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

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

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

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

Attitude to Health ; Civil Defense ; ethics ; Communicable Disease Control ; Communication ; Ethics, Medical ; Freedom ; Health Personnel ; ethics ; Health Planning ; ethics ; Health Priorities ; ethics ; Humans ; Influenza A Virus, H1N1 Subtype ; isolation & purification ; Influenza, Human ; epidemiology ; Mandatory Programs ; ethics ; Moral Obligations ; Pandemics ; ethics ; Patient Rights ; ethics ; Primary Health Care ; ethics ; Public Health ; ethics ; Public Health Administration ; ethics ; Resource Allocation ; ethics ; Singapore ; Trust

INTRODUCTIONSingapore had its first case of pandemic influenza A (H1N1) 2009 on 26 May 2009. As of 3 August 2009, 440 children with confirmed H1N1were admitted to KK Women's and Children's Hospital (KKH).

MATERIALS AND METHODSThis is a retrospective case control study of children admitted from 26 May 2009 to 19 July 2009 with H1N1infection. Cases and controls were first differentiated by whether they were complicated or non-complicated in nature, and subsequently analysed with regards to possible independent risk factors.

RESULTSWe analysed 143 admitted children; 48 cases and 95 controls (1: 2 ratio). Significant comorbidity was found in 20.3% (n = 29) of patients with the majority having asthma (n = 18, 12.6 %) followed by obesity (n = 7, 4.9%). Binary logistic regression analysis showed risk factors for complicated disease were comorbidity (adjusted OR 6.0, 95% CI, 2.5 to 14.6, P < 0.0001) and age <2 years (adjusted OR 9.8, 95% CI, 2.4 to 40, P = 0.001). Age less than 5 years was not found to be a risk factor.

CONCLUSIONIn the early stages of an evolving influenza epidemic when oseltamivir stocks are low, oseltamivir treatment for influenza can be streamlined and offered to those at highest risk who are under 2 years old or have significant comorbidity to prevent complicated disease.

Adolescent ; Age Factors ; Antiviral Agents ; therapeutic use ; Asthma ; complications ; Bronchitis ; complications ; Case-Control Studies ; Child ; Child, Preschool ; Cohort Studies ; Female ; Fluid Therapy ; Gastritis ; virology ; Gastroenteritis ; virology ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; isolation & purification ; Influenza, Human ; complications ; Male ; Obesity ; complications ; Oseltamivir ; therapeutic use ; Pneumonia, Bacterial ; complications ; Retrospective Studies ; Risk Factors ; Seizures ; complications ; Singapore

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

OBJECTIVETo investigate the epidemiological features, genetic drift in the epitopes of hemagglutinin (HA) of the novel influenza A (H1N1) virus and oseltamivir-resistant variants characterized by H275Y and N295S mutations in children in Shanghai since the outbreak.

METHODBetween June 2009 and May 2012, a prospective surveillance study was carried out in Shanghainese children who attended the outpatient clinic of Children's Hospital of Fudan University for influenza-like illness. One-step real-time fluorescence quantitative RT-PCR was performed to detect seasonal influenza A and influenza B virus and the novel influenza A (H1N1) virus in the respiratory samples. Genetic drift from the vaccine strain in HA epitopes of the novel influenza H1N1 virus and the molecular markers associated with oseltamivir resistance in neuraminidase (NA) were analyzed.

RESULTOut of 3475 enrolled cases, the novel influenza A (H1N1) virus was confirmed virologically in 222 (6.4%) otherwise healthy children with 133 (59.9%) being boys and 89 (40.1%) girls. The median ages of children with the novel influenza A (H1N1) virus infection during the first wave from August 2009 to February 2010 and the second wave from December 2010 to February 2011 were 53.5 months and 32.0 months, respectively (Z = -4.601, P = 0.000); 119 (46.9%) had the close contact with persons suffering from fever or respiratory infection, of whom, 68 (57.1%) contacts were family members and 47 (39.5%) contacts were classmates. During the outbreak in 2009-2010 season, 66 (40.9%) were exposed to primary index cases, school students were the major exposure subjects, accounting for 50.0%. The nucleotide sequences of HA1 gene were highly homologous between the vaccine strain A/California/07/2009 and Shanghai circulating novel influenza A (H1N1) strains and only S83P mutation in epitope E of HA was detected inclusively in the circulating strains. The H275Y and N295S amino acid mutations associated with oseltamivir resistance were not found in the circulating novel influenza (H1N1) strains.

CONCLUSIONTwo major waves of the novel influenza A (H1N1) outbreaks occurred in Shanghainese children during 2009-2011. Institutional children were the major affected individuals during the 2009 pandemic wave. Households and schools were the main sites of transmission among children during influenza pandemic. Influenza vaccination should be enhanced in children and their close family contacts. The novel influenza A (H1N1) virus in Shanghai has not undergone significant genetic changes. Oseltamivir is effective for the treatment of the novel influenza A (H1N1) virus.

Adolescent ; Amino Acid Sequence ; Antiviral Agents ; pharmacology ; Child ; Child, Preschool ; China ; epidemiology ; Drug Resistance, Viral ; Female ; Hemagglutinins, Viral ; genetics ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; drug therapy ; epidemiology ; pathology ; virology ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Neuraminidase ; genetics ; Oseltamivir ; pharmacology ; Pandemics ; Viral Vaccines ; genetics ; immunology