6种重要的鱼类病毒

病毒性传染病是当前渔业生产所面临的重要问题之一。文章主要介绍了鱼类几种重要病毒的病原学、流行、传播以及防控等相关情况。这些病毒包括水生双RNA病毒、β-野田村病毒、传染性鲑鱼贫血病毒、传染性造血组织坏死病毒、流行性造血器官坏死病毒、病毒性出血败血症病毒等。

鱼类病毒性出血性败血症病毒诊断胶体金免疫层析方法的建立

为建立一种快速检测鱼类病毒性出血性败血症病毒(Viral haemorrhagic septicaemia virus,VHSV)的胶体金免疫层析方法(GICA),采用柠檬酸三钠还原法制备胶体金颗粒,以标记纯化的VHSV G蛋白单克隆抗体(MAb)10A7为捕捉抗体,将纯化的抗VHSV N蛋白的MAb 10EN单克隆抗体和羊抗鼠IgG抗体包被在硝酸纤维素膜上,作为检测带与质控带,经试验条件优化,组装形成胶体金免疫层析试纸条。用本试验研制的胶体金试纸条检测VHSV感染的CHSE细胞培养物。结果显示:在检测线和质控线均呈现红色条带,而健康的CHSE细胞培养物对照仅在质控线呈现红色条带;试纸条检出病毒培养物的病毒量最低限为10^(4.0) TCID_(50);随机挑取3个不同批次的试纸条,对阳性样品和阴性样品进行重复试验,未发现差异。特异性试验表明,该试纸条与传染性造血器官坏死病毒(IHNV)、传染性胰坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、牙鲆弹状病毒(HRV)、草鱼呼肠孤病毒(GCRV)无交叉反应。将同一批次的试纸条在4℃条件下保存不同时间后进行检测,发现保存6个月的试纸条仍具有良好的稳定性。本试验研发的胶体金诊断试纸条具有良好的特异性、灵敏性和重复性,在快速辅助检测方面具有推广应用价值。

传染性造血器官坏死病毒RT-RAA快速检测方法的建立

为了给传染性造血器官坏死病(Infectious Hematopoietic Necrosisvirus,IHN)的防控提供特异、灵敏、快速的检测方法,根据NCBI基因库中传染性造血器官坏死病病毒(Infectious Hematopoietic Necrosis Virus,IHNV)保守序列设计特异性引物和探针,建立IHNV的逆转录重组酶介导扩增(Reverse Transcription Recombinase-aid Amplification,RT-RAA)检测技术。结果表明,该方法具有很高的特异性。其最低可检出100 copies/μL,检测灵敏度与OIE建议的RT-PCR方法相当。对35份虹鳟(含金鳟)养殖场样品检测并与RT-PCR方法进行比较,该方法的检测结果符合率达到96%以上。本文建立的RT-RAA方法具有操作简便、灵敏度高、特异性好、检测周期短、不需要昂贵仪器,结合荧光探针能够实现实时检测以及现场快速检测等优点。该方法的建立及应用可为IHN防控提供新的有用的检测工具。

鲤浮肿病毒环介导等温扩增(LAMP)检测方法的建立及应用

为了给鲤浮肿病(carp edema virus disease,CEVD)的防控提供特异、灵敏、快速的检测方法,针对鲤浮肿病毒(carp edema virus,CEV)的P4a基因序列设计5条特异性引物,建立CEV的环介导等温扩增检测方法(loop-mediated isothermal amplification,LAMP)。结果表明该方法具有很高的特异性。最低可检出6 000拷贝/μL,检测灵敏度与Real-time PCR方法相当。对30个鲤及锦鲤养殖场样品进行检测并与Real-time PCR方法进行比较,该方法的检测结果符合率达到96.7%以上。本方法的建立及应用为CEVD的防控提供依据。

Detection of Grass Carp Hemorrhage Virus (GCHV) from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as "Red Spot Disease", is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and comparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplification. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain induces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the 6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines, including CAB, FHM, CIK, EPC, CCO and GCO, infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHV strains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and the two strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-V viruses have been replicated and amplified despite there being no cytopathogenic effects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published recently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp fragment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is identical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously, the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product, and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.

Application of quantitative PCR method in detection of Lymphocystis disease virus China (LCDV-cn) in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease causes serious economic losses in the fish farming industry. The causative agent of the disease is Lymphocystis disease virus China (LCDV-cn), which has a wide range of hosts. Based on competitive quantitative PCR technology, we established a method to quantify the LCDV-cn in tissue. Results demonstrate that the average amount of LCDV-cn in the peripheral blood of infected flounder with evident tumors is about 106virions/ml while the average amount in those flounder with no evident tumor but cultured with the flounder with evident tumor is about 104virions/ml. No virus was found in the negative samples of flounder.

Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China

We isolated a strain of lymphocystis disease virus （LCDV） from Japanese flounder （Paralichthys olivaceus） cultured in northern China. Based on published sequences of major capsid protein （MCP） gene of LCDV-cn （GenBank： AF126405）, we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization （ISFI） to detect LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral supematant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus （OFIV and RSIV）, lridovirus （CzlV and W/V）, Ranavirus （TFV and FV3）, and Lymphocystivirus （8 LCDV）. Based on the alignment, we confirmed the PCR product was from Lymphocystivirus （GenBank accession number DQ279090 （LCDV-HD））. Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen, stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel＇s black rockfish （Sebastes schlegeli Higendorf）, redwing sea robin （Lepidotrigla microptera Gtinther） and turbot （Scophthalmus maximus） using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method.

