传染性造血器官坏死病病毒的重组及安全效价分析

本试验目的是构建转基因莱茵衣藻,用于鱼类口服疫苗的应用。通过提取传染性造血器官坏死病病毒(IHNV)-G蛋白RNA进行扩增,将其克隆到p MD18T并与p Chlamy-4载体相连,重组得到莱茵衣藻并提取总蛋白,将重组的总蛋白通过给小鼠灌胃并利用流式细胞仪测其免疫指标评价其安全效应。结果:扩增片段长度为1 500 bp,经SDS-PAGE电泳得到1条大约为58 k Da的蛋白条带,Westen Blot分析结果显示,重组莱茵衣藻表达的蛋白可以与6×HIS标签抗体特异性结合,具有良好的反应原性;流式细胞仪检测结果显示,灌胃转基因莱茵衣藻组CD4~+、CD8~+T淋巴细胞含量都有所增加,小鼠未出现死亡。表明转基因莱茵衣藻构建成功。其安全性稳定,可以用于鱼类口服疫苗的推广使用。     

Neutralizing antibodies for infectious hematopoietic necrosis virus in eggs of steelhead trout (Salmo gairdneri)

Neutralizing antibodies specific for infectious hematopoietic necrosis virus (IHNV) were isolated from eggs of spawning Steelhead trout (Salmo gairdneri), using sodium sulfate precipitation. The isolated material was used in place of the primary antibody (rabbit anti-IHNV) in a protein immunoblotting assay to detect IHNV proteins specifically. The egg component that bound specifically to IHNV proteins was determined to be trout antibody by using antiserum toward trout immune globulins as the second antibody conjugate in the protein immunoblotting assay. The antibody recovered from eggs neutralized IHNV infectivity in cell culture. An average of 87.5% decrease in infectivity was observed.     

Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe

Marine farming of Atlantic salmon (Salmo salar) is growing rapidly in Norway, and fish diseases have now become one of the biggest obstacles for this new industry. Infectious pancreatic necrosis virus (IPNV) is commonly found in fish from Norwegian sea farms. Diagnosis of IPNV infection is, at present, mainly based on virus isolation in cell cultures and identification by neutralization tests (NT). A DNA-RNA hybridization assay was developed using a 24 base DNA oligonucleotide probe. This is homologous to a part of the nucleotide sequence of the IPNV genome coding for a protease. RNA extracted from IPNV and harvest from infected cell cultures were fixed to nylon filters and hybridized with the 32P end-labelled probe. The results showed that the probe specifically identified IPNV from these two materials, both for the three different virus strains (Ab, Sp and VR-299) used, and for several different field isolates. It did not hybridize with reoviruses or non-infected cell cultures used as controls. These results indicate that the probe is not serotype specific, and furthermore that RNA extraction is not required before hybridization. This method may be a useful alternative to NT for routine identification of IPNV, particularly when non-radioactive labelling of the probe is introduced.     

传染性造血器官坏死病病毒G基因全长cDNA克隆及重组表达研究

本研究旨在克隆G基因全长并与其他传染性造血器官坏死病病毒(IHNV)株进行分析,为IHNV的预防检测提供依据。通过提取IHNV-G蛋白RNA并进行扩增,将其克隆到p MD-18T并与p Chlamy-4载体相连,重组到莱茵衣藻中并提取总蛋白,结果可得:扩增片段长度为1 500 bp,经SDS-PAGE电泳得到一条大约为58 k Da的蛋白条带,Westen Blot分析结果显示,重组莱茵衣藻所表达的蛋白可以与6×HIS标签抗体特异性结合;并且以重组表达的G蛋白为抗原,鼠抗IHNV血清为一抗,辣根过氧化物酶标记的驴抗鼠IgG为二抗,再次进行Westen Blot检验,其结果显示,经诱导后的G蛋白能与鼠抗IHNV血清反应,并在58 k Da处出现了一条明显的特异性条带,具有良好的反应原性。表明重组莱茵衣藻表达系统构建成功。     

