Abstract: To investigate the distribution of avian influenza virus in environmental samples from live poultry markets (LPM) in China, samples were collected and tested by nucleic acid during 2009-2013 season. Each sample was tested by real-time RT PCR using flu A specific primers. If any real-time PCR was positive, the sample was inoculated into specific-pathogen-free (SPF) embryonated chicken eggs for viral isolation. The results indicated that the positive rate of nucleic acid in enviromental samples exhibited seasonality. The positive rate of nucleic acid was significantly higher in Winter and Spring. The positive rate of nucleic acid in LPM located in the south of China was higher than in northern China. Samples of Sewage for cleaning poultry and chopping board showed that higher positive rate of nucleic acid than other samples. The Subtype identification showed that H5 and H9 were main subtypes in the enviromental samples. Viral isolation indicated H5 subtypes was more than H9 subtypes between 2009 and 2013 while H9 subtypes increased in 2013. Our findings suggested the significance of public health based on LPM surveillance and provided the basis of prevention and early warning for avian flu infection human.
Animals ; China ; Feces ; virology ; Fresh Water ; virology ; Influenza A virus ; classification ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Poultry ; Poultry Diseases ; virology ; Public Health ; Seasons ; Sewage ; virology

Animals ; Humans ; Influenza A Virus, H5N1 Subtype ; immunology ; pathogenicity ; Poultry ; virology

Infectious bursal disease virus (IBDV) is an important member of the Birnaviridae family. IBUV mainly targets the bursa of Fabricius, the central immune organ of chicken, resulting in chicken infectious bursal disease (IBD). IBD represents one of the great challenges for ongoing development of the poultry industry. Reverse genetics for IBDV emerged over twenty years ago. Since then, the technologies behind virus rescue have continually improved leading to a deep understanding of IBDV gene function and tailored vaccine development. Our lab has also been instrumental in the field of IBDV research. Here we review studies on the pathogenic mechanism and the effective prevention and control of IBD.
Animals ; Birnaviridae Infections ; virology ; Chickens ; Infectious bursal disease virus ; genetics ; physiology ; Poultry Products ; virology ; Reverse Genetics

Animals ; China ; Influenza A virus ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Nucleic Acids ; genetics ; Poultry ; virology

Five H9N2 avian influenza virus strains were isolated from the environmental samples in live poultry market in Qinghai Lake region from July to September, 2012. To evaluate the phylogenetic characteristics of these H9N2 isolates, the eight gene segments were amplified by RT-PCR and sequenced. The phylogenetic and molecular characteristics of the five strains were analyzed. The results showed that the HA genes of five strains shared 93. 2%-99. 1% nucleotide identities with each other, and the NA genes shared 94. 5%-99. 8% nucleotide identities. The HA cleavage site sequence of the A/environment/qinghai/ 017/2012 isolate was PSKSSRGLF, and the HA cleavage site sequences of the other four strains were all PSRSSRGLF. The HA receptor-binding site had the Q226L mutation. The M1 gene segment had the N30D and T215A mutations. The phylogenetic analysis showed that the five strains were similar to the virus A/chicken/Hunan/5260/2005 (H9N2) isolated in Hunan Province, China and were reassortant genotype viruses; the HA, NA, and NS genes belonged to the Y280-like lineage; the MP gene belonged to the G1-like lineage; the NP, PB1, PB2, and PA genes belonged to the F98-like lineage.
Animals ; China ; Genome, Viral ; Genotype ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Molecular Sequence Data ; Phylogeny ; Poultry ; Poultry Diseases ; virology ; Viral Proteins ; genetics

OBJECTIVETo survey the distribution of influenza A subtypes in external environment and investigate the infectious status of highly pathogenic avian influenza (H5N1) in poultry-exposed population in Wuhan.

METHODSSeventy-eight external environmental samples (water, cage surface and fecal samples) were collected from 3 habitats of wild migratory birds and 5 urban live-poultry markets in 2010. In 13 avian influenza monitoring points, 249 serum samples were collected from people living around habitats of wild migratory birds or working in live poultry markets. Real-time RT-PCR method was adopted to detect influenza A virus from external environmental samples; and multiple RT-PCR method and specific H3, H5, H7 and H9 primers were then applied to analyze the subtypes of the positive samples. The levels of H5N1 antibody in poultry-exposed population were tested by horse hemagglutination inhibition test and two avian influenza inactivated antigens: A/Hubei/1/10 and A/Anhui/1/05.

