Isolation and Identification of Human Parainfluenza Virus 1/2/3 from Hospitalized Children in China Using An Air-liquid Interface Human Airway Epithelial Organoid
Human parainfluenza viruses(hPIVs)are one of the most important pathogens causing human respiratory tract infections.The resources of relevant clinical strains in China are limited.This study aimed to obtain recently prevalent clinically isolated hPIVs with clear background.Primary human respiratory epithelial cells differentiated from human airway epithelial(HAE)organoid with air-liquid interface were applied to isolate hPIV1/2/3 from hPIVs-positive throat swab specimens of hospitalized children in Shenzhen Third People's Hospital from 2016 to 2017,named hPIV1/C-Tan/SZ01/2016,hPIV2/C-Tan/SZ01/2017,and hPIV3/C-Tan/SZ01/2017,respectively.hPIV1/C-Tan/SZ01/2017 was detected by fluorescence reverse transcription-quantitative polymerase chain reaction(RT-qPCR)to detect viral nucleic acid amplification.Immunofluorescence was employed to measure expression of viral proteins in HAE Transmission electron microscopy(TEM)of negative staining and ultrathin sections was carried out to observe viral morphology and cellular localization.We also analyzed viral activity,genetic stability,and genomic characterization during TEM.The results showed that the isolation of hPIVs based on HAE could efficiently obtain clinical isolates of hPIVs,and the obtained strains had typical viral morphological features.The three hPIVs replicated well and were genetically stable in HAE,and viral titers could reach to 107 TCID50/100 μL.The whole genome and HN gene of hPIV1/2/3 clinical isolates were sequenced.hPIV1,with a total length of 15,568 bp,belonged to hPIV1 C isoform.hPIV2,with a total length of 15,694 bp,belonged to the hPIV2 A3 isoform.hPIV3,with a full length of 15443 bp,belonged to hPIV3 C3 isoform.The results suggested that these three clinical isolates were consistent with the genotypes prevalent in China.In conclusion,we applied HAE to successfully isolate hPIV1/2/3 strains with clear biological characteristics and genomic background,as well as genetic stability,from clinical samples of recently hospitalized children.The isolation and identification of these clinically prevalent strains lay the foundation for:(i)in-depth study of the infectious characteristics and pathogenic mechanisms of hPIVs;(ii)antiviral-drug screening and vaccine development.
Human parainfluenza virusVirus isolation and identificationHuman airway epithelial organoid
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