Weiwei Zeng

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.

A one-step duplex rRT-PCR assay for the simultaneous detection of grass carp reovirus genotypes I and II

Hemorrhagic disease of grass carp, caused by grass carp reovirus (GCRV), leads to severe economic losses in the grass carp farming industry in China. GCRV has been divided into three genotypes based on genome sequence. Genotypes I and II (GCRV-1 and GCRV-II, respectively) are the dominant genotypes and co-infections of GCRV-I and GCRV-II are common in grass carp aquaculture. A one-step duplex real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay was developed for simultaneous detection of GCRV-I and GCRV-II. The PCR assay is suitable for early diagnosis of grass carp hemorrhagic disease and for epidemiological surveillance. The detection limit of the assay is 10 copies for both GCRV-I and GCRV-II, which is as high as single-target rRT-PCR and higher than conventional RT-PCR. No cross reactivity with other GCRV subtypes or other viruses was observed. One hundred and twelve samples from grass carp suspected of hemorrhagic disease were collected from South and Central China. Eleven samples were positive for GCRV-I by RT-PCR alone, and fourteen samples were positive by single-target and duplex rRT-PCR. Forty two samples were positive for GCRV-II by RT-PCR alone and forty seven samples were positive by single-target and duplex rRT-PCR. Mixed infections were found in eight samples when analyzed by RT-PCR alone and in ten samples analyzed by single-target and duplex rRT-PCR. The duplex rRT-PCR system provides a sensitive and specific method to detect and differentiate between GCRV-I and GCRV-II in a single sample. This rRT-PCR assay could be a useful tool for the routine diagnosis of these two viruses and for epidemiology studies in grass carp aquaculture.

Pathogenicity and tissue distribution of grass carp reovirus after intraperitoneal administration

Grass carp reovirus (GCRV) is the causative agent of grass carp hemorrhage and causes significant loss of fingerlings. However, little is known about how the virus is distributed in organs and tissues. The aim of the present study was to investigate the distribution of different GCRV stains in tissues and organs of grass carp. The pathogenicity and tissue distribution of GCRV were monitored after intraperitoneal administration. The study showed a distribution of GCRV in different tissues and organs, particularly in the liver, spleen, kidney, intestine, and muscle, which had a higher number of viral RNA copies during the sixth to ninth days. The kidney had the highest numbers of viral RNA copies, as high as 24000 copies. Until the fourteenth day, nearly no viral RNA copies could be detected. This study defined the virus distribution in different tissues of grass carp inoculated by i.p. and supplied clues for the pathogenesis of GCRV.

A one-step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain

Six reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primers designed against conserved regions of segment 6 (s6) gene were used for the detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 subtype. The entire amplification could be completed within 40 min at 62·3° C. The RT-LAMP showed higher sensitivity than reverse-transcription polymerase chain reaction (RT-PCR). The RNA detection limit was 10 copies µl⁻¹ for RT-LAMP assay and 100 copies µl⁻¹ for conventional RT-PCR. In specificity tests, no cross-reactivity was detected in other viruses from common aquatic animals. In addition, the reaction results can be visualized by using calcein fluorescent dye. Furthermore, a total of 86 samples were tested by RT-LAMP, RT-PCR and virus isolation. The results demonstrated that all 54 specimens identified as positive by virus isolation were also positive when detected by RT-LAMP. Seven out of 54 samples, however, were misidentified by RT-PCR. The RT-LAMP method is more accurate than conventional RT-PCR. The results indicate that RT-LAMP has potential as a simple and rapid diagnosis technique for the detection of GCRV HZ08 subtype infection.

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.

