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Objective:To isolate and culture WU polyomavirus (WUPyV), and to analyze the genome-wide evolutionary patterns, homology and population dynamics.Methods:Real-time quantitative PCR was used to detect the nasopharyngeal aspirate samples of hospitalized children with respiratory tract infection in Beijing Friendship Hospital during 2020 to 2022. Primary human airway epithelial cells cultured at the air-liquid interface were used to isolate and culture WUPyV. Whole genome sequence of the isolated strain was obtained by Sanger sequencing. For phylogenetic and evolutionary dynamics analysis, the whole genome was compared with the published whole genome sequences in GenBank database.Results:The detection rate of WUPyV was 4.7% (31/659) during 2020 to 2022, and a clinical strain BJ0593 of WUPyV type Ⅲc was successfully isolated. The homology of the whole genome and gene fragments of WUPyV was high. The average evolutionary rate of VP2 gene was about 1.256×10 -4 substitution/site every year, and the population dynamics of WUPyV tended to be flat in the last decade. Conclusions:This study successfully isolated a clinical WUPyV type Ⅲ strain for the first time, which provided the basis for further investigation on the molecular evolution and pathogenicity of WUPyV.
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Washington University(WU)Karolinska Institute(KI) polyomaviruses were first found from the respiratory tract secretion in 2007,successively have detected positive reports from all over the world.ZheJiang,ShenZhen and other areas from our country have also detected these two viruses recent years.WUPyv/KIPyv belong to the polyomavirus genus and infections always occurred in early childhood,the seasonal distribution across different areas,clinical features present for kinds of respiratory symptoms such as fever,cough,wheezing and so on,without specificity,and they often come with other respiratory viruses.The infection rate of WUPyv/KIPyv is higher in immunocompromised patients,patients with-HIV infection may lead to severe disease.However,the pathogenesis,and pathogenic characteristics of WUPyv/KIPyv are not clear,so whether they are respiratory pathogenic agents is still controversal.
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Objective To express the capsid proteins of WU polyomavirus(WUPyV) for research and find antigen for diagnostic value. Methods Coding sequences of capsid proteins of WU polyomavirus by PCR were cloned in prokaryotic expression vector PGEX-20T. Recombinant plasmids were transformed into E. coli BL21(DE3) and induced by IPTG for proteins expression. Recombinant proteins were identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed three bands with molecular relative mass of 69×103, 63×103 and 56×103. The recombinant proteins were recognized by anti-GST McAb. The antigenicity was tested by Western blot using 16 WU polyomavirus positive and 70 negative sera. Conclusion Recombinant VP1, VP2 and VP3 expressed in E. coli can combine with WUPyV-Ab and have good antigenicity. They can be used for further research.
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OBJECTIVE To develop a rapid, sensitive and specific real-time PCR assay for the detection of WU polyomavirus.METHODS The VP2 gene of WU polyomavirus was selected as target gene.The specific primers and TaqMan probes according to the principle of the TaqMan real-time PCR were designed and synthesized,we optimized the PCR reaction,and detected 394 clinical samples.The sensitivity,specificity,reproducibility and dynamic range of the methods were determined.RESULTS The real-time PCR was established and applied successfully to detect the WU polyomavirus.The real-time PCR assay showed excellent linearity between the log of target input and CT value,demonstrating that the assay had a dynamic range of at least 8 logs between 1.0?100 and 1.0?107 copies;the coefficient of variation of intra-assay and inter-assay was less than 5%.Among these 394 specimens,3(0.76%) were positive for WU polyomavirus.CONCLUSIONS The real-time TaqMan PCR is a rapid,specific and sensitive method to detect WU polyomavirus.It can be used in screening large numbers of samples at the same time and establish the solid technological base for clinical diagnosis and epidemiology study.