Haemagglutination as a low-cost detection method for gill-associated virus and by inference, yellowhead virus in Penaeus monodon Fabricius, 1798

A quantitative, low‐cost test based on haemagglutination (HA) using chicken erythrocytes was developed to indicate the viral load of Australian yellowhead‐like virus, gill‐associated virus (GAV), in Penaeus monodon. The study tested the haemolymph, gill, lymphoid organ, heart, sub‐cutaneous tissue, eye stalk, pleopods, uropods and the central nerve cord for agglutination activity in 100 prawns, with the haemolymph and gill tissue giving the highest end‐point titres of 1:1370 and 1:361 respectively. The sensitivity of the test was demonstrated by testing two different populations of P. monodon, which showed a highly significant difference (P<0.001) in HA activity, indicating a difference in viral load. By testing three other penaeid prawn species (n=20), Penaeus esculentus, Penaeus merguiensis and Penaeus longistylis, and the crayfish Cherax quadricarinatus, it was demonstrated that natural agglutinins were not causing the high agglutination in the population of P. monodon being tested. It was also demonstrated that there was no effect of freezing and thawing of samples on HA activity. The speed and low cost of this test makes it a very useful tool, particularly in the developing world, for on‐farm testing of penaeid prawns to indicate yellowhead virus and GAV loads which can contribute to management practices with respect to harvesting of ponds.     

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.     

罗非鱼湖病毒核蛋白的克隆表达、抗体制备及其组织分布

近年来罗非鱼湖病毒(TiLV)在多个国家流行,对世界罗非鱼养殖业造成严重威胁。中国是罗非鱼第一养殖大国,尽管我国大陆还没有TiLV的正式报道,鉴于吉富罗非鱼是我国重要的罗非鱼养殖品种,其对TiLV的感染特性研究具有重要意义。本实验采用TiLV对吉富罗非鱼进行人工感染,随后在肝脏组织中克隆和测定了TiLV第6片段基因。罗非鱼湖病毒第6片段基因cDNA全长1 044 bp,开放读码框(ORF)为954 bp,编码317个氨基酸,预测分子量为36.38 ku;5′非编码区(NCR)为19 bp,3′非编码区(NCR)为972 bp。系统进化树分析表明该蛋白属于TiLV核蛋白(NP)。随后在大肠杆菌中表达和提纯了GST融合NP蛋白,在新西兰大白兔上制备了多克隆抗体。通过酶联免疫吸附实验(ELISA)检测抗体效价为1∶51 200,且抗体可特异性识别感染组织中的病毒NP蛋白。对吉富罗非鱼不同组织进行苏木精—伊红(H.E)染色观察,发现肝脏组织坏死并形成合胞体,脾脏部分细胞出现空泡、坏死,含铁血黄素增多,头肾细胞坏死,鳃丝上皮细胞明显解离脱落,鳃小片黏连,脑组织细胞肿大。通过蛋白印迹法(WB)和免...     

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).     

南美白对虾白斑综合症病原检测与发病成因分析及防控对策

南美白对虾,学名凡纳滨对虾,头小身大,出肉率高,口味鲜美,作为海虾淡养的优良品种在全市得到了大力推广和迅猛发展。在杭州地区自2000年的1000多亩发展到2009年的10.6万亩,2009年全市南美白对虾总产量达到3.5万吨,     

In infectious pancreatic necrosis virus carrier Atlantic salmon, Salmo salar L., post-smolts, almost all kidney macrophages ex vivo contain a low level of non-replicating virus

