Cunbin Shi

Complete Genome Sequence of a Reovirus Isolated from Grass Carp, Indicating Different Genotypes of GCRV in China

ABSTRACT A widespread grass carp hemorrhagic disease (GCHD) caused by grass carp reovirus (GCRV) has been known in China since 1983. A virulent reovirus strain, HZ08, was isolated from diseased grass carp in Zhejiang Province, China. We sequenced and analyzed the complete genome of strain HZ08 and compared it with published GCRV genome sequences, contributing to the evidence of several genotypes of GCRV in China.

Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA)

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture.

Isolation and characterization of a rhabdovirus from snakehead fish(Ophicephalus striatus)

The diseased hybrid snakehead(Channa maculate ♀ × Channa argus ♂) were collected for analysis of the pathogen both in bacteriology and virology to make the pathologic agent clear.None of pathogenic bacteria w as obtained using routine bacteria separation method.One rhabdovirus w as isolated using cell culture technique,regression tests,electron microscopy,molecular biology techniques and others,w hich assigned hybrid snakehead rhabdovirus-C1207(HSHRV-C1207).HSHRV-C1207 could replicate and proliferate in EPC,FHM,GSB,SFC,CIK cells,and cause the significant cytopathic effect(CPE).The regression tests in w hich separated viruses w ere injected in the abdominal cavity w ere all presented w ith symptoms similar to the natural incidence,and the final mortality w as 90%.Electron microscopy observation of ultra-thin section samples of specimen-infected EPC cells revealed that the bullet-shaped virus replicated in cytoplasm w ith regular arrangement like other fish rhabdovirus,and had a non-enveloped structure w ith a diameter and length of approximately 60 nm and 145 nm respectively.The OIE recommended primers for Infectious haematopoietic necrosis virus(IHNV),spring viraemia of carp virus(SVCV),viral haemorrhagic septicaemia virus(VHSV) and three pairs of specific primers w ere designed by G gene conserved sequences of snakehead rhabdovirus(SHRV),siniperca chuatsi rhabdovirus(SCRV) and hirame rhabdovirus(HIRRV) respectively,w ere applied to the RT-PCR amplification for detecting the possible rhabdovirus in diseased fish and virus-infected cells.A specific ladder product about 350 bp only w as amplified w ith primers of SCRV.Sequencing analysis of the G gene of HSHRV-C1207 show ed that this gene shares the sequence identities 93.8% and 20.4% w ith the respective genes of SCRV and SHRV respectively,and the corresponding deduced amino acid sequence identity 93.7% and 18.3% respectively.Phylogenic relationship w ith other rhabdoviruses using the corresponding amino acid sequences of G protein indicates that HSHRV C1207 is most closely related to the vesiculovirus genus and shares the highest identity w ith SCRV,but w as far from the snakehead rhabdovirus(SHRV) and the other rhabdovirus.The above results indicated that the causes of hybrid snakehead diseases ceaseless breaking out w as the rhabdovirus,and this new isolates HSHRV-C1207 belongs to vesiculovirus genus.It may be a novel variant SCRV isolate or a new member.

Use of high-resolution melting curve analysis to differentiate vaccine and wild type strains of grass carp reovirus genotype II

Grass carp reovirus (GCRV) is the causative agent of a hemorrhagic disease that causes severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Discrimination between wild-type field and vaccine strains of GCRV is crucial for meaningful disease diagnosis and epidemiological investigation, yet current detection methods do not discriminate between these different viruses. This study exploited sequence differences between vaccine viruses and the virulent and field strains in the S6 gene that is present in all GCRV strains to develop a high resolution melting curve assay to differentiate between virus strains. The high resolution melting curve analysis was as analytically sensitive as real-time quantitative-Polymerase Chain Reaction (qPCR) detection and at least 10 times sensitive than the conventional PCR. This one-step assay will facilitate grass carp hemorrhagic disease outbreak responses and control.

Development of a VP38 recombinant protein‐based indirect ELISA for detection of antibodies against grass carp reovirus genotype II (iELISA for detection of antibodies against GCRV II)

Currently, serological assays for grass carp reovirus genotype II (GCRV-II) diagnosis are not available. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against GCRV-II was developed. The structural protein VP38 of GCRV-II was used as the coating antigen. Monoclonal antibodies (mAb) against IgM of grass carp labelled with HRP were used as a secondary antibody. The antigen concentration and serum dilution were optimized using chess board titration. Furthermore, the specificity of indirect ELISA assay was confirmed by cross check with sera positive for other grass carp pathogens. In comparison with results obtained from indirect immunofluorescence assay (IFA) and Western blot by testing of 60 serum samples to evaluate the sensitivity and specificity of the ELISA, agreement between 90% and 96.7% was reached, respectively. A serological survey was performed using the assay with grass carp field serum samples. The seropositive rate of the 242 serum samples was 69.8%. In conclusion, the developed indirect ELISA is a very specific and sensitive test that will be useful for large-scale serological surveys to detect indirectly GCRV II infections as well as to monitor the changes of antibody level after immunization.

Detection of koi herpesvirus (KHV) using a monoclonal antibody against Cyprinus carpio IgM

Koi herpesvirus disease (KHVD) is associated with high mortality in both common carp and koi carp (Cyprinus carpio L.) worldwide. The indirect detection of fish viruses based on the identification of antibodies has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against carp IgM. By using hybridoma-monoclonal antibody technology, one hybridoma cell line secreting MAbs against IgM from carp was established. In western blot analysis, the secreted MAb from cell line A5-E10 recognized the heavy chain of IgM from common carp or koi but did not react with immunoglobulins from three different fish species: grass carp (Ctenopharyngodon idella), tilapia (Oreochromis mossambicus) and Mandarin fish (Siniperca chuatsi). These results demonstrated that this MAb is highly specific for the IgM of carp and suggested that it can be used for monitoring the immunity level of carp, for example for indirect KHV diagnosis by antibody ELISA. We therefore established an indirect ELISA, which was tested using 200 serum samples from koi from three farms. The final results showed that 147 (73.5%) samples were confirmed to be KHV antibody negative and 53 (26.5%) were definitely positive, containing antibodies against KHV.

Identification of a novel envelope protein encoded by ORF 136 from Cyprinid herpesvirus 3

Cyprinid herpesvirus 3 (CyHV-3) is the pathogenic agent of koi herpesvirus disease (KHVD) afflicting common carp and koi (Cyprinus carpio L.) populations globally. As described previously, proteomic analyses of purified CyHV-3 particles have shown that at least 46 structural proteins are incorporated into CyHV-3 virions; among these ORF136 may encode a putative envelope protein. In this study, Western blotting analysis showed that a specific band with the predicted molecular weight of 17 kDa was detected both in purified virions and envelope components using a rabbit anti-ORF136 polyclonal antibody. Indirect immunofluorescence assay with confocal laser scanning microscopy indicated that the ORF136 protein was distributed in the cytoplasm of CCB cells infected with CyHV-3 and transfected with a pVAX1-ORF136 plasmid. Furthermore, immunogold electron microscopy confirmed that ORF136 protein localized to the CyHV-3 envelope.