To set up a new rapid method for the rapid determination of influenza virus H5N1 and H7N9 basing on the Multi-Analyte Suspension Array (MASA) technology. Sequence analysis and design of degenerate primers and specific probes were set in the comparison and analysis of H5, N1, H7 and N9 genes. In combination with MASA technology, these primers and probes were used for the determination of samples of H5N1 and H7N9 and other subtypes ( H1N1, PH1N1, H5N2, H3N2 and H9N2). We developed a rapid determination method. This method had high specificity and sensitivity that could detect H5N1 and H7N9 at one time, and could detect samples that containing 10 copies of H5N1 and H7N9. This determination method could be used for rapid determination of influenza virus H5N1 and H7N9 at one time.
Humans ; Influenza A Virus, H5N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H7N9 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Oligonucleotide Array Sequence Analysis ; methods

OBJECTIVETo analyze the etiology and genomic sequences of human infection of avian-origin influenza A(H7N9)virus from Zhejiang province.

METHODSViral RNA was extracted from patients of suspected H7N9 influenza virus infection and real-time RT-PCR was conducted for detection of viral RNA. All 8 segments of influenza virus were amplified by one-step RT-PCR and genomic sequences were assembled using the sequencing data. All the currently available HA and NA genes of the novel H7N9 virus, some other HAs from H7 subtype and NAs from N9 subtype were downloaded from public database for phylogenetic analysis, using the Mega 5.1 software. Mutations and variations were analyzed, using the genomic sequence data.

RESULTSReactions for influenza type A, subtype H7 and subtype N9 were all positive and all the genomic fragments were amplified for sequencing. After alignment, sequences were subjected for phylogenetic analysis. The results revealed highest homology with A/duck/Zhejiang/12/2011(H7N3)in HA gene and with A/wild bird/Korea/ A14/2011(H7N9)in NA gene of the H7N9 influenza virus. All 6 genes coding for internal proteins shared highest identities with H9N2 avian influenza which were circulated in the Chinese mainland, in the last two years. The sequenced virus showed Q226L mutation in HA protein, but E627K was not presented in PB2 protein of this virus. The E627K mutation was shared by all the other novel H7N9 viruses resulted in human infections through analysis on the currently available sequences.

CONCLUSIONUsing the clinical samples, both detection of the viral genes and amplification of all 8 segments of the novel H7N9 influenza virus were accomplished. High homology of the novel H7N9 influenza viruses was observed by phylogenetic test, using the currently available sequences. The virus showed Q226L mutation on HA protein but E627K did not present on PB2 protein of this virus.

China ; epidemiology ; Genome, Viral ; Humans ; Influenza A Virus, H7N9 Subtype ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Male ; Middle Aged ; Phylogeny ; RNA, Viral ; genetics ; Sequence Analysis, DNA

Animals ; Birds ; virology ; China ; epidemiology ; Computer Communication Networks ; Epidemiologic Methods ; Influenza A Virus, H7N9 Subtype ; isolation & purification ; Influenza in Birds ; epidemiology ; transmission ; virology

Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.
Animals ; Base Sequence ; Birds ; China/epidemiology* ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus/immunology* ; Hemagglutinins/genetics* ; Humans ; Influenza A Virus, H7N9 Subtype/isolation & purification* ; Influenza in Birds ; Influenza, Human/virology* ; Neuraminidase/genetics* ; Phylogeny ; RNA, Viral/genetics* ; Sequence Analysis, DNA

OBJECTIVETo trace the source of human H7N9 cases in Huai'an and elucidate the genetic characterization of Huai'an strains associated with both humans and birds in live poultry market.

METHODSAn enhanced surveillance was implemented when the first human H7N9 case was confirmed in Huai'an. Clinical specimens, cloacal swabs, and fecal samples were collected and screened by real-time reverse transcription-polymerase chain reaction (RT-PCR) for H7N9 virus. The positive samples were subjected to further RT-PCR and genome sequencing. The phylodynamic patterns of H7N9 virus within and separated from Huai'an and evolutionary dynamics of the virus were analyzed.

