Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of putative spring viraemia of carp viruses (SVCV) and pike fry rhabdoviruses (PFRV), including isolates from tench, grass carp, roach, bream and false harlequin, sheatfish and orfe. Phylogenetic analysis of a 550 nucleotide (nt) region of the glycoprotein gene identified 4 groups, I to IV. Significantly, the majority of viruses previously identified as PFRV formed a distinct cluster (Genogroup IV) which shared <80% nucleotide identity with the PFRV reference strain (Genogroup III). The similarity between another PFRV-like virus isolated from grass carp and representatives of Genogroups III and IV was also <80%, indicating that this virus belonged to a third group (Genogroup II). All of the putative SVC viruses were assigned to a 4th group (Genogroup I), sharing <61% nucleotide identity with viruses in Genogroups II to IV.     

Genotyping spring viraemia of carp virus and other piscine vesiculo-like viruses using reverse hybridisation

A simple nylon membrane-based DNA macroarray was developed to genotype spring viraemia of carp virus (SVCV) and related viruses. Twenty-six viruses were genotyped using the array, and the results were confirmed by phylogenetic analysis of a 426 bp partial glycoprotein gene sequence. The array was not only capable of discriminating between the 4 main genogroups of cyprinid vesiculo-type viruses described previously, but also accurately sub-type the SVC viruses assigned to Genogroup I. The assay offers a practical solution for diagnostic laboratories that currently lack a sequencing capability to confirm the nature of PCR products generated in suspected SVCV cases.     

Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA)

An ELISA was developed using a rabbit antiserum with high levels of binding antibody against SVC virus (SVCV). Modifications were made to the standard assay which increased the sensitivity and resulted in a rapid ELISA (rELISA) which could détéct SVCV antigen in cell cultures and in extracts from infected carp in approximately 1 h. When other cell-grown fish rhabdoviruses were tested, pike fry rhabdovirus (PFRV) antigen cross-reacted in the rELISA but only at a low level. Virus antigen was détécted by the rELISA in clinically and sub-clinically diseased carp during an SVC epizootic but the sensitivity of détéction of subclinical levels of virus was not as great as that of isolation of infectious virus in cell culture. However, antigen was détécted in some fish extracts which failed to yield infectious virus. Fresh extracts of organs from healthy carp were found to give false positive reactions in the rELISA. Kidney and liver extracts from these fish were found to contain a heat labile, non-specific binding factor and further modification of the rapid assay was undertaken which successfully reduced the effect of this factor.     

Spring viremia of carp (SVC)

Spring viremia of carp (SVC) is an important disease affecting cyprinids, mainly common carp Cyprinus carpio. The disease is widespread in European carp culture, where it causes significant morbidity and mortality. Designated a notifiable disease by the Office International des Epizooties, SVC is caused by a rhabdovirus, spring viremia of carp virus (SVCV). Affected fish show destruction of tissues in the kidney, spleen and liver, leading to hemorrhage, loss of water-salt balance and impairment of immune response. High mortality occurs at water temperatures of 10 to 17 degrees C, typically in spring. At higher temperatures, infected carp develop humoral antibodies that can neutralize the spread of virus and such carp are protected against re-infection by solid immunity. The virus is shed mostly with the feces and urine of clinically infected fish and by carriers. Waterborne transmission is believed to be the primary route of infection, but bloodsucking parasites like leeches and the carp louse may serve as mechanical vectors of SVCV. The genome of SVCV is composed of a single molecule of linear, negative-sense, single-stranded RNA containing 5 genes in the order 3'-NPMGL-5' coding for the viral nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase, respectively. Polyacrylamide gel electrophoresis of the viral proteins, and sequence homologies between the genes and gene junctions of SVCV and vesicular stomatitis viruses, have led to the placement of the virus as a tentative member of the genus Vesiculovirus in the family Rhabdoviridae. These methods also revealed that SVCV is not related to fish rhabdoviruses of the genus Novirhabdovirus. In vitro replication of SVCV takes place in the cytoplasm of cultured cells of fish, bird and mammalian origin at temperatures of 4 to 31 degrees C, with an optimum of about 20 degrees C. Spring viremia of carp can be diagnosed by clinical signs, isolation of virus in cell culture and molecular methods. Antibodies directed against SVCV react with the homologous virus in serum neutralization, immunofluorescence, immunoperoxidase, or enzyme-linked immunosorbent assays, but they cross-react to various degrees with the pike fry rhabdovirus (PFR), suggesting the 2 viruses are closely related. However, SVCV and PFR can be distinguished by certain serological tests and molecular methods such as the ribonuclease protection assay.     

Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK

Genetic relationships between 35 spring viremia of carp virus (SVCV) genogroup Ia isolates were determined based on the nucleotide sequences of the phosphoprotein (P) gene and glycoprotein (G) genes. Phylogenetic analysis based on P gene sequences revealed 2 distinct subgroups within SVCV genogroup Ia, designated SVCV Iai and Iaii, and suggests at least 2 independent introductions of the virus into the USA in 2002. Combined P- and G-sequence data support the emergence of SVCV in Illinois, USA, and in Lake Ontario, Canada, from the initial outbreak in Wisconsin, USA, and demonstrate a close genetic link to viruses isolated during routine import checks on fish brought into the UK from Asia. The data also showed a genetic link between SVCV isolations made in Missouri and Washington, USA, in 2004 and the earlier isolation made in North Carolina, USA, in 2002. However, based on the close relationship to a 2004 UK isolate, the data suggest than the Washington isolate represents a third introduction into the US from a common source, rather than a reemergence from the 2002 isolate. There was strong phylogenetic support for an Asian origin for 9 of 16 UK viruses isolated either from imported fish, or shown to have been in direct contact with fish imported from Asia. In one case, there was 100% nucleotide identity in the G-gene with a virus isolated in China.     

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections

Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in?vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases.     

用RT-PCR法快速检测鲤春病毒血症病毒基因

用逆转录多聚酶链式反应（RT-PCR）方法快速,灵敏、特异地检测鲤春病毒血症病毒（SVCV）,根据SVC病毒糖蛋白基因序列设计的引物经过RT-PCR和半嵌套PCR（semi-nested-PCR）引扩增出SVC病毒核酸中的714bp和606bp片段,与其他弹状病毒IHNV、VHSV、PFRV没有交叉,没有特异性,灵敏度检测,表明不小于1000个病毒就可检测出阳性结果。本文还对复性温度、引物、Mg^2 、Tag酶以及反转录酶的浓度对检测结果的影响进行了探讨。     

Spring viremia of carp virus RNA and virion-associated transcriptase activity

An RNA-dependent RNA polymerase activity has been demonstrated for spring viremia of carp virus (SVCV). The optimal temperature for in vitro synthesis of RNA was 20 to 25 degrees C. The SVCV enzyme activity was stimulated when the methyl donor S-adenosyl-L-methionine was included in the reaction mixture. S-adenosyl-L-methionine was not particularly effective in stimulating the virion RNA polymerase activity of vesicular stomatitis virus or pike fry rhabdovirus. The 5' nucleotide of the SVCV viral RNA is pppAp.     

Molecular Analysis of Spring Viraemia of Carp Virus in China: A Fatal Aquatic Viral Disease that Might Spread in East Asian

Spring viraemia of carp (SVC) is a fatal viral disease for cyprinid fish, which is caused by spring viraemia of carp virus (SVCV). To date, no SVC outbreak has been reported in China. Between 1998 and 2002, outbreaks of SVC were reported in ornamental and wild fish in Europe and America, imported from multiple sources including China. Based on phylogenetic analysis, the viral strain isolated from America was shown to be originated from Asia. These outbreaks not only resulted in huge economic losses, but also raise an interesting question as to whether SVCV really exists in China and if so, is it responsible for SVC outbreaks? From 2002 to 2006, we screened 6700 samples from ornamental fish farms using the cell culture method of the Office International des Epizooties (OIE), and further verified the presence of SVCV by ELISA and real-time quantitative RT-PCR. Two infected samples were found and the complete genome of SVCV was sequenced from one of the isolates, termed SVCV-C1. Several unique hallmarks of SVCV-C1 were identified, including six amino acid (KSLANA) insertion in the viral RNA-dependent RNA polymerase (L) protein and ten nucleotide insertion in the region between glycoprotein (G) and L genes in European SVCV strains. Phylogenetic tree analysis of the full-length G protein of selected SVCV isolates from the United Kingdom and United States revealed that G proteins could be classified into Ia and Id sub genogroups. The Ia sub genogroup can be further divided into newly defined sub genogroups Ia-A and Ia-B. The isolates derived from the United States and China including the SVCV-C1 belongs to in the Ia-A sub genogroup. The SVCV-C1 G protein shares more than 99% homology with the G proteins of the SVCV strains from England and the United States, making it difficult to compare their pathogenicity. Comparison of the predicted three-dimensional structure based on the published G protein sequences from five SVCV strains revealed that the main differences were in the loops of the pleckstrin homology domains. Since SVCV is highly pathogenic, we speculate that SVC may therefore pose a serious threat to farmed cyprinid fish in China.     

