[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu...[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.展开更多
[Objective] The aim is to develop the piezoelectric immunosensor to detect H9-subtype avian influenza virus（AIV）.[Method] The immunosensor chip was constructed by self-assembling mercaptopmpionic acid（MPA） to be m...[Objective] The aim is to develop the piezoelectric immunosensor to detect H9-subtype avian influenza virus（AIV）.[Method] The immunosensor chip was constructed by self-assembling mercaptopmpionic acid（MPA） to be monolayer on the silver-coated electrode of quartz crystal and coupling the monoclonal antibody to H9 subtype AIV with N-ethy-N＇-（3-dimethyl aminopropyl）carbodiimide hydrochloride（EDC） and N-hydroxysuccinimide（NHS）.The immunosensor to detect H9 subtype AIV was established.[Result] The results showed that the immunosensor displayed better specificity to H9 AIV and had no response to H5AIV and NDV when it was used for detection.The sensitivity test indicated the detection sensitivity for the H9 subtype AIV could reach 20-100 EID50.[Conclusion] The research provided a foundation for further research on the immunosensor for detecting AIV and it could be a new approach to detect other related viruses.展开更多
Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Metho...Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Methods Mice were infected with increasing virus titers.Viral load in the lungs and trachea was determined by EID50 assay.Pulmonary histopathology was assessed by hematoxylin‐eosin staining.Anti‐HI antibody titers and T‐cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.Results Mice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 （H9N2） influenza virus.Virus was detected in the trachea and lungs of mice harvested on days 3,6,and 9 post‐infection.A T‐cell response to viral HA was detected on day 6 and H9 HA‐specific CD 4＋ T‐cells predominated.Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.Conclusion Our results suggest that H9N2 （A/Guangzhou/333/99） can replicate in the murine respiratory tract without prior adaptation,and both humoral and cell‐mediated immunity play an important role in the immune response.展开更多
H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg producti...H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.展开更多
A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of...A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.展开更多
[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium ...[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium （ MEM-MD-611 ） and serum-free medium （SFE4Mega） were used to culture the MDCK monolayer ceils, which were then inoculated with different dilutions of AIV H9 subtypes, and the 3 kinds of media were al- so used as the maintenance solution to culture the virus. The cytopathic changes were observed at every 24 h, and the HA titers of the culture su- pernatants were also determined. [ Result] After culturing for 72 -96 h, the HA titers of the serum-free media were higher than that of low-serum culture media, while the HA titers were higher in the low-serum media than in the serum containing media. [ Conclusion] The 3 kinds of media can all used for the proliferation of AIV_ but the low-serum culture medium （MEM-MD-611 ） and serum-free medium （SFE4Meaa3 are preferred.展开更多
Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ）...Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ） carrying hemagglutinin （HA） gene of AIV H9 subtype. After cell fusion, one positive hybridoma cell strain was screened out by hemagglutination inhibition assay （ HI ）, and another positive hybddoma call strain was screened out by ELISA. After subcloning 3 times, the two cell strains could still secret antibodies against the HA of AIV H9 subtype. The mono- clonal antibodies did not react with Newcastle disease virus, AIV H5 subtype and duck adenovirus A. Their subtypes were IgG2b with kappa light chain. These two hybridoma cell strains may play an important role in rapid diagnosis and early-warning surveillance of AIV H9 subtype.展开更多
Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedur...Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.展开更多
Live poultry markets（LPMs） are crucial places for human infection of influenza A（H7N9 virus）.In Yangtze River Delta,LPMs were closed after the outbreak of human infection with avian influenza A（H7N9） virus,and t...Live poultry markets（LPMs） are crucial places for human infection of influenza A（H7N9 virus）.In Yangtze River Delta,LPMs were closed after the outbreak of human infection with avian influenza A（H7N9） virus,and then reopened when no case was found.Our purpose was to quantify the effect of LPMs＇ operations in this region on the transmission of influenza A（H7N9） virus.We obtained information about dates of symptom onset and locations for all human influenza A（H7N9） cases reported from Shanghai,Jiangsu and Zhejiang provinces by May 31,2014,and acquired dates of closures and reopening of LPMs from official media.A two-phase Bayesian model was fitted by Markov Chain Monte Carlo methods to process the spatial and temporal influence of human cases.A total of 235 cases of influenza A（H7N9） were confirmed in Shanghai,Jiangsu and Zhejiang by May 31,2014.Using these data,our analysis showed that,after LPM closures,the influenza A（H7N9） outbreak disappeared within two weeks in Shanghai,one week in Jiangsu,and one week in Zhejiang,respectively.Local authorities reopened LPMs when there was no outbreak of influenza A（H7N9）,which did not lead to reemergence of human influenza A（H7N9）.LPM closures were effective in controlling the H7N9 outbreak.Reopening of LPM in summer did not increase the risk of human infection with H7N9.Our findings showed that LPMs should be closed immediately in areas where the H7N9 virus is confirmed in LPM.When there is no outbreak of H7N9 virus,LPMs can be reopened to satisfy the Chinese traditional culture of buying live poultry.In the long term,local authorities should take a cautious attitude in permanent LPM closure.展开更多
文摘[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.
