Lingbing Zeng

Cyprinid herpesvirus 2 infection emerged in cultured gibel carp, Carassius auratus gibelio in China

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 110-120 nm in diameter with a 170-200 nm 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.

Ultrastructural Morphogenesis of an Amphibian Iridovirus Isolated from Chinese Giant Salamander (Andrias davidianus)

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.

Oligochitosan stimulated phagocytic activity of macrophages from blunt snout bream (Megalobrama amblycephala) associated with respiratory burst coupled with nitric oxide production

The immunostimulating effects of oligochitosan have been proven in several fish, however, the mechanisms underlying the stimulation are not characterized. In the present study, the effects of oligochitosan were investigated using macrophages isolated from blunt snout bream (Megalobrama amblycephala). The results showed that the phagocytic activity of the macrophages was enhanced by the addition of oligochitosan in vitro and in vivo. The two of the most important antimicrobial pathways of macrophages, NADPH oxidase and iNOS pathways were included for further studies. The amounts of superoxide anion and the mRNAs of the five subunits of NADPH oxidase genes were significantly enhanced in the oligochitosan-treated macrophages and macrophages isolated from fish fed with feed containing oligochitosan. In addition, the NO production, iNOS activity and iNOS gene expression were all significantly increased in the presence of oligochitosan. Furthermore, the mRNA levels of the TNF-α and IL-1β were also significantly increased in the macrophages derived from fish fed with oligochitosan. In conclusion, the stimulation effects of oligochitosan on the phagocytic activity of the fish macrophages were associated with respiratory burst coupled with nitric oxide production.

Supplemental diagnosis of a myxozoan parasite from common carp Cyprinus carpio: Synonymy of Thelohanellus xinyangensis with Thelohanellus kitauei

Thelohanellus kitauei Egusa et Nakajima, 1981, was described from common carp Cyprinus carpio L. in Japan. In China, a myxosporean infecting the intestinal tissue of the same host species was described as Thelohanellus xinyangensis Xie, Gong, Xiao, Guo, Li et Guo, 2000, despite many similarities to T. kitauei. To examine the potential conspecificity of these species, a morphological and molecular investigation of T. xinyangensis was conducted. Comparing myxospore morphology, the mean spore length and width of each species is not identical between species, but ranges of dimensions overlap. These data are more suggestive of intraspecific variation than distinct species. Comparison of relative ratios of spore length to polar capsule length and spore width to polar capsule width of T. xinyangensis and T. kitauei reveal no differences and scanning electron microscopy reveals a smooth spore surface of T. xinyangensis, which is consistent with that of T. kitauei. Most convincingly, DNA sequences of the small subunit ribosomal RNA (ssrRNA) gene of the two species were identical. From the morphological and molecular biological data, we propose T. xinyangensis from the intestine of common carp is not a distinct species and is synonymous with T. kitauei.

Inhibition of the replication of grass carp reovirus in CIK cells with plasmid-transcribed shRNAs

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.

Identification of Type I IFN in Chinese giant salamander (Andrias davidianus) and the response to an iridovirus infection

The type I IFNs play a major role in the first line of defense against virus infections. In this study, the type I IFN gene designated gsIFN was identified and characterized in the Chinese giant salamander (Andrias davidianus). The genomic DNA of gsIFN contains 5 exons and 4 introns and has a total length of 5622 bp. The full-length cDNA sequence of gsIFN is 1113 bp and encodes a putative protein of 186 amino acids that has a 43% identity to type I IFN of Xenopus tropicalis. The deduced amino acid sequence has the C-terminal CAWE motif, that is mostly conserved in the higher vertebrate type I IFNs. Real-time fluorescence quantitative RT-PCR analysis revealed broad expression of gsIFN in vivo and the highest level expression in blood, kidney and spleen. Additionally, the expression of gsIFN at the mRNA level was significantly induced in peripheral blood leucocytes after stimulation with poly I:C and after infection with the Chinese giant salamander iridovirus (GSIV). A plasmid expressing gsIFN was constructed and transfected into the Chinese giant salamander muscle cell line. Expression of the IFN-inducible gene Mx was up-regulated in the gsIFN-overexpressing cells after GSIV infection. The virus load and titer were significantly reduced compared with that in control cells. Additionally, a lower level of virus major capsid protein synthesis was confirmed by immunofluorescence assay compared to the control cells. These results suggest that the gsIFN gene plays an important role in the antiviral innate immune response.

Immunological responses and protection in Chinese giant salamander Andrias davidianus immunized with inactivated iridovirus

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.

Significant inhibition of two different genotypes of grass carp reovirus in vitro using multiple shRNAs expression vectors.

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.

A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cyprinid Herpesvirus 2 in Gibel Carp (Carassius auratus gibelio)

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.

Development of a loop-mediated isothermal amplification assay for rapid detection of iridovirus in the Chinese giant salamander

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 0.01 pg/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.