[Analysis on the whole genome of the influenza H1N1 virus of the mild and severe cases in Beijing in 2009].

OBJECTIVE: To explore the characteristics of the whole genome of the influenza H1N1 virus of the mild and severe cases in Beijing. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Establishment of a high-throughput respiratory virus detection technology without RNA purification and reverse transcription].

OBJECTIVE: To establish a convenient and high-throughput respiratory virus detection method to facilitate epidemiological viral monitoring. METHODS: 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. RESULTS: 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. CONCLUSION: We establish a sensitive, specific, convenient, and high-throughput multiplex detection method suitable for detecting multiple respiratory viral RNAs for epidemiological viral monitoring.

[Application of EARS in early-warning of influenza pandemic in Beijing].

OBJECTIVE: To illustrate the efficiency of cumulative sum (CUSUM) in pre-warning of the influenza peak in Beijing. METHODS: CUSUM was used to analyze the data of influenza like illness (ILI), and the results of the influenza laboratory surveillance was regarded as the gold standard to judge the approaching of the influenza peak. RESULTS: The surveillance was launched in 421 hospitals in Beijing during the 2009 to 2010 influenza season, while the influenza laboratory surveillance was launched by 7 collaborative laboratories. From Jun. 2009 to Apr. 2010, the average ILI percentage in the 421 hospitals was 2.56%. In the study, 19 262 pharyngeal swab samples were collected from the ILI cases in 11 hospitals and 5 045 of them were tested positive for the influenza virus, with the novel swine-origin influenza A H1N1 virus dominating. After analyzing of the ILI surveillance data with CUSUM, it was found that the ILI surveillance in Beijing could make a satisfactory early warning for the approaching of the influenza peak referring to the gold standard based on the influenza laboratory results. CONCLUSION: It could give the prediction and early warning for the influenza peak efficiently and precisely, by using CUSUM to analyze the influenza surveillance data of Beijing.

Serologic survey of pandemic influenza A (H1N1 2009) in Beijing, China.

OBJECTIVE: To examine the frequency and distribution of antibodies against pandemic influenza A (H1N1 2009) [H1N1] in populations in Beijing and elucidate influencing factors. METHODS: In January 2010, a randomized serologic survey of pandemic influenza A (H1N1 2009) was carried out. Six districts that were randomly selected with a total of 4601 participants involved in the survey have their antibody level tested by hemagglutination inhibition assay. RESULTS: Among the 4601 participants, the overall seropositive rate for pandemic influenza A (H1N1 2009) antibodies was 31.7%. The seropositivity prevalence in participants who received the pandemic H1N1 vaccination was 60.9%. Only 53.1% of the pandemic influenza A (H1N1 2009) seropositive individuals who had not received the vaccination experienced respiratory tract infection symptoms. Multivariate logistic regression revealed that factors such as age, occupation, dwelling type, whether the participant's family included students in school, and the vaccination history with pandemic influenza A (H1N1 2009) were associated with antibody titers (p<0.05). CONCLUSIONS: Our data indicated that almost 30.0% of the residents had appropriate antibody titers against pandemic influenza A (H1N1 2009) in Beijing, and these titers may provide an immune barrier.

[A survey on serological epidemiology of influenza A (H1N1) 2009 in Beijing].

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, 6 - 17, 18 - 55, ≥ 56 year olds were 14.5%, 19.4%, 17.4% and 8.0% respectively (P = 0.009). There was no significant difference in the sero-positivity between males and females (P = 0.693). The age-adjusted positive rate was 15.8% in the population of Beijing. By multivariate logistic regression 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].

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.

[The significance of different sample types in study of pandemic A (H1N1) influenza diagnosis].

OBJECTIVE: To explore the value of different types of samples, including throat swabs, stools, bloods in pandemic A (H1N1) influenza diagnosis and virus shedding patterns. METHODS: 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. RESULTS: 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. CONCLUSION: 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).

