Objective:To carry out the monitoring of H5N6 subtype avian influenza virus (AIV) in the population infection and live poultry markets (LPMs) environmental pollution in Changsha city from 2016 to 2018. Laboratory data were provided to prevent human infection with H5N6 subtype AIV.Methods:A total of 6 909 throat swab specimens from the population and 1 719 environmental specimens from LPMs were collected in Changsha city from 2016 to 2018. The real-time RT-PCR method was used for the detection of influenza virus in type A, H5, H7, H9 and N6 subtypes. Eighty-two AIV nucleic acid-positive specimens were subjected to high-throughput nucleotide sequencing, and then BLAST similarity and amino acid (aa) key site analyses were performed on the sequencing results.Results:One H5N6 subtype AIV nucleic acid was detected from 6 909 population throat swab specimens, 927 (53.93%) positive specimens of A-type AIV nucleic acids and 193 (11.23%) positive specimens of both H5 and N6 subtypes were detected from 1 719 LPMs environmental specimens. Fourteen genomic sequences of H5N6 subtype AIV were obtained with high-throughput nucleotide sequencing, and aa key site analyses showed that there were 6 basic aa in 338-347 aa of viral HA protein-linked peptide, which was highly pathogenic to birds. Receptor binding site (RBS) 238-240 aa (corresponding to H3 type influenza virus 226-228 aa) is QSG or QRG, and the receptor is characterized by avian origin. The 290th resistance gene locus of NA protein did not show R290K mutation, and was sensitive to NA inhibitor (Duffy/osoterosine phosphate). The E627K and D701N(I) mutations in the viral PB2 protein indicate that the virus is pathogenic.Conclusions:Human infected with H5N6 subtype AIV in Changsha city is sporadic, and the H5N6 subtype AIV of LPMs is seriously polluted. It is necessary to further strengthen the AIV surveillance of LPMs.

The aim of this study is to analyze the evolutional and molecular characteristics of Hemagglutinin (HA),Neuraminidase(NA)and non-structural (NS) genes of H5N1 avian influenza virus (AIV) from sewage in live bird markets (LBMs) in Changsha,2014.Five hundred and one specimens were collected from environment in LBMs in Changsha,2014,and real-time RT-PCR was used for influenza A typing and subtyping (H5,H7 and H9) detection.Sequencing were used for the positive of single H5.The sequence homology of HA,NA and NS genes of the viruses were analyzed with the online Basic Local Alignment Search Tool (BLAST).The phylogenetic trees for HA,NA and NS genes and the ClustalW Multiple alignments of amino acids were constructed using MEGA 5 and BioEdit software,respectively.Results showed that of 501 environmental samples,177 (35.33 ％) samples were positive for influenza A viruses and H5 subtype.Eight H5N1 subtype AIV were confirmed by sequencing from the samples of the positive of single H5.Phylogenetic analysis indicated that most of HA genes of the H5N1 subtype AIV strains isolated in Changsha city were located in 2.3.2 and clustered into new subclade,and the most of NA and NS genes in this study were clustered into subclade 2.3.2.1b.QSG of the HA protein of the receptor binding site were found in these H5N1 viruses,and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of AIV.In strains from this study,we did not found amino acid substitutions of the NA protein at H275Y and N295S,and sensitive to neuraminidases,and the high pathogenicity molecular characteristics of HA,NA and NS genes were showed in these viruses.In conclusion,molecular characteristics of the HA,NA and NS of these H5N1 subtype viruses in this study showed high pathogenicity,but that may not facilitate human infection.So,the prevalence and genetic evolution of this virus should be closely monitored.

Objective To establish a reverse transcription loop-mediated isothermal amplification RT-LAMP rapid detection method of enterovirus 71(EV71) in hand ,foot and mouth disease (HFMD) ,and to evaluate its application.Methods Four types of LAMP primers were designed by using the software needle based on the 6 distinct regions of the specific gene of EV71.The process of amplification was completed in the ordinary thermostatic water container by 65 ℃ for 1200 min.The results of amplification were judged by electrophoresis and naked-eye.Seventy EV71 type intestinal positive specimens were simultaneously detected by RT-LAMP and RT-PCR methods.EV71 type of intestinal virus RNA were made a series.After 10 times of dilution ,the RT-LAMP and RT-PCR methods were used to conduct the detection for comparing their sensitivities.Results The LAMP characteristic ladder bands of EV71 appeared ,then the results could judged by the naked-eye.The detection rate in 100 EV71 samples had no statistical difference between RT-LAMP and RT-PCR methods (P>0.05).The sensitivity (10.0 pg/μL) of RT-LAMP was same to that of RT-PCR method.Conclusion The RT-LAMP detection method for EV71 was established ,which can be used for nucleic acid am-plification in the ordinary thermostatic water container.The preliminary application verifies that this RT-LAMP assay has a certain application prospect.

