| Part I Development of high through-out liquid chip to detect common respiratory virusesObjectivesDeveloping quick and high through-out liquid chip technique to detect common respiratory viruses in order to response better to emergency respiratory disease outbreaks.Methods(1) Two groups were developed. One includes seasonal H1N1, H3N2, novel H1N1, avain Influenza H5 and Influenza B. The other group aimed to test 15 respiratory viruses, including the emerging ones, human metapneumovirus (HMPV), human bocavirus (HBoV), WU polyomovirus (WUPyV), Coronavirus NL63(Cov NL63) and Coronavirus HKU1(Cov HKU1).(2) Primers and probes design:5 sets of primer and probe were selected according to WHO recommedation for influenza virus testing group, and 15 sets of primer and probe were for respiratory virus. Gene sequences of various viruses were downloaded from GenBank, sequence alignment was proceeded by Clustal X to find conservative gene segments, and using Primier 5.0 and Oligo 6.0 softwares to design virus-specific primers and probes. Tm of different various viruses should be close to each other, and the size of PCR products shoule be in 100-300bp. Using Oligo6.0 to evaluate the primers secondary structure. Then the 15 sets of respiratory primers were divided into 2 pools to conduct multi-PCR. The specificity of probes was evaluated through blast alignment.(3) Coupling of probes with beads:According to manufacturer's instruction to conduct the coupling, and evaluate the coupling effects with validation oligos which were complementary with probes. Influenza virus group was evaluated with seasonal HI, H3, novel A(H1N1), avain H5 subtype and B influenza virus probe coplmeatary validation oligos. Common respiratory virus group was evaluated withCov OC43, HMPV, Flu A and Flu B probe complementary validation oligos.(4) Optimizing of multi-PCR and hybridization conditions:five pairs of primer- multiplex PCR were constructed for influenza virus.2 pools were developed for respiratory viruses,1 was seven pairs of primer-multiplex PCR, the other was 8 pairs of primer-multiplex PCR. Optimize the annealing temperature and concentration of primers through different combination. The hybridization temperature was optimized with mean fluorescence intensity (MFI) value of PCR products and beads hybridization. Specimens which MFI was 5 times or greater than negative control specimens were judged as positive.(5) Assay sensitivity of influenza virus liquid chip was evaluated using serial dilution of tissue culture infectious dose (TCID50) strains which were stored in inhouse laboratory. For respiratory virus liquid chip, the sigle specific primer PCR products (except PIV2) were quantified and serial diluted to evaluate the sensitivity. Different respiratory viruses positive samples were used to evaluate specificity.(6) Specimens detection to verify liquid chip method:108 throat swabs (8 were collected from Influenza Like Illness (ILI) cases in Janurary 2009,100 were collected in Feburary 2010) were tested with both influenza virus liquid chip technique and real-time PCR.88 NPAs (which were collected in September 2008 to December 2009) were tested by common respiratory virus liquid chip method, and the results were compared with conventional PCR (for HBoV and WUPyV testing) and commercial Multi-PCR kit (Seegeen, Korea).Results(1) Single pair primer-PCR to verify designed primers:all 15 pairs of primers were used to amplify specific respiratory viruses, anticipated products were obtained from these amplification. It indicated that these 15 primers can be used to develop the following multiplex PCR.(2) Effects of probe and bead coupling:MFI value of validation oligo was in proportion to its concentration, the minimum MFI value was about 700. All these meaned that probe and bead coupling succeeded.(3) Optimizing of multi-PCR and hybridization conditions:Optimized annealing temperatures of influenza virus group and respiratory virus groups (2 pools) multi-PCR were 60?and 62?respectivly. Opitimized hybridization temperatures of influenza virus and respiratory virus liquid chip methods were 54?and 50?respectivly.(4)Sensitivity of influenza liquid chip assay was as follows:seasonal H1N1 5TCID50/L1, seasonal H3N20.05TCID50/mL, novel A(H1N1) 0.05TCID50/mL, and B 5TCID5o/ml and H510-8ng/?l. Sensitivity of respiratory virus liquid chip assay was as follows:HMPV10"8ng/?L, HBoV10-9ng/?l, WUPyV10-8ng/?l, AdvlO-8ng/ul, Cov OC4310-8ng/?l, Cov 229E10-7ng/?l, Cov NL6310-8ng/?l, Cov HKU110-8ng/?l, PIV110-9ng/?l, PIV3 10-9ng/?l, HRV 10-7ng/?l, RSV 10-8ng/?l, FluA 10-9ng/?l, FluB 10"8ng/?l.