RESULTSOf the 50 external environmental samples collected from live poultry markets, 17 samples were determined to be influenza A virus positive (positive rate 34.0%), including specific subtypes as follows: 4 samples of H5 single-positive subtype, 3 samples of H9 single-positive subtype, 4 samples of H3 and H5 mixed-positive subtype, 2 samples of H3 and H9 mixed-positive subtype, 2 samples of H5 and H9 mixed-positive subtype, 2 samples of H3, H5 and H9 mixed-positive subtype, but no H7 positive subtype was found. The 28 external environmental samples collected from habitats of wild migratory birds were all influenza A virus negative. Considering different types of external environmental samples, the influenza A virus positive rates in water, cage surface and fecal samples were 37.5% (6/16), 16.7% (5/30) and 18.8% (6/32), respectively. There were total 100 samples of serum whose A/Hubei/1/10 antigen inhibiting titers ≥ 40, accounting for 40.2%; while 36 samples of serum (14.5%) whose A/Anhui/1/05 antigen inhibiting titers ≥ 40 were found. The difference had statistical significance (χ(2) = 41.433, P < 0.05). Among the 249 serum samples collected from poultry-exposed population, 5 samples were H5N1 antibody positive against A/Hubei/1/10 antigen (inhibition titer ≥ 160), which came from 4 different live poultry markets, however, no positive serum sample against A/Anhui/1/05 antigen was found.

CONCLUSIONMultiple subtypes of avian influenza virus simultaneously prevailed in Wuhan urban poultry markets. Moreover, results from the distribution of avian influenza virus in external environment were consistent with the level of H5N1 antibody in poultry-exposed population.

Animals ; Antibodies, Viral ; blood ; Birds ; virology ; China ; Environment ; Humans ; Influenza A Virus, H5N1 Subtype ; immunology ; Occupational Exposure ; Poultry ; virology

OBJECTIVETo understand the infection status and epidemic rule of new bunia virus in the livestock and poultry which are closely related with humans such as sheep, cattle, dogs, pigs and chicken in the hilly area of Jiaodong peninsula in Shandong province.

METHODSPenglai and Laizhou in the hilly area of Jiaodong peninsula in Shandong province where severe fever with thrombocytopenia syndrome cases occurred in 2010 were selected as experimental sites. During April to November in 2011, serum specimens of the sheep, cattle, dogs, pigs and chicken with ticks in endemic area were randomly collected by random number table.5 ml venous blood was collected in each livestocks or poultries and there were total 3576 samples.New bunia virus antibody in different species of livestocks or poultries serum was continuously detected using double antigen sandwich enzyme-linked immunosorbent assay, and the infection rates of new bunia virus between different species of livestocks or poultries and between Penglai and Laizhou were analyzed using chi-square test.

RESULTSTest results in 3576 samples of livestocks or poultries serum specimen showed that the infection rate was as high as 63% (636/1013) in sheep, 53% (444/841)in cattle, 46% (242/530) in chicken, 29% (104/362)in the dogs, and 1% (12/830) in pigs. There were significant differences of new bunia virus infection among different species (χ(2) = 815.26, P < 0.05).In Penglai, the infection rate was as high as 71% (400/563) in sheep, 57% (232/409)in cattle, 35% (93/266) in chicken, 44% (796/1819)in total, while in Laizhou, the infection rate was 53% (236/450)in sheep, 49% (212/432)in cattle, 56% (149/264)in chicken, 36% (642/1757)in total, their difference was statistically significant(χ(2) values were 37.04, 4.93, 24.63, 19.38, all P values were < 0.05).Infection rates of dogs and pigs showed no obvious fluctuation.However, there were two peaks of infection in sheep in summer and autumn, the infection rate was as high as 62% (68/110) in June and 86% (204/236) in November;There were two peaks of infection in cattle in spring and autumn, the infection rate was as high as 56% (53/94) in April and 73% (116/159) in November; there was only one peak of infection in chicken, the infection rate was as high as 65% (55/85) in September.

CONCLUSIONThe infection rate is higher in sheep, cattle, chickens and dogs in the hilly area of Jiaodong peninsula. The peak season is spring, summer and autumn.

Animals ; Bunyaviridae ; isolation & purification ; Bunyaviridae Infections ; epidemiology ; veterinary ; Cattle ; Chickens ; China ; epidemiology ; Dogs ; Livestock ; virology ; Poultry ; virology ; Sheep

OBJECTIVETo elucidate the variation in characterizations and genetic evolution of the matrix protein 2 or ion channel protein(M2) genes of avian influenza subtype H5N1 viruses in the boundary region of Yunnan province from 2008 to 2012.

