N Protein of Viral Hemorrhagic Septicemia Virus Suppresses STAT1-Mediated MHC Class II Transcription to Impair Antigen Presentation in Sea Perch, Lateolabrax japonicus.

Upon virus invasion of the host, APCs process Ags to short peptides for presentation by MHC class II (MHC-II). The recognition of virus-derived peptides in the context of MHC-II by CD4+ T cells initiates the adaptive immune response for virus clearance. As a survival instinct, viruses have evolved mechanisms to evade Ag processing and presentation. In this study, we discovered that IFN-γ induced endogenous MHC-II expression by a sea perch brain cell line through the STAT1/IFN regulatory factor 1 (IRF1)/CIITA signaling pathway. Furthermore, viral hemorrhagic septicemia virus infection significantly inhibited the IFN-γ-induced expression of IRF1, CIITA, MHC-II-α, and MHC-II-ß genes. By contrast, although STAT1 transcript was upregulated, paradoxically, the STAT1 protein level was attenuated. Moreover, overexpression analysis revealed that viral hemorrhagic septicemia virus N protein blocked the IFN-γ-induced expression of IRF1, CIITA, MHC-II-α, and MHC-II-ß genes, but not the STAT1 gene. We also found out that N protein interacted with STAT1 and enhanced the overall ubiquitination level of proteins, including STAT1 in Lateolabrax japonicus brain cells. Enhanced ubiquitination of STAT1 through K48-linked ubiquitination led to its degradation through the ubiquitin-proteasome pathway, thereby inhibiting the biological function of STAT1. Our study suggests that aquatic viruses target Ag presentation in lower vertebrates for immune evasion as do mammalian viruses.

Naturally occurring substitution in one amino acid in VHSV phosphoprotein enhances viral virulence in flounder.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Naturally occurring VHSV strains vary greatly in virulence. Until now, little has been known about genetic alterations that affect the virulence of VHSV in flounder. We recently reported the full-genome sequences of 18 VHSV strains. In this study, we determined the virulence of these 18 VHSV strains in flounder and then the assessed relationships between differences in the amino acid sequences of the 18 VHSV strains and their virulence to flounder. We identified one amino acid substitution in the phosphoprotein (P) (Pro55-to-Leu substitution in the P protein; PP55L) that is specific to highly virulent strains. This PP55L substitution was maintained stably after 30 cell passages. To investigate the effects of the PP55L substitution on VHSV virulence in flounder, we generated a recombinant VHSV carrying PP55L (rVHSV-P) from rVHSV carrying P55 in the P protein (rVHSV-wild). The rVHSV-P produced high level of viral RNA in cells and showed increased growth in cultured cells and virulence in flounder compared to the rVHSV-wild. In addition, rVHSV-P significantly inhibited the induction of the IFN1 gene in both cells and fish at 6 h post-infection. An RNA-seq analysis confirmed that rVHSV-P infection blocked the induction of several IFN-related genes in virus-infected cells at 6 h post-infection compared to rVHSV-wild. Ectopic expression of PP55L protein resulted in a decrease in IFN induction and an increase in viral RNA synthesis in rVHSV-wild-infected cells. Taken together, our results are the first to identify that the P55L substitution in the P protein enhances VHSV virulence in flounder. The data from this study add to the knowledge of VHSV virulence in flounder and could benefit VHSV surveillance efforts and the generation of a VHSV vaccine.

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

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.

The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout.

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout (Oncorhynchus mykiss). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral nucleoprotein (N), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric recombinant VHSV (rVHSV) by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout virulence phenotype of either parental strain. However, reciprocal exchanges of the viral N gene resulted in a partial gain of function in the chimeric trout-avirulent strain (22% mortality) and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and P genes together resulted in complete gain of function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout.IMPORTANCE Rainbow trout farming is a major food source industry worldwide that has suffered great economic losses due to host jumps of fish rhabdovirus pathogens, followed by evolution of dramatic increases in trout-specific virulence. However, the genetic determinants of host jumps and increased virulence in rainbow trout are unknown for any fish rhabdovirus. Previous attempts to identify the viral genes containing trout virulence determinants of viral hemorrhagic septicemia virus (VHSV) have not been successful. We show here that, somewhat surprisingly, the viral nucleocapsid (N) and phosphoprotein (P) genes together contain the determinants responsible for trout virulence in VHSV. This suggests a novel host-specific virulence mechanism involving the viral polymerase and a host component. This differs from the known virulence mechanisms of mammalian rhabdoviruses based on the viral P or M (matrix) protein.