(5) No interassay cross amplification of other influenza/respiratoryviruses was observed in both assays.(6)The results of influenza virus liquid chip assay to detect 108 throat swabs were the same as that of Real-time PCR.88 NPA were detected using respiratory virus liquid chip assay. Compared with multiplex PCR (Seegeen, Korean) and single primer PCR, the sensitivity of 15 respiratory viruses liquid chip assay ranged from 50%-100%, specificity was 96.4%-100%, positive expected value was 33.3%?100%, negative expected value was 94.8%?100%. and accordance rate was 93.2%?100%.ConclusionInfluenza/respiratory virus liquid chip assay were developed successfully. These assays can be used as a rapid, sensitive and specific method for major respiratory viruses detection and typing and subtyping influenza viruses. It only takes 6 hours to complete test and the respiratory virus liquid chip assay can test 15 prototype viruses at one time. Part II Analysis molecular characters of emerging respiratory virusesObjectivesTo investigate the prevalence of emerging respiratory viruses, novel influenza A(H1N1) surveillance in all age population and HMPV, HBoV and WUPyV in infants and young children presented with acute respiratory tract infection, and and to identify the molecular characters.Methods(1) Samples collected from Influenza Like Illness (ILI) cases and severe respiratory tract infection in April 2009 to March 2011 were detected through virus isolation or Real-time PCR for influenza virus including novel Influenza A (H1N1). HA genes of 14 novel Influenza A (H1N1) strains isolated from severe/death cases and NA genes of 6 stains and complete genome of 3 strains from death cases were sequenced and analyzed.(2) Nasopharyngeal aspirates (NPAs) were taken from 310 hospitalized pediatric patients in the spring in 2006 and 2008, and autume and winter in 2008 and 2009. And the N and F gene fragments of HMPV were detected by nested PCR, G gene by conventional PCR. Phylogenetic analysis of N, F and G genes was performed. The clinical materials of patients were collected and analyzed. All HMPV-positive samples were examined by multi-PCR for other respiratory viruses. PCR positive samples were used to isolate HMPV with Vero-E6 and LLC-MK2 cells.(3)NPAs were taken from 238 hospitalized pediatric patients in spring, autume and winter in 2008, and spring and winter in 2009. And the NP1 gene fragments of HBoV were detected by PCR. VP1/VP2 genes were sequenced to analyze the variation characters and construct phylogenetic tree. All HBoV-positive samples were examined by PCR and multi-PCR for other 12 respiratory viruses.(4)NPAs were taken from 238 hospitalized pediatric patients in spring, autume and winter in 2008, and spring and winter in 2009, throat swabs were collected from 68 children with upper respiratory tract infection (URTI) in winter in 2009, and 43 children without respiratory tract clinical manifestation in Feburary 2009 to November 2009(control group). WUPyV from all above samples were examined by PCR for VP2 gene segment. All WUPyV-positive NPAs were examined by PCR and multi-PCR for other respiratory viruses.Results(1) Influenza virus:Among 7931 throat swab specimens from patients with ILI and severe respiratory tract infection in Tianjin area from April 2009 to March 2010,1567 (19.8%) were influenza viruses positive. Seasonal H3 (60.8%,185/304) was predomiant subtype detected from April 2009 to September 2009. From October 2009 to March 2010, novel influenza A(H1N1) (76.8%,630/820) became the most frequently detected subtype. From April 2010 to September 2010, Seasonal H3 (77.6%,118/152) was the predominant subtype. Novel influenza A(H1N1) (51.5%,150/291) became predomiant subtypes again from September 2010 to March 2011. Little variation of seasonal H3 subtype influenza viruses was observed in above 4 surveillance seasons, this variation belonged to antigen drift.