The First Isolation in North America of Infectious,Hematopoietic Necrosis Virus (IHNV) and Viral Hemorrhagic Septicemia Virus (VHSV) in Coho Salmon from the Same Watershed

Abstract

In November 1989, infectious hematopoietic necrosis virus (IHNV) was found for the first time in the Soleduck River at the Washington Department of Fisheries Soleduck Hatchery. The virus was isolated from ovarian fluid and kidney-spleen tissue pools from chinook salmon Oncorhynchus tshawytscha and ovarian fluid pools from coho salmon O. kisutch returning to the Soleduck Hatchery. The virus was identified as IHNV by neutralization assays. In December 1989, the virus causing viral hemorrhagic septicemia (VHSV) was found in ovarian fluid and milt pools from wild coho salmon obtained from the Soleduck and Bogachiel rivers and held at the Soleduck Hatchery. The virus was identified as VHSV by neutralization and immunoblot assays. These findings and their implications for routine broodstock sampling are discussed.     

Glycoprotein from Infectious Hematopoietic Necrosis Virus (IHNV) Induces Protective Immunity against Five IHNV Types

Abstract

Infectious hematopoietic necrosis virus (IHNV) is a fish pathogen that kills young salmon and trout. Outbreaks of the disease among hatchery-reared fish are a problem in the northwestern USA from northern California to Alaska. At least five biochemical types and several strains of differing host specificity of IHNV exist. Any vaccine developed to immunize fish must be able to elicit a response that will neutralize all strains of IHNV. This report shows that a single type of IHNV can induce a protective immune response in vivo to the five biochemical types of IHNV and indicates that, of the IHNV isolates examined, there is at least one common major neutralization epitope. Therefore, a vaccine developed against this common neutralization epitope will provide cross-protective immunity against these IHNV variants.     

In Vitro Infection of Salmonid Epidermal Tissues by Infectious Hematopoietic Necrosis Virus and Viral Hemorrhagic Septicemia Virus

Abstract

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.     

Serological Evidence of Infectious Hematopoietic Necrosis in Rainbow Trout from a French Outbreak of Disease

Abstract

Following the detection of infectious hematopoietic necrosis virus (IHNV) in France in April 1987, a serological survey was conducted of the rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) from an infected cultured stock previously known to be contaminated with viral hemorrhagic septicemia virus (VHSV) for 3 years. The work lasted from April to December 1987, at which time all the remaining fish were slaughtered. Serum samples were assayed by a plaque-reduction test and a simplified neutralization test that is more suitable for processing large numbers of serum samples. Such investigations revealed that IHNV neutralization by trout antibodies depended on trout complement, as did neutralization of VHSV. Incubation for 16 h at 4°C increased the sensitivity of the test compared to incubation for 1 h at 20°C. During the course of clinical IHN from April to June, young fish did not display any neutralizing activity, but in September, 29 of 50 of them exhibited significant anti-IHN neutralizing antibody titers ranging from 21 to over 160, and 18 of 46 of these same fingerlings did so in December. Similarly, fish that had undergone VHS infection in August began to develop anti-VHSV antibodies in December (5 of 50), demonstrating that one fish can harbor neutralizing antibodies to both IHNV and VHSV, and that these antibodies had required 14 weeks to appear under fish culture conditions at 10°C. As could be expected from seroneutralization tests, neutralizing antibodies to IHNV did not result in protection against VHS. Sera from 13 of 20 adult fish sampled in mid-June revealed neutralizing antibodies to IHNV, suggesting that they harbored the virus prior to the clinical infection that affected their progeny. Only two of the fish showed low anti-VHSV antibody titers. Similarly, neutralizing antibodies to IHNV were detected in 53 of 73 other adult fish sampled in late October, 10 months after they had spawned and 7 months after mortality had occurred among their progeny. Given the prevalence, level, and persistence of neutralizing antibody titers, the seroneutralization test would be worth investigating more thoroughly to define the conditions that could make it a reliable tool for checking the virus status of trout carriers.     

