对虾白斑综合征杆状病毒同源性比较的研究

比较我国沿海不同海域对虾白斑综合征杆状病毒三个分离株：即唐海分离株（渤海湾）、宁波分离株（东海）,深圳分离株（南海）的同源性。三个WSSV分离株基因组的限制笥内切酶（Sac Ⅰ,HindⅢ,PstⅠ）酶切多态（RFLP）以及病毒结构蛋白图谱完全一致,证实造成我国从南对北对虾爆发性流行病的对虾白斑杆状病毒为同一种病毒。利用高保真Taq酶,分别以报道的日本对虾杆状病毒（RV－PJ－PRDV）,斑节对虾白斑综合征杆状病毒（WSBV－PmNOBⅢ）基因组核酸片段特异性引物进行PCR扩增,结果均能从中国一杆状病毒（WSSV）基因组中扩增得到相应大小的PCR产物,扩增产物序列分析表明中国对虾白斑杆状病毒（WSSV）与斑节对虾白斑综合征杆状病毒（WSBV－PmNOBⅢ）,日本对虾相状RV-PJ=PRDV)同源率分别为100％与97％,其结果为证实亚洲及太平洋地区对虾白斑综合征杆状病毒为同一种病毒或同一种病毒的不同株系提供了依据。     

多重RT-PCR同时检测鉴别三种对虾病毒的研究与应用

根据基因库中对虾桃拉综合征病毒（TSV）、白斑综合征病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）的基因序列,分别设计了三对特异性引物,通过对多重RT—PCR扩增条件的优化,研究建立了可同时检测鉴别TSV、WSSV和IHHNV的多重RT—PCR。该技术对同一样品中的TSV RNA、WSSV DNA和IHHNV DNA模板进行扩增,结果均同时得到3条大小与实验设计相符的231bp（TSV）、593bp（WSSV）和356bp（IHHNV）的特异性多重RT—PCR扩增带,对其它对虾病原核酸的扩增结果为阴性。敏感性试验结果表明,该技术最低能检测到10pgTSV RNA、100pg WSSV DNA和100pg IHHNV DNA。临床检测试验结果表明,该技术对TSV、WSSV和I—HHNV的检出率明显高于传统的临床症状观察和组织病理学检查,提示该技术适用于这三种病毒的临床快速检测和鉴别诊断。     

对虾白斑综合征病毒中国地方株变异区基因的比较

对虾白斑综合征病毒(White spot syndromevirus,WSSV)是对虾养殖的主要病原之一,它是目前发现的基因组最大的动物病毒,为环状双链DNA病毒[1,2],全基因组序列分析结果显示,对虾白斑综合征病毒和其他杆状病毒相差甚远,最新病毒分类报告已将该病毒划归新建立的线头病毒科(Nima-viridae)白斑病毒属(Whispovirus)[3,4]。目前Gen-Bank公布有3个版本的WSSV全序列[1,2],其基因组大小的测定结果相差较大。不同的WSSV毒株可能在形态结构、理化性质上无法区分,但病毒基因组限制酶切片段长度多态性(RFLP)可以将之区分开来,Marks等[6,7]通过计…     

注意杆状病毒科分类的变化

杆状病毒由于被广泛用作目的基因的表达载体倍受重视.近年来证实某些杆状病毒对虾具有致病性,从而成为研究热点之一.目前公认的最重要的对虾致病病毒是对虾白斑综合征病毒(white spot syndromviruS,WSSV),在我国养殖对虾中广为流行,引致重大经济损失.不少文献将WSSV称作对虾白斑病杆状病毒,其实并不准确.     

一种简便、快速从病毒感染鱼、虾组织中制备PCR模板的方法

以虹彩病毒(iridovirus)感染大黄鱼的脾组织、对虾白斑杆状病毒(whitespotsyndromebaculovirus,WSBV)感染中国对虾的肌肉组织为材料,采用一种简便、快速的方法获得了可满足病毒PCR检测的高质量模板DNA,分别以针对虹彩病毒、WSBV的特异性引物进行PCR扩增,均能有效扩增出预期的条带。与常规DNA病毒模板制备方法相比,具有简便、快速、提取率高、无污染等优点,尤其适用于水产动物病毒PCR检测试剂盒的商品化开发及生产实际应用。     

对虾白斑综合征病毒基因组快速定性定量方法的建立

对于对虾白斑综合征病毒(white spot syndrome virus,WSSV)全基因组样品,使用限制性内切酶酶切以及荧光绝对定量的方法进行分析,建立了WSSV全基因组快速定性定量的方法。定性实验通过对GenBank中WSSV基因组序列深度分析,选择确定合适的限制性内切酶BamHI对基因组进行酶切,通过比对实际酶切结果和软件模拟酶切结果,以定性待测样品。定量实验使用荧光定量试剂盒,通过建立标准曲线的方法对未知浓度的WSSV基因组样品进行绝对定量。实验结果表明,结合使用酶切分析和荧光定量的方法可以准确、快速、方便、经济地对WSSV基因组样品进行定性定量分析,为进一步深入研究WSSV基因组奠定坚实基础。     