RESULTSSix patients with H7N9 infection were previously exposed to live poultry market and presented symptoms such as fever (>38.0 °C) and headaches. Results of this study support the hypothesis that live poultry markets were the source of human H7N9 exposure. Phylogenetic analysis revealed that all novel H7N9 viruses, including Huai'an strains, could be classified into two distinct clades, A and B. Additionally, the diversified H7N9 virus circulated in live poultry markets in Huai'an. Interestingly, the common ancestors of the Huai'an H7N9 virus existed in January 2012. The mean nucleotide substitution rates for each gene segment of the H7N9 virus were (3.09-7.26)×10-3 substitutions/site per year (95% HPD: 1.72×10-3 to 1.16×10-2).

CONCLUSIONOverall, the source of exposure of human H7N9 cases in Huai'an was live poultry market, and our study highlights the presence of divergent genetic lineage of H7N9 virus in both humans and poultry specimens in Huai'an.

Adult ; Aged ; Aged, 80 and over ; Animals ; China ; epidemiology ; Evolution, Molecular ; Female ; Humans ; Influenza A Virus, H7N9 Subtype ; classification ; genetics ; isolation & purification ; Influenza in Birds ; epidemiology ; virology ; Influenza, Human ; epidemiology ; virology ; Male ; Middle Aged ; Molecular Epidemiology ; Phylogeny ; Poultry ; Prevalence

OBJECTIVETo investigate the origin and the recombinant model of H7N9 virus prevailing in China by sequence analysis.

METHODSThe sequences of H7N9 virus were collected and analyzed with the software BLAST and MEGA 5.0. The phylogenetic trees were constructed after multiple sequences alignment. The homologous sequences of H7N9 segments were determined and the model was inferred according to the origin of H7N9 segments.

RESULTSThe most relevant sequences of HA, NA, NS and PB2 segments were located at one branch of the phylogenetic tree, while the closest relevant sequences of PB1, PA, NP and MP contained two H9N2 virus origins. According to the analysis of sequence origin, H7N9 viruses might be divided into 5 genotypes: namely A, B, A/Shanghai/1/2013-H7N9, A/Pigeon/Shanghai/S1069-H7N9 and A/Zhejiang/HZ1/2013-H7N9, and the genotype A consisted of A1 and A2 subtypes.

CONCLUSIONThe prevailing H7N9 virus might be derived from 5 different viruses after 4 times of recombination, which resulted in the two major types of A and B. The subtypes of A1 and A2 were two different derivatives from one reassortant. The A/Pigeon/Shanghai/S1069-H7N9 strain might be the recombinant of type A H7N9 virus with a local H9N2 virus during the H7N9 epidemics. The A/Zhejiang/HZ1/2013-H7N9 strain could be the re-arrangement of subtype A2 with type B H7N9 virus.

China ; Genome, Viral ; Genotype ; Humans ; Influenza A Virus, H7N9 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Phylogeny ; Reassortant Viruses ; classification ; genetics ; isolation & purification ; Sequence Analysis, RNA ; Viral Proteins ; genetics

OBJECTIVETo understand the transmission mode of human infection with avian influenza A (H7N9) virus.

METHODSField epidemiological investigation was conducted for a family clustering of human infection with H7N9 virus in Hengxian county, Guangxi Zhuang Autonomous Region in February 2014. Two patients and their 82 close contacts were surveyed. The samples collected from the patients, environments and poultry were tested by using real time reverse transcriptase-polymerase chain reaction (rRT-PCR), and the samples from patients were used for virus isolation. The samples from 5 close contacts were tested with RT-PCR. The clinical data, exposure histories of the patients and the detection results of the isolates and their homology were analyzed.

