"Methylation-coupled" transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA

S-adenosyl-L-methionine (SAM) activated the virus-associated RNA polymerase of cytoplasmic polyhedrosis virus in vitro. Synthesis of single-stranded viral RNA (mRNA) proceeded depending on the presence of SAM.     

Spring viraemia of carp virus induces autophagy for necessary viral replication

Outbreaks of spring viraemia of carp virus (SVCV) in several carp species and other cultivated fish can cause significant mortality and jeopardize the billion‐dollar worldwide fish industry. Spring viraemia of carp virus, also known as Rhabdovirus carpio, is a bullet‐shaped RNA virus that enters and amplifies in gill epithelium and later spreads to internal organs. Young fish under stressed conditions (spring cold water, etc.) are more vulnerable to SVCV‐induced lethality because of their lack of a mature immune system. Currently, the host response of SVCV remains largely unknown. Here, we observed that autophagy is activated in SVCV‐infected epithelioma papulosum cyprini (EPC) cells. We demonstrated that the SVCV glycoprotein, rather than viral replication, activates the autophagy pathway. In addition, SVCV utilized the autophagy pathway to facilitate its own genomic RNA replication and to enhance its titres in the supernatants. Autophagy promoted the survival of SVCV‐infected cells by eliminating damaged mitochondrial DNA generated during viral infection. We further showed that SVCV induces autophagy in EPC cells through the ERK/mTOR signalling pathway. Our results reveal a connection between autophagy and SVCV replication and propose autophagy suppression as a novel means to restrict SVCV viral replication.     

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.     

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.     

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。     

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.     

Polymerase Activity of Pichinde Virus

Pichinde virus, a member of the arenavirus group, was examined for polymerase activity. Purified virus was found to contain RNA-dependent RNA polymerase but not RNA-dependent DNA polymerase activity. Since RNase but neither DNase nor actinomycin D inhibited the endogenous polymerase reaction, RNA of the virus appeared to be used as the template. The divalent cations Mg(2+) and Mn(2+) were required for optimal reactivity. The RNA product was partially resistant to RNase and the resistant portion had a sedimentation coefficient of 22 to 26S in sucrose gradients.     

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。     

Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus: association of VP1 protein with RNA polymerase activity.

The bluetongue virus core particles have been shown to contain an RNA-directed RNA polymerase (1). To identify the protein responsible for the virion RNA polymerase activity, the complete 3.9 Kb DNA clone representing the largest RNA segment 1 (L1) of bluetongue virus (BTV-10) was placed under control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The derived recombinant virus was used to infect Spodoptera frugiperda cells. As demonstrated by stained polyacrylamide gel electrophoresis and by the use of bluetongue virus antibody, infected insect cells synthesized the largest protein of BTV-10 (VP1, 150 k Da). Antibody raised in rabbit to recombinant VP1 protein recognized bluetongue virus VP1 protein. The recombinant virus infected cell lysate had significantly inducible levels of RNA polymerase enzymatic activity as determined by a poly (U)-oligo (A) polymerase assay. The availability of enzymatically active bluetongue virus RNA polymerase provides a system in which we can precisely delineate the role this protein plays in the regulation of bluetongue replication.     

Ribonuclease-Sensitive Deoxyribonucleic Acid Polymerase Activity in Uninfected Rat Cells and Rat Cells Infected with Rous Sarcoma Virus

Rat cells infected with the B77 strain of avian sarcoma virus [R(B77) cells] produced no virus-like particles but contained information for the production of infectious B77 virus. (3)H-labeled deoxyribonucleic acid (DNA) product of the B77 virus endogenous DNA polymerase system was used to determine the relative amounts of B77 virus-specific ribonucleic acid (RNA) in B77 virus-infected chicken and R(B77) cells. R(B77) cells were found to contain much less B77 virus RNA than did B77 virus-infected chicken cells. Ribonuclease-sensitive DNA polymerase activity was present in high-speed pellet fractions from Nonidet extracts of B77 virus-infected rat cells. Similar preparations from some uninfected rat cells contained lesser amounts of a similar ribonuclease-sensitive DNA polymerase activity. The endogenous template for the DNA polymerase activity in high-speed pellet fractions from R(B77) cells was not related to B77 virus RNA or to RNA of a rat C-type virus. The DNA product of the endogenous DNA polymerase in high-speed pellet fractions of R(B77) cells hybridized to a small extent with RNA from the same fraction and to a similar extent with RNA from uninfected rat cells.     

Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro

The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate. oligouridylate [oligo(U)], polycytidylate . oligoinosinate, and polyinosinate. oligocytidylate templates to about the same extent. The observed activity on polyuridylate. oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg(2+) concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested.     

Assessment of commercial test kits for identification of spring viraemia of carp virus

Two test kits for the identification of spring viraemia of carp virus (SVCV), one an enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antiserum, and the other an indirect fluorescent antibody test (IFAT) using a mouse monoclonal antibody, were assessed for specificity using a range of virus isolates. The test viruses were selected from 4 recently described genogroups of piscine rhabdoviruses: Genogroup I (SVCV), Genogroup II (grass carp rhabdovirus), Genogroup III (pike fry rhabdovirus) and Genogroup IV ('tench rhabdovirus'). The test viruses included SVCV isolates from all 4 subgroups of Genogroup I. The ELISA was non-specific for these viruses and did not distinguish between SVCV and isolates from the other 3 Genogroups. However, the IFAT was too specific and detected SVCV isolates from only 1 of the 4 SVCV subgroups. Reliance on these test kits alone could result in misidentification of this OIE notifiable disease.     

Isolation of a foot-and-mouth disease polyuridylic acid polymerase and its inhibition by antibody

A template-dependent polyuridylic acid [poly(U)] polymerase has been isolated from BHK cells infected with foot-and-mouth disease virus (FMDV). Enzyme activity in a 20,000 x g supernatant of a cytoplasmic extract was concentrated by precipitation with 30 to 50% saturated ammonium sulfate. The poly(U) polymerase was freed of membranes by sodium dodecyl sulfate and 1,1,2-trichlorotrifluoroethane extraction, and RNA was removed by precipitation with 2 M LiCl. The solubilized poly(U) polymerase required polyadenylic acid as template complexed to an oligouridylic acid primer and Mg2+ for activity, but was inhibited by Mn2+. Antisera from animals infected with FMDV had previously been shown to inhibit the activity of FMDV RNA replicase complexed to the endogenous RNA template. The same antisera also inhibited the activity of poly(U) polymerase. Antisera depleted of antibody by absorption with the virus infection-associated antigen of FMDV no longer inhibited replicase and polymerase activities. The evidence suggests that FMDV RNA replicase, poly(U) polymerase, and the virus infection-associated antigen share a common protein.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.     

RNA dependent RNA polymerase activity associated with the double-stranded RNA virus of Giardia lamblia.

Giardia lamblia, a parasitic protozoan, can contain a double-stranded RNA (dsRNA) virus, GLV (1). We have identified an RNA polymerase activity present specifically in cultures of GLV infected cells. This RNA polymerase activity is present in crude whole cell lysates as well as in lysates from GLV particles purified from the culture medium. The RNA polymerase has many characteristics common to other RNA polymerases (e.g. it requires divalent cations and all four ribonucleoside triphosphates), yet it is not inhibited by RNA polymerase inhibitors such as alpha-amanitin or rifampicin. The RNA polymerase activity synthesizes RNAs corresponding to one strand of the GLV genome, although under the present experimental conditions, the RNA products of the reaction are not full length viral RNAs. The in vitro products of the RNA polymerase reaction co-sediment through sucrose gradients with viral particles; and purified GLV viral particles have RNA polymerase activity. The RNA polymerase activities within and outside of infected cells closely parallel the amount of virus present during the course of viral infection. The similarities between the RNA polymerase of GLV and the polymerase associated with the dsRNA virus system of yeast are discussed.