A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate (A/Chicken/ Hebei/WD/98, abbreviated as WD98) by comparing with other reference strains. I...A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate (A/Chicken/ Hebei/WD/98, abbreviated as WD98) by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology (95.8%) to D/HK/Y280/97 and the lowest homology (91.1% ) to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.展开更多
[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus (AIV) H9N2 subtype in Madin- Darby canine kidney (MDCK) cells. [Method] Three AIV H9 subt...[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus (AIV) H9N2 subtype in Madin- Darby canine kidney (MDCK) cells. [Method] Three AIV H9 subtype isolates were inoculated on MDCK cells respectively. Then, DMEM containing different concentrations of trypsin as maintenance media were added to MDCK monolayer cells. The cytopathic effect (CPE) was observed once every 24 h, and the HA titer of the supematant was measured by HA assay. [Result] When the trypsin concentration was 10 -20 μg/ml in DMEM, the HA titer of virus culture reached 7 log2 (1:128). Almost all cells were cytopathic after 96 h post inoculation with 1:1 000 or 1:10 000 dilution of AIV culture, and the virus titer reached a peak after 72 -96 h. [ Conclusion] The optimal concentration of trypsin is 10 -20 pg/ml for proliferation of AIV H9N2 subtype in MDCK cells.展开更多
Background H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this ...Background H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics, Methods In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A). Results In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found. Conclusion The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.展开更多
To investigate the susceptibility of Chukars to duck avian influenza virus H9N2 and explore their role in interspecies transmission of influenza viruses.Chukars were inoculated with duck avian influenza viruses H9N2.
H9N2 avian influenza virus(AIV) infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin(HA) gene sequences of H9N2 AIV...H9N2 avian influenza virus(AIV) infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin(HA) gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.展开更多
OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonate...OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonated hen eggs, and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03) and Editseg (version 3.69) softwares. RESULTS: The amino acid sequences at the cleavage site between HA1 and HA2 domains of H9N2 viruses isolated in China are R-S-S-R. One pigeon strain contains seven potential glycosylation sites on the HA protein molecule, while all others have eight. There are 2 to 15 differences of amino acid sequences distributed at 24 different positions on the HA protein molecules among six H9N2 viruses. The H9N2 viruses with multiple lineages of HA genes were co-circulating in China recently. CONCLUSION: The highest possibility is that human influenza A (H9N2) virus was derived from Chicken H9N2 virus, and not derived from pigeon H9N2 virus. However, it is still unknown whether the H9N2 virus could transmit from person to person. The H9N2 viruses with multiple lineages of HA genes are co-circulating in China.展开更多
Background Southeast China is one of the sites of influenza origin. During 2003-2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential...Background Southeast China is one of the sites of influenza origin. During 2003-2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential human infection by human influenza (H1N1, H3N2) and avian influenza (H5N1, H7N7, H9N2) in the avian influenza epidemic area of Guangdong Province, China, we conducted a seroepidemiologic survey in the people of this area from April to June of 2004.Methods Three out of 9 H5N1 avian influenza affected poultry areas in Guangdong were randomly selected, and the population living within 3 kilometers of the affected poultries were chosen as the survey subjects. One thousand two hundred and fourteen people were selected from 3 villages at random. Human and avian influenza antibody titers were determined by hemagglutination-inhibition (HI) test and microneutralization test (MNT). Results The positive rate of antibody to H5N1 was 3.03% in the occupational exposure group and 2.34% in general citizens group; that of H9N2 was 9.52% in the occupational exposure group and 3.76% in the general citizens group. Moreover one case in the occupational exposure group was positive for H7N7. One year later, all previously positive cases had become negative except for one H5N1-positive case. Conclusion The observations imply that H5N1 and H9N2 avian influenza silent infections exist in Guangdong populations.展开更多