Biochemical and Antigenic Properties of the First Isolates of Infectious Hematopoietic Necrosis Virus from Salmonid Fish in Europe

Abstract

The first isolates of infectious hematopoietic necrosis virus (IHNV) recovered from rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) in France and Italy were compared to six representative strains from North America by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of virion polypeptides and neutralization by monoclonal antibodies (MAbs). All three IHNV isolates from Europe had similar polypeptide profiles when compared by SDS-PAGE. An analysis of the antigenic relatedness of the European isolates to representative strains from North America showed that they were clearly different from viruses obtained from salmonids in California. The RB/B5 MAb, which was developed against virus isolated from adult steelhead (anadromous rainbow trout) reared in central Oregon, neutralized all isolates examined. The 193–110/B4 MAb, developed against IHNV isolated from infected yearling rainbow trout in southern Idaho, neutralized all isolates tested except those from California. The SRCV/A4 MAb, developed against Sacramento River chinook virus (SRCV) isolated from adult spring chinook salmon O. tshawytscha in central California, was the least reactive, and strong neutralization was observed only with the SRCV strain of IHNV from California. However, partial reactivity of the virus isolates from France with the SRCV/A4 MAb distinguished them from the virus recovered from salmonids in Italy.     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Whole-Body Assay and Immunohistochemistry

Abstract

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.     

Production of Neutralizing Antisera against Viral Hemorrhagic Septicemia (VHS) Virus by Intravenous Injections of Rabbits

Abstract

Rabbit antisera against viral hemorrhagic septicemia virus (VHSV) produced by two immunization procedures were compared for neutralization and immunochemical properties against homologous and heterologous strains. The VHSV isolate used as the immunogen was a member of a serogroup not neutralized by previously available antisera. The results from this study suggested that frequent intravenous (IV) injections of rabbits with viral antigens were superior to adjuvant-mediated, combined subcutaneous and intraperitoneal (SC/IP) injections for the production of neutralizing antisera. All IV injected rabbits produced high neutralization titers against the homologous VHSV isolate but not against an isolate from a different serogroup. The SC/IP injected rabbits had no significant neutralization titers against either the homologous VHSV strain or two isolates of a heterologous VHSV strain. Sera from all injected rabbits reacted in indirect immunofluorescence (IF) assays with either strain; however, the SC/IP injected rabbits produced higher titers against the heterologous VHSV strain by ELISA (enzyme-linked immunosorbent assay). By Western blotting, neutralizing antisera primarily stained the viral glycoprotein (G) whereas the nonneutralizing sera stained all the viral structural proteins equally well. Our results demonstrate that immunization procedures to produce antisera against VHSV in rabbits determine whether the resultant antibodies will have primarily neutralizing or binding capabilities.     

Optimization of a Plaque Neutralization Test (PNT) to Identify the Exposure History of Pacific Herring to Viral Hemorrhagic Septicemia Virus (VHSV)

Abstract

Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations.

Received November 7, 2016; accepted January 14, 2017 Published online April 4, 2017     

Infectious Hematopoietic Necrosis Virus (IHNV) in Coho Salmon

Abstract

Infectious hematopoietic necrosis virus (IHNV) causes important losses of chinook salmon Oncorhynchus tshawytscha, sockeye salmon Oncorhynchus nerka, and rainbow trout and steelhead Oncorhynchus mykiss on the west coast of North America. Although coho salmon Oncorhynchus kisutch are considered resistant to IHNV infection, the virus was detected in numerous adult coho salmon returning to Trinity River Hatchery, California, in 1985 and 1986. The virus was isolated from internal organs and ovarian fluids of these fish. Antigenic and structural polypeptides of the viruses were identical in adult coho and chinook salmon collected at the same location. Chinook salmon and rainbow trout alevins exhibited high degrees of susceptibility to IHNV obtained from adult coho and chinook salmon. Coho salmon alevins were resistant to both virus isolants.     