Studies on pathogenesis following single and double infection with viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) were bath challenged with viral hemorrhagic septicemia (VHS) virus or infectious hematopoietic necrosis (IHN) virus or with both viruses simultaneously. The viral distribution and development of histologic lesions were examined using immunohistochemistry, while virus titer in kidney was determined by viral titration in cell culture. Single infections with VHS virus and IHN virus showed similar distributions of virus in internal organs. The early identification of virus in gill epithelium, 1 and 2 days postinfection (PI) for VHS virus and IHN virus, respectively, indicates that this organ is the point of entry for both viruses. The detection of VHS virus at 1 day PI and 3 days PI for IHN virus is indicative of kidney and spleen being the target organs for these viruses. A simultaneous infection of VHS virus and IHN virus resulted in both viruses establishing an infection. Further double infection did not result in a statistically significant lower titer of both viruses in kidney but a more restricted distribution of IHN virus in internal organs compared with the single infected group. The most striking finding is that, for IHN virus, virus was not detected in the brain in situ in the double-infected group. This study provides support for the conclusion that simultaneous infection with two piscine rhabdoviruses in a susceptible host results in some degree of interaction at the cell level, leading to a reduced systemic distribution of IHN virus.     

Evidence of existence of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp cultured in China

Infectious hypodermal and hematopoietic necrosis virus is the causative agent of a shrimp disease which causes economic losses on a global scale. A pair of primers, I2814F/I3516R, was designed from the IHHNV genomic sequence (GenBank) that encodes for structural protein corresponding to nucleotides 2814-3516, which amplifies a 703 base pair (bp) region from the virus genome. PCR amplification with the primers generated a product of the expected size from the purified IHHNV DNA of Litopenaeus vannamei and IHHNV-infected penaeid populations but not from the IHHNV-free shrimp, white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV). The PCR amplicon described above was labeled with digoxigenin (DIG)-11-dUTP as a probe used for dot blot hybridization and in situ hybridization test. Under the optimized PCR conditions, the primers were detected by as little as 20 fg of purified IHHNV DNA, which contained only 8.83 x 10(3) copies of IHHNV, a 1000-fold greater than using dot blot hybridization. Sections of histopathology showed eosinophilic intranuclear inclusions (Cowdry type A inclusions or CAIs) in infected tissues while in situ hybridization, cells displayed an intense reaction with the DIG-labeled probe. PCR assay was developed to detect IHHNV in penaeid shrimp and other crustaceans from the rearing ponds of China (March 2001-June 2004). The positive rate was 51.5% (154 out of 299) and 8.3% (2 out of 24) for penaeid shrimp and crab samples, respectively. The survey demonstrated the presence of IHHNV in China.     

Comparison of methods for detection of an infection with different isolates of infectious haematopoietic necrosis virus (IHNV)

The infectious haematopoietic necrosis (IHN) is beside the viral haemorrhagic septicemia (VHS) one of the viral fish diseases that have a considerable economic impact on German aquaculture. The measures actually in force are focused on control and spread prevention of the disease within the borders of the European Union (EU). The detection and confirmation of an outbreak is performed according to the pertinent EU legislation which allow the application of methods like the virus neutralisation test (VNT), the immunofluorescence test (IFT) and the enzyme-linked immunosorbent assay (ELISA). Besides the classic virological serology methods, further tests for the identification and confirmation of the fish pathogen like i.e. PCR and DNA probe techniques are recommended by the OIE. To compare diagnostic methods as ELISA, cell cultivation and RT-PCR, rainbow trout of the strain "Isle of Man" were infected with six IHNV strains. Samples were taken on day 7 (viraemia period) and at the day 28 of the trial. The ground organs were inoculated into EPC cells (Epithelioma papulosum Cyprini cells) and examined by ELISA as well as after RNA extraction by RT-PCR. Besides the determination of the isolate as well as the virulence for 20 g trout, significant differences in the demonstration of the viruses were observed. While the RT-PCR demonstrated to be the most sensitive method, antigen ELISA and virus cultivation results showed in dependence of the IHNV isolate that not all viruses were identifiable under the chosen experimental condition in the same manner.     

