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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.
ABSTRACT
Human metapneumovirus (hMPV) is a newly described paramyxovirus that was found in the Netherlands in 2001. It is an important pathogen of acute respiratory diseases in infants, young children, elderly and immunocompromised patients. hMPV infection could cause mild upper respiratory tract infection or severe lower respiratory tract disease, including bronchiolitis and pneumonia. hMPV was usually detected using direct immunofluorescence and RT-PCR, but the detection method were different according to the respective experiment requirements. In this paper we review the detection method of hMPV to provide a basis for further study of hMPV.
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Objective@#To express envelope protein of ZIKA virus in baculovirus expression system.@*Methods@#Full-length E gene of ZIKA virus was obtained by DNA synthesis and inserted into vector pFastBac1. The constructed recombinant baculovirus transfer vector pFB1-E was transformed to competent DH10Bac cells. The obtained skeleton plasmid rBacmid-E was transfected to sf9 cells, and the constructed recombinant baculovirus rBac-E was determined for titer, for insertion of E gene by PCR, and for expression of E protein by IFA and Western blotting.@*Results@#PCR proved that skeleton plasmid rBacmid-E was constructed correctly. The titer of rBac-E of passage 3 was 2.58×105pfu/ml. The genome of infected cells virus was extracted, the gene band at length of 3 830 bp was observed after PCR amplification. Indirect immunofluorescence of the infected cells showed the specific green fluorescence, 55×103specific band was determined by Western blotting identification in the cell pellet of the infected recombinant baculovirus rBac-E.@*Conclusions@#The recombinant baculovirus with E gene of ZIKA virus was successfully constructed, which laid a foundation of further study on the function of E protein and the vaccine of ZIKA virus.