| ObjectiveAcute respiratory infections(ARI)are always the most common and the highest incidence of human diseases,they have a complex and diverse etiology and are difficult to identify from the symptoms of patients.Guangzhou is always the warehouse of respiratory pathogens because of its special geographical location,hot and humid climate,adjacent to Southeast Asia,the high frequency of international and national personnel exchanges,it is very important for disease control and prevention to research on accurate diagnosis and epidemiology of respiratory pathogens.Methods(1)The throat swabs from patients with ARI(?14 years old)were collected and tested for 18 common respiratory pathogens detection with real-time PCR from July 2009 to June 2016 in Guangzhou,China,and local meteorological data were also collected including monthly mean temperature,average relative humidity,average wind speed,Mean air pressure,mean vapor pressure and sunshine hours.The data were used to study epidemiologic feature and correlation relationship with climate factors of respiratory pathogens.(2)RSV M2-1 gene is known as transcription anti-termination factor.M2-1 was cloned into plasmid pc DNA3.1(+),the stable Hep-2 cell expression M2-1 were obtained through transfection and screening.The growth characteristics and RSV infection characteristics of stable Hep-2 cell lines expression M2-1 were analyzed.(3)Based on the Taqman real-time PCR technique,multiple fluorescent quantitative PCR was performed on the RSV-A,RSV-B G gene specific conserved region and the housekeeping gene ACTB.The multiplex PCR was used to validate its specificity through various respiratory pathogens detection.Its sensitivity and standard curve were identified with transcription RNA in vitro.The throat swabs and clinical presentation of patients with ARI(?14 years old)were collected for RSV-A and RSV-B analysis from January 2013 to December 2015 in Guangzhou.The comparative viral load CVL method was used for the first time to analyze the infection of RSV-A/RSV-B and the clinical manifestations of patients,and to explore the feasibility of using internal reference gene for quantitative analysis.Results(1)In this study,11399 throat swabs from patients with ARI were collected over a seven-year period,and 18 common respiratory pathogens were detected.The highest detection rate was RSV(1690/5606,30.1%)?inf A(839/5606,15.0%)?MP(760/5606,13.6%)?ADV(621/5606,11.1%)?HRV(402/3789,10.6%),followed by OC43,HMPV,inf B,PIV3,HBo V with positive rate among 6.2%-4.4%.The positive rates of the remaining 7 pathogens PIV1,CP,PIV2,229 E,NL63,HKU1 and PIV4 were all lower than 4.0%.There were four patterns of pathogens distribution in different age patients: 1)The positive rate decreased with age,including RSV(p<0.001);2)increased with age,including inf A and inf B(p<0.01);3)Infection peak found in patients aged 0-14,including EV,HRV,ADV,MP,PIV3,OC43,HBo V,HMPV(p?0.025);4)No significant difference or too few to analyze,including PIV1,PIV2,PIV4,229 E,NL63,HKU1,CP.In general,the positive rate of the sample increased or decreased as the seasons changed: 1)Positive rates increased: between June and September(summer turning to autumn)and between April and May(spring turning to summer);2)positive rates decreased: between October and December(autumn turning to winter)and between January and March(winter turning to spring).According to multiple linear regression analysis,significant linear relationship were found between the positive rate of the specimen and mean temperature,mean relative humidity,sunshine hours(p?0.002);RSV and mean relative humidity,sunshine hours(p?0.034);MP and the relative humidity(P=0.003);ADV and sunshine hours(p=0.008);EV and mean air temperature,mean relative humidity(p?0.039);HMPV and sunshine hours(p=0.004);PIV3 and mean temperature,relative humidity,sunshine hours(p?0.049);HBo V and mean temperature,mean relative humidity(p?0.024).(2)In this study,Hep-2 2F5 cells stably expressing RSV M2-1 were constructed by transfecting and screening with pc DNA3.1(+)plasmid containing RSV M2-1.Compared with Hep-2 2F5 and Hep-2 cells in growth speed,Hep-2 2F5 proliferated faster than Hep-2;compared with Hep-2 2F5 and Hep-2 cells in RSV infection characteristics,Hep-2 2F5 was more sensitive and susceptible to RSV.However,the virus yield was lower than that of the original Hep-2 cells.(3)In this study,multiple real-time PCR methods were established according to the G-gene specific conserved region of RSV-A and RSV-B and ACTB gene.No non-specific amplification by detection a series of respiratory pathogens.In vitro using transcription RNA templates,RSV-A,RSV-B and ACTB were detected at the lowest 4 copies/?l,8 copies/?l,and 12 copies/?l,respectively,and good linearity were found among 10–1?1010 copies/?l(r2=0.9973),100–1?1010 copies/?l(r2 = 0.9989),and 100–1?1010 copies/?l(r2=0.9984)RNA template concentration for RSV-A,RSV-B and ACTB,respectively.A total of 5483 ARI patients(?14 years)were collected in three years,and 13.3%(729/5483)of total patients were RSV positive.RSV-A and RSV-B positive rate were 51.2%(373/729)and 48.8%(356/729).RSV-B circulated predominantly in 2013 and RSV-A in 2015,whereas in 2014,RSV-B and RSV-A co-circulated.There were significant differences in the frequency of clinical symptoms between RSV-A and RSV-B positive patients,including Coryza,Dyspnoea,Bronchiolitis,Chill,Headache,Myalgia,Debilitation,Vomiting,Poor appetite,Diarrhoea,Rash etc(p?0.01);Different RSV CVL patients were significantly different in the frequency of occurrence of Bronchiolitis,Fever(?38 oC)(p?0.009).Conclusions(1)The study investigated the distribution of 18 common respiratory pathogens in ARI patients under 14 years of age in Guangzhou: 1)understood the distribution of common respiratory pathogens in Guangzhou;2)obtained common respiratory pathogens' age distribution patterns of patients under 14 years of age;and 3)obtained the seasonal distribution of local respiratory pathogens and their correlation with climatic factors.(2)According to the function of RSV M2-1,1)Hep-2 2F5 with stable expression of M2-1 was established on the basis of Hep-2;2)The growth characteristics of Hep-2 2F5 and the characteristics of RSV infection were obtained;3)provided alternative cells for RSV culture isolation.(3)According to the characteristics of RSV subtype gene and ACTB,1)A new RSV subtype multiplex quantitative PCR method was established;2)The epidemiological data of RSV-A and RSV-B were obtained;3)For the first time using the CVL evaluation method for pathogen quantification in respiratory samples.In this study,we hope the work on rapid diagnosis of pathogens;isolation and epidemiology research of respiratory pathogens make some contribution to the prevention and control of disease,clinical diagnosis,drug,vaccine development. |
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