METHODSA total of swab samples were collected from foreign poultry such as the junction between Yunnan and Vietnam, Laos,myanmar and wild birds in boundary region of Yunnan province from 2008 to 2012 and screened by H5N1 subtype-specific multiplex RT-PCR. The M genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis of M2 genes were performed with sequences of the known reference strains.

RESULTSA total of 71 positive samples were found out of 1240 samples and the positive rate was 5.72%. A total of 14 different M2 sequences were obtained from 30 positive samples and were divided into 3 distinct clades or sub-clades(1.2.1, 1.2.2 and 2) by phylogenetic analysis, 5, 7 and 2, respectively. The M2 genes and Hemagglutinin(HA) genes of H5N1 viruses from the boundary region of Yunnan province had showed different relationship of genetic evolution. The substitution or mutation of key amino acids sites had been found among the domains of epitope, adamantane-resistance, and poultry or human original viral strains.

CONCLUSIONThe M2 genes of H5N1 subtype viruses in boundary region of Yunnan province from 2008 to 2012 showed genetic divergence and the virus of clade 1.2.2 had become dominant epidemic strain in this region.

Animals ; Birds ; virology ; Chickens ; virology ; China ; Evolution, Molecular ; Influenza A Virus, H5N1 Subtype ; classification ; genetics ; Influenza in Birds ; virology ; Phylogeny ; Poultry ; virology ; Viral Matrix Proteins ; genetics

The characteristics changes of apoptosis of Duck Embryo Fibroblasts (DEF) cells induced by New type gosling viral enteritis virus, NGVEV) were observed by means of HE staining, electron microscopy and Annexin V-FITC/PI fluorescent staining. During 24-48 h post infection (pi), the difference of morphological change between infected DEF cells and the mock infected cells was invisible. At 72 h pi, the nuclear chromatin was getting condensed through HE staining; apoptotic morphological change such as abnormal shape of the nucleus, condensation of the cytoplasm and chromatin were observed under electron microscope; and the early apoptotic cells (Annexin V-FITC positive and PI negative) were detected under fluorescence microscope. At 96-120 h pi, by means of HE staining and electron microscopy, the advanced morphological change of apoptosis such as formation of different kinds of apoptotic bodies, and shrink of the DEF cells and nucleus were detected; under fluorescence microscope the different stages of the apoptotic DEF can be easily distinguished: early apoptotic cells (Annexin V-FITC postive and pi negative), advanced or late apoptotic cells (both Annexin V-FITC and PI positive), necrosis cells or dead cells (Annexin V-FITC negative and PI positive). This investigation shows that NGVEV might induce apoptosis and form characteristic apoptotic morphological changes in the DEF cells. NGVEV inducement of apoptosis may be an important mechanism of efficient dissemination of virus progeny.
Adenoviridae ; physiology ; Animals ; Annexin A5 ; analysis ; Apoptosis ; Ducks ; embryology ; Enteritis ; veterinary ; virology ; Fibroblasts ; cytology ; virology ; Geese ; virology ; Microscopy, Electron, Transmission ; Poultry Diseases ; virology

Despite the existence of an active vaccination program, recently emerged strains of nephropathogenic infectious bronchitis virus (IBV) in Korea have caused significant economic losses in the poultry industry. In this study, we assessed the pathogenic and antigenic characteristics of a K-IIb type field strain of IBV that emerged in Korea since 2003, such as Kr/Q43/06. Specific pathogen free 1-week-old chickens exhibited severe respiratory symptoms (dyspnea) and nephropathogenic lesions (swollen kidneys with nephritis and urate deposits) following challenge with the recent IBV field strain. The antigenic relatedness (R value), based on a calculated virus neutralization index, of the K-IIb type field strain and K-IIa type strain KM91 (isolated in 1991) was 30%, which indicated that the recent strain, Kr/Q43/06, is a new variant that is antigenically distinct from strain KM91. This report is the first to document the emergence of a new antigenic variant of nephropathogenic IBV in chicken from Korea.
Animals ; Antigens, Viral ; *Chickens ; Coronavirus Infections/epidemiology/*veterinary/virology ; Infectious bronchitis virus/classification/*pathogenicity ; Korea ; Nephritis/*veterinary/virology ; Poultry Diseases/*virology ; Specific Pathogen-Free Organisms ; Virulence