Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015.

There have been many outbreaks of hydropericardium syndrome (HPS), which is characterized by pericardial effusion and hepatitis, in Chinese chicken farms since June 2015. HPS was mainly found in miscellaneous meat-type chickens, Ma chickens, layer chicks and Three-yellow chickens, while it was occasionally found in white broilers. To determine the specific causative pathogen and pathogenicity of HPS in chickens, we collected 25 suspected cases and performed clinical pathology and aetiology analyses. The results showed that the 25 cases exhibited multifocal hepatitis with intra-nuclear inclusion bodies and 70 nm-latticed viral particles in the cell nuclei. All samples were positive for fowl adenovirus (FAdV), and sequencing results showed that the hexon gene shared the highest nucleotide similarities with the hexon gene of group 1 serotype 4 (FAdV-4). FAdV-4 was highly pathogenic to embryos and specific pathogen-free chickens, causing 100 and 70 % mortality rates, respectively. Thus, FAdV-4 is associated with HPS outbreaks in China.

Convergent evidence from systematic analysis of GWAS revealed genetic basis of esophageal cancer.

Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with risk of esophageal cancer (EC). However, investigation of genetic basis from the perspective of systematic biology and integrative genomics remains scarce.In this study, we explored genetic basis of EC based on GWAS data and implemented a series of bioinformatics methods including functional annotation, expression quantitative trait loci (eQTL) analysis, pathway enrichment analysis and pathway grouped network analysis.Two hundred and thirteen risk SNPs were identified, in which 44 SNPs were found to have significantly differential gene expression in esophageal tissues by eQTL analysis. By pathway enrichment analysis, 170 risk genes mapped by risk SNPs were enriched into 38 significant GO terms and 17 significant KEGG pathways, which were significantly grouped into 9 sub-networks by pathway grouped network analysis. The 9 groups of interconnected pathways were mainly involved with muscle cell proliferation, cellular response to interleukin-6, cell adhesion molecules, and ethanol oxidation, which might participate in the development of EC.Our findings provide genetic evidence and new insight for exploring the molecular mechanisms of EC.

Prokaryotic expression and potential application of the truncated PCV-2 capsid protein.

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.

[Expression of bovine interferon gamma in recombinant baculovirus and determination of its antiviral activity].

The full-length bovine interferon gamma (BoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus honor vectors pFastBac 1 of Bac-To-Bac system. These recombinant plasmids, pFastBac 1-BoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-BoIFN-gamma. Recombinant baculovirus, rBac-BoIFN-gamma, was generated for expressing BoIFN-gamma, by transfecting recombinant Bacmid-BoIFN-gamma with Cellfectin Reagen into sf9 insect cells. BoIFN-gamma efficiently expressed by recombinant baculovirus in sf9 cells was testified by indirect immunofluorescence assay and indirect ELISA with monoclonal antibody against Bovine interferon-gamma. Furthermore, VSV * GFP, recombinant Vesicular Stomatitis Virus expressing green fluorescence protein and MDBK were used to determine the anti-viral activity of rBoIFN-gamma. The result shows rBoIFN-gamma could inhibit the replication of the VSV * GFP in MDBK cells and the antiviral activity of supernatant was 2 x 10(5) IU/mL. The antiviral activity of rBoIFN-gamma could be blocked by anti-BoIFN-gamma mouse serum. The results demonstrated that the recombinant baculovirus could express BoIFN-gamma efficiently and rBoIFN-gamma had high antiviral activity.

[Expression of porcine gamma-interferon in recombinant baculovirus and determination of its antiviral activity].

The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.

[Emerging of avian leukosis virus subgroup J in a flock of Chinese local breed].

Myeloid leukosis (ML) cases were first diagnosed in a chicken flock of Chinese local breed in Shan dong province. The main symptom included wasting, weight loss, anemia. It caused about 10% mortality of about 15000 birds at the age of 120-day. In the necropsy, gray-white nodules and protrusions in various sizes were commonly observed on the surface of the sternum, intestine and trachea. Almost all viscera tissues showed moderate to severe enlargement with diffuse gray-white nodules. Histological examination indicated that the tumor cells proliferated in tissues were myelocytes with eosinophilic granules in cytoplasm. In PCR with a pair of ALV-J-specific primers, 15 of 17 liver samples were positive. PCR product of one positive sample was sequenced and demonstrated 98.05% and 97.4% identity with ALV-J HPRS-103 strain at nuclei acid and amino acids level, respectively. By immunohistochemistry (IHC) technique with ALV-J monoclonal antibody, the most intense staining was in the tumor tissue, liver, spleen, kidney, bone marrow, and proventriculus. The results indicate that ALV-J already caused chickens infection and dead in Chinese local breed.