Analysis Of Hospitalized Neonatal Virome And Genetic Characteristics Of Novel Viruses

Background: The newborns are at the beginning of life,and their orgons and systems are also in the stage of rapid development.They have a special physiological state,and low immune function is one of their main characteristics.The hospitalized newborns with a variety of diseases are impaired in immune function compared to healthy newborns at full term.Hospitals are a gathering place for all kinds of microorganisms,including viruses.Newborns need to be in hospital for different lengths of time.So,the hospitalized newborns may be exposed to viral environments.Objective: Respiratory tract and gastrointestinal tract are important channels to the outside world.However,the blood system can be infected through many ways,for exemple funicle or skin etc.Therefore,we aim to explore and analyze the distribution characteristics of virome in respiratory tract,blood and intestinal tract of hospitalized newborns,and analyze the genetic characteristics and epidemic status of specific viruses.Methods: A total of 285 samples,including nasopharyngeal secretions,blood and fecal samples with 95 samples each(from 195 newborns),were randomly collected from hospitalized neonates in the Department of Neonatology,Affiliated Hospital of Jiangsu University from September to December 2018.The newborns included in this study had different diseases,including neonatal jaundice,neonatal respiratory syndrome,neonatal anemia and other common diseases.Forty-five nasopharyngeal secretions,45 blood samples and 45 fecal samples(from 45 newborns),were used to construct viral DNA library by viral metagenomics method.With the help of high-throughput sequencing and bioinformatics analysis,virus information in the library was obtained and further analyzed based on Gen Bank data in NCBI.According to the results of libraries analysis,large contigs were derived,and the Gap amplification primers were designed in Geneious software.The Gap nucleic acid segments of the newly emerged viruses were amplified by PCR technology.The phylogenetic analysis and recombination analysis of the newly emerged viruses were carried out,and their classification was identified.The primers were designed according to the genomes of newly emerged viruses,and 285 samples was used to screen for positive newly emerged viruses by PCR.Results:1.The distribution of neonatal respiratory,blood and gut virome at the family level: The Picornaviridae accounted for the largest proportion in the respiratory virome(60.03%),followed by the Retroviridae(10.03%);the Retroviridae accounted for the largest proportion(52.77%)in the blood virome,followed by the Poxviridae(9.94%);the Picornaviridae accounted for 97.62% of the gut virome,while other families accounted for very little.2.Genetic analysis and epidemiological investigation of specific viral genomes:(1)The genome length of Human Rhinovirus(HRV)is 7033 bp,and the GC content is 38.2%.An ORF of 6510 bp encoding a polyprotein is predicted.Phylogenetic analysis,recombination analysis and nucleic acid sequence similarity alignment were performed based on the whole genome construction.Based on phylogenetic analysis and nucleic acid sequence similarity comparison,it was identified as HRV-A31 and named as newborn_rhvzjlh.It was uploaded to Gen Bank and the Gen Bank accession number was MT157224.Nasopharyngeal secretions,blood and feces samples were screened for HRV,which each category consisted of 95 specimens.One nasopharyngeal secretion was positive for HRV-C,and four nasopharyngeal secretions were positive for HRV-A.The infection rate of respiratory HRV was 5.26%(5/95).One fecal specimen was positive for HRV-C,and two fecal specimens were positive for HRV-A.The infection rate of intestinal HRV was 3.16%(3/95).(2)The anellovirus genome length was2875 bp,GC content was 37%,and 4 open reading frames(ORF)were predicted.Among them,ORF1 was the largest ORF with a length of 2010 bp and encoded 670AA-associated proteins.Phylogenetic analysis and protein sequence similarity alignment based on ORF1 indicated that it might be a novel anellovirus,named anel-ch-zj,uploaded to Gen Bank,and obtained the Gen Bank accession number(MT157223).Nasopharyngeal secretions,blood and feces samples were screened for anellovirus,which each category consisted of 95 specimens.Three specimens of nasopharyngeal secretions were positive,and the rate of respiratory tract infection was3.16%(3/95).Eight fecal specimens were positive,and the rate of intestinal infection was8.42%(8/95).Two blood samples were positive,and the blood infection rate was 2.11%(2/95).Conclusion: By means of viral metagenomics,the composition of the virome in the respiratory tract,digestive tract and blood of the hospitalized newborns was analyzed successfully,which indicated that the virus diversity of the hospitalized newborns was low.In this study,a recombinant virus and a new virus were found,both of which have been uploaded to Gen Bank:A recombinant human rhinovirus was found in neonatal nasopharyngeal secretions,identified as HRV-A31,which had the highest rate of respiratory infection for an epidemiological survey;A novel anellovirus strain was found in nasopharyngeal secretions of the hospitalized neonates,which had the highest infection rate in intestinal tract for an epidemiological survey.