFN signaling pathway. These findings are important for better understanding of individual virus-host interactions in lytic as well as in persistent gammaherpesvirus replication and help us to elucidate IFN-III function in early events of virus infection.     

Expression analysis of cathelicidin and NK‐lysin in Dabry’s sturgeon (Acipenser dabryanus)

Antimicrobial peptides (AMPs) are a conserved component of the innate immune response in many species. In the present study, the cDNA sequences encoding two AMPs (cathelicidin and NK‐lysin, comprising 1,576 and 606 bp, respectively) were cloned from Dabry's sturgeon (Acipenser dabryanus). Phylogenetic analysis demonstrated that the two AMPs were clustered together with homologous protein sequences from other fish. NK‐lysin was highly expressed during early embryonic development, suggesting maternal transmission. Tissue distribution analysis showed that cathelicidin had the highest expression in the liver and NK‐lysin was most abundantly expressed in the spleen. In response to Poly I:C treatment, the expression of cathelicidin was upregulated at 12 and 24 hr post induction (hpi), but downregulated at 72 hpi. NK‐lysin mRNA expression increased after treatment with Poly I:C, reaching a peak at 24 hpi. Lipopolysaccharide treatment also induced the expression of two antimicrobial peptide genes. Lipopolysaccharide treatment significantly upregulated the expression of cathelicidin at 6, 24, and 48 hpi, and upregulated NK‐lysin expression at 6 and 12 hpi. These results suggested that two AMPs could participate in the immune response induced by poly I:C or LPS stimulation.     

Major Histocompatibility Complex Loci are Associated with Susceptibility of Atlantic Salmon to Infectious Hematopoietic Necrosis Virus

Infectious hematopoietic necrosis virus (IHNV) is one of the most significant viral pathogens of salmonids and is a leading cause of death among cultured juvenile fish. Although several vaccine strategies have been developed, some of which are highly protective, the delivery systems are still too costly for general use by the aquaculture industry. More cost effective methods could come from the identification of genes associated with IHNV resistance for use in selective breeding. Further, identification of susceptibility genes may lead to an improved understanding of viral pathogenesis and may therefore aid in the development of preventive and therapeutic measures. Genes of the major histocompatibility complex (MHC), involved in the primary recognition of foreign pathogens in the acquired immune response, are associated with resistance to a variety of diseases in vertebrate organisms. We conducted a preliminary analysis of MHC disease association in which an aquaculture strain of Atlantic salmon was challenged with IHNV at three different doses and individual fish were genotyped at three MHC loci using denaturing gradient gel electrophoresis (PCR-DGGE), followed by sequencing of all differentiated alleles. Nine to fourteen alleles per exon-locus were resolved, and alleles potentially associated with resistance or susceptibility were identified. One allele (Sasa-B-04) from a potentially non-classical class I locus was highly associated with resistance to infectious hematopoietic necrosis (p < 0.01). This information can be used to design crosses of specific haplotypes for family analysis of disease associations.     

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.     

Response to vaccination of Atlantic cod (Gadus morhua L.) progenies from families with different estimated family breeding values for vibriosis resistance

The purpose of the study was to elucidate whether responses to vibriosis vaccination and gene expressions in parts of the innate immune system were different in families of Atlantic cod (Gadus morhua). The fish were progenies of families with differences in estimated breeding values (EBV) for vibriosis resistance. Families of coastal cod (CC) and northeast Arctic cod (AC) responded well to vaccination with a relative percent survival of 72–95. No correlation between response to vaccination and vibriosis resistance were found (p = 0.146). The AC family with medium low (M) resistance had significant (p ≤ 0.019) lowest mortality among all the unvaccinated fish but the CC-M family. Further, when comparing the vaccinated fish the AC family with very high (VH) resistance had significant (p ≤ 0.004) higher mortality than all except the CC-VL and CC-H families.Parts of the innate immune response were studied by measuring the gene expression of innate immune genes 2 and 4 days post dip vaccination. Vaccinated fish from two families had a weak but significant higher innate immune response compared to control fish of the same family. In vaccinated fish, the gene expression of interleukin (IL) 1b, IL-10, IL-12p40 and hepcidin were significant up-regulated. While, no measureable activations of interferon gamma (IFNγ), IL-8, cathelicidin, LBP/BPI and G-type lysozyme were found.     

Theiler’s murine encephalomyelitis virus infection induces a redistribution of heat shock proteins 70 and 90 in BHK-21 cells,and is inhibited by novobiocin and geldanamycin

Theiler’s murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus.     

Genetic resistance to rhabdovirus infection in teleost fish is paralleled to the derived cell resistance status

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

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.     

