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

The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.
Animals ; China ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; pathogenicity ; Influenza in Birds ; virology ; Mice, Inbred BALB C ; Phylogeny ; Poultry

OBJECTIVETo provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

METHODSViruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

RESULTSNGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.

CONCLUSIONThe improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Clinical Laboratory Techniques ; DNA Barcoding, Taxonomic ; DNA Primers ; Enterovirus ; classification ; genetics ; isolation & purification ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Influenza B virus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods ; Sequence Analysis, RNA ; methods

OBJECTIVETo compare the epidemiological and clinical features of lower respiratory tract infection (LRTI) caused by influenza virus A (IVA) and influenza virus B (IVB) in children.

METHODSThe clinical data of 366 children with LRTI caused by influenza virus (IV), who were hospitalized in Yuying Children′s Hospital of Wenzhou Medical University between 2010 and 2014, were analyzed retrospectively, and there were 272 cases caused by IVA and 94 cases caused by IVB.

RESULTSIV was mainly prevalent from December to March of the next year, with the predominance of IVA. There were small peaks of IVA prevalence in July or September every other year, and IVB was prevalent from December to March of the next year every other year. The children with LRTI caused by IVA alone had a significantly higher white blood cell (WBC) count and significantly higher percentages of children with increased WBC, abnormal serum sodium, and abnormal serum potassium than those caused by IVB alone (P<0.05). However, there were no significant differences in age, sex, underlying diseases, clinical manifestations, and co-infection rate with bacteria or atypical pathogens between the two groups (P>0.05). The rate of co-infection with respiratory syncytial virus (RSV) was significantly higher in the IVB group than in the IVA group (P<0.01).

CONCLUSIONSIVA is prevalent in winter and spring every year and has small peaks in summer every other year, while IVB is prevalent in winter and spring every other year. Compared with IVB, IVA causes more cases of increased WBC and electrolyte disturbance. The children infected with IVB are more likely to be co-infected with RSV. The children with LRTI caused by IVA and IVB have similar clinical manifestations.

Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Influenza A virus ; genetics ; isolation & purification ; physiology ; Influenza B virus ; genetics ; isolation & purification ; physiology ; Influenza, Human ; diagnosis ; epidemiology ; virology ; Male ; Respiratory Tract Infections ; diagnosis ; epidemiology ; virology ; Retrospective Studies ; Seasons

Animals ; China ; Influenza A virus ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Nucleic Acids ; genetics ; Poultry ; virology

A quail-origin subtype of the influenza virus was isolated from a human-infecting H7N9 subtype of the avian influenza virus found in a live poultry market and was given the name A/Quail/Hangzhou/1/ 2013 (H9N2). We analyzed the whole genome of this virus and its biologic characteristics. Sequence analyses suggested that the: HA and NS genes belonged to a CK/BJ/1/94-like lineage; NA, NP, PA and PB1 genes belonged to a SH/F/98-like lineage; M and PB2 genes belonged to a G1-like lineage. Analyses of key amino acids showed that the cleavage site in HA protein was PSRSSR ↓ GL, and that the HA protein had a human receptor-binding site with Leu226. Deletion of amino acids 69 - 73 was detected in the stalk of NA protein, the M2 protein had an Asn31 mutation, and the NS1 protein had two mutations at Ser42, Ala149. The intravenous pathogenicity of this virus was 0.36. A study in chickens suggested that all inoculated birds shed the virus from the trachea and cloaca on the third day post-infection (p. i. ) until 11 days. All chickens that had direct contact shed the virus on the second day p. i. until 8 days. Results of virus reisolation suggested that lung and tracheal tissues could shed the virus in 5 days, whereas the other organs could shed the virus in 3 days. These results suggest that this virus strain is H9N2 subtype LPAIV, whose lineage is prevalent in mainland China. This research provides evidence on how to monitor and prevent the H9N2 subtype of the avian influenza virus.
Animals ; Chick Embryo ; Chickens ; China ; Genotype ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Phylogeny ; Quail ; virology

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

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.
Databases, Genetic ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Influenza A Virus, H1N1 Subtype/classification/*genetics/isolation & purification ; Influenza, Human/diagnosis/*virology ; Nasopharynx/*virology ; Nucleic Acid Amplification Techniques ; Phylogeny ; RNA, Viral/analysis/metabolism ; Sequence Analysis, RNA ; Viral Proteins/genetics

Data based on the antiviral-resistant phenotyping characteristics of 884 influenza B viruses circulating in mainland China from October 2013 to March 2014 were analyzed to assess the susceptibility of influenza B viruses to neuraminidase inhibitors. All 884 viruses were sensitive to oseltamivir; two viruses (0.23%) had reduced sensitivity to zanamivir and all other viruses were sensitive to zanamivir. Among the 38 viruses with a B/Victoria lineage, B/Shandong-Kuiwen/1195/2014 exhibited a half-maximal inhibitory concentration (IC50) for zanamivir that was elevated by 5. 12-fold (1.78 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D35G, N59D and S402T (39, 64 and 399 with N2 number) amino-acid substitutions in the NA gene were detected with no previously reported antiviral-resistant substitutions. Among viruses with the 846 B/Yamagata lineage, B/Hunan-Lingling/350/2013 exhibited a 7.99-fold elevated IC50 for zanamivir (2.72 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D197N (N2 number), a previously reported antiviral resistant-related amino-acid substitution in the NA gene, was detected in B/Hunan-Lingling/350/2013. These data suggest that recently circulating influenza B viruses in mainland China have retained susceptibility to neuraminidase inhibitors.
Amino Acid Substitution ; Antiviral Agents ; pharmacology ; China ; epidemiology ; Drug Resistance, Viral ; Enzyme Inhibitors ; pharmacology ; Humans ; Influenza B virus ; drug effects ; enzymology ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Microbial Sensitivity Tests ; Neuraminidase ; antagonists & inhibitors ; genetics ; metabolism ; Viral Proteins ; antagonists & inhibitors ; genetics ; metabolism

We investigated the genetic diversity and evolution of the M gene of human influenza A viruses in Hangzhou (Zhejiang province, China) from 2009 to 2013, including subtypes of A(H1N1) pdm09 strains and seasonal A(H3N2) strains. Subtypes of analyzed viruses were identified by cell culture and real-time reverse transcription-polymerase chain reaction, followed by cloning, sequencing and phylogenetic analyses of the M gene. Assessment of 5675 throat swabs revealed a positive rate for the influenza virus of 20.46%, and 827 cases were diagnosed as. infections due to influenza A viruses. Seventy-six influenza-A strains were selected randomly from nine stages during six phases of a virus epidemic. Sequences of the M gene showed high homology among six epidemics with identities of amino-acid sequences of 98.98-100%. All strains contained the adamantine-resistant mutation S31N in its M2 protein. Two of the A(H1N1)pdm09 strains had double mutants of V27A/S31N or V271/S31N. One of the seasonal A(H3N2) viruses had another form of double-mutant R45H/S31N. Evolutionary rate of the M gene was much lower than that of the HA gene and NA gene. Compared with A(H3N2) strains, higher positive pressure on the M1 and M2 proteins of A(H1N1) pdm09 viruses was observed. Separate analyses of M1 and M2 proteins revealed very different selection pressures. Knowledge of the genetic diversity and evolution of the M gene of human influenza-A viruses will be valuable for the control and prevention of diseases.
Amino Acid Substitution ; China ; epidemiology ; Evolution, Molecular ; Genetic Variation ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Phylogeny ; Selection, Genetic ; Viral Matrix Proteins ; genetics ; Viral Proteins ; chemistry ; genetics