[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; 2014:716413. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Haemorrhagic disease of Chinese giant salamanders (Andrias davidianus) (CGSs) is an emerging condition caused by an iridovirus of the genus Ranavirus within the family Iridoviridae. Several studies have described different biological properties of the virus, but some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe the morphogenesis of Chinese giant salamander iridovirus (GSIV) in an epithelioma papulosum cyprinid (EPC) cell line at the ultrastructural level. Cells were infected with GSIV at a multiplicity of infection (MOI) of 10 and examined at 1, 2, 4, 6, 12, 24, 48, 72, 84 and 96 h post infection. GSIV entered EPC cells by endocytosis or fusion after adsorption to the cell membrane. Following uncoating, the viral cores translocated to the nucleus and the virus began to replicate. Different stages of virus self-assembly were observed in the slightly electron-lucent viromatrix near the cell nucleus. In the late phase of virus infection, most nucleocapsids were mature and formed a typical icosahedral shape and aggregated in pseudocrystalline array at the viromatrix or were budding at the plasma membrane. Virus infection was readily detected by electron microscopy before cytopathic effect appeared in cell culture. The EPC cell line represents a suitable in-vitro model for study of GSIV morphogenesis and characterization of the GSIV replication cycle.
Journal of comparative pathology 11/2013; · 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.
Journal of virological methods 09/2013; · 2.13 Impact Factor
[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; · 3.13 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: To determine the pathogenic bacterium infecting giant salamander (Andrias davidianus).
Bacterium was isolated from the liver of diseased Chinese giant salamander and identified by the Biolog Microbial Identification System and molecular biology method. Healthy Chinese giant salamander and crucian carp were used for experimental infection with bacterial suspension.
A bacterial strain JZ01 was isolated and identified from diseased giant salamander. Infection with the bacterial suspension to healthy giant salamander could reproduce the diseased symptoms as occurred naturally and the same bacterium could be recovered from these infected giant salamanders. The isolated bacterium also has certain pathogenicity to crucian carp. Identification by the Biolog Microbial Identification System, and further 16S rDNA sequence and phylogenetic analysis demonstrated that the bacterium isolated from diseased giant salamander was Citrobacter freundii. The susceptibility test to antibiotics demonstrated that the bacterial strain JZ01 was susceptible to aztreonam, cefepime and cefotamine.
Citrobacter freundii is a pathogen for cultured Chinese giant salamander.
[show abstract][hide abstract] ABSTRACT: Two DNA constructs targeting the grass carp reovirus (GCRV) RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene that were each 64 bp in length were synthesized chemically and cloned into pSilencer2.1-U6 neo plasmid, named pSi-RdRp1286 and pSi-OCP117, respectively. After transfection of pSi-RdRp1286 and pSi-OCP117 plasmids into CIK cells, the inhibition of GCRV replication in the cells were detected by observing cytopathic effect (CPE), quantitating virus titers (TCID(50)/mL) and real-time quantitative RT-PCR analysis of viral RdRp and OCP genes. Five days after the cells were challenged with GCRV, both pSi-RdRp1286 and pSi-OCP117 reduced the viral titers by 5.47 lgTCID(50)/mL and 4.37 lgTCID(50)/mL, respectively. Compared to the positive control, CPE induced by GCRV in transfected cells was delayed and significantly less. Furthermore, the real-time quantitative RT-PCR analysis of the viral RdRp gene and OCP gene showed that the targeted gene expression were reduced by 89% and 73%, respectively. These results proved that the plasmid-transcribed shRNAs could inhibit effectively GCRV replication in CIK cells. These shRNAs provide potential tools for inhibiting GCRV infection and replication both in vitro and in vivo.
Journal of virological methods 08/2011; 175(2):182-7. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Myxobolus shantungensis Hu, 1965 infects gill arches of common carp Cyprinus carpio haematopterus (Temminck and Schlegel), causing serious pathological effects on host fish. An inadequate original description and absence of molecular data make accurate early diagnosis challenging. To augment the original description, M. shantungensis is redescribed here using morphological and molecular biological methods. Mature spores of M. shantungensis were ellipsoidal or apple shaped in frontal view and lemon shaped in lateral view, averaging 8.2 ± 0.3 μm (8.0-9.0 μm) × 10.1 ± 0.5 μm (9.2-11.1 μm) × 6.9 ± 0.3 μm (6.0-7.4 μm). Some spores had three to four "V"-shaped valve edge markings on the posterior of the spore. The two equal polar capsules were oval, measuring 4.3 ± 0.3 μm (3.8-5.0 μm) × 3.2 ± 0.2 μm (2.8-3.5 μm), situated at the anterior extremity of the spore. Polar filaments coiled with six to seven turns. Scanning electron microscopy revealed that the surface of mature spores of M. shantungensis was generally pitted with a number of irregular ridges in shape. M. shantungensis is also characterized on the molecular level using the sequence of the small subunit ribosomal RNA gene. A BLAST search revealed that this sequence did not match any available sequences in GenBank.
Parasitology Research 05/2011; 109(6):1619-23. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The type II interleukin-1 receptor (IL-1RII) cDNA was cloned from Japanese flounder (Paralichthys olivaceus) by mRNA differential display (DD-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA is 1793 bp in length, including a 100 bp 5'-terminal untranslated region (UTR), a 430 bp 3'-terminal UTR, and a 1263 bp open reading frame (ORF). The ORF encodes 420 amino acid residues with an estimated molecular mass of 46.65 kDa. The protein possesses signature features of the IL-R family, consisting of one N-terminal signal peptide, one transmembrane (TM) domain, two Ig-like domains in its extracellular region, one short cytoplasmic tail of 17 amino acids and four conserved proline residue sites. The predicted amino acid sequence has 65.3%, 52.5% and 51.6% identity with gilthead seabream, rainbow trout and Atlantic salmon IL-1RII, respectively. Phylogenetic analysis supported a close relation to mammalian IL-1RII. Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis indicated that the IL-1RII gene expression of Japanese flounder was weakly up-regulated and reached the peak expression in the kidney, spleen, and gill at 6 h after infection with Vibrio anguillarum, at 1.9, 2.0 and 1.5 times relative to the uninfected fish, respectively. These results suggest that IL-1RII has a signal transduction function and possibly plays a minor role in immune regulation against bacterial(s) infection in Japanese flounder.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 05/2010; 157(1):59-65. · 1.61 Impact Factor
[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.