RLRs介导的抗鲤春病毒血症病毒作用研究进展

鲤春病毒血症病毒(SVCV)是水生动物病毒中重要的病原体,常引起鲤科鱼类疾病暴发。近些年研究发现,维甲酸诱导基因I样受体家族(RLRs)信号通路在SVCV免疫过程中起到重要的作用。主要功能是在识别病原体相关模式,激活下游信号分子,诱导天然免疫的产生,以及控制病毒的早期复制。当病毒进入机体时会形成病毒-RLRs-IFN互联反馈回路,RLRs相关基因识别SVCV的RNA,最终引起Ⅰ型干扰素(IFN-I)表达量升高,并且RLRs族内成员相互作用增强抗病毒作用。RLRs不仅可以活化天然免疫信号通路,还可增强适应性免疫效应,在控制病毒感染过程中发挥重要作用。介绍RLRs家族,RLRs抗病毒信号调控因子,干扰素诱导的鱼类Mx (myxovirus resistant)蛋白对鲤春病毒血症病毒的抑制作用。     

Isolation and characterization of Scophthalmus maximus rhabdovirus

A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalmus maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.     

Fish rhabdoviruses: comparative study of protein structure.

Proteins from four fish rhabdoviruses have been studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viruses were: trout viral hemorrhagic septicemia (VHS), infectious hematopoietic necrosis virus (IHN), spring viremia virus of carp (SVC), and the pike fry rhabdovirus (PFR). For the two salmonid viruses (VHS-IHN), gel electrophoresis indicated the proteins, with molecular weights estimated to be 190,000, 80,000, 38,000, 25,000, and 19,000, respectively. The electrophoretic profile of the two other viruses (SVC-PFR) revealed four major proteins with molecular weights of 190,000 80,000 42,000 and 21,000, respectively. In this case a minor component with 50,000 daltons was found. For each virus only one protein was found to be glycosylated, i.e., the one with a molecular weight of 80,000. A major protein (molecular weight between 38,000 and 42,000) was found to be associated with the nucleocapsid. All these results revealed marked similarities in protein structure between the four fish rhabdoviruses and the previously well-characterized members of rhabdovirus group. However, one can distinguish two groups of viruses: the first one is composed of salmonid viruses (VHS and IHN) with a protein structure comparable to that of rabies virus and potato yellow dwarf virus; the second one is composed of carp and pike viruses, having a protein structure very similar to that of vesicular stomatitis virus.     

An enzyme-linked immunosorbent assay for detection of anti-Bacillus piliformis serum antibody in rabbits

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of rabbit serum antibody directed against the causative agent of Tyzzer's disease, Bacillus piliformis. Ninety-four percent agreement was found between the ELISA and an indirect fluorescent antibody test. The sensitivity of the ELISA was 95% and its specificity was 92% as compared to the indirect fluorescent antibody test (IFAT). The rabbit origin B. piliformis isolate used in this ELISA was found to be cross-reactive by ELISA and IFAT to B. piliformis isolates of rat, gerbil and horse origin. This suggests that a single B. piliformis isolate may be used as antigen for an ELISA utilizable for multiple species.     

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty-one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme-linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)-, antigen-coated plate (ACP)-, triple antibody sandwich (TAS)- with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS-ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.     

A new coumarin derivative plays a role in rhabdoviral clearance by interfering glycoprotein function during the early stage of viral infection

Coumarin forms an elite class of naturally occurring compounds that possess promising antiviral therapeutic perspectives. In this study, a coumarin derivative 7-[6-(2-methylimidazole) hexyloxy] coumarin (D5) was designed and synthesized to evaluate antiviral activity on a rhabdovirus, spring viraemia of carp virus (SVCV). Our results demonstrated that D5 had a robust antiviral activity with >90% inhibitory rate of SVCV expression in the host cells. And D5 significantly reduced viral-induced apoptosis and recovered virus-activated caspase-3/8/9 activities. Further data determined that SVCV could alter the cytoskeletal structure of EPC cells, characterized by a circumferential ring of microtubules and a disrupted microfilament organization, whereas cytoskeleton structure in D5-treated cells kept the normal morphology. Mechanistically speaking, D5 could interfere with SVCV replication inside or outside of cells through two different approaches. Before the process of virus entry into EPC cells, D5 had an impact on SVCV glycoprotein structure so as to disrupt viral binding to the cell surface or translocation to the cytosol. Another strategy for D5 to against SVCV was that D5 significantly suppressed SVCV-activated autophagy, which was beneficial for the host cells to restrict SVCV viral replication, accompanied by a higher phosphorylation of Akt-mTOR. In summary, our results revealed that D5 was effective in weakening SVCV infection and regulating SVCV-induced undesirable conditions, and this compound provided new therapeutic implications for the treatment of rhabdoviruses.