从噬菌体展示抗体文库筛选对虾白斑综合征病毒的单链抗体

对虾白斑综合征是一种严重危害对虾养殖业的病毒性疾病.由于目前对其病原体对虾白斑综合征病毒（WSSV）的研究不够深入,所以对WSSV的有效防治仍然是一大难题.为此,用完整的对虾白斑综合征病毒粒子作为靶抗原固相包被,淘选噬菌体展示单链抗体文库,得到两个能够与WSSV结合的单链抗体：E2和H4.单链抗体H4能够结合病毒并抑制病毒对原代培养的对虾淋巴细胞的感染,这些结果表明此单链抗体具有开发为诊断试剂盒和抗病毒药物的潜力.     

中国对虾杆状病毒基因克隆及探针制备与检测

从收集的中国对虾（Ｐｅｎａｅｕｓｃｈｉｎｅｎｓｉｓ）病虾样品中分离到一种杆状病毒,经负染在电镜下观察,完整病毒粒子大小为１１０×２８０～３２０ｎｍ。病毒核酸经ＥｃｏＲＩ酶切,克隆到ｐＵＣ１８质粒上,经筛选得到两个克隆Ｌ４６、Ｍ１３,克隆片段分别为２．１４ｋｂ和３．５８ｋｂ。将这两个基因片段和对虾白斑综合征杆状病毒（ＷｈｉｔｅＳｐｏｔＳｙｎｄｒｏｍｅＢａｃｕｌｏｖｉｒｕｓ,简称ＷＳＳＶ）基因克隆片段Ａ２６制备探针,共同用点杂交法对我国沿海地区的病虾样品进行检测,以了解我国沿海地区对虾杆状病毒的分布,并确定中国对虾杆状病毒与ＷＳＳＶ的同源性。     

养殖对虾病毒致病性的初步研究

用从日本对虾分离的杆伏病毒扣长毛对虾分离的球伏病毒分别感染健康无病毒的日本对虾,对虾死亡率分别为６５％（杆状病毒组）和５０％（球状病毒组）,而对照组的死亡率仅为２５％,在诸环境因素中,温度的上升和低压可能是对虾病毒病发作的最重要诱因之一。     

油桐尺蠖核型多角体病毒（羊楼洞株）基因组特性

用多种限制性内切酶单酶切、双酶切分析了油桐尺蠖核型多角体病毒（羊楼洞株）基因组DNA,同时用[α－32p）－dATP对几种酶的酶切产物进行末端标记。结果表明,此株病毒基因组约129kb,组成比较单一。与国内其它分离株基因组大小及酶切电泳图谱均有较大差别。     

White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.     

对虾白斑病毒感染的电子显微镜观察及DNA杂交证据

中国大陆养殖的中国对虾从1993年开始至今连年爆发病 毒性流行病,俗称“白斑病”(WSBV),死亡率近100%,造成巨大经济损失。为进一步明确虾病暴发原因,利用电子显微镜技术,对1993年至1998年收集的发病中国对虾组织进行观察,发现病原体为直径(125±76)nm、长约(345±16)nm大小的带包膜的非包涵体型杆状病毒。 经氯化铯密度梯度超速离心技术获得了纯化的病毒核衣壳,大小为(80±13)nm×(380±24)nm。形态学特征与台湾地区发现的白斑杆状病毒(WSBV)相似。利用地高辛标记的WSBV DNA探针对病虾标本及纯化病毒DNA进行斑点杂交检测,均呈阳性反应,而与正常对虾组织无杂交反应。从形态学及分子生物学角度证明WSBV感染是造成中国大陆养殖对虾连年暴发流行病的重要原因。     

Hepatopancreatic nuclease of black tiger shrimp Penaeus monodon unlikely to be involved in viral triggered apoptosis

Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT-PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT-PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas (P<0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.     

Quasi-immune response of Penaeus japonicus to penaeid rod-shaped DNA virus (PRDV)

A quasi-immune response was demonstrated in kuruma prawn Penaeus japonicus infected naturally or experimentally with PRDV (penaeid rod-shaped DNA virus, also called white spot syndrome virus or WSSV), the causative agent of PAV (penaeid acute viremia). In the first step of this study, natural survivors 4 mo after a PAV outbreak demonstrated 94 % relative percent survival (RPS) upon experimental PRDV challenge. Mortalities after challenge were confirmed by PRDV detection to be due to PAV using a PCR method. In the second step, experimental PAV survivors were produced by intramuscular (IM) injection of PRDV into naive shrimp subsequently reared collectively in a tank (A group) or individually in chamber units (B group). Survival was 41 and 90% in the A and B groups, respectively. A subsequent IM re-challenge of these PRDV survivor groups with PRDV made 32 d after the first challenge revealed a protective response with high RPS of 77 and 64%, respectively. These high survival rates suggested that PAV survivors (natural or experimental) were able to resist PRDV infection and that the resistance was not due to selection of naturally resistant shrimp during a PAV outbreak, but due to enhancement of an immune-like system (quasi-immune response) after exposure to PRDV. No PRDV neutralizing activity was revealed in the serum of the 4 mo natural survivors of the PRDV outbreak. However, it was found in their serum 17 d after they had been experimentally challenged with PRDV.     