Virulence marker candidates in N-protein of viral haemorrhagic septicaemia virus (VHSV): virulence variability within VHSV Ib clones.

Four major genotypes of viral haemorrhagic septicaemia virus (VHSV), which have been isolated from many marine and freshwater fish species, are known to differ in virulence. While fast and low-cost genotyping systems based on monoclonal antibodies (MAbs) have been developed for typing of VHSV virulence, there is a need for supplementing the knowledge. In particular, 2 field isolates from viral haemorrhagic septicaemia (VHS) outbreaks in sea-reared rainbow trout Oncorhynchus mykiss in Sweden, SE-SVA-14 and SE-SVA-1033 (both genotype Ib), have yielded contradictory reactions. In the present study, upon cloning by limited dilution, both isolates appeared to be heterogeneous in terms of reactivity with nucleo (N)-protein-specific MAbs as well their gene sequences. Infection trials in rainbow trout further revealed differences in the virulence of these virus clones derived from the same primary isolate. Based on a comparative analysis of the entire genome of the clones tested, we suggest that the differences in virulence are tentatively linked to substitutions of amino acids (aa) in the N-protein region covered by aa 43-46 and aa position 168, or a combination of the two. The fact that such minor naturally occurring genetic differences affect the virulence implies that even low-virulent VHSV isolates in the marine environment should be considered as a potential threat for the trout farming industry. The described MAbs can represent useful tools for initial risk assessment of disease outbreaks in farmed trout by marine VHSV isolates.

NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction.

Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

Viral hemorrhagic septicemia virus glycoprotein production in tobacco.

Viral hemorrhagic septicemia virus (VHSV) causes mortality in numerous marine and freshwater fish species resulting in heavy losses in fish farming. The glycoprotein gene of VHSV was fused with the cholera toxin B subunit (CTB) and expressed transiently in leaf tissues of Nicotiana benthamiana via the agroinfiltration method. The glycoprotein gene was divided into two parts to improve assembly of CTB fusion proteins (CTB-VHSV99-235 and CTB-VHSV258-417). Production of CTB fusion proteins was confirmed in the agroinfiltrated leaf tissue by western blot analysis. The plant-produced CTB fusion proteins showed biological activity to GM1-ganglioside, a receptor for biologically active CTB, on GM1-ELISA. The expression level of the CTB-VHSV fusion proteins was 0.86% (CTB-VHSV99-235) and 0.93% (CTB-VHSV258-417) of total proteins in agroinfiltrated leaf tissue, as determined by GM1-ELISA. These results suggest that Agrobacterium-mediated transient expression of CTB fusion antigens of VHSV is a rapid and convenient method and demonstrate the feasibility of using agroinfiltrated plant leaf tissues expressing CTB-fusion antigens as a plant-based vaccine to prevent VHSV infection.

Complete Protection against Influenza Virus H1N1 Strain A/PR/8/34 Challenge in Mice Immunized with Non-Adjuvanted Novirhabdovirus Vaccines.

