Viromics Study Of Respiratory Samples From Hospitalized Children With Acute Respiratory Infection

Acute respiratory infection is one of the main causes of acute disease and death in humans worldwide.Among them,acute viral respiratory infection is the main cause,accounting for about 80%.Common respiratory viruses include influenza virus,respiratory syncytial virus,coronavirus,adenovirus and rhinovirus.Mycoplasma pneumoniae is also a common pathogen that causes respiratory infections.Next-generation sequencing(NGS)is a high-throughput unbiased sequencing technology that can detect thousands of pathogens at the same time,enabling comprehensive and quantitative analysis of all microorganisms present in clinical samples.NGS detection mainly includes two parts:experimental operation(wet laboratory)and bioinformation analysis(dry laboratory).Wet laboratory operations include sample pretreatment,nucleic acid extraction,library construction and sequencing.This study explored the pretreatment methods and nucleic acid amplification methods of NGS samples,and provided technical references for metagenomics analysis of clinical respiratory samples.Since the relationship between Mycoplasma pneumoniae infection and viral infection is still unclear,this study clarified the virus spectrum of Mycoplasma pneumoniae positive respiratory samples and common respiratory virus mixed infections through metagenomic analysis of collected nasopharyngeal aspirate samples.In addition,the complete genome sequences of two HRS V strains and one HAdV strain were obtained,and the whole genome feature analysis was carried out to lay the foundation for the formulation of disease treatment and prevention and control strategies.The results are as follow:A.Exploring the pretreatment method of second-generation sequencing of clinical respiratory samplesExploring the mNGS pretreatment and amplification methods of clinical respiratory tract samples,using HAdV and HRV respiratory nasopharyngeal aspirate samples as the detection template,filtering,nuclease treatment and amplification(MDA and SIPA),compare different sample treatments the sequencing results matched the reads percentage,coverage and sequencing depth of HAdV and HRV.The results showed that the matching rate of HRV and HAdV whole gene sequences in the respiratory tract sample filtering group was higher than that of the unfiltered group,but the difference in coverage was not obvious.Nuclease pretreatment of clinical respiratory tract samples can increase the matching rate and sequencing depth of HRV and HAdV reads.For HAdV and HRV,there is no significant difference in the virus matching rate between the sequencing results amplified by SIPA and MDA,but the whole genome coverage rate after MDA amplification is higher than that of SIPA.For HAdV,MDA has a more uniform sequencing depth than SIPA.B.Study on Mycoplasma Positive and Negative Pathogen spectrum of Respiratory Tract Samples from Hospitalized Children1.In this study,the MP infection rate among hospitalized children under 6 years old in Beijing was 43.7%.The infection rate of MP in children aged 5-6 is the highest,and there is no obvious seasonal epidemic.2.Pathogen profile analysis results showed that a variety of common virus-related sequences were detected in both MP positive and negative respiratory samples,matching 11 virus families,and the most reads were pneumoviridae,and a large number of reads matched to ring virus family-related viruses and phage related sequence.3.Real Time PCR method detects common respiratory viruses co-infected with MP.Among 83 MP-positive samples,47 cases(56.63%)were combined with viral infections,including 38 cases(45.78%)with single virus infection and 7 cases(8.43%)with double infection and 2 cases of triple infection(2.41%).Among them,the most were infected with IFV(20 cases,24.1%).Among 102 MP-negative samples,66 cases(64.7%)were infected with the virus,of which HRSV infection was the most common(21 cases,20.6%).4.There was no significant difference between the length of hospitalization,the length of fever,serum creatine kinase isoenzyme and blood white blood cell count of patients with MP combined with virus infection and those with MP single infection.C.Analysis of whole genome characteristics of HADV and HRSV1.In this study,two HRSV strains and one HAdV genome sequence were obtained from the Beijing area through second-generation sequencing.Both HRSV strains were of the ON1 genotype,and HAdV belonged to the B3 genotype.2.Simplot analyzed the G protein,F protein and L protein of the two HRSV whole genome sequences with large variation.Many sites have amino acid sense mutations,and some sites are located in the P27 peptide and the antigen site(?).Both HRSV F proteins have 6 N-glycosylation sites,and G protein has 3 N-glycosylation sites.3.Based on Penton base,Hexon and Fiber genes,SNP analysis of HAdV showed that the sequences of the three genes are highly conserved.Simplot analysis showed that no recombination event occurred in HAdV.Conclusions:1.The sequencing results of the filtered samples were better than the unfiltered samples;the sequencing results of the nuclease-treated group were better than those of the non-nuclease-treated group;the sequencing results using different amplification methods had no significant difference in virus matching rate.2.Using mNGS technology to obtain the virus spectrum of MP-positive and negative children.The virus families with the highest number of reads in the two groups were all pneumoviridae.The number of reads matched to HRSV in the MP-positive group was much higher than that in the MP-negative group,and the MP-positive combination had the most cases of IFV infection.3.The genome sequences of two HRSV strains and one HAdV strain in Beijing area were obtained.The two HRSV strains have large variations in the G,F and L protein regions;HAdV is highly conserved in the three gene sequences,and no recombination event has occurred.