The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.
China ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Molecular Sequence Data ; Phylogeny

Pdm09 virus outbreak occurred in Mainland China in May 2009, a few months later, the prevalence of seasonal H1N1(sH1N1) influenza virus that already circulated in human for tens of years began to decline and disappeared afterwards. To identify the reason for the rapid decline of sH1N1 in mainland China, we sequenced the HA1 of sH1N1 during 2006-2011, and then analyzed the selective pressure in different phases. Our results showed before Pdm09 outbreak, the omega value was 0. 36 while after Pdm09 outbreak the omega value was 0. 28 and significant difference (t test, P<0. 05) was identified. We concluded that sH1N1 obtained stronger purifying selection after Pdm09 outbreak in China. This might one of the major reasons causing the disappearance of sH1N1 in human.
China ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Phylogeny ; Seasons ; Selection, Genetic

OBJECTIVETo analyze the type and subtype distribution of influenza virus and the genetic evolution of hemagglutinin (HA) in Shanghai area during 2004 to 2008.

METHODSAll 962 throat swabs were collected from influenza-like patients in 5 influenza sentry hospitals and influenza outbreaks. Influenza viruses were isolated in MDCK cell lines, and then viral types and subtypes were identified. The HA of influenza A isolates selected by outbreak or sporadic patients in different areas and epidemic seasons were sequenced and analyzed by phylogenetic trees.

RESULTSA/H3N2, accounting for 54.9% (162/295), was the dominate subtype in recent years, but less popular in the end of 2005 to the middle of 2006 with 0% (0/16)and 23.5% (8/34) of positive specimen, respectively. There were more A/H1N1 isolates in 2005 - 2006 with 21.4% (12/56), 43.8% (7/16) and 76.5% (26/34) of positive specimen, respectively, but declined obviously in 2007 - 2008 accounting for only 0% (0/44) and 5.0% (7/139). Influenza B virus was more popular in 2004 to 2005 with 42.9% (24/56) and 56.2% (9/16), respectively, and not isolated from 2006 to 2007, then increased in 2008 accounting for 34.5% (48/139). Phylogenetic tree of HA showed that A/H1N1 isolates in the same year clustered from 2005 to 2008, and most A/H3N2 isolated were homologous in the same year during 2004 - 2008 while some were inserted to the clusters of near years and more distinguished sequences appeared. A/H1N1 and A/H3N2 isolates were all similar to the vaccine strains recommended by WHO.

CONCLUSIONThe distribution of influenza type and subtype kept on changing each year, but A/H3N2 dominated in most years. A/H1N1 and A/H3N2 in the same year clustered, but some A/H3N2 of near years were and evolved faster with more distinguished strains appeared in same interval. Generally, HA of influenza A isolates in Shanghai during 2004 to 2008 were similar to the WHO reference strains.

China ; epidemiology ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology

OBJECTIVETo isolate and identify the influenza virus that caused four influenza-like-illness outbreaks in Jining city of Shandong Province in 2009 and analyze the genetic characteristics of hemagglutinin (HA) and neuraminidase (NA) gene, the variation of these genes were studied.

METHODS34 nasopharyngeal swabs from fever patients of four influenza-like-illness outbreaks were collected and diagnosed by real time quantitative RT-PCR method. The positive samples were incubated and cultured for virus. HA and NA genes of isolated pandemic influenza A (H1N1) virus were sequenced, the homology analysis was done with DNAStar software and the genetic evolution and amino acid substitutions were performed with Mega 4.0 software. The sequences were compared with WHO recommended vaccine virus, native reference virus.

RESULTSSeventeen of 34 nasopharyngeal swabs were positive, 11 pandemic influenza A (H1N1) viruses were isolated and HA and NA genes of 7 strains were sequenced. Phylogenetic analysis for hemagglutinin and neuraminidase gene of Shandong outbreak strains showed that there were 98.4% - 99.6% and 99.2% - 100.0% sequence identity. Compared with WHO-recommended vaccine strain, the reference virus in mainland China strain, eleven amino acids were changed for HA protein, including position 38, 40, 56, 90, 100, 145, 172, 173, 220, 303 and 338, and 38, 40, 303 of HA protein were located in the antigenic determination C cluster, 172, 173 in the D cluster, 56 in the E cluster, site 40 of HA protein were glycosylated. In NA protein, seven amino acids were changed, including position 80, 106, 241, 248, 351, 369 and 386, site 40 of NA protein were glycosylated. No mutations of 275 in NA protein were found.

CONCLUSIONThe HA and NA genes of the epidemic strains showed high homology, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process.