Novirhabdoviruses like Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) are fish-infecting Rhabdoviruses belonging to the Mononegavirales order. By reverse genetics, we previously showed that a recombinant VHSV expressing the West Nile Virus (WNV) E glycoprotein could serve as a vaccine platform against WNV. In the current study, we aimed to evaluate the potential of the Novirhabdovirus platform as a vaccine against influenza virus. Recombinant Novirhabdoviruses, rVHSV-HA and rIHNV-HA, expressing at the viral surface the hemagglutinin HA ectodomain were generated and used to immunized mice. We showed that mice immunized with either, rVHSV-HA or rIHNV-HA, elicited a strong neutralizing antibody response against influenza virus. A complete protection was conferred to the immunized mice when challenged with a lethal dose of influenza H1N1 A/PR/8/34 virus. Furthermore we showed that although acting as inert antigen in mice, since naturally inactivated over 20°C, mice immunized with rVHSV-HA or rIHNV-HA in the absence of adjuvant were also completely protected from a lethal challenge. Novirhabdoviruses platform are of particular interest as vaccines for mammals since they are cost effective to produce, relatively easy to generate and very effective to protect immunized animals.

Ultra-deep sequencing of VHSV isolates contributes to understanding the role of viral quasispecies.

The high mutation rate of RNA viruses enables the generation of a genetically diverse viral population, termed a quasispecies, within a single infected host. This high in-host genetic diversity enables an RNA virus to adapt to a diverse array of selective pressures such as host immune response and switching between host species. The negative-sense, single-stranded RNA virus, viral haemorrhagic septicaemia virus (VHSV), was originally considered an epidemic virus of cultured rainbow trout in Europe, but was later proved to be endemic among a range of marine fish species in the Northern hemisphere. To better understand the nature of a virus quasispecies related to the evolutionary potential of VHSV, a deep-sequencing protocol specific to VHSV was established and applied to 4 VHSV isolates, 2 originating from rainbow trout and 2 from Atlantic herring. Each isolate was subjected to Illumina paired end shotgun sequencing after PCR amplification and the 11.1 kb genome was successfully sequenced with an average coverage of 0.5-1.9 × 10(6) sequenced copies. Differences in single nucleotide polymorphism (SNP) frequency were detected both within and between isolates, possibly related to their stage of adaptation to host species and host immune reactions. The N, M, P and Nv genes appeared nearly fixed, while genetic variation in the G and L genes demonstrated presence of diverse genetic populations particularly in two isolates. The results demonstrate that deep sequencing and analysis methodologies can be useful for future in vivo host adaption studies of VHSV.

Specific nucleotides at the 3'-terminal promoter of viral hemorrhagic septicemia virus are important for virulence in vitro and in vivo.

Viral hemorrhagic septicemia virus (VHSV), a member of the Novirhabdovirus genus, contains an 11-nucleotide conserved sequence at the terminal 3'- and 5'-untranslated regions (UTRs) that are complementary. To study the importance of nucleotides in the 3'-UTR of VHSV for replication of novirhabdoviruses, we performed site-directed mutagenesis of selected residues at the 3'-terminus and generated mutant viruses using a reverse genetics approach. Assessment of growth kinetics and in vitro real-time cytopathogenicity studies showed that the order of two nucleotides (A4G5) of the 3'-terminus of VHSV directly affects growth kinetics in vitro. The mutant A4G-G5A virus has reduced total positive-strand RNA synthesis efficiency (51% of wild-type) at 48h post-transfection and 70h delay in causing complete cytopathic effect in susceptible fish cells, as compared to the WT-VHSV. Furthermore, when the A4G-G5A virus was used to challenge zebrafish, it exhibited reduced pathogenicity (54% lower end-point mortality) compared to the WT-VHSV. From these studies, we infer that specific residues in the 3'-UTR of VHSV have a promoter function and are essential to modulate the virulence in cells and pathogenicity in fish.

FinTRIMs, fish virus-inducible proteins with E3 ubiquitin ligase activity.

TRIM proteins have recently emerged as novel players in antiviral defense. TRIM proteins contain a tri-partite motif, composed of a RING zinc finger, one or two B-boxes and a coiled-coil domain. Many members of this large protein family of E3 ubiquitin ligases catalyze the attachment of ubiquitin to a substrate protein, an activity dependent on the RING domain. We earlier made a full description of the TRIM gene family in zebrafish and pufferfish and identified three multigene TRIM subsets, a feature unique to fish. To determine their biological role, we further characterized members of the finTRIM subset. FinTRIM gene expression was studied during development and in multiple tissues in adult rainbow trout. Upregulation of a large number of finTRIM upon viral stimulation suggests they are involved in antiviral immunity. We also demonstrate that two finTRIM members display E3 ubiquitin ligase activity, indicating that finTRIMs could regulate antiviral signaling through ubiquitination.

