Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O. nerka salmon

The level of protective immunity was determined for Chinook Oncorhynchus tshawytscha and sockeye/kokanee salmon (anadromous and landlocked) O. nerka following intramuscular vaccination with a DNA vaccine against the aquatic rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A DNA vaccine containing the glycoprotein gene of IHNV protected Chinook and sockeye/kokanee salmon against waterborne or injection challenge with IHNV, and relative percent survival (RPS) values of 23 to 86% were obtained under a variety of lethal challenge conditions. Although this is significant protection, it is less than RPS values obtained in previous studies with rainbow trout (O. mykiss). In addition to the variability in the severity of the challenge and inherent host susceptibility differences, it appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine. Neutralizing antibody titers were detected in both Chinook and sockeye that had been vaccinated with 1.0 and 0.1 pg doses of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish.     

Evaluation of the protective immunogenicity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout oncorhynchus mykiss using DNA vaccines.

The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow trout Oncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naive fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.     

Effectiveness of bivalent vaccines against Aeromonas hydrophila and Lactococcus garvieae infections in rainbow trout Oncorhynchus mykiss (Walbaum)

Lactococcus garvieae and Aeromonas hydrophila are bacterial pathogens affecting salmonids and other fish species and cause of heavy losses in aquaculture. Diseases caused by these bacteria can be controlled satisfactory by immunization using monovalent vaccines. In this study, the protective efficacy of two bivalent vaccines against L. garvieae and A. hydrophila was evaluated in rainbow trout (Oncorhynchus mykiss). Bivalent formulations, containing formalin-inactivated bacteria, were prepared as an aqueous bacterin and as an adjuvanted vaccine using montanide ISA-763. Protection against L. garvieae and A. hydrophila was tested at day 30 and 90 post-vaccination. High levels of protection were achieved for the aqueous and adjuvanted bivalent vaccines against L. garvieae (RPS of 100% and 95.3%) and A. hydrophila (RPS of 100% and 95.3%) at day 30 post-vaccination. Significant differences (p < 0.05) were found between the RPS at days 30 and 90 post-immunization with a decrease in the protection levels for the aqueous bivalent vaccine against L. garvieae (RPS 76.2%) and A. hydrophila (RPS 85%), but not for the adjuvanted vaccine (RPS of 90% against L. garvieae and 95% against A. hydrophila). In addition, high antibody levels were observed in the vaccinated fish at day 15 post-immunization using both vaccines. Our results demonstrate that these bivalent vaccines can effectively protect rainbow trout against L. garvieae and A. hydrophila and could offer an appropriate strategy to prevent these infections in rainbow trout farms.     

Naked DNA vaccination of Atlantic salmon Salmo salar against IHNV

A naked plasmid DNA encoding the glycoprotein (pCMV4-G) of a 1976 isolate of infectious hematopoietic necrosis virus (IHNV) obtained from steelhead Oncorhynchus mykiss was used to vaccinate Atlantic salmon Salmo salar against IHNV. Eight weeks post-vaccination the fish were challenged with a strain of IHNV originally isolated from farmed Atlantic salmon undergoing an epizootic. Fish injected with the glycoprotein-encoding plasmid were significantly (p < 0.05) protected against IHNV by both immersion and cohabitation challenge. Survivors of the first challenges were pooled and re-challenged by immersion 12 wk after the initial challenge. Significant (p < 0.05) protection was observed in all of the previously challenged groups including those receiving the complete vaccine. Fish injected with the glycoprotein-encoding plasmid produced low levels of virus-neutralizing antibodies prior to the first challenge. Neutralizing antibodies increased in all groups after exposure to the IHNV. Passive transfer of pooled sera from pCMV4-G vaccinates and IHN survivors provided relative survivals of 40 to 100% compared to fish injected with sera collected from fish immunized with control vaccines or left unhandled. In this study, DNA vaccination effectively protected Atlantic salmon smolts against challenges with IHNV.     

DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish

Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.     

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.     

Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

Infectious hematopoietic necrosis virus (IHNV), a member of the genus Novirhabdovirus, causes a highly lethal disease of salmonid fish. In the present study, G gene nucleotide sequences of 9 Japanese IHNV isolates obtained from 1971 to 1996 were analyzed to evaluate the genetic diversity and compared with IHNV isolates from North America and Europe. A radial phylogenetic tree revealed 5 major clusters including 3 genogroups (U, M and L) for North American isolates and 1 genogroup for European isolates. Five Japanese isolates from 1971 to 1982 appeared in the cluster for genogroup U, while the remaining Japanese isolates from 1980 to 1996 formed a new genogroup, JRt (Japanese rainbow trout). Maximum nucleotide diversity among the Japanese isolates was 4.5%, which was greater than that within the North American isolates (3.6%), and the degree of nucleotide diversity within Japanese isolates was increased by inclusion of the genogroup JRt isolates. It was concluded that Japanese isolates shared a common source with the genogroup U of the North American isolates and that there were large divergences between Japanese isolates before and after the 1980s.     

DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout

DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.     

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

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

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

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

Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.     

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

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

Polymorphisms in major histocompatibility complex class IIα genes are associated with resistance to infectious hematopoietic necrosis in rainbow trout,Oncorhynchus mykiss (Walbaum, 1792)

Infectious hematopoietic necrosis is a serious viral disease of salmonids, including rainbow trout Oncorhynchus mykiss, and causes tremendous economic losses to the rainbow trout farming industry. Major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity in vertebrate organisms and have been linked with the resistance to numerous pathogenic diseases. In this study, polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by cloning and sequencing were used to examine polymorphisms in the DAA genes (specifically DAA exon 2 of MHC class IIα) of rainbow trout and investigate their association with the infectious hematopoietic necrosis virus (IHNV) resistance in rainbow trout. Seventeen alleles were resolved, including 13 novel alleles. Individuals possessed between two and five alleles, indicating that the genome harbours at least three closely‐related DAA exon 2 loci. The ratio of non‐synonymous to synonymous nucleotide substitutions suggested that DAA exon 2 is under positive selection. A greater variability of amino acids and non‐synonymous nucleotide substitution rate was evident in the peptide‐binding region (PBR) than in the non‐PBR (27.75%). Importantly, the analyses revealed that certain MHC class IIα alleles appear to confer resistance to IHNV in rainbow trout, while others confer susceptibility. The most common alleles in the resistant populations of rainbow trout, Onmy‐DAA*1301 and Onmy‐DAA*0304, confer resistance to IHNV and were not present in the susceptible population. Hence, these alleles may be ideal molecular markers that can assist the breeding of IHNV resistance in rainbow trout.     

一例虹鳟低死亡率疾病的病原学及病理学研究

为确定病原类型和造成虹鳟低死亡率的原因,研究对患病虹鳟进行了病理学观察、病毒的分离和鉴定以及动物感染实验。临床检查发现发病虹鳟体色变黑,肌肉和腹壁点状出血。病理学观察发现虹鳟造血器官脾和肾间组织严重坏死。通过反转录PCR法检测坏死组织和病变细胞中传染性造血器官坏死病毒、出血性败血病毒和传染性胰腺坏死病毒,并对得到的371 bp大小片段进行测序和构建进化树分析,发现感染病原为传染性造血器官坏死病毒。同时,给体重为1.5 kg健康虹鳟腹腔注射104 TCID50的组织滤液,累计死亡率达到35%。除此之外,将组织滤液接种到鲤鱼上皮瘤细胞系后出现了特征性病变。在实验过程中未发现细菌或寄生虫感染。结果证实引起虹鳟低死亡率的病原为传染性造血器官坏死病毒。     

Immunity to viral haemorrhagic septicaemia (VHS) following DNA vaccination of rainbow trout at an early life-stage

Rainbow trout fry of average weight 0.5 g were vaccinated against viral haemorrhagic septicaemia (VHS) by intramuscular injection of 1 microg of plasmid DNA encoding the VHS virus glycoprotein gene. Challenge with a lethal dose of virus at two different time points, 9 and 71 days post-vaccination respectively, revealed that a highly protective and lasting immunity was established shortly after vaccination, in accordance with earlier experiments with larger fish. The defence mechanisms activated by the DNA vaccine are thus functional at an early life-stage in rainbow trout.     

Polymorphisms in MHC class Ia genes and resistance to IHNV in rainbow trout (Oncorhynchus mykiss)

Infectious haematopoietic necrosis virus (IHNV) is detrimental to the farming of rainbow trout (Oncorhynchus mykiss) and other salmonids in the Northern hemisphere. The major histocompatibility complex (MHC) plays a key role in immune response in invertebrates, as evidenced by the close correlation of MHC polymorphisms with disease resistance/susceptibility. To analyse the correlation between rainbow trout resistance and susceptibility to IHNV and genetic variation in exon 2 of MHC class Ia gene, UBA, we employed two approaches, namely, polymerase chain reaction-single strand conformation polymorphism analysis and cloning/sequencing. From 102 resistant and 82 susceptible individuals, a total of 12 alleles in UBA exon 2 (GenBank: JX136662–JX136673) were identified, including 11 novel alleles. The maximum number of these alleles in a single individual was four, suggesting that UBA exon 2 most likely resides on at least two loci in the genome. Most of the variations in UBA exon 2 were located in the peptide-binding region and were determined to have been subject to positive selection during evolution. Correlation analysis revealed that Onmy-UBA*0111 and Onmy-UBA*0107 are highly associated with IHNV susceptibility (P = 0.001), whereas Onmy-UBA*0101, Onmy-UBA*0102, and Onmy-UBA*0103 are highly related to IHNV resistance (P = 0.000). In addition, the three resistant alleles were predominant in the IHNV disease-resistant population; thus, these molecular markers can be used for anti-IHNV breeding of rainbow trout.