Establishment of a high-throughput respiratory virus detection technology without RNA purification and reverse transcription / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a convenient and high-throughput respiratory virus detection method to facilitate epidemiological viral monitoring.</p><p><b>METHODS</b>We used high-throughput microsphere-based flexible multi-analyte profiling technology (xMAP) coupled with signal amplification molecules to simultaneously detect RNAs of 8 viruses including influenza viruses A and B, parainfluenza viruses type 1, 2 and 3, respiratory syncytial viruses A and B, and metapneumovirus in a 96-well plate format. The sensitivity and specificity of the method for the synthetic viral RNAs were evaluated.</p><p><b>RESULTS</b>There was no cross-reactivity among the 8 respiratory viral target RNAs. The detection limits for the 8 viral in intro-transcribed RNAs ranged from 1204 to 4695 RNA copies.</p><p><b>CONCLUSION</b>We establish a sensitive, specific, convenient, and high-throughput multiplex detection method suitable for detecting multiple respiratory viral RNAs for epidemiological viral monitoring.</p>

Analysis on the whole genome of the influenza H1N1 virus of the mild and severe cases in Beijing in 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the characteristics of the whole genome of the influenza H1N1 virus of the mild and severe cases in Beijing.</p><p><b>METHODS</b>A total of 21 samples of throat swabs were collected from surveillance-designated hospitals between June and December in 2009, including 10 severe cases (4 death cases) and 11 mild cases. RNA of the virus were extracted,and the amplified primers of the whole genome were designed.Reverse transcription and PCR were performed to the RNA and then the PCR product was sequenced by software to analyze the evolution of the viral genes and the variation of the amino acids.</p><p><b>RESULTS</b>Compared with the reference vaccine strain A/California/07/2009 (H1N1), the genetic nucleotide homology in the eight segments of the pandemic H1N1 virus in Beijing in 2009 was higher than 99%, without significant variation. Among them,the genetic distance of hemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) was comparatively far, separately 0.0050, 0.0040 and 0.0040.The gene of HA, P83S, the gene of NA, N248D, the gene of polymerase (PA), P224S and the gene of NP, V100I and L122Q were found to mutate in all the samples. Genes of HA, NA, NP, PA, PB 2 and nonstructural protein (NS1) in severe cases showed obviously clustered evolution. The mutation of gene S128P and S203T of HA, gene R269R and D547E of PA, gene T588I of PB 2 and gene I123V of NS mainly happened in severe cases, separately counting 6, 9, 6, 7, 9 and 6 cases. The relevance between the mutation happened in S203T of HA, R269K and D547E of PA and the severeness of the cases showed statistical significance (P < 0.05). The mutations of HA gene were mainly on the Ca and Cb antigene domains. No drug resistant mutation was found on NA gene but happened on matrix protein 2 (M2 gene). None of the mutations were found on the virulence related genes.</p><p><b>CONCLUSION</b>A high homology was found between the pandemic H1N1 virus in Beijing in 2009 and the reference vaccine strain A/California/07/2009(H1N1). Mutational sites related with the severe and fatal cases were found, but not the virulence related mutation.</p>

The significance of different sample types in study of pandemic A (H1N1) influenza diagnosis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the value of different types of samples, including throat swabs, stools, bloods in pandemic A (H1N1) influenza diagnosis and virus shedding patterns.</p><p><b>METHODS</b>From May to June in 2009, 135 samples were collected from 23 confirmed cases of pandemic influenza A (H1N1) infection, including 99 throat swabs, 14 stools, 11 bloods, 1 respiratory tract washing from 13 confirmed cases and 10 blood samples from other confirmed cases. The virus was detected by real-time RT-PCR, the antibody was detected by haemagglutination inhibition assay.</p><p><b>RESULTS</b>For 99 throat swabs of 13 patients, the median time of the first positive real-time RT-PCR was 1 day (ranged from 0 to 7 days) after the onset of the symptoms of illness; the median length of time duration of positive real-time RT-PCR results from throat swabs was 3 days (ranged from 1 to 15 days). Four cases intermittently released virus. One respiratory tract washing sample was positive. In 14 stools, 8 stools were real-time RT-PCR positive, the positive rate was 57.14%. The median time of the positive real-time RT-PCR was 3 days (ranged from 1 to 4 days) after the onset of the symptoms of illness. In 21 blood samples collected at 2 to 9 days of onset, 1 blood sample was real-time RT-PCR positive, the positive rate was 4.76%. All these 21 blood samples were antibody negative.</p><p><b>CONCLUSION</b>Throat swabs and stools samples can be used as A (H1N1) influenza early diagnosis. The length of time duration of positive real-time RT-PCR in throat swabs was longer than stool samples and intermittently releasing of virus were found in throat swabs. Influenza A H1N1 cases showed the presence of small amount of viremia and antibody was negative in early blood samples (< 9 days).</p>