Rainbow trout Oncorhynchus mykiss ladderlectin, but not intelectin, binds viral hemorrhagic septicemia virus IVb.

The innate immune system of fish is critical for rapid detection and immediate response to infection, as well as to orchestrate the adaptive branch of the immune system. Rainbow trout Oncorhynchus mykiss ladderlectin and intelectin are plasma pattern recognition receptors (PRR) for bacterial and fungal pathogens of rainbow trout, but their role as PRRs for virus is unknown. Viral hemorrhagic septicemia virus (VHSV) IVb is a recently described fish pathogen in the Great Lakes, and rainbow trout can be experimentally infected. Using an indirect enzyme-linked immunosorbent assay, rainbow trout plasma ladderlectin significantly (p < 0.05) bound purified VHSV while intelectin did not. In addition, plasma ladderlectin but not intelectin was eluted from a VHSV-conjugated Toyopearl column using EDTA. Protein identification was confirmed with polyclonal antiserum used with slot immunoblot, 1-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis, and Western immunoblot.

An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein.

An enzyme linked immunosorbent assay (ELISA) method to study serum antibodies to viral haemorrhagic septicemia virus (VHSV) was designed by using recombinant fragments of their G protein. By using this fragment-ELISA, we describe the binding of antibodies against recombinant G fragments of 45-445 amino acids present in VHSV-hyperimmunized trout sera. Fragments were designed by taking into account their tridimensional pH-dependent structure and functional domains. Sera were obtained from hyperimmunized trout following 4-5 intraperitoneal injections of VHSV antigens by using Freund's or saponin adjuvants. Sera from different hyperimmunized trout differed quantitatively rather than qualitatively in their recognition of solid-phase frg11 (56-110), frg12 (65-109), frg13 (97-167), frg14 (141-214), frg15 (65-250), frg16 (252-450) and G (G21-465) by Western blot and ELISA. However, titres were higher when using frg11, frg15 or frg16, rather than G21-465, suggesting higher accessibility to G epitopes. Further knowledge of the antigenicity of the G protein of rhabdoviruses by using fragments might be used to improve current vaccines. On the other hand, they might be used to dissect the trout antibody response to VHSV infections, to complement in vitro neutralizing assays, and/or to quantitate anti-VHSV antibodies in VHSV-infected/vaccinated trout, other fish and/or other body fluids such as mucus.

Evolutional conservation of molecular structure and antiviral function of a viral RNA receptor, LGP2, in Japanese flounder, Paralichthys olivaceus.

LGP2 is an important intracellular receptor that recognizes viral RNAs in innate immunity. To understand the mechanism of viral RNA recognition, we cloned an LGP2 cDNA and gene in Japanese flounder (Paralichthys olivaceus). Viral hemorrhagic septicemia virus-induced expressions of LGP2 mRNA were evaluated in vivo and in vitro by quantitative real-time PCR (Q-PCR) using primers based on the clone sequences. The expression of LGP2 mRNA in the kidney dramatically increased at 3 d postinfection. The expression of LGP2 mRNA also increased in the head kidney leukocytes stimulated with artificial dsRNA (polyinosin-polycytidylic acid) in vitro. To evaluate the antiviral activity of the flounder LGP2, three expression constructs containing pcDNA4-LGP2 (full-length), pcDNA4-LGP2ΔRD (regulatory domain deleted), and pcDNA4-Empty (as a negative control) were transfected into the hirame (flounder) natural embryo (hirame natural embryo) cell line. Forty-eight hours after transfection, the transfected cells were infected with ssRNA viruses, viral hemorrhagic septicemia virus, or hirame rhabdovirus. The cytopathic effects of the viruses were delayed by the overexpression of Japanese flounder LGP2. The Q-PCR demonstrated that mRNA expression levels of type I IFN and IFN-inducible genes (Mx and ISG15) in the hirame natural embryo cells overexpressing LGP2 were increased by polyinosin-polycytidylic acid and viral infections. These results suggest that Japanese flounder LGP2 plays an important role in the recognition of both viral ssRNA and dsRNA to induce the antiviral activity by the production of IFN-stimulated proteins.