RESULTSPatient A became ill 4 days after her last exposure to poultry in Zhongshan, Guangdong province, and returned to her hometown in Hengxian 2 days after onset. Patient B was patient A's 5 years old son, who had no known exposure to poultry but slept with patient A for 4 days. He developed symptoms 4 days after last contact with his mother. Two strains of H7N9 virus were isolated from the two patients. The 2 isolates were highly homogenous (almost 100%) indicated by gene sequencing and phylogenetic tree. None of the other 81 close contacts developed symptoms of H7N9 virus infection.

CONCLUSIONPatients B was infected through close contact with patient A, indicating that avian H7N9 virus can spread from person to person, but the transmissibility is limited and non-sustainable.

Animals ; Child, Preschool ; China ; Cluster Analysis ; Contact Tracing ; Family ; Female ; Homozygote ; Humans ; Influenza A Virus, H7N9 Subtype ; genetics ; isolation & purification ; Influenza, Human ; transmission ; virology ; Male ; Phylogeny ; Poultry ; virology ; Real-Time Polymerase Chain Reaction ; Sleep

This study aimed to establish a method for the detection and identification of H7N9 avian influenza viruses based on the NA gene by pyrosequencing. According to the published NA gene sequences of the avian influenza A (H7N9) virus, a 15-nt deletion was found in the NA gene of H7N9 avian influenza viruses. The 15-nt deletion of the NA gene was targeted as the molecular marker for the rapid detection and identification of H7N9 avian influenza viruses by pyrosequencing. Three H7N9 avian influenza virus isolates underwent pyrosequencing using the same assay, and were proven to have the same 15-nt deletion. Pyrosequencing technology based on the NA gene molecular marker can be used to identify H7N9 avian influenza viruses.
Animals ; Base Sequence ; Birds ; Chickens ; High-Throughput Nucleotide Sequencing ; methods ; Influenza A Virus, H7N9 Subtype ; classification ; enzymology ; isolation & purification ; Influenza in Birds ; virology ; Molecular Sequence Data ; Neuraminidase ; genetics ; Phylogeny ; Poultry Diseases ; virology ; Viral Proteins ; genetics

In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.
Animals ; Birds ; virology ; Influenza A Virus, H7N9 Subtype ; genetics ; isolation & purification ; physiology ; Influenza in Birds ; prevention & control ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Species Specificity ; Taq Polymerase ; metabolism ; Time Factors

Objective: To understand the epidemiological characteristics of human infection with avian influenza A (H7N9) virus in China, and provide evidence for the prevention and control of human infection with H7N9 virus. Methods: The published incidence data of human infection with H7N9 virus in China from March 2013 to April 2017 were collected. Excel 2007 software was used to perform the analysis. The characteristics of distribution of the disease, exposure history, cluster of the disease were described. Results: By the end of April 2017, a total of 1 416 cases of human infection with H7N9 virus were confirmed in China, including 559 deaths, the case fatality rate was 39.5%. In 2016, the case number was lowest (127 cases), with the highest fatality rate (57.5%). The first three provinces with high case numbers were Zhejiang, Guangdong and Jiangsu. The median age of the cases was 55 years and the male to female ratio was 2.3∶1. Up to 66% of cases had clear live poultry exposure history before illness onset, 31% of cases had unknown exposure history and only 3% of the cases had no live poultry exposure history. There were 35 household clusters (5 in 2013, 9 in 2014, 6 in 2015, 5 in 2016, 10 in 2017), which involved 72 cases, accounting for 5% of the total cases. Conclusions: The epidemic of human infection with H7N9 virus in China during 2013-2017 had obvious seasonality and spatial distribution. There was limited family clustering. Infection cases were mostly related to poultry contact.
Adult ; Age Distribution ; Aged ; Aged, 80 and over ; Animals ; China/epidemiology* ; Cluster Analysis ; Disease Outbreaks ; Epidemics ; Female ; Humans ; Incidence ; Influenza A Virus, H7N9 Subtype/isolation & purification* ; Influenza, Human/virology* ; Male ; Middle Aged ; Poultry ; Sex Distribution