(2) Novel influenza A(H1N1):Among 1567 influenza viruses,56.3%were novel influenza A(H1N1).25.5% (192/754) severe respiratory tract infections cases were novel influenza A(H1N1) positive. The proportion of novel influenza A(H1N1) in different surveillance seasons were as follows:33.6%(102/304) from April 2009 to September 2009, 76.8%(630/820) from April 2009 to March 2010,0.6%(1/152) fromOctober 2009 to March 2010,51.5%(150/291) from September 2010 to March 2011. Novel influenza A(H1N1) was the predominant subtype in two surveillance seasons (September 2009 to March 2010 and September 2010 to March 2011). Phylogenetic analysis showed that HA, NA, M, NP and NS genes of novel influenza A(H1N1) viruses gathered together with classical swine influenza A(H1N1). PB2 and PA genes came from avain influenza virus. PB1 gene came from human seasonal influenza virus. The high identities(98.2%-99.5%) of HA gene were observed between novel influenza A(H1N1) isolated in Tianjin and WHO recommended vaccine strain A/California/07/2009(H1N1) and China representative strain A/Sichuan/1/2009(H1N1). No variation of receptor binding sites was observed in HA protein. NA amino acid sequence analysis showed that novel A(H1N1) influenza viruses in Tianjin was sensitive with Neuraminidase Inhibitor (such as Oseltamivir).(3) HMPVOf 310 pediatric patients,20(6.5%) were positive for HMPV. The median age of HMPV infected children was 15 months(from 16 days to 9 years old),90%(18/20) of the cases were under 2 months. Phylogenetic analysis of 17 N and 18 F gene fragments showed that 13 HMPV strains were A2b subtype. G gene was more variable than N and F genes. All the 20 HMPV-positive children were subjected to pneumonia. The common clinical manifestations of HMPV infected patients were cough, wheezing, shortness of breath and fever. No significant difference of clinical manifestations between type A and B were displayed. Two patients were coinfected with adenovirus and rhinovirus respectively. HMPV were successfully isolated from PCR positive specimens though it was so difficult, and Vero-E6 was better than LLC-MK2 to isolate HMPV.(4)HBoVOf 238 pediatric patients,17(7.1%) were positive for HBoV. All HBoV infected children was equal or less than 6 years old, the highest infection frequency age group was 6m-12m, infection rate was 16.0%(8/50). All HBoV-positive children were subjected to pneumonia and asthmatic bronchitis.82.4%(14/17) HBoV-positive patients were co-infected with other respiratory viruses.13 HBoV VP1/VP2 genes were successfully sequenced, phylogenetic analysis of VP1/VP2 genes showed that they were all belong to Ib cluster.(5)WUPyVAmong 238 hospitalized pediatric patients,45(18.9%) were positive for WUPyV.The median age of WUPyV infected children was 9 months(from 6 days to 6y), the highest infection frequency age group was=6 months(37.8%,17/45). Among them,28 were male,17 were female.36 cases(80%) were coinfected with other respiratory viruses, the most commonly co-detected with RSV B (35.6%).3 of 68 (4.4%) throat swabs from URTI children were WUPyV positive, significantly lower than the infection rate (18.9%)of low respiratory tract illness(LRTI)(?2= 8.4, P=0.0037). No WUPyV was detected in 43 samples collected from asymptomatic control patients. Conclusions(1) Influenza virus was the most important respiratory virus in spring and winter seasons. The predominant subtypes of the 4 surveillance seasons in April 2009 to March 2011 were as follows:seasonal H3, novel influenza A(H1N1), seasonal H3, and novel influenza A(H1N1). Seasonal H3 variation belonged to antigen drift.(2) Novel Influenza A (H1N1) was first detected in Tianjin in June 2009, and it attained prevalence peak in October 2009 to March 2010(76.8%), then decreased dramatically in April 2010 to September 2010(0.6%), and became the predominant subtype in October 2010 to March 2011(51.5%). Novel Influenza A (H1N1) can lead to severe respiratory tract infection such as pneumonia. Consistent with other reports, novel influenza A (H1N1) viruse was a tri-rearrangment virus. HA, NA, M, NP and NS genes of novel influenza A (H1N1) viruses originated from swine influenza viruses. PB2 and PA originated from avain influenza viruses. PB1 originated from huamn influenza viruses. The gene variation of novel influenza A (H1N1) viruses was not so significant, no Oseltamivir-resistant strain was observed.(3) HMPV, HBoV and WUPyV were detected as high frequency in LRTIs, and coinfection was common with them. A2b was the predominant subtype of HMPV, and all HBoV in Tianjin was clustered to Ib. |
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