Engineered virus-encoded pre-microRNA (pre-miRNA) induces sequence-specific antiviral response in addition to nonspecific immunity in a fish cell line: convergence of RNAi-related pathways and IFN-related pathways in antiviral response.

Transfection with synthesized virus-specific small interfering RNAs (siRNAs) efficiently inhibits viral replication in viral-infected fish cell lines, implying the involvement of RNA interference (RNAi)-related pathways in the antiviral response of fish cells. Here, we demonstrate that plasmid expressing virus-encoded pre-microRNAs (pre-miRNAs) can also inhibit viral replication through these pathways. By incorporating sequences encoding miRNAs specific to major capsid protein (MCP) gene of red sea bream iridovirus (RSIV) and a miRNA specific to hirame rhabdovirus (HIRRV) genome into a murine miR-155 pre-miRNA backbone, we were able to intracellularly express viral pre-miRNAs (miR-MCPs and miR-HIRRV) in a fish cell line. The miR-MCPs and miR-HIRRV, delivered as pre-miRNA precursors in transfected cells, inhibited viral replication when these cells were infected with the target virus. Although this may suggest sequence-specific interference, inhibitory effect on viral replication was also observed in cells transfected with a plasmid expressing pre-miRNA targeting beta-galactosidase gene (miR-LacZ) that served as a specificity control. Expression of pre-miRNAs was found to activate interferon (IFN)-related pathways, correlating with upregulation of the antiviral IFN-induced Mx protein. The antiviral effects of viral-miRNAs observed here were partly the result of the antiviral miRNA-related pathways and partly the result of the antiviral IFN-related pathways. We propose that engineered virus-encoded pre-miRNA can engage not only RNAi-related pathways but also IFN-related pathways to induce potent antiviral responses in fish cells.

Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination.

Japanese flounder, Paralichthys olivaceus juveniles were vaccinated against viral hemorrhagic septicemia (VHS) by intramuscular injection of 10 microg of a plasmid DNA vector which encodes the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene under the control of the cytomegalovirus promoter. Experimental challenge of two viral doses (1 x 10(2) TCID50 and 1 x 10(3) TCID50) one month post-vaccination revealed that the G gene was able to induce protective immunity against VHS and this lasted until 21 days after the challenge. The VHSV G-protein gene DNA vaccine had a high protective efficiency, giving relative percentage survival (RPS) values of at least 93%. The defense mechanisms activated by the DNA vaccine were further elucidated by microarray analysis. Non-specific immune response genes such as NK, Kupffer cell receptor, MIP1-alpha and Mx1 protein gene were observed to be up-regulated by the VHSV G-protein DNA vaccine at 1 and 3 days post-immunization. Also, specific immune-related genes including the CD20 receptor, CD8 alpha chain, CD40 and B lymphocyte cell adhesion molecule were also up-regulated during that time. We observed significant up-regulation of some immune-related genes that are necessary for antiviral defense. Significant up- and/or down-regulation of unknown genes was also observed upon DNA vaccination. Our results confirm previous reports that the VHSV G gene elicits strong humoral and cellular immune responses which may play a pivotal role in protecting the fish during virus 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.

Neutralisation and binding of VHS virus by monovalent antibody fragments.

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (Vkappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIAcore analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted molecular analysis of the theoretical influence of each mutation on antigen binding, led to the identification of a single mutation at position 35a in the VH domain as having the most marked impact on viral neutralisation.