Interaction of the attenuated recombinant rIHNV-Gvhsv GFP virus with macrophages from rainbow trout (Oncorhynchus mykiss)

One of the most important threats to the salmonid aquaculture industry is infection caused by novirhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV). Using reverse genetics, an avirulent recombinant rIHNV-Gvhsv GFP strain was generated, which was able to replicate as effectively as wild type IHNV in a fish cell line and in macrophages. Although this recombinant virus induced protective responses against IHNV and VHSV, the response did not involve the production of antibodies or modulate the expression of some antiviral genes. To determine the immune mechanisms underlying the protection conferred by the rIHNV-Gvhsv GFP virus, different immune parameters (NO production, respiratory burst activity and the induction of apoptosis) were assessed in the macrophage population. The results obtained in the present work may indicate that the Nv protein could be important in the modulation of NO and ROS production. rIHNV-Gvhsv GFP did not appear to have a clear effect on nitric oxide production or apoptosis. However, an increased respiratory burst activity (with levels induced by the recombinant virus significantly higher than the levels induced by the wild type virus), suggests a stimulation of the macrophage population, which could be related to the protection against virulent viruses.     

Differential reactivity of a monoclonal antibody directed to the membrane protein of porcine reproductive and respiratory syndrome virus.

A monoclonal antibody (2C12) against the 19 kDa membrane (M) protein of a Canadian isolate of porcine reproductive and respiratory syndrome (PRRS) virus was produced. By indirect immunofluorescence (IIF) cytoplasmic fluorescence was observed in infected cells, but the pattern of fluorescence was generally different and intensity was weaker than that observed using the nucleocapsid protein-directed monoclonal antibody SDOW17. When tested by IIF towards a total of 26 PRRS virus isolates from Canada, 122 isolates from the US and 13 isolates from Europe the 2C12 MAb reacted with all the North American isolates tested including the VR-2332 isolate and the vaccine (RespPRRS) isolate. However no reactivity was observed towards the European isolates tested including the Lelystad virus. This reactivity pattern suggests that the epitope recognized by this MAb on the M protein of PRRS virus appears highly conserved among North American isolates but absent or weakly expressed on European isolates of PRRS virus.     

Characterisation of monoclonal antibodies to Epizootic Hemorrhagic Disease Virus of deer ( ) and Bluetongue Virus by immunisation of mice with recombinant VP7 antigen

Immunisation of mice with recombinant VP7 antigen of epizootic hemorrhagic disease virus of deer ( ) induced serum antibody responses to . However, from the 19 monoclonal antibodies (Mab) produced from these mice, 15 were specific for and four for bluetongue virus ( ). No Mabs were identified with the specificity for an epitope of VP7 shared by both and in spite of the fact that they share a large portion of homology in VP7 amino acids composition. These Mabs were divided into five groups based on their specificity and interaction with each other. Group II Mabs, consisting of 13 Mabs, recognises a potential serogroup specific, linear epitope of VP7 antigen. One of the Mabs to (Group V) was identified as VP7 specific with the possibility of being the serogroup specific and recognizes a potential conformational epitope. Two Mabs from these VP7 specific groups were further analysed and found to be useful in a competitive enzyme-linked immunosorbent assay ( - ) for detection of specific antibodies against and in bovine sera.     

Susceptibility of Four New Salmonid Cell Lines to Infectious Hematopoietic Necrosis Virus

Abstract

Four salmonid cell lines, CoE 45, CoE 115, CoE 345, and RBTE 45, were established from embryonic tissues of coho salmon Oncorhynchus kisutch and rainbow trout O. mykiss. In vitro challenges of the new lines were conducted with four isolates of infectious hematopoietic necrosis virus (IHNV). Two of the IHNV isolates used for the challenges were derived from infected tissues of rainbow trout, one was derived from chinook salmon O. tshawytscha, and the other isolate was derived from coho salmon. To standardize the virus challenges of the new cell lines, several established piscine cell lines (EPC, CHSE 214, CSE-119, RTH-149, RTG, and RTS) were challenged in the same way as the new lines. Each of the lines was challenged with virus at a single low multiplicity of infection (0.01 plaque-forming unit per cell). Virus yields were quantitated by plaque assay on epithelioma papulosum cyprini (EPC) cells on day 3. Results of the challenge experiments revealed different levels of production of virus for each isolate on the various cell lines. Overall, the new cell line derived from rainbow trout, RBTE 45, was quite susceptible to all viruses tested. The three cell lines newly derived from coho salmon embryo were not as resistant to the replication of IHNV as was the established coho salmon cell line, CSE-119. An established cell line, EPC, derived from an epithelial tumor of common carp Cyprinus carpio, remained the most susceptible to all four IHNV isolates tested.     