Background PB1-F2 protein has been proven to increase the pathogenicity of influenza A virus (IAV) strains in primary infection and in secondary bacterial infection. It can also regulate the activity of viral polyme...Background PB1-F2 protein has been proven to increase the pathogenicity of influenza A virus (IAV) strains in primary infection and in secondary bacterial infection. It can also regulate the activity of viral polymerase. However, it was shown in another retrospective study that a portion of IAVs do not express full-length PB1-F2 protein during virus development; different kinds of stop codons cause exits in the open reading frames and form PB1-F2 gene products with the corresponding genotypes. Truncated PB1-F2 in human H3N2 IAVs has long been detected in North America but its evolution in China is still unclear. Methods Influenza-like illnesses (ILls) from the whole of Jiangsu Province were collected and inspected to determine the type and subtype of the viruses. A portion of isolates collected in the epidemic period were selected as samples for later whole-genome sequencing, and the exact sequences were determined and analyzed. Results H3N2 influenza virus was one of the epidemical strains which had been prevalent during 2009-2010, in Jiangsu. Five H3N2 isolates with truncated PB1-F2 protein (25aa) were detected in influenza samples from Nanjing and Xuzhou, while seven similar H3N2 isolates were also reported in Niigata, Japan. Conclusion This emergence indicates the possibility that there has been transmission of the H3N2 virus between the two countries.展开更多
Influenza A(H3N2)virus has a faster evolution rate than other types of influenza viruses.In this study,whole genome sequencing was performed to better understand themolecular evolution of influenzaH3N2 and the protect...Influenza A(H3N2)virus has a faster evolution rate than other types of influenza viruses.In this study,whole genome sequencing was performed to better understand themolecular evolution of influenzaH3N2 and the protective effect of influenza virus vaccine in Qinghai Province,China,in 2017.Complete sequences of eight gene segments of two seasonal influenza H3N2 isolates were sequenced and analyzed using DNASTAR and MEGA 6.06 software.Additionally,the three-dimensional structure of the HA protein was predicted using the SWISS-MODEL server.Phylogenetic and amino acid sequence analysis revealed that two Qinghai H3N2 isolates were typical low-pathogenic influenza viruses,and were relatively closely related to the 2016–2017 vaccine strain,3C.2a-A/Hong Kong/4801/2014.The presence of several antigenic site substitutions(T131K,G/R142K,K160T and R261Q in the HA protein)were specific for the two Qinghai H3N2 virus strains.In addition,amino acid substitution of K160T at the glycosylation site of HA and H75P in PB1-F2 in Qinghai isolatesmight affect the antibody binding ability and virulence of the influenza virus.The presence of several antigenic site mutations in the Qinghai H3N2 isolates confirmed the evolution of circulating H3N2 strains.展开更多
基金supported by subproject of National Program on Key Basic Research Project (973 Program )(2005CB523001)
文摘A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate (A/Chicken/ Hebei/WD/98, abbreviated as WD98) by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology (95.8%) to D/HK/Y280/97 and the lowest homology (91.1% ) to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.
基金funded by the Beijing Academy of Agriculture and Forestry Sciences (2010A007)
文摘[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus (AIV) H9N2 subtype in Madin- Darby canine kidney (MDCK) cells. [Method] Three AIV H9 subtype isolates were inoculated on MDCK cells respectively. Then, DMEM containing different concentrations of trypsin as maintenance media were added to MDCK monolayer cells. The cytopathic effect (CPE) was observed once every 24 h, and the HA titer of the supematant was measured by HA assay. [Result] When the trypsin concentration was 10 -20 μg/ml in DMEM, the HA titer of virus culture reached 7 log2 (1:128). Almost all cells were cytopathic after 96 h post inoculation with 1:1 000 or 1:10 000 dilution of AIV culture, and the virus titer reached a peak after 72 -96 h. [ Conclusion] The optimal concentration of trypsin is 10 -20 pg/ml for proliferation of AIV H9N2 subtype in MDCK cells.
文摘Background H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics, Methods In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A). Results In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found. Conclusion The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.
基金the National Natural Science Foundation of China[31260033,31660041]
文摘To investigate the susceptibility of Chukars to duck avian influenza virus H9N2 and explore their role in interspecies transmission of influenza viruses.Chukars were inoculated with duck avian influenza viruses H9N2.
基金supported by the National Modern Agricultural Industry Technology System Project of China(CARS-41)the Science and Technology Plan Project of Guangdong Province,China(2012B020306002 and 2012B091100078)
文摘H9N2 avian influenza virus(AIV) infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin(HA) gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.
