Microbiome Analysis Of Children With Acute Respiratory Tract Infection And Phylogenetic Analysis Of Seasonal Coronaviruses

Part I-Epidemiology and microbiome analysis of children with acute respiratory infections in Guangzhou from 2020 to 2021BackgroundAcute Respiratory Tract Infection(ARTI)is one of the leading causes of hospitalization and death in children.The main pathogens that cause respiratory infections include bacteria,viruses,and mycoplasma or chlamydia,and most cases are viral in origin.Common respiratory viruses include respiratory syncytial virus(RSV),parainfluenza virus(HPIV),human metapneumovirus(HMPV),coronavirus(HCoV-OC43/229E//NL63/HKU1),influenza virus,boca virus(HBoV),rhinovirus(HRV),adenovirus(HAdV),etc.In addition to the known pathogens,there are many unknown pathogens that are poorly understood and require further study to determine whether their infection alone or in combination with known pathogens can cause respiratory disease in children.During the COVID19 epidemic,the epidemiological patterns of conventional respiratory pathogens are poorly understood due to the control policies that reduced population movement,making it particularly important to monitor the epidemiological and phylogenetic patterns of conventional respiratory pathogens,identify bacterial resistance genes,and study respiratory microecological components.Multiplex PCR technology and high-throughput sequencing are the most commonly used tools to identify respiratory pathogens,enabling rapid identification of known and unknown pathogens,and the use of this technology can provide important support for early warning and diagnostic prevention and control of respiratory infectious diseases.ObjectiveThe purpose of this research is to identify and analyze the epidemiological patterns of common respiratory pathogens in children in 2020-2021,to fill the gaps in the pathogenic spectrum of childhood respiratory infections in the late stage of the SARS-CoV-2 epidemic,and to deeply analyze the phylogenetic patterns of respiratory viruses with high infection rates and to explore the respiratory microbiome characteristics of children with acute respiratory infections.MethodsA total of 590 respiratory samples from children with respiratory tract infections in Guangzhou Women and Children’s Medical Center from 2020-2021 were collected in this study,specifically including 505 upper respiratory nasopharyngeal swab(NS)samples and 85 lower respiratory bronchoalveolar lavage fluid(BALF)samples,as well as 24 nasopharyngeal swab samples from healthy children.Corresponding clinical information was also collected from patients and healthy children,including symptoms,clinical diagnosis,gender,age,and length of hospitalization.Nucleic acids were extracted from all respiratory samples by magnetic bead method,and 22 common respiratory pathogens were detected by multiplex PCR,including 16 RNA viruses(FluA,FluB,H1N1,RSV-A/B,HPIV-1/-2/-3/-4,HCoV-OC43/229E/NL63/HKU1,HRV/EV HMPV),four bacterial species(CP,MP,Lp,Bp),and two DNA viruses(HAdV and HBoV)to resolve changes in the pathogenic spectrum and epidemiological patterns of respiratory viruses.Genotyping primers were used to identify genetic subtypes of major respiratory viruses to further elucidate the molecular variation trends of viruses.In addition,based on the results of multiplex PCR,the respiratory samples were divided into conventional respiratory pathogen infection group and non-conventional respiratory pathogen infection group,and also divided into severe pneumonia group and mild pneumonia group based on clinical symptoms,and the differences between conventional and non-conventional respiratory pathogen infection groups,severe and mild pneumonia groups were comparatively analyzed in terms of virulence,microbiomics and bacterial drug resistance genes using high-throughput sequencing technology and the CLC Genomics Workbench biomimetic analysis platform.ResultsIn this study,505 NS samples,85 BALF samples and 24 NS samples from healthy children were successfully tested.The positive rates of respiratory pathogens in 505 NS samples were: HMPV(137/27.13%),HRV/EV(134/26.53%),HBoV(48/9.50%),RSV-B(39/7.72%),HPIV3(33/6.53%),HAdV(27/5.35%),HPIV4(24/4.75%),HPIV1(19/3.76%),RSV-A(12/2.38%),HCoV-229E(10/1.98%),MP(7/1.39%),HPIV2(5/0.99%)HCoV-NL63/HKU1(5/0.99%),HCoV-OC43(4/0.79%),Lp(2/0.40%),BP(1/0.20%),CP(1/0.20%),FluB(1/0.20%),FluA(0%),H1N1(0%).The positive rates of respiratory pathogens in the 85 BALF samples were HRV/EV(28/32.94%),HAdV(13/15.29%),HMPV(10/11.76%),HBoV(10/11.76%),RSV-B(4/4.71%),HPIV3(4/4.71%),HPIV1(2/2.35%),MP(2/2.35%),HCoV-OC43(2/2.35%),HPIV4(1/1.18%),RSV-A(1/1.18%),HCoV-229E(1/1.18%),HPIV2(1/1.18%),HCoV-NL63/HKU1(1/1.18%),Lp(0%),BP(0%),CP(0%),FluB(0%),FluA(0%),H1N1(0%).The above pathogens were not detected in healthy children.The results showed that HMPV,HRV/EV,HBoV,RSV-B,HPIV3,and HAdV were the most positive in the upper and lower respiratory samples,and the sequencing results showed that the main prevalent genotypes of HMPV were B2 by using typing primers for PCR or RT-PCR analysis of HMPV,HRV/EV,HBoV,RSV-B,and HAdV genotype,HRV was mainly HRV-A genotype,HBoV was HBoV-1 genotype,RSV-B was BA9 genotype,and HAdV was C genotype.Based on the 22 heavy pathogen detection results,the respiratory samples were divided into conventional