The level of infection by infectious pancreatic necrosis virus (IPNV) of kidney macrophages from 12 asymptomatic carrier Atlantic salmon post-smolts was studied. Kidney leucocytes were fractionated on 34/51% Percoll gradients, allowed to adhere to plastic wells overnight, washed to remove non-adherent cells and cultured for up to 7 days with or without renewal of medium on day 3. On day 1, supernatants were harvested, macrophages were counted, lysed and IPNV in the supernatants and lysates was titred in chinook salmon embryo (CHSE-214) cells. The multiplicity of infection ranged between 1:2.2 and 1:7.4 (virus:macrophages). On day 3, the titres of IPNV in macrophage lysates decreased and in wells where the medium was renewed on day 3, IPNV was no longer detectable on day 7. In the supernatants, one fish was positive for IPNV on day 1, four fish on day 3 but none were detectably positive on day 7. In parallel wells in which the medium was not renewed, on day 7 IPNV was detected in macrophage lysates of three fish and the supernatants were also IPNV positive in two of these fish. This suggests that virus might be shed from infected macrophages and then reinfect other macrophages. When macrophages were serially diluted in wells and cultured for 24 h, IPNV could be cultured from macrophage lysates of wells containing between two and 70 macrophages. These results indicate that a very high proportion of the adherent kidney macrophages must be infected with very few non-replicating virions.     

Epizootic haematopoietic necrosis virus: detection by ELISA, immunohistochemistry and immunoelectron-microscopy

Abstract. Epizootic haematopoietic necrosis virus (EHNV) was isolated from cultured rainbow trout, Oncorhynchus mykiss (Walbaum). Antibodies to the virus and to associated capsid subunits have been produced and used in immunohistochemistry, immunoelectron-microscopy and an antigcn-capture-immunosorbent assay (ELISA). The results show that both antibodies can be used by various immuno-procedures to detect both redfin perch, Perca ftuviatilis L., and rainbow trout isolates of EHNV. The procedures described provide for the first time rapid and specific tests for the detection of EHNV in cultured and clinical material.     

美洲地区对虾TSV、IHHNV、WSSV和YHV病毒流行现状、诊断方法及防治对策(一)

从１９７４年在佛罗里达的墨西湾北部桃红对虾中首次发现对虾杆状病毒以来，感染这类海洋无脊椎动物的病毒种类已发现近２０种。这些病毒作为主要病原体已经在世界各地养虾地区出现。一目前已知有９种病毒在西半球对虾中流行，５种已作为严重的病原出现于一种或多种养殖对虾中。病毒病也严重影响东半球对虾养殖业，在印－太地区及东亚的对虾养殖地区，至少发现１２种（群）对虾病毒。１２种（群）对虾病毒中的５种已被证实（报导）能引起严重的区域性流行病，其中的两种病毒已造成印－太和东亚地区大面积的虾病流行。尽管有近２０种不同的对…     

Multiple passage of infectious salmon anaemia virus in rainbow trout,Oncorhynchus mykiss (Walbaum), did not induce increased virus load

The infectious salmon anaemia virus (ISAV) has not been observed to cause natural disease in farmed rainbow trout, Onchorhynchus mykiss (Walbaum), but may cause high mortality in farmed Atlantic salmon, Salmo salar L. In this study, ISAV was passaged 10 times in succession by intraperitoneal injections of serum from previous passage into naïve rainbow trout. The serum viraemia was monitored by real‐time qPCR. The rainbow trout in this study became infected but did not develop ISA. No clinical signs were observed in the rainbow trout in any passage, but replication of ISAV was detected from Day 4 post‐infection (p.i.). Neither increased relative virus loads nor histopathological and immunohistochemical findings consistent with ISA were observed. However, the expression of interferon type I and Mx genes were slightly up‐regulated in the hearts of some individual fish at day 17 p.i. Sequencing of all open reading frames in the ISAV genome of the 10th passage revealed two nucleotide mutations, one in segment 6 coding for the haemagglutinin–esterase (HE) and one in segment 1 coding for the basic polymerase 2 (PB2). The mutation in HE resulted in an amino acid substitution T/K₃₁₂.     

