The Second Generation Sequencing Technology Was Applied To The Study Of The Pathogen Spectrum Of Hand, Foot And Mouth Disease

Hand,foot and mouth disease(HFMD),caused by various enteroviruses(EV),is a common contagious childhood disease that usually attacks children younger than 5 years old.In recent years,numerous epidemics or outbreaks of HFMD have been reported in China,and non-EV71 and non-CVA 16 enteroviruses(other EVs)have frequently emerged to replace the main roles of EV71 and CVA16 in causing outbreaks and have gradually become a predominated pathogen of HFMD.It poses an enormous challenge for accurate identification of viral pathogens in HFMD cases,because of the variety of genotypes of other EVs and the complication of single-or mixed-infectious events.Moreover,some cases are tested negative for specific pathogens(EV71/CVA16/other EVs)both in severe and mild HFMD.The case numbers of negative specimens have largely increased and their pathogenic information is still unclear and less reported.Therefore,to clarify the pathogen spectrum of severe and mild HFMD cases using an accurate diagnostic technology is urgently needed.Next generation sequencing(NGS)has been an important method in precision medicine field.It is a powerful tool without requiring a priori knowledge of sequence information of the potential infectious agents,and enables to parallelly sequencing and comprehensively analyse the nucleic acids sequences of all microbes in a certain biotope.Especially,NGS is an appropriate technique to identify various viruses from complicated cases,such as HFMD.However,enormous limitations impede the clinical use of common NGS,such as high cost,complicated procedure,tremendous data analysis,as well as high backg,round noise in clinical samples.In the present study,an improved NGS platform,a simplified,cost-effective,time-saving and direct identification of viral pathogens procedure,has been constructed using a strategy of barcoded oligonucleotide primers(random hexamers or non-ribosomal hexanucleotides)and one-tube fragmentation-free library of DNA pool.Thereafter,three known representative viruses were used in a model experiment via the NGS platform.We found that all the barcoded oligonucleotide primers enabled to capture viral sequences,and the ratios of virus-related reads captured by hexanucleotide primers were higher than those by hexamer primers,as well as the genome coverage of model viral reads.These results indicate that the non-ribosomal hexanucleotide primers are more suitable for identification of viruses in samples.Then the verified experiment was executed using 20 HFMD clinical specimens via the barcoded non-ribosomal hexanucleotide primers-based NGS approach.We found that this approach acquired viral pathogen information of HFMD and provided further detailed genotypes of enteroviruses,as well as identified other potential viruses or differentiated misdiagnosis event.So these results indicate that the NGS approach can work efficiently to directly detect viral pathogens in clinical specimens.In order to fully understand the virome of severe and mild HFMD cases,study the difference in pathogen components between severe and mild cases,and attempt to analyze the severity on etiology,94 severe cases and 150 mild cases were respectively sequenced by above NGS platform.The data revealed that thirteen mammalian-and plant-virus families were found including Picornaviridae,Caliciviridae,Paramyxoviridae,Orthomyxoviridae,Parvoviridae,Astroviridae,Adenoviridae,Reoviridae,Anelloviridae,Virgaviridae,Alphaflexiviridae,Tymoviridae and Tombusviridae.And the viral populations identified from mild cases were more diverse than that of severe cases.A total of 21 viruses from 7 virus families were identified in severe cases,versus 38 viruses from 12 virus families in mild cases.Moreover,mixed-infectious events among multiple enterovirus serotypes or non-enterovirus were found.All viruses were validated by molecular methods.Then the positive detected cases of viruses were analyzed by Pearson’s chi-square test,and the results indicated that co-existence with respiratory viruses probably tended to be found in severe HFMD(P=0.001),whereas co-existence with diarrheal-related viruses tended to be found in mild HFMD(P<0.001).In addition,the complete genome sequences of sapovirus assembled from NGS data were used to phylogenetic analysis by bioinformatic techniques.The phylogenetic trees showed that this virus was currently circulating in China.Taken together,we successfully constructed an improved NGS platform which is appropriate for direct identification of viral pathogens,and firstly conducted comparative analysis of viral enrichment performance of random hexamer primers and non-ribosomal hexanucleotide primers.Furthermore,we presented the first preliminary descriptions of virus constitutions in pediatric patients with mild and severe HFMD,and revealed the diversity of enteroviruses causing HFMD and the complexity of mixed-infections.These studies provide an accurate method for the clinical diagnosis of the HFMD,and provide preliminary data supports or references for confirmation of HFMD pathogens or relevant epidemics,which show a great significance for prevention and control of HFMD.