南美白对虾(Litopenaeus vannamei)幼虾和杜拉对虾(Farfantepenaeus duorarum)稚虾对六种白斑综合病病毒(WSSV)地理隔离株的免疫比较

白斑综合病病毒(WSSV)是一种对对虾养殖危害性最大的病原体之一。它广泛分布在大多数养殖对虾的亚洲国家以及墨西哥湾、美国。我们采用南美白对虾(Litopenaeus vannamei)幼虾和杜拉对虾(Farfantepenuaeus duorarum)稚虾对六种WSSV地理隔离株的病毒性进行免疫比较。这6组WSSV地理隔离株分别取自中国、印度、泰国、美国德克萨斯州、南卡来罗纳州的对虾以及美国国家动物园养殖的淡水龙螯虾。为了进行免疫性试验研究，我们把病毒感染组织接种到南美白对虾幼虾和杜拉对虾稚虾中。通过组织检查结果证实发生WSSV感染。在这次免疫性试验中，被六组WSSV地理隔离株感染的南美白对虾幼虾的死亡率为100％。其中，德克萨斯州的病毒比其它的病毒厉害，最快引起实验对象死亡，而淡水龙虾的WSSV病毒致病性最慢。与之形成显著对比，杜拉对虾稚虾的死亡率仅为35％--60％，并且对不同的病毒其死亡率也有所不同。值得注意的是，杜拉对虾对德克萨斯州病毒感染也是最为严重，而对淡水龙虾病毒的感染最轻。这个实验结果表明：不同地区的WSSV病毒对实验对象的致病性略有不同，不同品种的对虾及同一种对虾在其生长的不同阶段对病毒的易感性也有所不同。

Prevalence of Three Shrimp Viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV),Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province,China,in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations,8 farms were positive for WSSV,8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV,while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples:Bingjiang (93.3%),liuao (66.7%),Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus （WSSV） in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based mieroarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the mieroarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62μg/mL, with a woven long shelf life for 6 months at 4℃ or 8 months at -20℃. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.

Identification of a novel C-type lectin from the shrimp Litopenaeus vannamei and its role in defense against pathogens infection

Acting as one of the pattern recognition receptors （PRRs）, C-type lectin is believed to mediate pathogen recognition and plays an important role in the clearance of pathogens as part of the innate immune system. In this work, a novel C-type lectin gene （named LvLecl） was cloned from the shrimp Litopenaeus vannamei, The ORF of LvLecl is 510 bp, encoding 169 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain （CRD） at the C-terminal. LvLecl was mainly expressed in the hepatopancreas. Real-time PCR analysis indicated that the level of LvLecl transcripts significantly changed in the hepatopancreas after the shrimp were artificially challenged with LPS, Micrococcus lysodeikticus and white spot syndrome virus （WSSV）. RNAi-based silencing of LvLecl resulted in increases in mortality when the shrimp were challenged with WSSV, and the median lethal time was reduced compared with controls. Although there was no characteristic ＂EPN＂ （Glu-Pro-Ser） or ＂QPD＂ （Gin-Pro-Asp） motif, the recombinant LvLecl, expressed in Escherichia coli BL21 （DE3）, could also agglutinate M. lysodeikticus and Vibrio anguillarum. The agglutinating activities were calcium-dependent and could be inhibited by D-mannose, D-glucose, D-galactose and N-Acetyl-D-mannose. These results suggest that LvLecl might be involved in the immune response against WSSV and bacterial infections and contribute to non-self recognition as a pattern recognition receptor in the innate immune system of the shrimp L. vannamei.

Influence of White Spot Syndrome Virus Infection on Hepatopancreas Gene Expression of ‘Huanghai No.2' Shrimp(Fenneropenaeus chinensis)

To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus(WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of ‘Huanghai No. 2'(Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60 K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early(48–96 h), peak(168–192 h) and late(264–288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes(1916 annotated) expressed at all three phases, and most of the annotated were either up-or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp's response to WSSV.

White spot syndrome virus inactivation study by using gamma irradiation

The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus （WS SV）. White spot syndrome virus is a pathogen of major economic importance in cultured penaeid shrimp industries. White spot disease can cause mortalities reaching 100% within 3-10 days of gross signs appearing. During the period of culture, immunostimulant agents and vaccines may provide potential methods to protect shrimps from opportunistic and pathogenic microrganisms. In this study, firstly, WSSV was isolated from infected shrimp and then multiplied in crayfish. WSSV was purified from the infected crayfish haemolymph by sucrose gradient and confirmed by transmission electron microscopy. In vivo virus titration was performed in shrimp, Penaeus semisulcatus. The LD50 of live virus stock was calculated 1054/mL. Shrimp post-larvae （1-2 g） were treated with gamma-irradiated （different doses） WSSV （10^o to 10^-4 dilutions） for a period of 10 days. The dose/survival curve for irradiated and un-irradiated WSSV was drawn; the optimum dose range for inactivation of WSSV and unaltered antigenicity was obtained 14- 15 kGy. This preliminary information suggests that shrimp appear to benefit from treatment with gamma- irradiated WSSV especially at 14-15 KGy.