基金Supported by the Supporting Program of the"Eleventh Five-year Plan"for Sci&Tech Research of China(2006BAK20A29)Strategical Project for Science and Technology of Guangdong Province(2004A2090102)~~
文摘[Objective] The aim is to develop the piezoelectric immunosensor to detect H9-subtype avian influenza virus（AIV）.[Method] The immunosensor chip was constructed by self-assembling mercaptopmpionic acid（MPA） to be monolayer on the silver-coated electrode of quartz crystal and coupling the monoclonal antibody to H9 subtype AIV with N-ethy-N＇-（3-dimethyl aminopropyl）carbodiimide hydrochloride（EDC） and N-hydroxysuccinimide（NHS）.The immunosensor to detect H9 subtype AIV was established.[Result] The results showed that the immunosensor displayed better specificity to H9 AIV and had no response to H5AIV and NDV when it was used for detection.The sensitivity test indicated the detection sensitivity for the H9 subtype AIV could reach 20-100 EID50.[Conclusion] The research provided a foundation for further research on the immunosensor for detecting AIV and it could be a new approach to detect other related viruses.
基金supported by the National Basic Research Program of China (973 program: 2005CB523006)
文摘Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Methods Mice were infected with increasing virus titers.Viral load in the lungs and trachea was determined by EID50 assay.Pulmonary histopathology was assessed by hematoxylin‐eosin staining.Anti‐HI antibody titers and T‐cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.Results Mice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 （H9N2） influenza virus.Virus was detected in the trachea and lungs of mice harvested on days 3,6,and 9 post‐infection.A T‐cell response to viral HA was detected on day 6 and H9 HA‐specific CD 4＋ T‐cells predominated.Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.Conclusion Our results suggest that H9N2 （A/Guangzhou/333/99） can replicate in the murine respiratory tract without prior adaptation,and both humoral and cell‐mediated immunity play an important role in the immune response.
基金supported by the National High-Tech R&D Program of China(2012AA101303)
文摘H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.
文摘A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.
基金funded by the General Project of Beijing Academy of Agricultural and Forestry Sciences ( 2010A007)
文摘[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium （ MEM-MD-611 ） and serum-free medium （SFE4Mega） were used to culture the MDCK monolayer ceils, which were then inoculated with different dilutions of AIV H9 subtypes, and the 3 kinds of media were al- so used as the maintenance solution to culture the virus. The cytopathic changes were observed at every 24 h, and the HA titers of the culture su- pernatants were also determined. [ Result] After culturing for 72 -96 h, the HA titers of the serum-free media were higher than that of low-serum culture media, while the HA titers were higher in the low-serum media than in the serum containing media. [ Conclusion] The 3 kinds of media can all used for the proliferation of AIV_ but the low-serum culture medium （MEM-MD-611 ） and serum-free medium （SFE4Meaa3 are preferred.
基金funded by the National Key Technology R&D Program(2006BAK20A29)the Shenzhen Entry-Exit Inspection and Quarantine Project(sz2008102)
文摘Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ） carrying hemagglutinin （HA） gene of AIV H9 subtype. After cell fusion, one positive hybridoma cell strain was screened out by hemagglutination inhibition assay （ HI ）, and another positive hybddoma call strain was screened out by ELISA. After subcloning 3 times, the two cell strains could still secret antibodies against the HA of AIV H9 subtype. The mono- clonal antibodies did not react with Newcastle disease virus, AIV H5 subtype and duck adenovirus A. Their subtypes were IgG2b with kappa light chain. These two hybridoma cell strains may play an important role in rapid diagnosis and early-warning surveillance of AIV H9 subtype.
文摘Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.
文摘Live poultry markets（LPMs） are crucial places for human infection of influenza A（H7N9 virus）.In Yangtze River Delta,LPMs were closed after the outbreak of human infection with avian influenza A（H7N9） virus,and then reopened when no case was found.Our purpose was to quantify the effect of LPMs＇ operations in this region on the transmission of influenza A（H7N9） virus.We obtained information about dates of symptom onset and locations for all human influenza A（H7N9） cases reported from Shanghai,Jiangsu and Zhejiang provinces by May 31,2014,and acquired dates of closures and reopening of LPMs from official media.A two-phase Bayesian model was fitted by Markov Chain Monte Carlo methods to process the spatial and temporal influence of human cases.A total of 235 cases of influenza A（H7N9） were confirmed in Shanghai,Jiangsu and Zhejiang by May 31,2014.Using these data,our analysis showed that,after LPM closures,the influenza A（H7N9） outbreak disappeared within two weeks in Shanghai,one week in Jiangsu,and one week in Zhejiang,respectively.Local authorities reopened LPMs when there was no outbreak of influenza A（H7N9）,which did not lead to reemergence of human influenza A（H7N9）.LPM closures were effective in controlling the H7N9 outbreak.Reopening of LPM in summer did not increase the risk of human infection with H7N9.Our findings showed that LPMs should be closed immediately in areas where the H7N9 virus is confirmed in LPM.When there is no outbreak of H7N9 virus,LPMs can be reopened to satisfy the Chinese traditional culture of buying live poultry.In the long term,local authorities should take a cautious attitude in permanent LPM closure.
