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

OBJECTIVETo understand the epidemiological characteristics of influenza outbreaks in China from 2005 to 2013.

METHODSThe data of influenza-like illness outbreaks involving 10 or more cases were collected through Public Health Emergency Management Information System and National Influenza Surveillance Information System in China, and the influenza outbreaks were identified according to the laboratory detection results. Descriptive epidemiological analysis was conducted to understand the type/subtype of influenza virus and outbreak time, area, place and extent.

RESULTSFrom 2005 to 2013, a total of 3 252 influenza-like illness outbreaks were reported in the mainland of China, in which 2 915 influenza outbreaks were laboratory confirmed, and influenza A (H1N1) pdm09 virus and influenza B virus were predominant. More influenza outbreaks were reported in the influenza A (H1N1) pandemic during 2009-2010. Influenza outbreaks mainly occurred during winter-spring, and less influenza outbreaks occurred in winter and summer vacations of schools. More influenza outbreaks were reported in southern provinces, accounting for 79% of the total. Influenza outbreaks mainly occurred in primary and middle schools, where 2 763 outbreaks were reported, accounting for 85% of the total. Average 30-99 people were involved in an outbreak.

CONCLUSIONA large number of influenza outbreaks occur during influenza season every year in China, the predominant virus type or subtype varies with season. Primary and middle schools are mainly affected by influenza outbreaks.

China ; epidemiology ; Disease Outbreaks ; Humans ; Influenza A Virus, H1N1 Subtype ; isolation & purification ; Influenza B virus ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Population Surveillance ; Schools ; Seasons

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

Laggera pterodonta is commonly used for treating influenza in Southwest China, especially in Yunnnan province. The main clinical effects of L. pterodonta include anti-influenza, anti-microbial, anti-inflammatory. To investigate the anti-influenza A (H1N1) virus effect of L. pterodonta, neutralization inhibition and proliferation inhibition tests were performed. MDCK culture method was used to observe the cytopathic effect (CPE) of extracts from L. pterodonta in inhibiting influenza A (H1N1) virus and haemagglutination titre of H1N1 virus in vitro. The culture medium were collected at 24 h, 48 h, 72 h, 96 h, and detected by Real time RT-PCR, in order to compare the effect of different extracts from L. pterodonta on in vitro proliferation of H1N1, virus. The result of neutralization inhibition test showed that hemagglutination titer of ethyl acetate extract were 8 times lower at 72 h; in proliferation inhibition test, hemagglutination titer of ethyl acetate extracts reduced by 2 and 4 times. According to the results of Real time RT-PCR test, the H1N1 inhibition ratio of ethyl acetate extract was 72.5%, while the proliferation inhibition ratio of ethyl acetate extract was 25.3%; as for petroleum ether extracts, the H1N1 inhibition ratio was 60.2%, while the proliferation inhibition ratio was 81.4%. In conclusion, both ethyl acetate extract and petroleum ether extract of L. pterodonta have significant neutralization and direct proliferation inhibition effects on influenza A virus.
Asteraceae ; chemistry ; China ; ethnology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Influenza, Human ; drug therapy ; virology ; Medicine, Chinese Traditional

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

Three cyclotides were isolated from the whole plant of Viola yedoensis in this study. The two, vary peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately. The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again. Besides, the IC50 value of cycloviolacin VY1 against influenza A H1N1 virus was (2.27 +/- 0.20) microg x mL(-1). It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.
Antiviral Agents ; isolation & purification ; pharmacology ; Cyclotides ; pharmacology ; Influenza A Virus, H1N1 Subtype ; drug effects ; Tandem Mass Spectrometry ; Viola ; chemistry

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 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 understand and master the dynamic variation of the pandemic influenza A (H1N1) 2009 in Hunan province from 2009 to 2011, and to know the genetic characteristics and drug resistance of the pandemic (H1N1) 2009 viruses. Throat swab specimens of influenza-like illness patients were collected from sentinel hospitals and tested for influenza by fluorescent PCR or virus isolation methods. Partial isolates were selected for sequencing. The sequences were used for phylogenetic analysis by MEGA 5. 05 software. From the 20th week of 2009 to the 52nd week of 2011, 17 773 specimens were tested. 3 831 specimens were influenza-positive with a positive rate of 21. 6%, of which 1 794 were positive specimens of pandemic (H1N1) 2009, accounting for 46. 8%00 of the influenza-positives. There were 2 epidemic peaks of pandemic (H1N1) 2009, which were in the 41st-53rd week of 2009 and the 1st-12nd week of 2011, respectively. The HA genes of 23 strains that were selected for sequencing had close relationship; the distribution of strains in the phylogenetic tree was basically in chronological order. The complete genome sequence analysis showed that all of 8 gene segments of 7 strains were homologous to the vaccine strain, and there was no gene reassortment. The HA amino acid sites of the 23 strains were highly similar to the vaccine strain (98. 2% - 100. 0% in homology), but all 23 strains had P83S, S203T and 1321V mutations. The 222 site mutation that may lead to enhanced virulence was found in the A/Hunan/YQ30/2009 strain. The mutation was D222E. There was no oseltamivir resistance mutation found in all strains. The pandemic (H1N1) 2009 in Hunan province from 2009 to 2011 had a bimodal distribution. There was no large-scale variation of virus genes. The clinical use of oseltamivir was still effective. Key words: Pandemic (H1N1) 2009; Surveillance; Genetic characteristics
Amino Acid Sequence ; China ; epidemiology ; Humans ; Influenza A Virus, H1N1 Subtype ; chemistry ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Molecular Sequence Data ; Pandemics ; Phylogeny ; Public Health Surveillance ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

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