[Show abstract][Hide abstract] ABSTRACT: The emergence of an infectious viral disease caused by the Chinese giant salamander iridovirus (GSIV) has led to substantial economic losses. However, no more molecular information is available for the understanding of the mechanisms associated with virus–host interaction. In this study, de novo sequencing was used to obtain abundant high-quality ESTs and investigate differentially-expressed genes in the spleen of Chinese giant salamanders that were either infected or mock infected with GSIV. Comparative expression analysis indicated that 293 genes were down-regulated and 220 genes were up-regulated. Further enrichment analysis showed that the most enriched pathway is “complement and coagulation cascades”, and significantly enriched diseases include “inherited thrombophilia”, “immune system diseases”, “primary immunodeficiency”, “complement regulatory protein defects”, and “disorders of nucleotide excision repair”. Additionally, 30 678 simple sequence repeats (SSRs) from all spleen samples, 26 355 single nucleotide polymorphisms (SNPs) from the spleens of uninfected animals and 36 070 SNPs from the spleens of infected animals were detected. The large amount of variation was specific for the Chinese giant salamanders that were infected with GSIV. The results reported herein provided significant and new EST information that could contribute greatly in investigations into the molecular functions of immune genes in the Chinese giant salamander.
Veterinary Research 12/2015; 46(1). DOI:10.1186/s13567-015-0279-8 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyprinid herpesvirus 2 (CyHV-2) infection is a newly emerged infectious disease of farmed gibel carp (Carassius gibelio) in China and causes huge economic losses to the aquaculture industry. In this study, the three membrane proteins encoded by genes ORF25, ORF25C, and ORF25D of CyHV-2 were truncated and expressed in yeast, Pichia pastoris. Screening of the recombinant yeasts was done by detecting the truncated proteins using Western blot. Through immunogold labeling, it was shown that proteins binding the colloidal gold were presented on the surface of cells. In the experiment of inhibition of virus binding by the recombinant truncated proteins, the TCID50 of the tORF25 group (10(4.1)/ml) was lower than that of tORF25C (10(4.6)/ml) or tORF25D groups (10(5)/ml). These results suggested that the proteins may be involved in attachment of the virus to the cell surface. Healthy gibel carp were immunized with 20 μg of tORF25, tORF25C, and tORF25D proteins, and the control group received PBS. Interleukin 11 (IL-11) expression in the spleens of the immunized fish peaked at day 4 and the complement component C3 (C3) genes were significantly up-regulated at day 7 post-immunization. Specific antibodies were measured in the three immunized groups and the titer detected in the tORF25 group reached 327, that was significantly higher than the tORF25C (247) or tORF25D (228) groups. When the immunized fish were challenged with live CyHV-2 by intraperitoneal injection the relative percent survival (RPS) of the tORF25, tORF25C, and tORF25D immunized groups was 75%, 63%, and 54%, respectively. The feasibility of the Pichia pastoris yeast expression system for the production of the recombinant truncated proteins and their apparent bioactivity suggests that tORF25, tORF25C, and tORF25D are potential candidate vaccines against Cyprinid herpesvirus 2 infection in gibel carp.
[Show abstract][Hide abstract] ABSTRACT: The aquaculture of penaeid shrimp has grown rapidly during the past three decades. However, shrimp aquaculture continues to be plagued by infectious disease, particularly of viral aetiology. Urgently needed are in vitro biological tools to facilitate early detection and diagnosis of penaeid shrimp viruses. An immediate need is the establishment of stable or continuous shrimp cell lines, which remains to be an elusive goal. Researchers have made significant advancements in the methodologies for primary shrimp cell culture. Also, new media and technologies including cell immortalization are presently under development. The principal goal of this review paper is to collate current attempts in the establishment of permanent shrimp cell lines by summarizing the latest research progress on penaeid shrimp cell culture studies, including general cultivation conditions, methods of cell preparation, types of media and their utilization and the development of immortal cell lines. In addition, current and potential applications of penaeid shrimp cell cultures in the various related research fields are also discussed in this review.