A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus

Since it first appeared in 1992, white spot syndrome virus (WSSV) has become the most threatening infectious agent in shrimp aquaculture. Within a decade, this pathogen has spread to all the main shrimp farming areas and has caused enormous economic losses amounting to more than seven billion US dollars. At present, biosecurity methods used to exclude pathogens in shrimp farms include disinfecting ponds and water, preventing the entrance of animals that may carry infectious agents and stocking ponds with specific pathogen-free post-larvae. The combination of these practices increases biosecurity in shrimp farming facilities and may contribute to reduce the risk of a WSSV outbreak. Although several control methods have shown some efficacy against WSSV under experimental conditions, no therapeutic products or strategies are available to effectively control WSSV in the field. Furthermore, differences in virulence and clinical outcome of WSSV infections have been reported. The sequencing and characterization of different strains of WSSV has begun to determine aspects of its biology, virulence and pathogenesis. Knowledge on these aspects is critical for developing effective control methods. The aim of this review is to present an update of the knowledge generated so far on different aspects of WSSV organization, morphogenesis, pathology and pathogenesis.     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

Mortality patterns in infectious salmon anaemia virus outbreaks in New Brunswick, Canada

Mortality levels attributed to infectious salmon anaemia viral (ISAV) infections were examined at the net pen and site level in the 1996 smolt year class in three areas of New Brunswick, Canada. The year class in this region was the first known to have potential exposure to ISAV beginning at the time of seawater transfer. There was considerable variability in mortality patterns among net pen groups of fish. Net pen outbreak definitions were based on at least seven high mortality days in which there were at least 100 per 100 000 fish per day or >5% cumulative mortality for the study period. There were 106 net pen outbreaks in a study population consisting of 218 net pens. Although the number of new cases decreased as water temperature decreased, overall mortality levels at the study sites did not decrease noticeably. The median peak daily mortality rate during outbreaks was 492 per 100 000 fish per day, with 10% of cases experiencing >5200 mortalities per 100 000 fish per day. The median duration of outbreaks in net pens for which the fish were not slaughtered during the outbreak was 33 days and the median total loss in those outbreaks was 6600 per 100 000 fish.     

Erythrocytic inclusion body syndrome virus in wild Atlantic salmon, Salmo salar L

An investigation was undertaken to establish aspects of the epizootiology of erythrocytic inclusion bodies in wild Atlantic salmon, Salmo salar L., in Scotland. From 1992 to 1995, adult and juvenile salmon, from Scottish rivers, were screened for the presence of erythrocytic inclusion bodies and haematological parameters measured. The nature of the inclusion bodies was assessed through transmission electron microscopy, negative-staining and blood smear-staining techniques and was demonstrated to be viral in origin with characteristics similar to a member of the family Togaviridae. Specifically, these were a viral genome of single-stranded RNA, spherical virion morphology with an icosahedral core, average size of 70 nm and a buoyant density of 1.15-1.20 g cm(-3). The cytoplasmic inclusions were either large, single inclusions (1-2 microm) or smaller multiple inclusions (0.5-1 microm). A total of 4.2% (n=48) and 27.7% (n=213) of the parr and adult salmon, respectively, were positive for the presence of the inclusions. The intensity of the inclusions, when present, varied from light in parr to moderate and heavy in adults, when graded according to the number of inclusions per field of view. Neither haematological variations nor clinical disease was associated with the presence or absence of viral inclusions.     

Inhibition of infectious pancreatic necrosis virus infectivity by extracts of rainbow trout, Salmo gairdneri Richardson, tissue

Abstract. Extracts of healthy rainbow trout liver, kidney, spleen and whole fry inhibited plaque production of infectious pancreatic necrosis virus (IPNV) in cell cultures. The mode of inhibition is not known, although it appears not to be manifest at the cellular level, as pre-treatment of the cell cultures with tissue extracts did not inhibit plaque production. Any effect on the virus itself was not permanent as the inhibition could be mitigated by treatment of virus/extract mixtures with 1,1,2-trichloro-1,1,2-trifluoroethane. The inhibition may be caused by prevention or reduction of virus attachment to the cell surface or, alternatively, the tissue extract may cause aggregation of the virus and thereby reduce the number of available infectious units. The inhibitory effect is also lost by dilution of the extract, reinforcing the claim that adequate dilutions of fish extracts prior to attempted virus isolation are necessary, especially for the detection of carrier fish.