Application Of Q-PCR And Sequencing To Analyze The Virology And Clinical Characteristics Of Children With Respiratory Adenovirus Infections

Research background:Human adenovirus(HAdV)is one of the most common pathogens caused respiratory diseases worldwide.Of the whole pathogens that cause respiratory infections in children,HAdV accounts for at least 5% to 10%.The incidence of pneumonia in newborns and young children with HAdV-infected is more than 20%.Lung and systemic inflammation caused by HAdV is heavier than other viruses.Among children with adenoviral pneumonia,14% to 60% of children have lung-related sequelae.To date,about 90 adenovirus serotypes have been found.The literatures show that there are differences in the types of HAdV circulating in different countries or regions and in the same region at different times.At present,there is a shortage of accurate detection methods for adenovirus infections,and the correlations between adenovirus types and disease severity are still not very clear.Objective:(1)Establish adenoviral qPCR detection method.(2)Investigate the occurrence and subtype distribution of HAdV in children with ARTI in Shezhen,China from 2018 to 2019.(3)Analyze the correlations between virological characteristics and clinical characteristics of children with adenovirus infections.Subjects and Methods:(1)Recruited outpatients or inpatients(? 14 years old)with acute respiratory infections in Shenzhen Third People's Hospital from May 2018 to November 2019.(2)All specimens were detected for HAdV by qPCR.(3)Human adenovirus-positive samples were cultured in Hep-2 cell,isolated strains and original samples were sequenced by Hexon gene PCR,and phylogenetic tree was constructed.(4)Used statistical methods to analyze the correlation between virological characteristics and clinical characteristics of HAdV-positive children.Results:(1)We have successfully established a universal qPCR detection method with the detection sensitivity of 50 copies DNA per reaction.(2)In the research.Positive rate of adenovirus in children in Shenzhen from 2018 to 2019 was 9.98%,of which the highest positive rate in children aged 3 to 6 years was 12.5%,and the lowest positive rate of HAdV(1.85%)was detected in children under 6 months of age.Among 144 HAdVs-positive patients,99 were male,45 were female,and the male to female ratio was 2.2:1.HAdVs were detected every month during the research,with a monthly distribution rate of 3.68%-44.23%.(3)Of 144 HAdV-positive cases,141 samples were sequenced successfully(containing 132 were from live viruses and 9 were from original samples).The types of 141 HAdV were 58 HAdV-B7,50 HAdV-B3,10 HAdV-E4,6 HAdV-C1,6 HAdV-C2,6 HAdV-C6,2 HAdV-C5,2 HAdV-B55 and 1 HAdV-B21.(4)Of the 144 HAdV-positive children,fever and cough were the most common symptoms.39 children had gastrointestinal symptoms in addition to respiratory symptoms.In the diagnosis of HAdV-positive children,pneumonia account for up to about 29%.and severe HAdV-positive patients had ARDS and extrapulmonary complications.Among children with adenovirus infections,66 patients had increased WBC count,94 cases also had increased CRP,and the decreased PLT was in 66 cases.Adenovirus B line is slightly higher than the C and E lines in the incidence of pneumonia in children,but there was no significant difference.The rate of pneumonia and severe pneumonia caused by HAdV-B7 was significantly higher than that of HAdV-B3.Conclusions:(1)Our HAdV detection method had good sensitivity and specificity.(2)The positive rate of HAdV was 9.98% in children with respiratory tact infections from 2018 to 2019 in Shenzhen,China.HAdV infections could occur throughout the whole year with the highest positive rate was in autumn.Most HAdV-positive children were below 6 year-old.The detection rate of male to female was 2.2:1 with significant gender differences.(3)Human adenovirus B,C and E species were prevalent in Shenzhen from 2018 to 2019 with B specie predominant.The major epidemic strains were HAdV-B7 and HAdV-B3.HAdV-B55 type was detected in Shenzhen for the first time.(4)HAdV-B7 had a greater potential causing severe illness,and it is necessary to focus highly on patients infected with HAdV-B7.