Reviews in Aquaculture 08/2015; DOI:10.1111/raq.12106 · 3.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Chinese giant salamander iridovirus (GSIV), belonging to the genus Ranavirus in the family Iridoviridae, causes severe hemorrhagic lesions and nearly 100% mortality in naturally infected Chinese giant salamanders Andrias davidiamus. However, the replication and distribution of the virus has not been well characterized in vivo. Using in situ hybridization, the expression of the GSIV major capsid protein (MCP) was detected in the cytoplasm of cells of the spleen, kidney, liver and gut tissues. MCP expression in the spleen and kidney appeared to fluctuate significantly during the acute phase of infection. Using an immunofluorescence assay, GSIV antigens were abundant in the spleen and kidney tissues but appeared to be at relatively low levels in the liver and gut. Additionally, there were significant changes in the expression of the pro-inflammatory cytokines macrophage migration inhibitory factor (MIF), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in different tissues in response to infection with GSIV. The expression of MIF, TNF-α and IL-1β had significantly increased in the spleen at 3 d post-infection; this correlated with a decrease in virus replication in the spleen. These results suggest that the spleen and kidney are the major target tissues of GSIV, and the increased expression of MIF, TNF‑α and IL-1β may contribute to a reduction of virus replication in the spleen.
[Show abstract][Hide abstract] ABSTRACT: Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry in China. Recently, a fatal bacterial disease occurred in farmed Chinese sturgeon. The clinical signs of the diseased fish were systemic blood loss and anemia. The gills were very pale with swollen and hemorrhagic gill filaments. Discrete blood spots were apparent in the oral cavity. Small petechial hemorrhages were present in pale livers and the digestive tract. Hemorrhages were also present in the kidney and gonad. The spleen was red and swollen. Additionally, a small amount of clear ascites fluid was present in the body cavity. A bacterial pathogen was isolated from the internal organs of diseased fish and subsequently identified as Pseudomonas alcaligenes by morphology, biochemical testing and phylogenetic analysis of the 16S rDNA. Experimental infection in healthy hybrid sturgeon confirmed the pathogenicity of the bacterial isolate. The results of this study represent the first isolation and identification of Pseudomonas alcaligenes in cultured Chinese sturgeon in China and provide a foundation for the diagnosis, prevention and treatment of this new disease.
[Show abstract][Hide abstract] ABSTRACT: Chinese giant salamander hemorrhage is a newly emerged infectious disease in China and has caused huge economic losses. The causative pathogen has been identified as the giant salamander iridovirus (GSIV). In this study, the immunological responses and protection in Chinese giant salamander immunized with β-propiolactone inactivated GSIV are reported. Red and white blood cell counting and classification, phagocytic activity, neutralizing antibody titration, immune-related gene expression and determination of the relative percent survival were evaluated after vaccination. The red and white blood cell counts showed that the numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly on days 4 and 7 post-injection (P < 0.01). Additionally, the differential leukocyte count of monocytes and neutrophils were significantly different compared to the control group (P < 0.01); the percentage of lymphocytes was 70.45 ± 7.52% at day 21. The phagocytic percentage and phagocytic index was 38.78 ± 4.33% and 3.75 ± 0.52, respectively, at day 4 post-immunization which were both significantly different compared to the control group (P < 0.01). The serum neutralizing antibody titer increased at day 14 post-immunization and reached the highest titer (341 ± 9.52) at day 21. The quantitative PCR analysis revealed that the immunization significantly up-regulated the expression of immune related genes TLR-9 and MyD88 the first two weeks after immunization. The challenge test conducted at day 30 post-injection demonstrated that the immunized group produced a relative survival of 72%. These results indicate that the inactivated GSIV could elicit significant non-specific and specific immunological responses in Chinese giant salamander that resulted in significant protection against GSIV induced disease.