Development of indirect immunofluorescence assay for TCID 50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells

Grass carp reovirus (GCRV) caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus, in southeast Asian. It was first isolated in 1983 in China, and clade analysis of the different GCRV isolates indicates there are at least three different genotypes I, II, and III. In recent years, GCRV genotype II has been determined as a dominant virus type which cause severe obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. TCID50 is one of standard method to quantity infectious virus particles. In the present study, an indirect immunofluorescence assay (IFA) was developed using antibody against a protein encoded by segment 10 of GCRV genotype II. Moreover, the specific assay to differentitate GCRV of different genotypes and a sensitive assay for determination of GCRV genotype II were developed respectively. The results showed the IFA only can recognize genotype II virus at the lowest initial concentration of 550 genomic copies/ml. Furthermore, comparison of results obtained from qPCR and the TCID50 assay combined IFA was conducted. The results indicated that TCID50 of GCRV isolates JX0901 and HZ08 differs with 2 log steps reduction in the numbers of viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is sensitive, specific, and the TCID50 combined with IFA will be a standardizable technique for the quantitation and detection of infectious GCRV in cell culture without cytolysis.

Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA)

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.

A Cross-Sectional Study of the Association between Risk Factors and Hemorrhagic Disease of Grass Carp in Ponds in Southern China

A cross-sectional survey of 215 Grass Carp Ctenopharyngodon idella ponds was conducted in southern China between May 2010 and November 2011. An in-depth questionnaire was developed to evaluate a series of biosecurity practices, environmental factors, and management factors at the farm level. Fish samples with clinical hemorrhagic signs were also collected from each pond to assess the clinical disease of Grass Carp reovirus by using reverse transcription (RT) PCR assay. The association between the incidence of Grass Carp hemorrhagic disease (GCHD) and risk factors was analyzed using logistic regression. Of the 215 ponds, 144 showed GCHD-positive responses to RT-PCR assay. In addition, survey results revealed that inferior environmental conditions occurred in most ponds with an incidence of GCHD; such conditions included a thick mud layer; no cleansing and restoration practices (CRPs) before culture; and poor water quality (i.e., high ammonia nitrogen and nitrite concentrations and low transparency). Logistic regression model results suggested that decreased risks were associated with fry vaccination, a safe water source, and deepening of the water level, whereas increased risk factors mainly included no CRPs, excessive rearing density, disease history, and inferior water quality. Presently, control efforts are restricted to immunization of Grass Carp as the best management option for farms. Deepening the water levels and improving water sources can also effectively reduce the incidence of GCHD by diluting the pond rearing densities.

Genetic Variability of Koi Herpesvirus In vitro—A Natural Event?

Worldwide koi herpesvirus (KHV) causes high mortalities in Cyprinus carpio L. aquaculture. So far, it is unknown how the different variants of KHV have developed and how they spread in the fish, but also in the environmental water bodies. Therefore, a phylogenetic method based on variable number of tandem repeats (VNTR) was improved to gain deeper insights into the phylogeny of KHV and its possible worldwide distribution. Moreover, a VNTR-3 qPCR was designed which allows fast virus typing. This study presents a useful method for molecular tracing of diverse KHV types, variants, and lineages.

Is There Any Species Specificity in Infections with Aquatic Animal Herpesviruses?-The Koi Herpesvirus (KHV): An Alloherpesvirus Model

Most diseases induced by herpesviruses are host-specific; however, exceptions exist within the family Alloherpesviridae. Most members of the Alloherpesviridae are detected in at least two different species, with and without clinical signs of a disease. In the current study the Koi herpesvirus (KHV) was used as a model member of the Alloherpesviridae and rainbow trout as a model salmonid host, which were infected with KHV by immersion. KHV was detected using direct methods (qPCR and semi-nested PCR) and indirect (enzyme-linked immunosorbant assay; ELISA, serum neutralization test; SNT). The non-koi herpesvirus disease (KHVD)-susceptible salmonid fish were demonstrated to transfer KHV to naive carp at two different temperatures including a temperature most suitable for the salmonid (15°C) and cyprinid (20°C). At 20°C KHVD was induced in carp cohabitated with infected trout. KHV was also detected virologically and serologically at the end of the experiment in both rainbow trout and carp.