Bing Yang

Complete genome sequence of a Megalocytivirus (family Iridoviridae) associated with turbot mortality in China.

BackgroundTurbot reddish body iridovirus (TRBIV) causes serious systemic diseases with high mortality in the cultured turbot, Scophthalmus maximus. We here sequenced and analyzed the complete genome of TRBIV, which was identified in Shandong province, China.ResultsThe genome of TRBIV is a linear double-stranded DNA of 110,104 base pairs, comprising 55% G + C. Total 115 open reading frames were identified, encoding polypeptides ranging from 40 to 1168 amino acids. Amino acid sequences analysis revealed that 39 of the 115 potential gene products of TRBIV show significant homology to other iridovirus proteins. Phylogenetic analysis of conserved genes indicated that TRBIV is closely related to infectious spleen and kidney necrosis virus (ISKNV), rock bream iridovirus (RBIV), orange-spotted grouper iridovirus (OSGIV), and large yellow croaker iridovirus (LYCIV). The results indicated that TRBIV belongs to the genus Megalocytivirus (family Iridoviridae).ConclusionsThe determination of the genome of TRBIV will provide useful information for comparative study of Megalocytivirus and developing strategies to control outbreaks of TRBIV-induced disease.

A new nodavirus is associated with covert mortality disease of shrimp.

A new nodavirus, named covert mortality nodavirus (CMNV), is associated with covert mortality disease of shrimp which has caused serious loss in China since 2009. Histopathological examination of shrimp suffering the disease revealed coagulative necrosis of striated muscle similar to typical histopathology features of infectious myonecrosis virus (IMNV), Penaeus vannamei nodavirus (PvNV) and Macrobrachium rosenbergii nodavirus (MrNV). However, shrimp suffering this disease tested negative for IMNV, MrNV and PvNV by reverse transcription (RT)-PCR. Additionally, eosinophilic inclusions were found in epithelium of the tubules in the hepatopancreas and lymphoid organ, and mass karyopyknotic nuclei existed in the muscle and lymphoid organ. The tubular epithelium of the hepatopancreas showed significant atrophy. A cDNA library was constructed from total RNA of infected shrimp. Sequencing and alignment analysis showed that one clone with an 1185 bp insert (designated CMNV-7) shared 54, 53 and 39% identity with the amino acid sequences of RNA-dependent RNA polymerase from Flock House virus, black beetle virus and MrNV. The results of fluorescence in situ hybridization showed that the hepatopancreas, striated muscle and lymphoid organ were positively reacting tissues. The mean size of negative-stained virus particles was 32 nm. In addition, a nested RT-PCR assay was developed for CMNV, and the RT-PCR detection results revealed that Fenneropenaeus chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were CMNV-positive.

Characterization of a new member of Iridoviridae , Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp ( Litopenaeus vannamei )

A newly discovered iridescent virus that causes severe disease and high mortality in farmed Litopenaeus vannamei in Zhejiang, China, has been verified and temporarily specified as shrimp hemocyte iridescent virus (SHIV). Histopathological examination revealed basophilic inclusions and pyknosis in hematopoietic tissue and hemocytes in gills, hepatopancreas, periopods and muscle. Using viral metagenomics sequencing, we obtained partial sequences annotated as potential iridoviridae. Phylogenetic analyses using amino acid sequences of major capsid protein (MCP) and ATPase revealed that it is a new iridescent virus but does not belong to the five known genera of Iridoviridae. Transmission electron microscopy showed that the virus exhibited a typical icosahedral structure with a mean diameter of 158.6 ± 12.5 nm (n = 30)(v-v) and 143.6 ± 10.8 nm (n = 30)(f-f), and an 85.8 ± 6.0 nm (n = 30) nucleoid. Challenge tests of L. vannamei via intermuscular injection, per os and reverse gavage all exhibited 100% cumulative mortality rates. The in situ hybridization showed that hemopoietic tissue, gills, and hepatopancreatic sinus were the positively reacting tissues. Additionally, a specific nested PCR assay was developed. PCR results revealed that L. vannamei, Fenneropenaeus chinensis, and Macrobrachium rosenbergii were SHIV-positive, indicating a new threat existing in the shrimp farming industry in China.