Monoclonal antibodies to bovine viral diarrhea virus: cross-reactivities to field isolates and hog cholera virus strains.

Monoclonal antibodies to bovine viral diarrhea virus (BVDV) were examined for binding with a large number of North American BVDV isolates and eight strains of the serologically related pestivirus, hog cholera virus (HCV). No single BVDV monoclonal antibody reacted with all BVDV isolates. The most cross-reactive monoclonal antibody was an anti-p80/p125 antibody which showed a positive reaction with 173 of 180 (96%) North American isolates. From a fewer number of isolates tested, one anti-gp53 monoclonal antibody also showed a high cross-reactivity (94%). All BVDV isolates showed a positive reaction with at least one of the seven monoclonal antibodies in the panel. Thus, the results indicated that a pool of these monoclonal antibodies may be used in place of polyclonal antisera for the detection of BVDV contamination of cell lines or for virus isolation. For HCV, all three anti-p80/p125 monoclonal antibodies reacted positively with all eight virus strains. In contrast, none of the anti-gp53 monoclonal antibodies were reactive to HCV strains. Thus, the anti-gp53 monoclonal antibodies may be useful for distinguishing between usually innocuous BVDV infections and the highly significant HCV infections in swine for foreign animal disease surveillance.     

H9N2亚型禽流感病毒中和单克隆抗体的制备与应用

为建立H9N2亚型禽流感病毒（Avian influenza virus,AIV）免疫层析快速检测技术,本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠,将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养;以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法,通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体;以胶体金标记HA单克隆抗体,配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线,制备H9N2亚型AIV快速检测试纸条,测定其特异性和敏感性。结果显示,获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞,其单克隆抗体腹水IPMA效价在1.28×10^-4至2.56×10^-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性,其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性,中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示,该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2,灵敏度与经典血凝试验（HA）相当,与其他亚型AIV （H1、H3、H5、H7）,以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体,并初步研制了H9N2亚型AIV检测试纸条,为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。     

Experimental Infection of Brook Trout with Infectious Hematopoietic Necrosis Virus Types 1 and 2

Abstract

Fry of brook trout Salvelinus fontinalis became infected and diseased after immersion exposure to infectious hematopoietic necrosis virus (IHNV), but a long-lasting IHNV carrier state was not induced. Duplicate groups of 100 fish were immersed for 6 h in baths containing a type 1 (Round Butte, RB) or a type 2 (Rangen, RA) IHNV isolate at a high or low dose. Brook trout mortalities induced by immersion in a bath of the RB or RA IHNV isolate at 10² plaque-forming units (pfu) per milliliter were equivalent (1 and 0%), but fish were more susceptible to infection with RA IHNV. Only the single dead fish in the RB group was infected, but 24% of the RAexposed fish were infected 1 week after exposure. At a dose of 10⁶ pfu/mL, exposure to RB IHNV resulted in a higher mortality (35%) and prevalence of infection (89% of live fish sampled at 1 week postexposure), but no infectious virus was detectable by 5 weeks after exposure. In contrast, RA IHNV exposure at a dose of 10⁴ pfu/mL resulted in only 5% mortality, and live fish killed at 1 week postexposure had a 22% prevalence of infection, but infectious virus was not detectable by week 3. Although brook trout have been previously considered to be resistant to IHNV, this study has shown that brook trout become diseased and die after exposure to a high dose of one type I IHNV isolate and can be infected after immersion exposure to even a low dose of type 1 or type 2 IHNV.     