A survey on serological epidemiology of influenza A (H1N1) 2009 in Beijing / 中华流行病学杂志

Objective To investigate the immunological level against influenza A (H1N1)2009 in Beijing and provide evidence to evaluate the developing trend of the disease. Methods Between Nov. 27,2009 and Dec. 23,2009, subjects were randomly selected from patients in hospitals (infectious and respiratory diseases related departments were excluded) ,volunteers in blood donation center and healthy subjects attending the physical examination center. Questionnaire survey was conducted and serum samples were collected to detect the hemagglutination-inhibition (HI) antibody against influenza A(H1N1) 2009 virus. Results 856 subjects participated in this survey, and 127 showed positive HI antibody to this pandemic virus. The proportions of sero-positivity among 0-5 ,was no significant difference in the sero-positivity between males and females (P=0.693). The analysis, factors as age, acute respiratory symptoms and the rate of pandemic (H1N1) 2009 vaccination were significantly associated with sero-positivity of HI antibody to the influenza A (H1N1) 2009 virus. Conclusion Above 15% of the population in Beijing showed protective antibody against influenza A (H1N1) 2009 virus, indicating the development of immunological barrier to this disease had been formed, to some extent.

Etiological characteristics of influenza A (H1N1) 2009 virus in Beijing / 中华流行病学杂志

Objective To analyze the results of detection on influenza A (H1N1) 2009 virus in Beijing from May 2009 to December 2009 and to understand the epidemiologic characteristics during the pandemic period. Methods The study was conducted from the May 1 to December 27,2009. A total of 101 852 throat swab samples were detected with the real-time RT-PCR assay by the Beijing Network Laboratory. Data was statistically analyzed. Results 9843 samples showed influenza A (H1N1) 2009 positive, with an overall positive rate as 9.66%. In terms of the positive rates, they were 2.85% from May to June, 3.32% from July to August and 8.35% from September to October. The peak month fell in November (29.67%) and December (24.33%). The positive rates among the following subpopulations were: 8.40% among the suspected cases, 4.75% among close contact cases, 11.46% among the influenza-like illness cases and 7.33% among the cluster cases with fever. Positive cases mainly fell in age groups 5-14 and 15-24. The ratio of male to female was 1.5:1.Conclusion During the pandemic period of influenza A (H1N1) 2009, positive cases gradually increased during May to November but slowly decreasing in December.

Investigation on the source of the first human of avian influenza A (H5N1) case in Beijing / 中华流行病学杂志

Objective To investigate the source of the first human case of avian influenza A (H5N1) infection in Beijing. Methods Interviewing the relatives of the case and other key persons, collecting and detecting samples of related biological, epidemiological and environmental data of the case were conducted. Later, the infection source was thoroughly investigated. Results The case ever contacted a slaughtered duck 5 days prior to the onset of illness, and the duck was bought from a stall of a wet market in Yanjiao area of Hebei province. Ten environmental samples were collected in this stall and the neighboring stall of the market. Another 6 samples were tested positive for H5N1 virus by PCR method, with 5 virus strains isolated. The whole-genome sequencing indicated that the amino acid homology between the H5N1 virus strains from the environment and the virus isolated from the case reached 99.8%-100%. Conclusion From both epidemiological and virological evidence, it was proved that the first human case of avian influenza A (H5N1) infection in Beijing was infected by a duck that carrying H5N1 virus the case contacted 5 days proceeding the onset of illness.