文摘OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonated hen eggs, and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03) and Editseg (version 3.69) softwares. RESULTS: The amino acid sequences at the cleavage site between HA1 and HA2 domains of H9N2 viruses isolated in China are R-S-S-R. One pigeon strain contains seven potential glycosylation sites on the HA protein molecule, while all others have eight. There are 2 to 15 differences of amino acid sequences distributed at 24 different positions on the HA protein molecules among six H9N2 viruses. The H9N2 viruses with multiple lineages of HA genes were co-circulating in China recently. CONCLUSION: The highest possibility is that human influenza A (H9N2) virus was derived from Chicken H9N2 virus, and not derived from pigeon H9N2 virus. However, it is still unknown whether the H9N2 virus could transmit from person to person. The H9N2 viruses with multiple lineages of HA genes are co-circulating in China.
文摘Background Southeast China is one of the sites of influenza origin. During 2003-2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential human infection by human influenza (H1N1, H3N2) and avian influenza (H5N1, H7N7, H9N2) in the avian influenza epidemic area of Guangdong Province, China, we conducted a seroepidemiologic survey in the people of this area from April to June of 2004.Methods Three out of 9 H5N1 avian influenza affected poultry areas in Guangdong were randomly selected, and the population living within 3 kilometers of the affected poultries were chosen as the survey subjects. One thousand two hundred and fourteen people were selected from 3 villages at random. Human and avian influenza antibody titers were determined by hemagglutination-inhibition (HI) test and microneutralization test (MNT). Results The positive rate of antibody to H5N1 was 3.03% in the occupational exposure group and 2.34% in general citizens group; that of H9N2 was 9.52% in the occupational exposure group and 3.76% in the general citizens group. Moreover one case in the occupational exposure group was positive for H7N7. One year later, all previously positive cases had become negative except for one H5N1-positive case. Conclusion The observations imply that H5N1 and H9N2 avian influenza silent infections exist in Guangdong populations.
基金This work was supported by grants from the Natural Science Foundation of Jiangsu Province (No. BK20131450), Jiangsu Province Key Medical Talent Foundation (No. RC2011084) and National Natural Science Foundation of China (No. 81273143).
文摘Background PB1-F2 protein has been proven to increase the pathogenicity of influenza A virus (IAV) strains in primary infection and in secondary bacterial infection. It can also regulate the activity of viral polymerase. However, it was shown in another retrospective study that a portion of IAVs do not express full-length PB1-F2 protein during virus development; different kinds of stop codons cause exits in the open reading frames and form PB1-F2 gene products with the corresponding genotypes. Truncated PB1-F2 in human H3N2 IAVs has long been detected in North America but its evolution in China is still unclear. Methods Influenza-like illnesses (ILls) from the whole of Jiangsu Province were collected and inspected to determine the type and subtype of the viruses. A portion of isolates collected in the epidemic period were selected as samples for later whole-genome sequencing, and the exact sequences were determined and analyzed. Results H3N2 influenza virus was one of the epidemical strains which had been prevalent during 2009-2010, in Jiangsu. Five H3N2 isolates with truncated PB1-F2 protein (25aa) were detected in influenza samples from Nanjing and Xuzhou, while seven similar H3N2 isolates were also reported in Niigata, Japan. Conclusion This emergence indicates the possibility that there has been transmission of the H3N2 virus between the two countries.
基金supported by Doctor research startup foundation of Changzhi Medical College(BS201912,BS201921)Key Project of Qinghai Health and Family Planning Commission(2017-wjzd-08)and Qinghai Thousand People Plan.
文摘Influenza A(H3N2)virus has a faster evolution rate than other types of influenza viruses.In this study,whole genome sequencing was performed to better understand themolecular evolution of influenzaH3N2 and the protective effect of influenza virus vaccine in Qinghai Province,China,in 2017.Complete sequences of eight gene segments of two seasonal influenza H3N2 isolates were sequenced and analyzed using DNASTAR and MEGA 6.06 software.Additionally,the three-dimensional structure of the HA protein was predicted using the SWISS-MODEL server.Phylogenetic and amino acid sequence analysis revealed that two Qinghai H3N2 isolates were typical low-pathogenic influenza viruses,and were relatively closely related to the 2016–2017 vaccine strain,3C.2a-A/Hong Kong/4801/2014.The presence of several antigenic site substitutions(T131K,G/R142K,K160T and R261Q in the HA protein)were specific for the two Qinghai H3N2 virus strains.In addition,amino acid substitution of K160T at the glycosylation site of HA and H75P in PB1-F2 in Qinghai isolatesmight affect the antibody binding ability and virulence of the influenza virus.The presence of several antigenic site mutations in the Qinghai H3N2 isolates confirmed the evolution of circulating H3N2 strains.