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
[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
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
[ 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
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.
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.
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[ 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.