China ; epidemiology ; Disease Outbreaks ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Neuraminidase ; genetics

BACKGROUNDThrough the analysis of internal genes of viruses to understand whether the internal genes of two strains of swine influenza A(H1N1) virus contain the gene segment deriving from avian influenza A viruses, and whether the reassortment of the internal genes occurred between swine H1N1 and swine H9N2 viruses.

METHODSViruses were passaged in embryonated hen eggs and virion RNA was extracted from allantoic fluids 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).

RESULTSAll the six, but PB2 gene segment internal gene segments were the same between A/swine/Beijing/1/2002(H1N1) and A/Swine/Beijing/3/2002(H1N1) viruses. Whereas all the six internal gene segments in two trains of swine (H1N1) virus were similar to those of swine H1N1 viruses, but different from those of classical strain of swine (H1N1) virus.

CONCLUSIONTwo strains of Beijing swine H1N1 influenza A virus were not reassortant, All the six internal gene segments were closely related to swine influenza A (H1N1) viruses.

Animals ; Chick Embryo ; DNA, Complementary ; chemistry ; genetics ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H9N2 Subtype ; genetics ; Phylogeny ; RNA, Viral ; genetics ; Reassortant Viruses ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Swine ; virology

In order to visually detect H1, N1 and N2 subtype of avian influenza virus (AIV), three reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed. According to the sequences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 subtype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were fin-ished. Fourteen specimens of H1N1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMEP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field.
Animals ; Chickens ; DNA Primers ; genetics ; Ducks ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H1N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza A virus ; classification ; genetics ; isolation & purification ; Influenza in Birds ; diagnosis ; virology ; Nucleic Acid Amplification Techniques ; methods ; Poultry Diseases ; diagnosis ; virology ; Reverse Transcription ; Turkeys

To analyze influenza pathogen spectrum in Yunnan province during 2009-2014 years, and analyze HA and NA genes of influenza A H1N1. Analysis was made on the monitoring date of influenza cases in Yunnan province in recent 6 years, 23 strains of influenza virus of HA and NA gene was sequenced and analyzed by MEGA 5 software to construct phylogenetic tree. 4 times of influenza AH1N1 epidemic peak were monitored from 2009-2014 years in Yunnan Province, as the nucleic acid detection results of influenza A H1N1 accounted for 28.8% of the total. The sequencing result showed that HA and NA gene were divided into 3 groups, one was detected with H275Y mutation strains. Influenza A H1N1 is one of the important subtypes in Yunnan province and their genes have divided into three branches during the period of 2009-2014 years, the vast majority of influenza a H1N1 are still sensitive to neuraminidase inhibitors.
China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; enzymology ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Molecular Sequence Data ; Mutation ; Neuraminidase ; genetics ; metabolism ; Phylogeny ; Viral Proteins ; genetics ; metabolism

OBJECTIVETo characterize the prevalence and antigenic drift of influenza A viruses isolated during the period from 2001 to 2005 in infants and young children in Beijing.

METHODSMDCK cell culture, indirect immunofluorescence assay (IFA) and hemagglutination inhibition (HI) assay were used to isolate and identify type A influenza viruses (H1N1 and H3N2) from clinical samples collected from outpatients and inpatients who visited the Affiliated Children's Hospital because of acute respiratory infections from Oct. 2001 to Aug. 2005. The HA1 regions of hemagglutinin gene of H3N2 isolates were amplified by using RT-PCR followed by sequencing.

RESULTSOut of 7338 clinical samples collected during this surveillance period, 347 (4.7%) were positive for influenza A viruses, including 48 (13.8%) of H1N1, 273 (78.7%) of H3N2 and 26 (7.5%) of subtype-unidentified influenza A viruses. Although there was a prevalence season of influenza A from October each year to April of next year during the 2001-2004 period, it was worth noting that a consecutive influenza A activity was detected from Aug. 2004 to Aug. 2005, when some influenza A viruses were detected even in summer. The positive rate of H3N2 was 14.2% in August, 2005, which was equal to that of the peak season of 2003-2004. H3N2 were predominant in most of the influenza seasons during the surveillance period, and H1N1 was detected only in the influenza seasons of the 2001-2002 and 2004-2005 along with H3N2. The positive rates for both H3N2 and H1N1 were higher in specimens from outpatients than those from inpatients. A total of 46.6% (110/236) of the H3N2 were detected from children younger than 2 years of age, and 14.0% (33/236) were from children older than 5 years, whereas, more H1N1 was found in children older than 5 years (48.0%, 12/31) than in those younger than 2 years (6.5%, 2/31) during a period from Nov. 2003 to Aug. 2005. Sequence analysis of the HA1 regions of hemagglutinin of H3N2 isolated in a series of years revealed amino acid changes in the HA1 domain of H3N2 isolates in the antigenic sites (A-E) each year.

