Study On The Virus Group Of Clinical Respiratory Infectio

Respiratory tract infections(RTI),including the upper respiratory infection(URI)and lower respiratory tract infections(LRI),is a common respiratory infectious disease.Respiratory tract infections are considered to be a major cause of morbidity and mortality,which cause epidemics worldwide and affect people of different ages.According to the 2015 global cause of death ranking,among the top 30 causes of death,respiratory infections ranked in the top five.In addition,lower respiratory tract infection is also considered to be the main cause of morbidity and mortality in children under the age of five.Therefore,the economic burden of hospitalization and public health caused by respiratory infections cannot be ignored.The pathogens that cause respiratory tract infections include viruses,bacteria and fungi,of which the viruses accounted for 68%.Besides,there are nine common viruses that cause human respiratory tract infections,including influenza virus(IFV),human coronavirus(HCoV),human respiratory syncytial virus(HRSV),human rhinovirus(HRV),human parainfluenza virus(HPIV),Human Enterovirus(HEV),Human Metapneumovirus(HMPV),Human Bocavirus(HBoV)and Human Adenovirus(HAdV),etc.Viruses infected humans through different ways,either single infection,co-infection with different types of the same virus or various viruses.In addition,due to the differences of three distributions(time,location and population),there also exist some differences in the prevalence of respiratory viruses.Therefore,clarifying the epidemiological characteristics and composition of the respiratory virome,and obtaining the genome sequence are contribute to make full understanding of the virus evolution,which finally provide a solid foundation for subsequent viral diagnosis,monitoring,prevention,clinical treatment,and the development of drugs and vaccines.With the development of sequencing technology,high-throughput sequencing(HTS)technology has shown obvious advantages in obtaining metagenomic information,and it was increasingly used in the study of clinical samples.However,the parallel disadvantage is that it brings a large amount of host information,resulting in redundant sequencing data,and the whole genome information of the virus can only be obtained in a few samples,which exhibit low cost-effective.Therefore,there have two technical difficulties(ie,pre-processing of clinical samples and subsequent analysis of biological information)need to be overcome before conducting study on the respiratory viruses,especially in obtaining the full sequence of the virus.Considering above problem,this study first established an optimized metagenomic approach for virome detection of clinical pharyngeal samples with respiratory infection.Secondly,we processed the 291 samples with clinical respiratory tract infection that collected during 2012-2018 based the optimized approach,and sequenced by NGS platform.The obtained clean reads were assembled in order to clarify the composition of the virome(including virus species and relative abundance)and to obtain virus-related genome information.Of them,we focused on the genomes of nine respiratory viruses.Finally,we performed in-depth molecular evolution analysis of human coronaviruses(HCoV-NL63,HCoV-229E,HCoV-HKUl and HCoV-OC43)and human parainfluenza virus(HPIV1-4),involving nucleotide and amino acid identity,recombination event,temporal signal,evolution rate,phylogenetic analysis and demographic history,as well as the relationship among important sites(including selection site,glycosylation site,key site mutation and amino acid conservation,etc.),and the potential pathogenicity of sites.First,we selected three pharyngeal samples with COPD and each sample was processed in parallel by the Ml to M4 methods.We evaluated the efficient of viral enrichment under different methods and found that the M3 method was significantly better than M4 in removing human host information.The viral reads obtained by the M3 method are about 22 times higher than those obtained by the M4 method.In terms of sequencing depth and coverage of each site of the genome,the genome coverage without enrichment(M4 method)is 4-32%,while the genome coverage is as high as 99.8%(97.8-99.9%)when enriched by the M3 method.Comparison between the M3 method and M-d method(five times sequencing depth),we found that merely increasing the sequencing depth cannot effectively improve the efficiency of virus detection.By contrast,it added the burden of subsequent analysis.All the above results indicated that the M3 method was superior to the M1,M2 and M4 methods in studying the virome and obtaining the virus sequences.Secondly,based on the M3 method,we processed 291 clinical samples with respiratory tract infection.There are two types of samples,including the samples with known pathogens,and the samples with unknown pathogens.Virome analysis was performed according to the results of NGS.The data of most samples is about 1.5G and the proportion of median value of virus reads is 4.49%,the proportion of maximum value of virus reads is 89.73%,while the number of virus reads of a certain kind of virus accounts over 99%.Various types of viruses were detected in the samples,including DNA viruses,RNA