We analyzed the evolutional and molecular characteristics of Hemagglutinin(HA),Neuraminidase(NA) and nonstructural(NS) genes of avian influenza A(H9n2) viruses from environment in poultry markets in Changsha,China,2014,providing laboratory data for prevention and control of human infection with avian influenza A(H9N2) virus.Five hundred and one specimens (263 poultry drinking water specimens,226 poultry sewage specimens and 17 others specimens) were collected from environment in poultry markets in Changsha,2014,and real-time RTPCR was used for influenza A typing and subtyping (H5,H7 and H9) detection.HA and NA universal primer sets for conventional RT-PCR and sequencing were used for the positivity of single H9.The sequence homology of HA,NA and NS genes of the viruses were analyzed with the online Basic Local Alignment Search Tool (BLAST).The ClustalW multiple alignments of amino acids and the phylogenetic trees for HA,NA and NS genes were constructed using the BioEdit and MEGA 5 software,respectively.Results showed that among 501 environmental samples,350 samples were positive for influenza A virus,191 (38.12％) for H9 subtype,177 (35.33％) for H5 subtype,11 (2.20％) for H7 subtype and 68 (13.57％) for H5 and H9 subtypes co-detection.Twenty-three H9N2 subtype AIV were confirmed by conventional RT-PCR and sequencing from the samples of the positivity of single H9.Phylogenetic analysis revealed that most of HA,NA and NS genes of the H9N2 subtype AIV isolated in Changsha City had gene constellations of genotype S,and these virues might have acquired their HA,NA and NS from A/Chicken/Shanghai/F/1998-like (H9N2).L235 (correspond to H3 numbering 226) of the HA protein of the receptor binding site (RBS) were found in these H9N2 viruses,and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of human influenza virus,and the low pathogenicity molecular characteristics of HA,NA and NS genes were showed in these viruses.The positive rate of nucleic acid of the H9 subtype of avian influenza virus from environment was the highest in poultry markets in Changsha,2014,and molecular characteristics of the HA,NA and NS of these H9N2 subtype AIV showed low pathogenicity,but that may facilitate human infection.So,the prevalence and genetic evolution of this virus should be closely monitored.

Objective To establish a polymerase chain reaction (PCR) method for detecting Brucella spp .and to evaluate its ap‐plication .Methods The PCR primer aiming at the outer membrane protein(omp‐2) coding gene were designed and the PCR method for detecting Brucella spp .was established .By using DNA of 19 kinds of common bacteria and Brucella spp .,the specificity and sensitivity were evaluated .Three strains of suspected Brucella spp .were performed the nucleic acid detection .Then ,3 suspected strains of Brucella spp .were amplified by the PCR method with omp‐2 gene ,and the results were compare to the those by the cul‐ture method .Results The established Brucella spp .PCR detection method could only detect the Brucella spp .positive strain ,the control bacterium DNA did not appear the target band ;the sensitivity was 100 copies/response;the PCR method detected omp‐2 gene band in 3 strains of suspected Brucella spp .,which was identified as Brucella spp .and was same to the results by the isolation culture method .Conclusion The established PCR method for detecting Brucella spp .is accurate and rapid compare with the isola‐tion and culture method ,which is suitable for the rapid detection of brucellosis epidemic situation .

Objective To establish a multiplex polymerase chain reaction (PCR) method for rapid detection of three kinds of di‐arrheagenic Escherichia coli(EPEC ,EIEC ,EHEC)simultaneously .Methods The eae gene of EPEC ,ipaH gene of EIEC and stx1 gene of EHEC were selected to design primers ;the reaction system and condition were adjusted to optimize the multiplex PCR sys‐tem .Results The target gene fragments were amplified correctly with these primers .The three target bacteria could be detected at the same time by multiplex PCR .Conclusion A rapid multiplex PCR system were successfully established for detection of three di‐arrheagenic Escherichia coli ,and this system could be suitable for rapid screening in food safety .