[Show abstract][Hide abstract] ABSTRACT: The hemorrhagic disease of grass carp (Ctenopharyngodon idellus), caused by grass carp reovirus (GCRV), is the most severe disease of the fish that leads to huge economic losses. GCRV, belonging to the genus Aquareovirus of the family Reoviridae, has been classified into three genotypes based on their phylogenetic relationship. It is essential to develop an effective method to inhibit the replication of different genotypes of GCRV simultaneously. In this report, two multiple-shRNAs expression vectors, named pMultishVP2/2 and pMultishVP6/7, were generated and investigated. pMultishVP2/2 targeted the VP2 gene of GCRV-JX0901 (genotype I) and the VP2 gene of HGDRV (Hubei grass carp disease reovirus; genotype III). pMultishVP6/7 targeted the VP7 gene of GCRV-JX0901 and the VP6 gene of HGDRV. These two multiple-shRNAs expression vectors can simultaneously, significantly inhibit the replication of GCRV- JX0901 and HGDRV in vitro. Compared to the positive control, CPE induced by GCRV-JX0901 or HGDRV in cell transfected with shRNA transcribing vector was significantly delayed. The quantitative PCR analysis of the GCRV genomic RNA revealed that the pMultishVP2/2 could simultaneously inhibit the GCRV-JX0901 and HGDRV VP2 coding genes by 89.02% and 89.84%, respectively. The pMultishVP6/7 could simultaneously inhibit the GCRV-JX0901 VP7 coding gene and HGDRV VP6 coding gene by 80.63% and 86.78%, respectively. Furthermore, compared to the positive control, the indirect immunofluorescence assay and western blot demonstrated that the protein expression of the two genotypes of GCRV decreased significantly. The results in this study indicated that this multiple-shRNAs expression system could be used as a cross-reactive antiviral agent for treating the hemorrhagic disease of grass carp caused by multiple genotypes of GCRV.
Virus Research 05/2014; 189. DOI:10.1016/j.virusres.2014.05.009 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay for Cyprinid herpesvirus 2 (CyHV-2) detection in gibel carp was developed. Following cloning and sequencing of the putative DNA helicase gene of CyHV-2 isolate from China, a set of four specific primers was designed based on the sequence. The MgCl2 concentration and the reaction temperature were optimized to 6 mM, 64°C, respectively. LAMP products were detected by visual inspection of a color change due to addition of SYBR Green I stain. The specificity and sensitivity of the LAMP assay were determined. No cross-reaction was observed with other fish DNA viruses including eel herpesvirus, koi herpesvirus, and Chinese giant salamander iridovirus. The LAMP assay was found to be equally sensitive as nested PCR. A comparative evaluation of 10 fish samples using LAMP and nested PCR assays showed an overall correlation in positive and negative results for CyHV-2. These results indicate that the LAMP assay is simple, sensitive, and specific and has a great potential use for CyHV-2 detection in the laboratory and field.
The Scientific World Journal 01/2014; 2014(2-3):716413. DOI:10.1155/2014/716413 · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Chinese giant salamander (Andrias davidianus) iridovirus (GSIV) is an emerging infectious pathogen responsible for severe hemorrhagic disease and high mortality in cultured Chinese giant salamanders. A loop-mediated isothermal ampliﬁcation (LAMP) assay based on the major caspid protein (MCP) gene has been developed to detect this virus. Primer pairs for the LAMP assay were designed based on the GSIV MCP gene sequence. Amplification results indicate that under optimized conditions the LAMP assay has the ability to specifically detect the virus in both diseased animals and infected epithelioma papilloma cyprinid (EPC) cells. The assay was shown to be 10-fold more sensitive than nested PCR and was able to detect concentrations of 10(-9) (approximately 0.01pg/μL). The LAMP assay is relatively easy to perform in situ and the amplification products can be observed directly under UV light or via staining with SYBR Green I. The LAMP assay is also rapid and cost-effective. This study establishes the use of a LAMP assay for rapid detection of GSIV, which is a novel and important tool for the diagnosis of GSIV infection in laboratory or farmed Chinese giant salamanders.