The VP2 protein of grass carp reovirus(GCRV) expressed in a baculovirus exhibits RNA polymerase activity

The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA-dependent RNA polymerase （RdRp）.In previous work,we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity （denoted as rVP2390-900） in E.coil and have prepared a polyclonal antibody against VP2.To characterize the GCRV RNA polymerase,a recombinant full-length VP2 （rVP2） was first constructed and expressed in a baculovirus system,as a fusion protein with an attached His-tag.Immunofluorescence （IF） assays,together with immunoblot （IB） analyses from both expressed cell extracts and purified Histagged rVP2,showed that rVP2 was successfully expressed in Sf9 cells.Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly（C）-dependent poly（G） polymerase activity.The RNA enzymatic activity required the divalent cation Mg2＋,and was optimal at 28 ℃.The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic losses.In this study,we aimed to develop the DNA vaccine against LCDV,using DNA vaccination technology.We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate.The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line.The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR,RT-PCR and fluorescent microscopy.Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver,6 and 25 days after vaccination.The vaccine plasmids disappeared 100 d post-vaccination.Fluorescent microscopy revealed green fluorescence in the injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver of fish 48 h post-vaccination,green fluorescence did not appear in the control treated tissue.Green fluorescence became weak at 60 days post-vaccination.RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days post-vaccination.These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish.The antigen would therefore potentially initiate a specific immune response.The plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated.The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD.Further studies are required for the development and application of this promising DNA vaccine.

Localization and Dynamic Expression of a 27.8kDa Receptor Protein for Lymphocystis Disease Virus Infection in Sea Bass(Lateolabrax japonicus) Tissues

Lymphocystis disease virus(LCDV) infects target cells by attaching to a 27.8 k Da receptor(27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies(MAbs) have been developed. However, the 27.8R existence in tissues of sea bass(Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.

Potency of Nannochloropsis oculata as Antibacterial, Antioxidant and Antiviral on Humpback Grouper Infected by Vibrio alginolyticus and Viral Nervous Necrotic

The aim of this study was to explore the potency of N. oculata extracts as antibacterial, antioxidant and antiviral on grouper Cromileptes altivelis （C. altivelis） infected by Vibrio alginolyticus （V. alginolyticus） and Viral Nervous Necrotic （VNN）. Dilution test was used to measure antibacterial activity of N. oculata extracts. Antioxidant activity of extracts was expressed by levels of MDA （Malondialdehyde） and SOD （superoxide dismutase） in the brain and kidneys of fish. Antiviral capability of N. oculata was determined by the expression of cellular immune cells Major Histocompatibility Complex （MHC） Class I for proliferation and inhibition of VNN in blood cells with immunocytochemistry. The results showed that N. oculata extract was able to suppress the growth of V. alginolyticus at a concentration of 40%. Increasing levels of SOD and reducing level of MDA indicated that N. oculata extracts may serve as an antibacterial and antioxidant. Providing cellular response of MHC class I cell expressed on C. altive/is blood cells, N. oculata demonstrated its antiviral activity.

Detection of Fish Disease Caused by Iridovirus on Grouper （Epinephelus sp.） and Pomfret Star （Trachinotus blochii） with Co-agglutination Method in Tanjungpinang, Indonesia

Iridovirus infection often causes death and considerable economic losses in the aquaculture industry. This research applies the co-agglutination method that is fast, cheap and accurate in confirming the diagnosis of the cause of an outbreak of illness caused by iridovirus in the field, so that remedial action can be taken quickly and appropriately to minimize the impact of wider losses. Samples were taken from the grouper and pomfret star farms that are experiencing outbreaks of infectious diseases in the months from May to August 2015, Tanjungpinang, Indonesia. The sick and allegedly attacked by iridovirus samples showed abnormal swimming clinical symptoms, weakness and the swollen spleen. The swollen spleen of sick fish created suspension in phosphate buffered saline （PBS） with pH 7.2, and then centrifuged at 8,000 rpm for I0 rain. The supernatant after centrifuge was used as the test sample. On a clean object glass, 50 μL of the supematant was treated with 50 μL kit co-agglutination pre-prepared. The positive results were shown by the agglutination reaction after 10-15 rain, while as a negative control, PBS was reacted with co-agglutination kit that looked homogeneous （no agglutination）. It was showed that the grouper （Epinepkelus sp.） on four farms and pomfret star （Thracinotus blochii） on one farm that experienced an outbreak of infectious disease in Tanjungpinang showed positively infected iridovirus. The same positive iridovirus result was also demonstrated by examination using polymerase chain reaction （PCR） at 570 bp. So, the causative agent of plague on grouper and pomfret star was iridovirus. In addition, the co-agglutination test based on serology is more quick, cheap and accurate.

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV （lymphocystis disease virus）-infected Japanese flounder （Paralichthys olivaceus）, serum proteins from self-healing and sick Japanese flounder were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Protein spots demonstrating changes greater than two-fold in the expression level were digested and further identified in capillary liquid chromatography tandem mass spectrometry （LC-MS/MS）. Two immunityrelevant proteins were thus identified as transferrin and the complement component C3 of Japanese flounder. These findings suggest that the two proteins may play important roles in the self-healing of lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing in LCDV-infected Japanese flounder by improving their innate immunity.