respiratory pathogen infection group and non-conventional respiratory pathogen infection group,and the relative abundance of viruses in the above two groups was found to be significantly different by NGS sequencing,in which the viral spectrum of the conventional respiratory pathogen infection group was mainly Picornaviridae,Pulmonaviridae,Paramyxoviridae,Coronaviridae and Parvoviridae,and the non-conventional respiratory pathogen The infection groups were mainly plant-associated viruses;there were no significant differences in virological and microbiomic aspects between the severe pneumonia group and the mild pneumonia group.The upper and lower respiratory samples showed significant differences in the distribution of genera with relative abundance in the top 10(Top 10),with Veillonella being enriched in the upper respiratory tract and Pseudomonas being more abundant in the lower respiratory tract,while most children with respiratory infections did not differ significantly from healthy individuals in the microbial composition of the upper respiratory tract;drug-resistant bacteria were only present in a small number of samples.Conclusions1.In 2020-2021,HMPV,HRV,HBoV,RSV-B,HPIV3 and HAdV are the main pathogens causing respiratory tract infections in children in Guangzhou area,which need to be focused on prevention.2.Phylogenetic analysis showed that the prevalent of HMPV were mainly B2 genotypes,HRV/EV were mainly HRV-A genotypes,HBoV were HBoV-1 genotypes,RSV-B were BA9 genotypes and HAdV were subgenus C.3.The results of NGS revealed that there was no significant difference in the distribution of upper and lower respiratory virus profiles in children with ARTI,and the composition of respiratory viruses in children with mild and severe pneumonia was similar,with Picornaviridae,Pulmonaviridae,Paramyxoviridae,and Coronaviridae predominating.There were significant differences in the microbial community structure of the upper and lower respiratory tracts of children with ARTI,as well as in the respiratory tract of patients with mild and severe pneumonia.4.In this study,eight drug resistance genes: lnu C,Erm B,tet M,mel,Cfx A,Cfx A3,APH(6)-Id were identified from children with ARTI,which provided theoretical basis for the development of drug resistance control measures.Part II-Phylogenetic analysis of seasonal coronavirusesBackgroundAn outbreak of coronavirus disease 2019(COVID-19)caused by SARS-CoV-2 has drawn public attention to coronaviruses.The three highly pathogenic coronaviruses,SARS-CoV,MERS-CoV and SARS-CoV-2,pose an important threat to people’s health and social stability.In addition,seasonal coronaviruses that have been circulating among humans for many years(HCoV-OC43,HCoV-229 E,HCoV-NL63 and HCoV-HKU1)also pose a potential threat to public health.There are no effective antiviral drugs available for the time being and no commercially available vaccines to treat and prevent infection with the four seasonal coronaviruses,and virus genotyping is continuously updated and molecular epidemiological features are not yet clear.The ability of coronaviruses to spontaneously mutate at high frequencies in the spike to generate adaptations to different hosts makes it easier for coronaviruses to spread across species.Therefore,studies on the genomic diversity and evolutionary features of seasonal coronaviruses are urgently needed.ObjectiveThe aim of this study was to determine the genotypes of epidemic seasonal coronaviruses HCoV-OC43,HCoV-229 E,HCoV-NL63 and HCoV-HKU1 in Guangzhou from 2017-2021,and to resolve their genomic diversity and evolutionary features.MethodsA total of 40 HCoV-OC43,5 HCoV-229 E,8 HCoV-NL63 and 8 HCoV-HKU1-positive patients in Guangzhou from 2017-2021 were included in this study.All infected patients were tested positive for low pathogenic coronavirus by RT-PCR.All samples were sequenced by NGS technology,and finally,multiple bioinformatics analysis software was used to resolve viral phylogenetic features and mutation patterns.Results1.Evolutionary analysis of the whole genome sequences of the 40 HCoV-OC43 strains with the sequences in the NCBI database revealed that HCoV-OC43 formed two new genotypes,Genotype J and Genotype K.The results of divergence time analysis of novel genotypes of HCoV-OC43 indicated that the common ancestor of the two new genotypes may have appeared in 2014-2016.Genetic recombination analysis indicated that Genotype J originated from the recombination of Genotype H and I,while Genotype K probably originated from the progressive evolution of Genotype I.2.Phylogenetic analysis of 5 HCoV-229 E spike sequences showed that the prevalent strain in 2017-2021 was mainly genogroup 6.3.Phylogenetic analysis of the whole genome sequences of 8 HCoV-NL63 strains showed that C3 and B genotypes are the main prevalent strains at present.4.Phylogenetic analysis of the whole genome sequences of the 8 HCoV-HKU1 strains showed that genotypes A and B are the current dominant strains.Conclusions1.With the evolutionary of HCoV-OC43,two new genotypes,Genotype J and Genotype K have evolved.2.HCoV-229 E,HCoV-NL63 and HCoV-HKU1 epidemic strains have no new genotypes.HCoV-229 E prevalent strains are mainly and genogroup 6;HCoV-NL63 prevalent strains are mainly C3 and B genotypes;HCoV-HKU1 prevalent strains are mainly A and B genotypes.