Immunosuppressive effect of infectious pancreatic necrosis virus on rainbow trout (Oncorhynchus mykiss)

The infectivity of infectious pancreatic necrosis virus (IPNV) for rainbow trout (Oncorhynchus mykiss) mononuclear leukocyte subpopulations was investigated to determine the mechanisms of immunosuppression caused by the virus. IPNV was recovered from nylon wool-adherent, surface immunoglobulin (Ig)-positive leukocytes of head kidney, spleen and peripheral blood collected from virus-inoculated fish with higher titers than non-adherent, Ig-negative cells. Non-adherent cell population showed mitogenic response to phytohemagglutinin and concanavalin A but not to lipopolysaccharide. Conversely, the responses of adherent cells to these mitogens were weak. Mitogenic response and non-specific cytotoxicity of head kidney leukocytes significantly decreased by the inoculation of fish with the virus. These results suggest that the suppression of immune responses is involved in the establishment of carrier state in fish after infection with IPNV.     

Persistence of infectious pancreatic necrosis virus (IPNV) in Scottish salmon (Salmo salar L.) farms

Infectious pancreatic necrosis is a disease that is causing increasing loses to Scottish Atlantic-salmon farms. I asked the question: is infection of individual salmon farms persistent or transient? Using Fisheries Research Services' Fish Health Inspectors' data, conditional probabilities that a farm would (time 0) be infected were estimated for farms that had been infected [P(I(0)|I(-T))] or free [P(I(0)|F(-T))] from infectious pancreatic necrosis virus (IPNV) when a sample was taken at some earlier time (-T). A logistic-regression model was used to estimate these conditional probabilities with T; this model was multilevel to account for regional and inter-annual level differences in prevalence of IPNV. In freshwater, conditional probabilities remained substantially different for periods of at least 4 years, so, although many farms did change infection status, IPNV either persisted or recurred at specific freshwater farms. Marine farms showed similar conditional probabilities after about 2 years following a positive or a negative sample, indicating that infection was transient. Management of larger areas and exchanges between farms might be more effective than farm-level management at controlling marine IPNV.     

Characterization of an infectious pancreatic necrosis (IPN) virus carrier cell culture with resistance to superinfection with heterologous viruses

A state of persistence of a non susceptible fish cell line with infectious pancreatic necrosis virus (IPNV) was established in vitro by experimental infection. The persistently infected culture showed sustained production of infectious virus and could be continuously passaged for months. A distinct feature of this culture is that only a very small fraction of the cells harbours virus replication, in contrast to other reported IPNV-persistently infected cells from salmonid fish, where nearly all the cells express viral antigens. In spite of the small number of detectable IPNV-infected cells, the carrier culture shows resistance to superinfection with homologous as well as heterologous viruses. Temperature shift-up experiments indicate that viral interference is due to continuous replication of IPNV in the culture. Quantitation of Mx gene expression suggested that the interference phenomenon could be mediated by the activation of the interferon (IFN) system. However, conditioned medium from the IPNV-infected cell cultures only marginally protected other cells against VHSV infection, indicating that other type I IFN-independent mechanism may be underlying the resistance of the persistently infected culture to infection with heterologous viruses. Our study defines a novel in vitro model of IPNV persistence and contributes to the understanding of the widespread distribution of aquabirnaviruses in marine and fresh water environments by establishing a carrier state in non susceptible fish species.     

Immunohistochemical identification of infectious pancreatic necrosis virus in paraffin-embedded tissues of Atlantic salmon (Salmo salar)

Infectious pancreatic necrosis virus serotype Sp was identified by immunohistochemistry in formaldehyde-fixed and paraffin-embedded tissue of Atlantic salmon (Salmo salar). The immunoreaction was present in degenerating and necrotic cells in exocrine pancreatic cells. Cross reactions were observed with rabbit antisera against serotypes Sp, Ab, and VR-299 in neutralization tests and western blotting. Immunohistochemically, only Sp antiserum produced positive immunostaining to Sp antigens, whereas antisera to serotypes Ab and VR-299 were negative.     

