[Risk related to the transmission of H5N1 subtype avian influenza virus in the environment of poultry markets in Changsha, China].

OBJECTIVE: To investigate the risk of H5N1 subtype avian influenza virus (AIV) transmission in the poultry market environment in Changsha city. H5N1 antibody levels among the groups related occupational exposure and AIV nucleic acid in the environment of poultry markets were detected. The characteristics of haemagglutinin (HA) genes of H5N1 AIV in the environment were analyzed. METHODS: One district and one county from Changsha city were selected randomly and two poultry markets at inner city or township levels were selected in the same district or county respectively. H5N1 antibody of the occupational exposure groups in the poultry market was tested and AIV nucleic acid in the poultry market environment monitored. One hundred and two blood samples of the occupational exposure groups were tested for H5N1 antibody with single radioimmunoassay diffusion hemolysis (SRH) while 160 environment samples (from sewage, birds stools, feathers and smearing samples of poultry cages) in the poultry market were also detected for AIV nucleic acid with real-time PCR method. Four sewage samples of H5N1 subtype AIV were collected from poultry markets in Changsha, and the HA genes of H5N1 subtype AIV amplified by RT-PCR and then sequenced with TA cloning. Amino acid sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega 5.0 software. RESULTS: The results through H5N1 antibody monitoring program showed that H5N1 antibody positive rates from workers were 25.5% (26/102), 50.0% (9/18) and 25.4% (17/67) respectively in the poultry markets of township and inner cities. H5N1 antibody positive rate in the township poultry markets was higher than in the inner cities poultry markets. RESULTS: from the surveillance on AIV nucleic acid showed that the overall H5 subtype positive rate in Changsha poultry markets was 31.3% (50/160), and the positive rate of townships poultry markets was 37.3% (31/83), which were both higher than those from the inner cities poultry markets (24.7%, 19/77). H5 subtype AIV positive rate was different in the tested specimens, with ranking of positive rates were sewage (50.0%, 24/48), feathers (44.5%, 4/9), birds stools (29.8%, 14/47) and smearing samples of poultry cages (14.3%, 8/56), with statistically significant differences (P < 0.01). Four H5N1 HA genes TA cloning were successfully constructed and identified as Eurasian branch, similar to viruses isolated in mainland China and Hong Kong in the same group, according to genetic analysis. Sequence data of the four HA genes showed the same feature of high pathogenicity, compared to the H5N1 AIV from mainland China of human origin. The receptor specificities were still with avian influenza origin (QSG) and the connecting peptide between HA1 and HA2 possessing the polybasic motif (RERRRKK or RERRGKK). CONCLUSION: One of the reasons for H5N1 antibody positive rate of 25.5% among poultry markets workers was that there were large numbers of H5N1 subtype AIV detected in the environment of poultry markets and HA genes of H5N1 subtype AIV in the poultry markets environment carried molecular characteristics of highly pathogenic which could increase the risk for H5N1 subtype AIV transmission in the environment of poultry markets.

[Genetic analysis of the NS genes of H5N1 avian influenza viruses isolated from sewage in poultry markets].

In order to investigate the transmission risk of H5N1 avian influenza viruses (AIV) from sewage in Changsha poultry markets, the evolution relationship and molecular characteristics of non-structural (NS) genes of H5N1 AIV from sewage were analyzed. Nine H5N1 AIV environmental sewage specimens were collected from Changsha poultry markets. The NS genes were amplifyed by PCR and then sequenced with TA cloning. Amino acid(aa) sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega5 software. Eight NS genes TA cloning were constructed successfully. Phylogenetic tree indicated that they were belonged to the allele A subgroup. Aa homology analysis showed 90.1% 92.5% identity in NS1 proteins and 91.0% - 92.6% identity in NS2 proteins compared with reference viruses of the allele A (A/chicken/ Hubei/ w h/ 1999). The homologies of the amino sequences of NS1 and NS2 in this study were 93.8%-100.0% and 98.4%-100.0%, respectively. The C terminal of all eight H5N1 NS1 proteins from sewage in poultry markets carried a ESEV of PL motif and the 92 amino acids were E, furthermore, the 80 to 84aa were missed which were the characteristics of highly pathogenic AIV. The NS genes of H5N1 AIV from sewage in poultry markets have molecular characteristics of highly pathogenic and have the potential risk of H5N1 virus spreading.

[Rapid detection of norwalk GII with reverse transcription loop-mediated isothermal amplification].

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.