Detection of Infectious Pancreatic Necrosis Virus from the Leeches Hemiclepsis marginata and Hirudo medicinalis

Leeches have been reported to harbor several important fish pathogens, including spring viremia of carp virus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV), and also may contain blood protozoa. In the present study, leeches were collected from water bodies located in Kurdistan province, Iran. The specimens were tested for IHNV, VHSV, and infectious pancreatic necrosis virus (IPNV) using the PCR method. The results showed that two different species of leeches, Hemiclepsis marginata and Hirudo medicinalis, were infected by IPNV among the seven species studied. The infected leeches were found in areas that were polluted with untreated sewage coming from upstream fish farms culturing Rainbow Trout Oncorhynchus mykiss. In addition, the fish at fish farms in the vicinity had been infected with IPNV 9 months previously. Our results showed that the virus causing infectious pancreatic necrosis is present in the leeches H. marginata and H. medicinalis, suggesting that leeches are a potential source of IPNV in fish farms.

Received October 14, 2015; accepted June 1, 2016 Published online September 29, 2016     

DNA Vaccination of Rainbow Trout against Viral Hemorrhagic Septicemia Virus: A Dose–Response and Time–Course Study

Abstract

Viral hemorrhagic septicemia (VHS) in rainbow trout Oncorhynchus mykiss is caused by VHS virus (VHSV), which belongs to the rhabdovirus family. Among the different strategies for immunizing fish with a recombinant vaccine, genetic immunization has recently proven to be highly effective. To further investigate the potential for protecting fish against VHS by DNA vaccination, experiments were conducted to determine the amount of plasmid DNA needed for induction of protective immunity. The time to onset of immunity and the duration of protection following administration of a protective vaccine dose were also analyzed. The dose–response analysis revealed that significant protection of rainbow trout fingerlings was obtained following intramuscular injection of only 0.01 μg of plasmid DNA encoding the VHSV glycoprotein gene. In addition, higher doses of DNA induced immunity to a virus isolate serologically different from the isolate used for vaccine development. Following administration of 1 μg of a DNA vaccine, significant protection against VHS was observed in the fish as early as 8 d postvaccination. At 168 d postvaccination, the fish had increased in size by a factor of 10 and protection against a lethal dose of VHSV was still evident. The results confirm the great potential for DNA vaccination in inducing efficient immunoprophylaxis against viral diseases in aquacultured fish.     

Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). Western blotting analysis revealed that three mAbs (1H5, 1E10, and 1H7) recognized specifically to a single protein of 47kDa (N), the mAb 3G4 reacted with two SVCV0504 proteins of 69kDa (G) and 47kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69kDa (G), 50kDa (P), and 47kDa (N). By indirect ELISA, two mAbs (1H5 and 1H7) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID(50) (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28h after inoculation with the virus (0.01PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus.     

Construction of Baculovirus Expression System and Antigenicity Analysis of Infectious Hematopoietic Necrosis Virus Glycoprotein

To study the major structural protein glycoprotein (G) of infectious hematopoietic necrosis virus (IHNV), glycoprotein gene (1 380 bp) was amplified by RT-PCR from IHNV. In order to construct a recombinant plasmid pFB-LIC-Bse-G, G gene was cloned into the baculovirus vector pFB-LIC-Bse. Then, the constructed plasmid pFB-LIC-Bse-G was transformed into E.coli DH10Bac. The recombinant bacmids rBacmid-G was got, and then it was transfected to insect Sf9 cells,and the recombinant baculovirus that contained G gene was obtained. Western blotting analysis and indirect immunofluorescence assay (IFA) showed that the recombinant G protein could be recognized by histidine monoclonal antibody (Anti-His) and anti-IHNV antibody of mouse. The results indicated IHNV G protein had been expressed correctly in Sf9 cells. The study laid a foundation for further studying the G protein structure, function and immunological characteristics.