Objective To understand the origin and variation of the hemagglutinin gene of isolates viruses from 3 novel influenza A( H1N1 ) deaths in Changsha ( A/Hunan Kaifu/SWL4142/2009 ( H1N1 ) , A/Hunan Changsha/SWL4346/2009 ( H1 N1 ) and A/Hunan Furong/SWL4224/2009( H1N1 )). Methods The nasopharyngeal swab specimens from the 3 novel influenza A( H1N1 ) deaths in Changsha were tested by RT-PCR and influenza viruses were isolated simultaneously. With the sequencing primers recommended by World Health Organization (WHO), the HA gene of sequences of 3 novel influenza A( H1N1 ) deaths were tested by CEQTM 8000 Genetic Analysis System, through dye terminator cycle sequencing. The sequencing results were submitted to GenBank, then the results were analyzed for amino acid alignment and phylogenetic tree analysis with ClustalX and Mega4.1 software. Results All the nucleotide homologies of HA gene sequences in A/Hunan Kaifu/SWL4142/2009 ( H1N1 ), A/Hunan Changsha/SWL4346/2009 ( H1N1 ) and A/Hunan Furong/SWL4224/2009( H1N1 ) are 99% as compared with the novel influenza A( H1N1 ) virus strains of A/NewYork/3502/2009 ( H1N1 ), A/Shanghai/71T/2009 ( H1N1 ) and A/Chita/01/2009 ( H1N1 )The nucleotide homology of the 3 HA gene sequences are more than 99. 5% the same compared with the novel influenza A( H1N1 ) virus strain ( A/Sichuan/1/2009( H1N1 ) ) in China. Phylogenetic tree analysis reveals that 2009 novel influenza A(H1N1 ) viruses including 3 HA gene sequences of A/Hunan Kaifu/SWL4142/2009 ( H1 N1 ), A/Hunan Changsha/SWL4346/2009 ( H1N1 ), A/Hunan Furong/SWL4224/2009( H1N1 ) had a close evolutionary relationship with the swine H1 virus isolates in North America ( A/Swine/Indiana/P12439/00), but a distant evolutionary relationship with those human seasonal A( H1 N1 ) influenza virus and avian. After comparing with genes of A/Swine/Indiana/P12439/00, we found that the HA gene sequences of the 3 viruses isolated had 28,30 and 27 amino acids with mutation respectively, but only one (R53K) amino acids mutation at 21 important antigenic sites in the 3 viruses isolated. Multiple alignment of 364 HA genes sequences of novel influenza A ( H1N1 ) viruses in the world showed they had 119 nonconserved amino acids, 5 non-conserved position at important antigenic sites. Conclusions The HA gene sequences from 3 viruses isolated in this study and other influenza A ( H1N1 ) viruses might originate from swine A( H1N1 ) in North America by variation. The 3 HA gene sequences of viruses isolated have high homology as compared with the novel influenza A ( H1N1 ) virus strains worldwide, and the 3 HA gene sequences of viruses isolated are in stable condition as the vast majority of novel influenza A( H1N1 ) virus strains in the world.

Objective To detect Pneumocystis carinii (Pc) DNA by loop-mediated isothermal amplification (LAMP). Methods After injected with hydrocortisone acetate for 8 weeks, the bronchoalveolar lavage fluid (BALF) of Wistar rats were collected and a portion of BALF were examined for identifying Pc organisms using microscope. Then Pc DNA was extracted by phenol-chloroform extraction. Four primers which recognized 6 distinct regions on the mtrRNA gene of Pc were designed and used for LAMP assay. To evaluate the specificity of the assay, M. tuberculosis, M. pneumoniae, C. pneumoniae, P. gondii and rat leucocyte were used as negative controls. To compare the sensitivity of the LAMP to that of conventional PCR, Pc DNA were 10-fold serially diluted and was amplified by LAMP and PCR. LAMP results were judged by naked eye, electrophoretic analysis and restriction digestion. Results Pc organisms were detected from BALF of rats injected with hydrocortisone acetate. After LAMP reaction, positive signal was observef rats injected with hydrocortisone acetate. By contrast, no positive signal was observed for the negative controls in the study. The amplified product digested by restriction enzyme demonstrated 3 bands (82, 135, 189 lip) upon agarose gel electrophoresis, in good agreement with the predicted sizes. The detection limit of LAMP assay was 1 pg/μl of Pc DNA per reaction and that of PCR was 10 pg/μ1 of Pc DNA per reaction. Conclusion LAMP assay has usefulness for rapid detection of Pc.

Objective To evaluate protective function of ischemia preconditioning for myocardium during percutaneous transluminal coronary angioplasty (PTCA), using cardiac troponin I (cTnI) as myocardial injury marker. Methods One hundred and fifty patients with coronary artery disease (CAD) undergoing PTCA were divided into two groups: Group Usual Cure (G-UC), including 120 cases, and Group Ischemia Precondition (G-IP), including 30 cases. Serum cTnI were measured before and 6, 12, 24, 48 and 72 hours after the procedure respectively. The relative factors were analyzed. The cardiac events were followed-up.Results The serum cTnI levels of 29 cases elevated in G-UC, while those of 2 cases elevated in G-IP. There was statistical difference on elevated cTnI levels between the two groups (P

Objective To evaluate the possible effect of percutaneous transluminal coronary angioplasty(PTCA) on myocardial injury. Methods Serum cTnI and CK MB levels were measured in 173 patients with coronary artery disease undergone PTCA before and 6, 12, 24, 48 and 72 hours after the procedure. Cardiac events during follow up in these patients were recorded. Results Serum cTnI level was increased after PTCA in 42 patients, remained normal in 84, and was over baseline level before and after the procedure in 47. Serum CK MB level was above baseline before and after the procedure in one patient and increased in 10. Compared with normal cTnI group, elevated cTnI was related to total balloon inflation time, total pressure, number of dilation and stents deployed, contrast medium dose and occurrence of angina during balloon inflation ( P 0.05). Conclusion cTnI was more sensitive and specific than CK MB in identifying minor myocardial injury during PTCA. This injury was related to the intensity of PTCA, but not enough to make worse influence on overall outcome.