Detection and quantification of hepatopancreatic parvovirus in penaeid shrimp by real-time PCR assay

As one of the major pathogens, hepatopancreatic parvovirus (HPV) can cause severe diseases in penaeid shrimp. We developed a TaqMan-based real-time PCR assay for the HPV detection in China. A pair of primers (HPVF and HPVR) and a TaqMan probe were designed according to the HPV genomic sequence of Chinese isolate (GenBank: GU371276). Our data showed that the primers and TaqMan probe were specific for HPV, and they exhibited no cross-reaction with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and specific pathogen free (SPF) shrimp DNA. The assay had a detection limit of four plasmid HPV DNA copies per reaction. Furthermore, HPV was detected in 16 of 21 Fenneropenaeus Chinensis, 3 of 52 Litopenaeus vannamei and 2 of 2 Marsupenaeus japonicus penaeid shrimp samples. In addition, HPV was also detected in crabs. Therefore, this assay could be successfully used as a sensitive and rapid molecular-based diagnostic method to screen HPV-free animals and survey the prevalence of HPV in cultured populations of penaeid shrimp in China.

Prevalence and distribution of covert mortality nodavirus (CMNV) in cultured crustacean

An emerging covert mortality nodavirus (CMNV) was proved to be the infectious agent of shrimp viral covert mortality disease (VCMD). Prevalence and distribution of CMNV were investigated by using the methods of reverse transcription loop-mediated isothermal amplification (RT-LAMP), nested reverse transcription PCR, gene sequencing, histopathology, in situ RNA hybridization (ISH) and transmission electron microscope (TEM) in this study. RT-LAMP results showed that CMNV positive samples appeared in the cultured crustaceans including Litopenaeus vannamei, Fenneropenaeus chinensis, Marsupenaeus japonicus, Penaeus monodon, and Macrobrachium rosenbergii, and mostly distributed the coastal provinces in China. The prevalence rates of CMNV among the collected samples in 2013, 2014 and 2015 were 45.93% (130/283), 27.91% (84/301) and 20.85% (54/259), respectively. CMNV infection in M. japonicas and P. monodon was verified by ISH. The presence of CMNV particles were confirmed by TEM analysis in the CMNV positive samples diagnosed by RT-LAMP. The high prevalence and wide epidemic distribution of CMNV in this investigation revealed that it was necessary to pay close attention to the high risk of CMNV transmission in farmed crustaceans.

Complete genome sequence of shrimp hemocyte iridescent virus (SHIV) isolated from white leg shrimp, Litopenaeus vannamei

Infection with shrimp hemocyte iridescent virus (SHIV), a new virus of the family Iridoviridae isolated in China, results in a high mortality rate in white leg shrimp (Litopenaeus vannamei). The complete genome sequence of SHIV was determined and analyzed in this study. The genomic DNA was 165,809 bp long with 34.6% G+C content and 170 open reading frames (ORFs). Dotplot analysis showed that the longest repetitive region was 320 bp in length, including 11 repetitions of an 18-bp sequence and 3.1 repetitions of a 39-bp sequence. Two phylogenetic trees were constructed based on 27 or 16 concatenated sequences of proteins encoded by genes that are conserved between SHIV homologous and other iridescent viruses. The results of this study, suggest that SHIV should be considered a member of the proposed new genus “Xiairidovirus”.

Detection and quantification of shrimp hemocyte iridescent virus by TaqMan probe based real-time PCR

Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R2 = 0.998) in the range of 4 × 109 to 4 × 100 DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei, Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (μg DNA)-1. Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load.