Shuqin Wu

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.

Genotype and host range analysis of infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a disease causing high mortality in mandarin fish, Siniperca chuatsi. In this study, complete major capsid protein (MCP) genes of nine ISKNV isolates were sequenced and compared with other known megalocytiviruses to evaluate genetic variation and host range of the viruses. Comparison of nucleotide sequences of MCP gene revealed 92.6–100% identity among nine ISKNV isolates. A phylogenetic tree revealed that 33 megalocytiviruses were divided into three genotypes, and there was a strong host species signal in three genotypes: for genotype I, the host was mainly marine fish; for genotype II, the host was freshwater fish; and for genotype III, the host was mainly flatfish. Nine ISKNV isolates belonged to genotype I or genotype II, suggesting mandarin fish may be a mixing vessel host for megalocytivirus.

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.

An outbreak of granulomatous inflammation associated with Francisella noatunensis subsp. orientalis in farmed tilapia (Oreochromis niloticus × O. aureus) in China

In 2013, a novel disease was detected in tilapia (Oreochromis niloticus × O. aureus) in Guangzhou, South China. To identify the causative pathogen, we conducted histological examination, bacteria isolation, and purification, and sequenced the 16S rRNA gene of isolates. Infected fish had a large number of white granulomas in the kidney, liver, heart, and spleen. The head kidney and spleen were markedly swollen. A bacterium strain designated as gz201301 was recovered from the spleen of infected tilapia. The 16S rRNA sequence of gz201301 revealed that it was highly similar to F. noatunensis subsp. orientalis. This is the first report of a Francisella-like infection in farmed tilapia in China.

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.

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.

The complete mitochondrial genome of jade perch, Scortum barcoo (Perciformes: Terapontidae: Scortum)

Abstract This is the first documentation of the complete mitochondrial genome sequence of the jade perch, Scortum barcoo. The 16,843 bp mitogenome containes 37 genes (13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes) and two main non-coding regions (the control region and the origin of the light strand replication) as do other vertebrate mitogenomes. The overall base composition of S. barcoo is 27.39% for A, 24.74% for T, 31.14% for C and 16.73% for G, with a slight A + T bias of 52.13%. All protein-coding genes are initiated with ATG except for COX1, which begin with GTG instead. Meanwhile, the termination codons of 13 protein-coding genes are varied with TAA, TAG, TA, T or AGG. The measure of complete mitogenome sequence of S. barcoo will provide fundamental data for the phylogenetic and biogeographic studies of the Terapontidae.

Identification of a transparent mutant tiger barb Puntius tetrazona and its use for in vivo observation of a Pleistophora sp. (Microsporidia) infection.

A transparent mutant tiger barb Puntius tetrazona was identified and characterized by its transparent body, which allows clear visualization of internal organs. Hybridization of this mutant with the albino variant produces a transparent and albinoid double phenotype, and the transparency of this mutant is controlled by a recessive allele. Light microscopic and ultrastructural examinations show that in contrast to normal individuals, transparent mutants lack iridophores, and light penetrates unimpeded through the body. Pleistophora sp. infection was observed in vivo, allowing live observation of parasite dissemination and the consequent pathological alterations in the fish body as well as the simultaneous acquisition of data on the dynamics and spatial pattern of pathogenic invasion. It is superior to common fish models, as dynamic experimental data can be obtained from individual fish.

[Construction and application of bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer].

As a key component of life science, bioinformatics has been widely applied in genomics, transcriptomics, and proteomics. However, the requirement of high-performance computers rather than common personal computers for constructing a bioinformatics platform significantly limited the application of bioinformatics in aquatic science. In this study, we constructed a bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer. The platform consisted of three functional modules, including genomic and transcriptomic sequencing data analysis, protein structure prediction, and molecular dynamics simulations. To validate the practicability of the platform, we performed bioinformatic analysis on aquatic pathogenic organisms. For example, genes of Flavobacterium johnsoniae M168 were identified and annotated via Blast searches, GO and InterPro annotations. Protein structural models for five small segments of grass carp reovirus HZ-08 were constructed by homology modeling. Molecular dynamics simulations were performed on out membrane protein A of Aeromonas hydrophila, and the changes of system temperature, total energy, root mean square deviation and conformation of the loops during equilibration were also observed. These results showed that the bioinformatic analysis platform for aquatic pathogen has been successfully built on the MilkyWay-2 supercomputer. This study will provide insights into the construction of bioinformatic analysis platform for other subjects.