CONCLUSIONH3N2 and H1N1 prevailed in each influenza season during the surveillance period in Beijing, and H3N2 strains were predominant. The data from all-year around surveillance of influenza in Beijing indicate that continuous surveillance throughout a year and use of both antigenic and molecular analysis will be more helpful for early identification of any antigenic variants as well as prevention and control of influenza by promoting development of vaccines.

Age Factors ; Animals ; Antigenic Variation ; genetics ; Cell Culture Techniques ; Child ; Child, Preschool ; China ; epidemiology ; Dogs ; Female ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; isolation & purification ; Influenza A virus ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Inpatients ; Male ; Outpatients ; Prevalence ; Respiratory Tract Infections ; virology ; Retrospective Studies ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVETo investigate characteristics of the whole-genome of influenza A H1N1 virus circulated in Qingdao from year 2009 to 2011.

METHODSRNA of 35 influenza A H1N1 virus isolates circulated in Qingdao between year 2009 and 2011 was extracted and all segments were amplified by RT-PCR. The sequence was then detected and assembled by software Sequencher.25 HA full-length sequences published on GenBank were selected as reference. While MEGA 5.0 software package was explored for phylogenetic analysis to characterize the molecular feature with reference to the whole-genome sequence and the hemagglutinin (HA).1068 HA sequences of influenza A H1N1 virus isolated worldwide from August 2010 to March 2011 were downloaded for amino acid mutation analysis.

RESULTSOn the HA genes phylogenetic tree, the virus were separately divided into 4 clades in 2009-2010 and 2010-2011 surveillance season, each with a preponderant epidemic clade. The homogeneity of nucleotide and amino acids of HA isolates were 99.6%-99.9% and 99.1%-99.8% respectively in 2009-2010 surveillance season; 99.1%-99.6% and 98.2%-99.1% respectively in 2010-2011 surveillance season. The homogeneity of nucleotide and amino acids of the preponderant isolates were separately 98.8%-99.8% and 98.0%-99.6%. Compared with the vaccine strain, there were separately 14 and 12 variant amino acids of virus HA in the two surveillance season, involving 10 antigen sites and 5 positive selected sites. The sequence analysis of neuraminidase protein showed that the positions 247, 274 presented serine and histidine(S247, H274) respectively. The sequence analysis of M2 protein showed that the isolated A H1N1 viruses presented asparagine in amino acid site 31 (N31).

CONCLUSIONAll the A H1N1 influenza virus circulated in Qingdao from year 2009 to 2011 presented continual variation and therefore caused antigenic drift. All the isolations were adamantane-resistance, but susceptible to inhibitors of neuraminidase.

Amino Acid Sequence ; China ; epidemiology ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Neuraminidase ; genetics ; Phylogeny ; RNA, Viral ; Sequence Analysis, Protein

In 2006, a swine influenza virus (SIV) isolate was isolated from 30 nasal swabs samples collected from pigs with clinical syndromes of swine influenza in a pig farm of Liaoning Province. The virus isolate was studied and identified by the growth in 9-11 days old chicken embryo, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, reverse transcription-polymerase chain reaction assays (RT-PCR) for its genetic subtype, whole gene sequence analysis and animal trial for its virulence. The virus isolate demonstrated the hemagglutination activity. Result of HI test against H1 subtype of SIV was positive, however, the results were negative when the HI studies were conducted using SIV H3 subtype virus and Newcastle Disease Virus (NDV). Eight gene segments of the virus isolate were amplified by RT-PCR. Phylogenetic analysis of the gene sequence of the virus isolate by using DNAstar software program revealed that the isolate have the H1 HA gene, by comparing to the sequences of H1-H16 in the GenBank. Furthermore, sequencing results also demonstrated that the virus isolate's NA gene belongs to N1 subtype. Therefore, the subtype of the SIV isolate is H1N1. The results of sequence analysis indicated that the genome of the SIV-H1N1 LN strain includes 8 fragments, among which only M protein gene is not swine originated. All other 7 fragments have close relationship with the domestic standard swine H1N1 strains. Results suggested that the SIV isolate LN strain might be created by a possible triple reassortants among the classic swine influenza virus, human influenza-like virus, and avian influenza-like virus. Piglets were inoculated with the SIV LN strain virus preparations and the virus caused the typical clinical symptoms of swine influenza in the inoculated piglets. This study, the isolation, identification and genetic analysis of the SIV LN strain provided useful information and basic data for the further investigation of epidemic principles and patterns of swine influenza virus in China.
Animals ; Hemagglutination Inhibition Tests ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Lung ; virology ; Orthomyxoviridae Infections ; virology ; Phylogeny ; Swine ; Swine Diseases ; virology