[Investigation on the source of the first human of avian influenza A (H5N1) case in Beijing].

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.

RecA-ssDNA filaments supercoil in the presence of single-stranded DNA-binding protein.

Using atomic force microscopy (AFM), we find that RecA-single-stranded DNA (RecA-ssDNA) filaments, in the presence of single-stranded DNA-binding (SSB) protein, organize into left-handed bundles, which differ from the previously reported disordered aggregates formed when SSB is excluded from the reaction. In addition, we see both left- and right-handedness on bundles of two filaments. These two-filament supercoils, individual filaments, and other smaller bundles further organize into more complicated bundles, showing overall left-handedness which cannot be explained by earlier arguments that presumed supercoiling is absent in RecA-ssDNA filaments. This novel finding and our previous results regarding supercoiling of RecA-double-stranded DNA (RecA-dsDNA) filaments are, however, consistent with each other and can possibly be explained by the intrinsic tendency of RecA-DNA filaments, in their fully coated form, to order themselves into helical bundles, independent of the DNA inside the filaments (ssDNA or dsDNA). RecA-RecA interactions may dominate the bundling process, while the original conformation of DNA inside filaments and other factors (mechanical properties of filaments, concentration of filaments, and Mg(2+) concentration) could contribute to the variation in the appearance and pitch of supercoils. The tendency of RecA-DNA filaments to form ordered supercoils and their presence during strand exchange suggest a possible biological importance of supercoiled filaments.

Methods of stretching DNA molecules using flow fields.

Using fluorescence microscopy, we compare the degree of adsorption and stretching of DNA onto surfaces achieved by published stretching methods that use fluid flow: molecular combing, spin-stretching, and air-blowing. Molecular combing uses a receding meniscus to stretch out and deposit the DNA onto a hydrophobic surface. In spin-stretching, we find that the effect of radial hydrodynamic flow created by the centrifugal force of the rotating disk is minimal and that the DNA is stretched out on a hydrophobic substrate by the moving meniscus. In air-blowing, a jet of gas pushes liquid across a substrate, depositing stretched DNA molecules along the way. In our study, DNA molecules either combed or spin-stretched onto hydrophobic surfaces stretch to a greater degree than those that are air-blown; fewer are deposited at pH 8.0 than at lower pH, apparently because at pH 8.0 DNA adhesion occurs primarily only at the DNA extremities and so avoids trapped regions of incompletely stretched DNA, with the side effect that more molecules avoid adhesion altogether. We find by high-speed video microscopy that there is complex droplet deformation and motion during air-blowing, which complicates the deposition and stretching process, leading to radial alignment. Our results are a first step toward understanding and optimizing the various proposed methods of DNA stretching and anchoring onto surfaces, which is important in studying their interactions with proteins.

Atomic force microscopic study of aggregation of RecA-DNA nucleoprotein filaments into left-handed supercoiled bundles.

RecA and its complexes with double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) are responsible for homologous recombination and DNA repair. In this study, we have observed, by atomic force microscopy (AFM), two-filament left-handed superhelices of RecA-dsDNA filaments that further interwind into four- or six-filament bundles, in addition to previously reported left-handed bundles of three or six filaments. Also revealed are four-filament bundles formed by further interwinding of two intrafilament superhelices of individual filaments. Pitches of superhelices of RecA-DNA filaments are similar to each other regardless the number of component filaments, and those formed on Phix174 RFII dsDNA and pNEB206A dsDNA are measured as 339.3 +/- 6.2 nm (690 counts of pitch/2) and 321.6 +/- 11.7 nm (101 counts of pitch/2), respectively, consistent with earlier measurements made by electron microscopy with a much smaller sample size. The study of these structures provides insight into the self-interactions of RecA and RecA-like proteins, which are present in all living cells, and into the general phenomenon of bundling, which is relevant to both biological and nonbiological filaments.