Development Of A Method For The Detection Of Pan-human Parechoviruses And Study Of The Whole Genome Sequence Characterization

Objective:1.Since laboratory-diagnosed cases of HFMD samples account for a low proportion of clinically diagnosed cases,and many of the symptoms following parvovirus infection are similar to those of HFMD disease,this study tested samples with clinically diagnosed HFMD cases but EV(-)for HPeV.2.Despite evidence of the presence of HPeVs in a variety of serious paediatric infectious diseases,there is a lack of surveillance for HPeVs in China.This study establishes a test for HPeV to fill the gap in surveillance of HPeV,which has important public health significance and practical application.3.In China,there are few studies addressing the genomic characteristics of HPeV,so this study investigates the molecular typing as well as the evolutionary and recombination patterns of HPeV detected in stool samples from healthy children in Beijing in 2019.Methods:A single-tube real-time RT-PCR assay for all serotypes of HPeV was developed and tested for sensitivity,specificity and reproducibility.The assay was validated using 748 samples from healthy children in Beijing in 2019 and 1097 samples from HFMD in Gansu Province in 2019.Primer sequences for molecular typing were designed for VP1 sequence amplification,and the whole-genome sequences of positive samples were determined using high-throughput sequencing technology,and evolutionary and recombination analysis was performed on VP1 regional sequences and whole-genome sequences.Results:1.A single-tube real-time RT-PCR assay for all genotypes of HPeV was developed.The assay was specific for HPeV only,with the limit of detection(LOD)of 102 copies/?L and a coefficient of variation(CV)of less than 1.5%.2.Of the 748 stool samples from healthy children in Beijing,11 samples tested positive for nucleic acid and all successfully amplified the VP1 region,with molecular typing results for 8 strains of HPeV-1,1 strain of HPeV-4 and 2 strains of HPeV-6.3.Nucleic acid positive samples were isolated using four cell lines,HT-29,A549,Vero and RD,of which two strains(18.2%)showed cytopathic effect(CPE)on A549 cells,three strains(27.3%)showed CPE on Vero cells and eight strains(72.7%)showed CPE on HT-29 cells.-29 cells,and no CPE was observed in RD cells.4.Whole genome sequencing was performed on 11 nucleic acid positive samples using high-throughput sequencing technology,and seven whole sequences were obtained,including six HPeV-1 and one HPeV₆.Recombination analysis revealed that the six HPeV-1 strains had three different recombination patterns,and no recombination was found for HPeV-6.5.1097 HFMD samples from Gansu Province in 2019 were tested for HPeV.The nucleic acid positivity rate was found to be 4.19%,and HPeV was not isolated.Conclusion:1.The real-time RT-PCR assay established in this experiment has good specificity,sensitivity and reproducibility.2.The highest proportion of HPeV infections among healthy children in Beijing in 2019 was type 1,followed by type 6,in line with the worldwide prevalence.3.The most sensitive cell line for HPeV virus isolation was HT-29,but it is worth noting that HPeV-3 could not be effectively replicated on this cell.4.Seven full sequences were obtained using high-throughput sequencing,six for HPeV-1 and one for HPeV-6.Three different recombination patterns existed for the six HPeV-1 strains,with extensive recombination with HPeV-5 and HPeV-7;no recombination was detected for the one HPeV-6 strain.5.Preliminary exploration of the relationship between HPeV and HFMD revealed little association between HPeV and HFMD.