Age‐0 striped bass,Morone saxatilis (Walbaum, 1792), response to immunostimulation

Young‐of‐the‐year (age‐0) striped bass, Morone saxatilis, were studied to characterize their responses to inflammatory stimuli. There were two studies, with the hypotheses that (i) <5 g striped bass would respond to inflammatory insult within 6 h, and (ii) response to antigens would be maintained for >24 h in larger fish. Study I was conducted to understand the cytokine expression pattern (IL‐1β, TNF‐α, Nramp and TGF‐β) in response to lipopolysaccharide (LPS) or Freund's complete adjuvant (FCA) stimulation in age‐0 striped bass (3.44 ± 1.68 g, 70.6 ± 10.3 mm fork length) up to 24 h post injection (hpi). Study II was similar to Study I, but striped bass were sampled over a longer time frame (by 48 hpi) and larger age‐0 striped bass were used (20.6 ± 5.9 g, 129.2 ± 10.9 mm fork length). It was confirmed that immunostimulants such as LPS and FCA induce production of inflammatory cytokines and Nramp, which are important in innate immune response to bacterial infection. The responses were rapidly stimulated with LPS (IL‐1β, TNF‐α, TGF‐β >3‐fold increase, compared to PBS) or FCA (IL‐1β >3‐fold and TGF‐β >2‐fold, compared to PBS) within 6 hpi and maintained in most cases 48 hpi (spleen Nramp and TGF‐β 2‐fold >PBS, at 24 and 48 h), similar to other teleosts. Intraperitoneal injection with PBS also simulated inflammatory gene expression, but was delayed (IL‐1β, TNF‐α, TGF‐β and Nramp, FCA and LPS< at 6 h; 24 h >LPS and PBS) in comparison to LPS and FCA, suggesting that this procedure and possibly the volume used can be stimulatory and potentially harmful in age‐0 fish. Therefore, this study suggests that age‐0 striped bass are capable of strong cytokine induction in response to immunological stimulation within a very short period of time.     

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.     

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.     

Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream

The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.     

A Reverse Genetics System for the Great Lakes Strain of Viral Hemorrhagic Septicemia Virus: the NV Gene is Required for Pathogenicity

Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus in the family of Rhabdoviridae, causes a highly contagious disease of fresh and saltwater fish worldwide. Recently, a novel genotype of VHSV, designated IVb, has invaded the Great Lakes in North America, causing large-scale epidemics in wild fish. An efficient reverse genetics system was developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The recombinant VHSV (rVHSV) was comparable to the parental wild-type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified recombinant VHSV was generated in which the NV gene was substituted with an enhanced green fluorescent protein gene (rVHSV-ΔNV-EGFP), and another recombinant was made by inserting the EGFP gene into the full-length viral clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV; however, the rVHSV-ΔNV-EGFP grew 2 logs lower. In yellow perch challenges, wtVHSV and rVHSV induced 82-100% cumulative per cent mortality (CPM), respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-ΔNV-EGFP caused only 15% CPM. No reversion of mutation was detected in the recovered viruses and the recombinant viruses stably maintained the foreign gene after several passages. These results indicate that the NV gene of VHSV is not essential for viral replication in vitro and in vivo, but it plays an important role in viral replication efficiency and pathogenicity. This system will facilitate studies of VHSV replication, virulence, and production of viral vectored vaccines.     

Larval and juvenile Pacific herring Clupea pallasii are not susceptible to infectious hematopoietic necrosis under laboratory conditions

Infectious hematopoietic necrosis (IHN) leads to periodic epidemics among certain wild and farmed fish species of the Northeast (NE) Pacific. The source of the IHN virus (IHNV) that initiates these outbreaks remains unknown; however, a leading hypothesis involves viral persistence in marine host species such as Pacific herring Clupea pallasii. Under laboratory conditions we exposed specific pathogen-free (SPF) larval and juvenile Pacific herring to 10(3) to 10(4) plaque-forming units (pfu) of IHNV ml(-1) by waterborne immersion. Cumulative mortalities among exposed groups were not significantly different from those of negative control groups. After waterborne exposure, IHNV was transiently recovered from the tissues of larvae but absent in tissues of juveniles. Additionally, no evidence of viral shedding was detected in the tank water containing exposed juveniles. After intraperitoneal (IP) injection of IHNV in juvenile herring with 10(3) pfu, IHNV was recovered from the tissues of sub-sampled individuals for only the first 5 d post-exposure. The lack of susceptibility to overt disease and transient levels of IHNV in the tissues of exposed fish indicate that Pacific herring do not likely serve a major epizootiological role in perpetuation of IHNV among free-ranging sockeye salmon Oncorhynchus nerka and farmed Atlantic salmon Salmo salar in the NE Pacific.