viruses,retroviruses,enveloped viruses,non-enveloped viruses,viruses from vertebrates or arthropods,which often in the state of co-infection.In the known pathogens of 75 samples,the composition of the virome was dominated by human parainfluenza virus 1-3(mainly HPIV3)infection,and the co-infected respiratory viruses also included IFV-A(H3N2 and H1N1),IFV-B,IFV-C,HRSV(A and B),HMPV,HCoV(229E and OC43),HRV(A and C),HBoV,HEV(B and C)and HAdv,etc.The unknown pathogens of 216 samples(H1-H82 and R1-R134)were mainly infected with HAdv,HRSV(A and B),and HRV(A,B,and C),and the co-infected respiratory viruses also included IFV-A(H3N2),IFV(B and C),HMPV,HCoV(NL63,229E,HKU1 and OC43),HPIV,HBoV and HEV(A and C),etc.It can be found that TTV virus was detected in most samples,involving three types of α,β and y.Only a few samples not have the existence of TTV virus.In addition,another virus that frequently detected was the Endogenous retrovirus K113.For the 9 common respiratory viruses,we obtained 181 genome sequences or complete CDS,including 4 IFA,25 RSV,78 HPIV,7 HMPV,18 HCoV,36 HRV,1 HEV,2 HBoV and 10 HAdv.Among them,human rhino virus involves 3 genotypes and 36 subgenotypes,mainly including 24 HRV-A,1 HRV-B,and 11 HRV-C.Notably,we found the reads that associated with human poliovirus strain Sabin I(attenuated vaccine strain),measles virus and Norovirus GIV.Finally,in-depth analyses of HCoVs and HPIVs were carried out based on the obtained sequence information.For the human coronaviruses,18 genome sequences were obtained,including 2 HCoV-NL63,8 HCoV-229E,2 HCoV-HKU1 and 6 HCoV-OC43.Based on the whole genome,we observed two phenomena of recombination events in the obtained HCoVs:intraspecies recombination and interspecies recombination.Accoording to the complete gene S,only HCoV-229E was found to have a strong temporal signal,and the evolution rate was at the level of～×10-4 substitutions/site/year.Based on the MCC tree of full length gene S,we named the generated H78 as a new subgenotype C2 of HCoV-HKU1,and found that the obtained sequence P43 belonged to the reported novel genotype K of HCoV-OC43 in Guangzhou,China.Furthermore,in the RBD region of RMB2 of HCoV-NL63,an amino acid substitution(G534V)was found in the genotype C2 of H45,and the residue G534 was considered to be the key site for the binding of RBD to human ACE2.In the CTD of the S1 subunit of HCoV-HKU1,another amino acid substitution site(H512R)was found in the genotype A of R63,residue H512 was thought to be necessary for binding neutralizing antibodies.For the human parainfluenza virus HPIV1-4,a total of 68 genome sequences were obtained,including 4 for HPIV1,5 for HPIV2,58 for HPIV3 and 1 for HPIV4.We conducted 68 genome sequences with 24 sequences that obtained in previous study.Based on the complete CDS of F gene,HPIV1 was distributed in clade 2 and clade 3,HPIV2 was classified into Gla and G3,HPIV3 belonged to cluster C and distributed in C3a and C3b,and HPIV4 sequence belonged to cluster 4A.Based on the complete CDS of HN gene,HPIV1 was divided into clade 2,which was close to the strains circulated in the United States and Malaysia;HPIV2 was close to the strains circulated in the United States,Malaysia and Croatia,and is distributed in G1a,G1c and G3;HPIV3 sequences belonged to the cluster C and distributed in subclusters C3a,C3b,C3d and C3f;HPIV4 sequences fall into cluster 4A,which was close to the strains that circulated in Henan,China.Above results indicated that HPIV1-4 co-circulated in China,and different subtypes of the same genotype were co-circulated.Among them,HPIV3 was mainly distributed in C3.In addition,potential recombination signal was only found in the HN gene of 1 HPIV3,which indicated that the evolution of human parainfluenza virus is relatively stable.Two models Analysis of the site selection using The MEME model detected two positive selection sites in the F gene and HN gene of HPIV3,namely 10,73 and 5,25,respectively.The FUBAR model showed that there were 3 positive selection sites(amino acid positions 7,73 and 488)and 154 negative selection sites in the F gene of HPIV3,while there was one positive selection site(amino acid position 25)and 148 negative selection sites in the HN gene of HPIV3.Furthermore,in the obtained HPIV3 sequence,we found that two amino acid substitution sites(R73K in F protein and A281V in HN protein)and one negative selection site(amino acid position 398 in F protein)corresponded to neutralization-related sites that previously reported.In conclusion,based on the optimized approach for clinical pharyngeal samples with respiratory infection,and relatively easy-to-implement data processing and analysis process,this study established a standardized and programmed processes from sample preparation to data analysis,which effectively and accurately improved the ability to detect and identify respiratory virome.This study significant to clarify the composition and characteristics of the virome,and to detect the existence of potential viruses,which can provide sufficient evidence support for further basic research,and effectively provide key technical support for the prevention and control of infectious disease outbreaks,and ultimately benefit the development of clinical treatment and public health.