Molecular Epidemiology Study Of Human Adenovirus Infection And Anti-adenovirus Research In Children In Guangzhou Area In 2019

Part 1 Molecular epidemiological characteristics and phylogenetic analysis of children’s human adenovirus infection in Guangzhou area in 2019Background:Human adenovirus(HAd V)is a highly contagious pathogen that can cause acute respiratory diseases(ARD)after infection,such as community-acquired pneumonia.Although adults with normal immune function usually show "flu"-like symptoms or no obvious symptoms after being infected with HAd V.But infection with the HAd V in children often causes lower respiratory infections and even develops into severe,life-threatening pneumonia.The most common pathogen of respiratory infections in children are HAd V-B,C,and E,among which HAd V-3 and HAd V-7 of group B are the most common.This article aims to provide clinical characteristics and molecular epidemiological data of HAd V infection in children in Guangzhou.Methods:First,specific primers were designed for HAd V subtypes to establish PCR typing methods for different HAd V subtypes.Second,respiratory tract samples were collected from children admitted to Guangzhou Women and Children’s Medical Center with respiratory tract infections in 2019,and all of them were positive for HADV by fluorescence quantitative PCR;then,samples were amplified by PCR using type-specific primers,and the results were interpreted by agarose gel electrophoresis.A sample from each subtype was randomly selected for PCR amplification using hexon universal primers,and amplified products were sent to Shanghai Shenggong for sequencing.The obtained sequence and reference sequences of each subtype were used to construct a phylogenetic tree using MEGA 6.0 to confirm the molecular subtype.We also amplified Hexon,Penton base and Fiber of different subtypes of HAd V,and amplified products were sent to Shanghai Shenggong for sequencing.The obtained sequences and reference sequences were analyzed by using Bio Edit and MEGA 6.0 software,and the phylogenetic tree were beautified by using i TOL online software.Clinical data were collected,and SPSS software was used for statistical analysis of clinical features.Results:Among 1203 HAd V-positive samples,1128(93.77%)were detected as HAd V-B group,and the most prevalent subtype of HAd V was HAd V-7,accounting for 46.30%,followed by HAd V-3(40.57%).In this study,there were 286 cases(23.77%)of HADV co-infection with other pathogens,among which Mycoplasma pneumoniae(MP)co-infection was the most common.Most of the children were 1-3 years old,followed by3-6 years old,1 year old and below.Among hospitalized children,HAd V-7 was the main epidemic subtype,and the peak period of infection was from April to June;among outpatients,HAd V-3 was the main epidemic subtype,and the peak of infection was from September to December.For severe inpatients,their mean hospital stays was 24.65 days,which was approximately twice that of mild inpatients(P<0.001).Compared with mild inpatients,the serum levels of AST(P<0.001),ALT(P<0.001)and LDH(P<0.001)in severe inpatients were significantly higher;albumin(P<0.001),serum calcium Ions(P<0.001)and alkaline phosphatase(P<0.001)were significantly decreased.Phylogenetic analysis shows that the genome of HAd V subtypes in this study is relatively stable during transmission,and has a very close relationship with virus strains circulating in many places in China in recent years.Conclusions:In 2019,the main subtype of HAd V infection among children in Guangzhou area was HAd V-7,followed by HAd V-3.Children aged 1-3 were the main population affected by HADV.Inpatients were mainly infected with HAd V-7,while outpatients were mainly infected with HAd V-3.Severe inpatients with HAd V infection showed longer hospital stays and higher serum levels of AST,ALT and LDH.Phylogenetic analysis showed that the HAd V subtypes in this study were closely related to the virus strains circulating in China in recent years.Part Two: Preliminary study on the effect of selenocystine against human adenovirus 7 in vitro Background:HAd V-7 is one of the most common pathogens that cause respiratory tract infections in children,and it has a high morbidity and mortality rate.At present,there is no vaccine and specific antiviral drugs for HAd V-7 available for clinical use.Therefore,it is necessary to increase the research of antiviral drugs for HAd V-7.The effect of selenium-containing compound selenocysteine on the replication and proliferation of HADV-7 was investigated.Methods:The rAD7EGFP(HAd V-7 virus strain with fluorescent label)was used to infect A549 cells.Cytopathic effect(CPE)and fluorescence intensity were used to judge the inhibitory effect of selenocystine on HAd V-7.The protein expression of p53 was detected by Western blot assay.Results:72 hours after rAD7 EGFP infects A549 cells,a large number of cells showed green fluorescence under the inverted fluorescence microscope,indicating that rAD7 EGFP infects host cells and replicates and proliferates to produce progeny viruses;.Two hours after Rad7 e GFP infection,host cells were treated with selenocysteine(2 μM,4 μM,8 μM)for 72 hours,and the green fluorescence in cells was significantly decreased under fluorescence inverted microscope,indicating that selenocystine can effectively inhibit rAD7 EGFP infection of A549 cells.Western blot results showed that rAD7 EGFP infected A549 cells up-regulated the expression of cellular p53 protein.Two hours after rAD7 EGFP infected A549 cells,treated with selenocystine(8 μM),selenocystine(8 μM)was significantly down-regulated Cell p53 protein expression.Conclusions:In this study,it was preliminarily explored that selenocysteine could significantly inhibit the replication and proliferation of HADV-7 and regulate the expression of p53 protein in cell models in vitro.