Multiple viral infection in Penaeus monodon shrimp postlarvae in an Indian hatchery

Moribund Penaeus monodon postlarvae (PL8-PL10) in a hatchery in India were found to be simultaneously infected by 3 different viruses. They were highly infected with monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) by histology and with white spot syndrome virus (WSSV) by non-nested polymerase chain reaction (PCR). Apparently healthy postlarvae tested from the same hatchery were positive for MBV and WSSV by nested PCR only. Tissue sections of such postlarvae did not show any histopathological changes. The simultaneous occurrence of these 3 viruses in hatchery-reared postlarval P. monodon is being reported for the first time.     

Polistes formosanus (Hymenoptera: Vespidae), a good species supported by both morphological and molecular phylogenetic analyses, and a key social wasp in understanding the historical biogeography of the Nansei Islands

Polistes formosanus Sonan, 1927 is closely related to P. japonicus de Saussure, 1858, and has been treated variously as a good species or subspecies or synonym of P. japonicus. We designate the lectotype of P. formosanus. Detailed examination of morphological characters of specimens from continental Asia, Taiwan, the Nansei Islands and main islands of Japan showed that P. formosanus is a good species different from P. japonicus. Molecular phylogenetic analyses using mitochondrial genes also supported this conclusion. Polistes formosanus is distributed in northern and central Taiwan and in the Nansei Islands and extends northward to the Amami Islands, while P. japonicus occurs in continental Asia, central Taiwan, Korean Peninsula, Honshu to Kyushu of Japan and the Osumi Islands (Yakushima and Tanega-shima Island) of the Nansei Islands. The speciation and biogeography of P. formosanus are briefly discussed.     

Differences in the susceptibility of some penaeid prawn species to gill-associated virus (GAV) infection

Four species of penaeid prawn cultured in Australia (Penaeus monodon, Penaeus esculentus, Marsupenaeus japonicus and Fenneropenaeus merguiensis) were injected with a virulent preparation of gill-associated virus (GAV). P. monodon (average weight = 8.9, 13.9 and 19.2 g), P. esculentus (average weight = 19.5 g), F. merguiensis (average weight = 10.5 g), and small (average weight = 5.8 g) M. japonicus displayed overt signs of disease and mortalities which reached 82 to 100% within 23 d post-injection. Cumulative mortalities in P. esculentus and F. merguiensis were significantly lower than for P. monodon of the same size class. Medium (average weight = 13.0 g) M. japonicus also developed overt signs of disease but cumulative mortalities were not significantly higher than uninfected controls. Large (average weight = 20.3 g) M. japoncius did not display symptoms of disease and there were no significant mortalities up to 23 d post-injection.     

Polychaete worms--a vector for white spot syndrome virus (WSSV)

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.     

Detection of Taura syndrome virus (TSV) strain differences using selected diagnostic methods: diagnostic implications in penaeid shrimp

Positive results were obtained with nested white spot syndrome virus (WSSV) diagnostic PCR performed on 5 commercial brands of dry-packed Artemia cysts using several WSSV genomic sequence-specific primers. In 2 brands, PCR and nucleotide sequence analysis found C-->T and C-->G point mutations in the pms 146 WSSV amplicon, but in all 5 brands, the nucleotide sequences that were successfully amplified by the rrl, rr2 and tk-tmk gene-specific primer sets were identical to those of Penaeus monodon WSSV. However, despite the inarguable presence of WSSV or WSSV-like template DNA, we were unable to detect WSSV by PCR in hatched nauplii derived from PCR-positive cysts or in P. monodon postlarvae fed Artemia nauplii hatched from such cysts. Most simply, these results suggested that the cysts were externally contaminated with WSSV or WSSV-like template material that was removed during hatching and washing of the nauplii. Given the small sequence variations found, it may also have been a variety of WSSV non-infectious for P. monodon or Artemia and derived from other crustaceans or arthropods in the Artemia environment. However, we could not establish this conclusively and a small possibility remained that the PCR template in these tests was derived from WSSV template present internally in the cysts and derived from infected Artemia adults. However small, this possibility must be vigorously tested, given the impact that a positive outcome could have on the shrimp industry.