Detection of vaccine-like infectious bursal disease (IBD) virus in IBD vaccine-free chickens in Japan

The prevalence of infectious bursal disease virus (IBDV) was studied in chickens, which had not been vaccinated against IBD. Fifty sera and forty-six bursae of Fabricius from chickens showing impaired growth, collected from 7 IBD vaccination-free farms in Japan were used for virus neutralization (VN) tests and RT-PCR for detection of IBDV genome corresponding to the VP2 hypervariable region. Of the fifty sera, 39 sera (78%) from 6 farms were VN antibodies positive. Of the forty-six bursae, 37 bursae (80.4%) from 6 farms were positive in the RT-PCR assay. The sequences of all the RT-PCR products detected in this study were closely related or identical to those of the vaccine strains. These results show that vaccine-like IBDV is prevalent even in IBD vaccine-free chicken farms in Japan.     

The persistence of infectious pancreatic necrosis virus in Atlantic salmon

Atlantic salmon leucocytes from Infectious Pancreatic Necrosis Virus (IPNV) carriers showed a suppressed response to phytohemagglutinin stimulation compared with uninfected controls. A significant degree of inhibition of DNA synthesis was observed using 3H-thymidine incorporation. IPNV was isolated from 41% of the stimulated leucocyte cultures supernatants, while only 6% of the unstimulated cultures were found to be positive.     

Isolation and identification of the infectious pancreatic necrosis virus in trout

The first isolation and identification of the infectious pancreatic necrosis virus in trout in Czechoslovakia is described. The RTG-2, PG and CHSE cell lines were used for isolation and the identification was made by the methods of electron microscopy, cross virus-neutralizing test, and immunofluorescence. As demonstrated, all the isolates in the Czechoslovak territory are serologically identical and are closely related (or perhaps congruent) with the Sp reference strain which has the highest frequency in Europe. The described methodical procedure is also applicable to the diagnostics of other virus diseases of fish.     

传染性造血器官坏死病病毒(IHNV)分离株病原学研究及全基因组序列分析

为初步了解东北地区某虹鳟养殖场的一株传染性造血器官坏死病病毒株的病原学特征,将该病毒株进行虹鳟鱼苗人工回接感染试验。结果显示,一周内人工感染试验鱼均出现明显的临床症状。将病死虹鳟鱼苗研磨过滤除菌后接种到胖头鱼肌肉细胞系(FHM),出现了特征性病变(CPE),用传染性造血器官坏死病病毒(IHNV)4组水生动物病毒的6对特异性引物对该毒株进行race-PCR,扩增其全长。结果显示,该病毒基因组全长为11 132 nt,基因组序列中4种核苷酸G、A、T、C含量分别为24.28%、28.67%、19.46%、27.59%。将序列与多株GenBank中已发表的IHNV毒株相应基因进行比较。结果显示,分离株G基因与韩国株ChYa07和PcKw11同源性较高,分别为97.8%和97.5%,其进化树分析结果显示,CJ-13株与韩国株和日本株在遗传进化关系上较近。     

Validation of betapropiolactone (BPL) as an inactivant for infectious bovine rhinotracheitis (IBR) virus

Infectious bovine rhinotracheitis (IBR) virus causes vulvovaginitis, abortion and respiratory disease in cows and heifers. Betapropiolactone (BPL) is a disinfectant, effective against bacteria, fungi and viruses. It is also used to prepare inactivated vaccines because it destroys the nucleic acid core of viruses but does not damage the capsid. For the validation of BPL when used as an inactivant, it is more important to assure the quality of inactivating agent and the validity of the inactivation process. In the present study, the inactivation kinetics of IBR virus was determined with different concentration of BPL (1:250, 1:500, 1:1000, 1:1500, 1:2000 and 1:2500) at 4 and 37 °C. The result indicated that the BPL at 4 °C was able to inactivate the IBR virus within 4, 5 and 12 h with the concentration of 1:250, 1:500 and 1:1000, respectively. BPL at 37 °C was able to inactivate virus within 30 min with the concentration of 1:250. BPL with the concentration of 1:500 and 1:1000 were able to inactivate the virus within 120 min at 37 °C. Based on the kinetic study seven formulations were prepared and a sero conversion study of IBR inactivated vaccine was carried out. Serological response in animals to different formulations did not differ significantly (P > 0.05).