Chuangfu Chen

Detection of genetic variations in Orf virus isolates epidemic in Xinjiang China.

Orf is an exanthemous viral disease seriously threatening the goat and sheep industry and widely epidemic in the goat and sheep populations in Xinjiang, China. In order to investigate the genetic variability of the orf virus (ORFV), three virus isolates (SHZ1, SHZ2, and SHZ3) were isolated by PCR and Vero cell culture using the clinical samples from the lips of the lambs suspected of ORFV infection. The isolates were further verified by electron microscopy and animal infection experiments. The protective antigen genes B2L, F1L, and virulence genes VIR, GIF, and VEGF in the isolates were cloned, sequenced and analyzed for genetic evolution. The results showed that B2L and F1L were relatively conservative with homology 86.7–97.9%, while VIR, GIF, particularly VEGF were considerably variable with homology 71.5–97.9% at amino acid sequence level, respectively. Phylogenetic tree analysis based on B2L and VIR showed that the isolates SHZ1 and SHZ2 were closely related with the Taiwan isolates. This is the first report to confirm that there have been genetic variations in the Xinjiang ORFV isolates. The findings provide molecular evidence about the genetic variability of the major antigenic and virulence genes in the virus isolates epidemic in Xinjiang.

RsbV of Listeria monocytogenes contributes to regulation of environmental stress and virulence.

SigmaB factor is an important regulatory factor for stress response in Gram-positive bacteria such as Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus and Bacillus subtilis. However, the activity of SigmaB factor is regulated by RsbV factor. Currently, the functional studies of RsbV factor are mostly focused on non-pathogenic B. subtilis, but the roles of RsbV factor in pathogenic L. monocytogenes during the regulation of environmental stress and virulence are still unclear. In the study, a ∆RsbV mutant of L. monocytogenes was constructed to explore the regulatory role of RsbV in environmental stress and virulence. The environmental stress experiments indicated that the growth and survival capability of ∆RsbV mutant obviously decreased in stress of low temperature, osmotic pressure, alcohol and acid, compared with EGD strain. The macrophage infection experiment indicated that ∆RsbV mutant had weaker survival capability than EGD strain, and the expression of PrfA, actA, PlcA and LLO was down-regulated in infected cells. Animal inoculation experiments indicated that RsbV deletion significantly reduced the pathogenicity of L. monocytogenes. Our data demonstrate that, in addition to regulating tolerance under environmental stress conditions, RsbV also contributes to regulation of L. monocytogenes virulence.

Rapid detection of Akabane virus by a novel reverse transcription loop-mediated isothermal amplification assay (RT-LAMP)

BackgroundAkabane disease, caused by Akabane virus, is an insect-transmitted disease of ruminants that is primarily characterized by fetal damage.Methods and resultsIn this study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of Akabane virus was successfully developed. The primers were designed to target the highly conserved fragment of nucleoprotein from the Akabane virus. The results indicate that the assay is highly specific and sensitive with a detection limit of 5.0 TCID50 /mL within a 60-min incubation time. A total of 126 abortive samples collected from Xinjiang province were detected by the established RT-LAMP. The results of RT-LAMP assay showed 96.8% agreement with the semi-nested RT-PCR.ConclusionThis study is to first to develop a rapid, sensitive, and accurate method for the detection of Akabane virus, which may be used to screen clinical samples in developing countries or regions.

Rapid detection of Betacoronavirus 1 from clinical fecal specimens by a novel reverse transcription loop-mediated isothermal amplification assay.

Betacoronavirus 1 (BCoV-1) is an important pathogen causing diarrhea in calves. In the current study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of BCoV-1 was successfully developed. The primers were designed to target the highly conserved fragment of BCoV-1 nucleocapsid gene. The assay displayed high specificity detecting only BCoV-1 with no cross reaction with other viruses. When 418 clinical samples from 6 different geographical areas of Xinjiang province were tested by the RT-LAMP method, the results indicated that this test is a simple, rapid, accurate, and sensitive method for the detection of BCoV-1.

Isolation of Arthrobotrys oligospora from soil of the Chinese Northern Tianshan Mountain slope pasture show predatory ability against Haemonchus contortus larvae

To understand the biocontrol ability of Arthrobotrys oligospora isolated from the Northern Tianshan Mountain slope pasture, Xinjiang region, China, on livestock gastrointestinal nematode diseases, we acquired 68 soil samples and isolated 26 nematode-trapping fungi using Haemonchus contortus L3. Eight isolates were identified based on their morphological and molecular identification. The predacious activity against H. contortus was detected before and after passage through the sheep gastrointestinal tract. As a result, these eight isolates were identified as A. oligospora. They displayed predacious activities ranging from 90% to 97%. Six of the isolates could pass through the sheep gastrointestinal tract significantly reducing the number of H. contortus larvae by 76–79%. This study shows that A. oligospora isolated from the northern slope pasture of Tianshan Mountain has high predacious activity against H. contortus larvae and partly passing through the sheep gastrointestinal tract. This study also shows that conidial suspensions have no toxic side-effects on the sheep, indicating that they have the potential for the development of oral biological agents which prevent and control livestock gastrointestinal nematode diseases.

Mixed Nipple Infections Caused by Variant of BPV3 and a Putative New Subtype of BPV in Cattle

Bovine papilloma is a chronic and proliferative skin and mucosal wart caused by Bovine papillomavirus (BPV). In June, 2013, a leaf-and flat-shaped wart disease was observed on the nipple skins in a cattle farm in Xinjiang. To diagnose the disease, we collected the diseased skins for pathological biopsy and DNA analysis by PCR amplification using a pair of degenerate primers FAP59 and FAP64. Sequencing and phylogenetic analysis showed that the infection was caused by a variant of BPV3 and putatively a new subtype of BPV (BPV/CHI-SW1, belonging to the Xi papillomavirus genus). This is the first report of mixed infection caused by variant of BPV3 and BPV (putatively new subtype) in China, and would be of importance for the molecular epidemiological study of the disease.