[Show abstract][Hide abstract] ABSTRACT: A novel fish reovirus, Hubei grass carp disease reovirus (HGDRV, formerly grass carp reovirus strain 104, GCRV104), was isolated from diseased grass carp in China in 2009. Thereafter, the full genome sequence was determined. This reovirus was propagated in grass carp kidney cell line with typical cytopathic effect. The total size of the genome is 23,706 bp with a 51% G+C content and the eleven dsRNA segments encode twelve proteins (two proteins encoded by segment eleven). Nucleotide sequence similarity search using Blastn found no significant matches except for the segment two, which partially matched with RNA-dependent RNA polymerase (RdRp) from several viruses in the genera Aquareovirus and Orthoreovirus, of the Reoviridae family. At the amino acid level, seven segments (Seg-1 to Seg-6, and Seg-8) matched with species in Aquareovirus (with 15-46% identities) and Orthoreovirus (12-44%) genera, while for four segments (Seg-7, Seg-9, Seg-10, and Seg-11) no similarities in these genera were found. Conserved terminal sequences, 5'-GAAUU----UCAUC-3', were found in each HGDRV segment at the 5' and 3' ends, and the 5'-terminal nucleotides were different from any known species in Aquareovirus. Phylogenetic analysis based on RdRp amino acid sequences from Reoviridae showed that HGDRV clustered with aquareoviruses prior to joining a branch common with orthoreoviruses. Based on these observations, we propose that HGDRV is a new species of Aquareovirus that is distantly related to any known species within this genus.
Journal of General Virology 07/2013; 94(Pt_10). DOI:10.1099/vir.0.054767-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An epizootic with severe mortality has emerged in cultured gibel carp, Carassius auratus gibelio, in China since 2009, and caused huge economic loss. The signs and epidemiology background of the disease were investigated. Parasite examination, bacteria and virus isolation were carried out for pathogen isolation. The causative pathogen was obtained and identified as Cyprinid herpesvirus 2 (CyHV-2) by experimental infection, electron microscopy, cell culture, PCR assay and sequence alignment, designated as CyHV-2-JSSY. Experimental infection proved the high virulence of CyHV-2-JSSY to healthy gibel carp. Electron microscopy revealed that the viral nucleocapsid was hexagonal in shape measuring 110-120nm in diameter with a 170-200nm envelope. The virus caused significant CPE in Koi-Fin cells at the early passages, but not beyond the fifth passages. Sequence alignment of the partial viral helicase gene (JX566884) showed that it shared 99-100% identity to the published sequences of other CyHV-2 isolates. This study represented the first isolation and identification of CyHV-2 in cultured gibel carp in China and laid a foundation for the further studies of the disease.
[Show abstract][Hide abstract] ABSTRACT: A reovirus, designated as CCRV-730, was isolated from channel catfish, Ictalurus punctatus, fingerlings suffering a severe hemorrhage in Hubei province in China. Experimental infection confirmed the pathogenicity of the virus and proved it to be the causative pathogen. Signs of the diseased channel catfish included abdominal distention, eyes bulging and hemorrhages of operculum, lower jaw, skin and fin bases. The CCK cell line was used for viral pathogen isolation. The electron microscopy observation of the virus infected cells revealed that there were a large number of reovirus-like particles measuring 60–70 nm in diameter in cytoplasm arrayed in crystalline. The viral genomic RNA was extracted and analyzed by SDS-PAGE and the complete S-class (S7–S11) segments of genome RNA were cloned and sequenced. The sequence alignment analysis of the S-class segments of CCRV-730 with corresponding sequences of other Aquareovirus members in the genetic sequence database of NCBI indicated that the virus had high similarity to grass carp reovirus, especially shared 99%–100% nucleotide sequence identity with the grass carp reovirus 873 strain (GCRV-873). The results implied that the genetic variation of GCRV-873 potentially arose in natural environment and resulted in the viral host conversion or expansion and made it pathogenic to channel catfish.
Aquaculture 01/2013; s 372–375:39–44. DOI:10.1016/j.aquaculture.2012.10.011 · 1.88 Impact Factor