| ?Objective?The studies hope to obtain deeper understanding of epidemiology and clinical presentations of respiratory syncytial virus and mycoplasma pneumoniae,four types of human coronavirus,and hope to improve clinical diagnoses for these pathogens infection.To lay foundation for the development of therapeutic antibodies for HAdv-3 infection,we tried to humanize the murine anti-HAdv-3 neutralization monoclonal antibody which secreting in hybridoma cell strain 3D7.?Methods?1.Throat swabs were collected from children(?14 years old)with acute respiratory tract infections(ARTI)in Guangzhou from January 2012 to January 2014.Nucleic acid was extracted and tested in RSV and MP samples by real-time PCR.Clinical presentations of the patients were recorded for further analysis.2.We collected and tested 11,399 throat swabs from children with ARTI in Guangzhou for the four types of HCoV(HCoV-229 E,HCoV-NL63,HCoV-OC43 and HCoV-HKU1)from July 2009 to June 2016 using real-time PCR.Eleven other respiratory pathogens were also tested for HCoV-positive patients.Clinical presentations of the patients were recorded for further analysis.3.Total RNA was extracted from the murine mAb 3D7 cell lines.The first strand DNA was reversed transcript.Then the kappa and heavy chain variable region gene were amplified by PCR with the upstream and downstream primers.L chain was constructed by cloning the kappa chain of murine mAb into pa-L5 vector which containing the human light chain constant region and signal peptide.Similarly,H chain was constructed by cloning the heavy chain of murine mAb into pa-H2 vector which containing the human heavy chain constant region and signal peptide.The L and H chain were cloned into pcDNA3.1 vector,respectively,and then transfected CHO-S cells to build a stable cell line.?Results?1.Of 3760 patients tested,392 cases were RSV positive(10.4%),and 339 cases were MP positive(9.0%).In RSV-infection pediatric patients,the peak positivity rate of RSV [19.6%(181/923 cases)] was found in patients at the age of less than 9 months.Monthly peak of RSV infection occurred from February to April 2012 [26.8%(136/507 cases)] and from January to April 2013 [18.0%(151/839)],respectively.In MP-positive patients,the peak positivity rate [20.1%(97/484 cases)] was detected from pediatric patients at the age of 6 to 10 years old.Monthly peak of MP infection occurred from July to October 2012 [16.3%(92/564 cases)] and from June to October 2013 [13.4%(124/922 cases)],respectively.More nasal obstruction [28.6%(112/392 cases)] and sneezing [33.2%(130/392 cases)] presented in RSV-positive patients than those in MP-positive patients [15.0%(51/339 cases),20.6%(70/339 cases)] in the upper respiratory tract illness,and there were significant differences(all P<0.001).Less cough [78.1%(306/392 cases)] and expectoration [20.4%(80/392 cases)] presented in RSV-positive patients than those in MP-positive patients [90.0%(305/339 cases),27.1%(92/339 cases)] in the upper respiratory tract illness,and there were significant differences(P<0.001,P=0.032).More wheezing [33.2%(130/392 cases)],shortness of breath [13.5%(53/392 cases)],wheeze rale [29.3%(115/392 cases)],and phlegm rale [18.9%(74/392 cases)] presented in RSV-positive patients than those in MP-positive patients [11.5%(39/339 cases),6.5%(22/339 cases),11.5%(39/339 cases),10.6%(36/339 cases)] in the low respiratory tract,and there were significant differences(P<0.001,P=0.002,P<0.001,P=0.002).More Sound coarse [59.2%(201/399 cases)] presented in MP-positive patients than that in RSV-positive patients [40.1%(157/392 cases)] in the low respiratory tract illness,and there was significant difference(P<0.001).2.Of the 11,399 patients tested,489(4.3%)patients were positive for HCoV,comprising 346 patients positive for OC43(3.0%),65 for 229E(0.6%),60 for NL63(0.5%),38 for HKU1(0.3%).Furthermore,some patients were co-infected with multiple HCoV.The total HCoV detection rate was between 3.3%–5.5% across all age groups investigated.The highest detection frequencies of total HCoV and OC43 were in pediatric patients between 7–12 months of age(p<0.001).The peak seasons for total HCoV,OC43,229 E,NL63 and HKU1 occurred in April 2012(15.3%,25/163),April 2012(14.1%,23/163),February 2011(10.4%,8/77),September 2009(7.7%,3/39)and June 2010(6.2%,5/81),respectively.Fever(?38°C)(p=0.014)and abnormal pulmonary breath(p=0.043)were found to be significant clinical symptoms when single HCoV and co-infections with other types of HCoV or other respiratory pathogens were compared.Furthermore,significant differences in clinical presentation among HCoV types were found to be coughing(p=0.032),pneumonia(p=0.026)and abnormal pulmonary breath(p=0.002).The most common co-infecting respiratory viruses were influenza A virus(21.6%,50/231)and respiratory syncytial virus(21.6%,50/231).3.After three rounds of selection,the CLEIA result showed that the antibody expression level was 20mg/L,and Western blotting showed that the chimeric antibody had the light and heavy chain region.The chimeric antibody had a neutralizing activity by neutralization tests in vitro.?Conclusion?1.RSV infection is mostly found in patients less than 9 months from January to April each year and MP is mostly detected in children at the age of 6 to 10 years old in June to October each year in Guangzhou,and more attention should be paid.However,control and prevention of infection should be conducted all the year round to decrease the hospitalization rate caused by RSV and MP infection.2.Total HCoV and OC43 infected group are mostly found in children between 7–12 months of age and more attention should be paid in spring and autumn to prevent infection.229 E infection is mostly detected in patients between 4–6 months of age and mostly found in spring,and NL63 infection is mostly found in children between 7–12 months of age and high frequncy found in autumn,which should be concerned.In addition,HKU1 infection is mostly found in summer.3.We successfully constructed the eukaryotic expression vector of anti-HAdv-3 mouse/human chimeric neutralizing antibody,and transfected CHO-S to build a stable cell line.The antibody was confirmed having neutralizing activity. |
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