| Acute respiratory infection(ARI)is one of the most common infectious diseases in the world.It is a major threat to children's health and has become the second leading cause of death among children under 5 years old.Viruses are main causes of respiratory tract infections,and common viral pathogens include influenza virus,respiratory syncytial virus,parainfluenza virus,rhinovirus,human metapneumonia virus,human coronavirus,human bocavirus and adenoviru.Human metapneumonia virus(HMPV)is a new member of paramyxovirus subfamily and was isolated in 2001.People of all ages can be infected with HMPV,mainly in infants,the elderly and the immunosuppressive population,and there are no approved vaccines and treatments.WU polyomavirus(WUPyV)was detected in nasopharyngeal aspirates of children with pneumonia by next generation sequencing in 2007,the detection rate of WUPyV was relative high in respiratory samples,and the infection mechanism is unclear due to lack of cell culture model.In the present study,HMPV and WUPyV were isolated and identified on tranditional cell culture and HAE 3D model,and the epidemiology and genomics of HMPV and WUPyV were investigated to lay a foundation for research of infection mechanism,drug evaluation and disease control and prevention of HMPV and WUPyV viral infections.The results are as follow:A.Molecular epidemiology of HMPV and WUPyV in hospitalized children with ARIs in Beijing,China1.A total of 1,276 nasopharyngeal aspirate samples of hospitalized children with ARIs were collected from April 2017 to March 2018,the positive rates of HMPV and WUPyV infection detected by Real time PCR were 4.08%(52/1276)and 5.96%(76/1276),respectively.2.HMPV infection mostly occurred in winter and spring,and peaked in March.90.38%(47/52)of children aged ?4 years,36.54%(19/52)were co-infection,and most of them were double infection(73.68%,14/19).HMPV infection mainly diagnosed as pneumonia(55.77%,29/52)and bronchitis(44.23%,23/52).WUPyV has no significant seasonal variation.92.11%(70/76)of children aged ?4 years,and 60.53%(46/76)were co-infected,double infection and triple infection were easy to see.WUPyV infection mainly diagnosed as bronchitis(68.42%,52/76)and pneumonia(30.26%,24/76).Addtionally,the main clinical sighs in the positive patients for HMPV and WUPyV were fever,cough.3.The viral loads of HMPV positive samples were ranged from 7 copies/?l to 9.61 × 104 copies/?l,there was no relation between viral loads and clinical characteristics,and there was also no statistical difference in the viral loads between HMPV mono-infection and co-infection(P=0.398).The viral loads of WUPyV positive samples were ranged from 3 copies/?l to 1.36×108 copies/?l,there was no relation between viral loads and clinical characteristics,and there was also no statistical difference in viral loads between WUPyV mono-infection and co-infection(P=0.251).4.Evolution analysis of HMPV N gene showed that A2b,B1 and B2 subtypes were prevalent in Beijing from April 2017 to March 2018,subtype of B1 was the most common(50.00%,26/52),clinical characteristics and viral loads were not associated with HMPV subtypes.The dominated subtype of WUPyV was gene cluster I(96.05%,73/76)based on phylogenetic analysis of WUPyV VP2 gene.B.Genome amplification of HMPV and WUPyV and their basic genomic characteristics analysis1.6 HMPV complete genomes and 2 WUPyV complete genomes were obtained by next generation sequencing and sanger sequencing.5 HMPV complete genomes were classified as A2b subtypes,which were most closed to gz01 strian in genetic distance,another HMPV complete genomes was classified as B1 subtypes,which was the first reported in China.2 WUPyV complete genomes were classified as cluster I,belong to the same branch with WUPyV derived from the other areaes in China before.2.Complete genomes analysis showed that N gene of HMPV is the most conservative and G gene has the largest variation.All WUPyV protein coding regions were highly conservative and VP2 is the most conservative gene.Phylogenetic tree analysis indicated that the topological structure of the HMPV N gene and M2-2 gene were different from that of the whole genome sequence.No recombination was found for both HMPV and WUPyV by the Simplot analysis.C.Isolation and identification of HMPV and WUPyV1.HMPV was isolated from clinical samples using LLC-MK2 and Vero-E6 cells culture and HAE 3D model,which was proved by nucleic acid detection and immunofluorescence.HMPV has similar proliferation characteristics on passage cell and HAE 3D culture model.2.Nucleic acid detection has showed that WUPyV can be isolated from clinical samples in HAE 3D model.No viral proliferation was detected in the culture of the WUPyV on the five passage cells.3.HMPV is a kind of irregular spherical particle with envelope and spikes,diameter between 150-600nm observed under transmission electron microscopy.Additionally,WUPyV was a regular spherical particle with no envelope and spikes,and about 45-50nm in diameter.Conclusions:1.HMPV and WUPyV are common virus in RTIs pediatric patients in Beijing,China,especially in patients aged ?4 years.HMPV were prevalent in winter and spring,and WUPyV has no seasonal characteristics.Both HMPV and WUPyV infection were accompanie with other respiratory viruses.There was no relation between viral loads and clinical characteristics,and the presence or absence of mixed infection with other viruses was independent of viral load.HMPV-B1 and WUPy V-I were the most frequent subtypes during the one-year study period,but the clinical characteristics was not related to subtypes of HMPV.2.6 HMPV complete genomes and 2 WUPyV complete genomes were obtained.HMPV genome is highly variable and WUPyV genome is very conservative.Two or more genes of HMPV(P,M,F,SH,G and L)should be at detected simultaneous for HMPV subtype identification.3.HMPV strain was successfully isolated from clinical samples using HAE 3D model and passage cells.WUPyV strain was isolated for the first time using HAE 3D model. |
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