| Nowadays,world is highly globalized,and new outbreaks of viral infectious diseases seriously endanger the lives,health and safety of people all over the world.There are a large number of unknown virus species in the biological world.They may develop and evolve over time to become an infectious pathogen and cause serious new infectious diseases.Due to the lack of knowledge of the genetic information of unknown viruses,the existing clinical testing methods are unable to detect and respond.In recent years,molecular biological research and detection methods of viruses have been intensified,and next-generation sequencing technologies have developed rapidly.High-throughput detection methods are often used in the discovery and detection of new unknown viruses.The metagenomic detection of viruses has become an important means to discover and obtain genetic information related to unknown viruses.Yunnan is an important border of our country,bordering Burma,Laos,and Vietnam.Due to geographical,historical and cultural reasons,Yunnan has close contacts with neighboring countries and the population is highly mobile.Moreover,due to economic development,the population flows into our country in one direction.The influx of the virus into China caused the spread of the disease in Yunnan Province because of the pathogenic virus carried by the entry personnel.In recent years,due to imported cases from countries with severe dengue fever epidemics such as Myanmar,Laos,Vietnam,etc.,dengue fever cases continue to appear in Yunnan Province.Moreover,the climate and geographical location of Yunnan are also suitable for the prevalence and spread of Zika virus,and cases of Zika virus infection have also been reported in neighboring countries.Therefore,the serovirus group of fever patients in the Yunnan border port area was researched and analyzed,so as to prevent the transmission of viral diseases at the source and realize the border shift.Methods:(1)Five groups were selected for pre-experiment,after exploring the best plan for serum sample preparation;(2)Viromics analysis method combined with bioinformatics analysis using NGS technology to investigate dengue in the border areas of Yunnan,my country Fever patients with negative Dengue virus screening are carrying the virus;(3)Using semi-nested PCR,genome walking,and terminal RACE to obtain the whole genome sequence of key viruses;(4)Using evolutionary analysis,the research obtains the genetic evolution characteristics of the virus(5)Use recombination analysis to study the possible recombination relationship between newly identified viruses and known viruses to analyze the recombination and disease risk of the virus.Results:(1)After passing five sets of pre-experiments,according to the sequencing results,the serum samples were centrifuged and filtered,followed by enzymatic digestion to extract viral RNA,and then random PCR amplification.After electrophoresis,the length of more than 500bp was cut and recovered.After preparing the prepared serum samples and then establishing a cDNA library for metagenomic sequencing,the virus sequences obtained are the most.(2)270 serum samples are divided into 93 groups for virus enrichment,and NGS is performed to obtain the Raw data 160G,quality control data(HQ data)113G,after removing human sequences and bacterial sequences,the remaining virus sequences accounted for 8%;the average virus sequence obtained for each sample:43682(median 6198).(3)Among the 93 samples,the viruses were mainly concentrated in single positive-stranded RNA viruses,mainly Flaviviruses and Enteroviruses.Among the detected viruses,the main pathogenic viruses are Dengue virus(DENV),mainly DENV 4;Herpesvirales;Retroviridae;Hepatitis B(HBV);Enterovirus(EV).This batch of samples also contains animal viruses,such as Murine leukemia virus;Parvovirus;Iridovirida virus.At the same time,this batch of samples also contains virus sequences that cause plants to disease,such as Tobacco mosaic virus and Melon necrotic spot virus.(4)Because 47 of the 93 samples contained Enteroviruses,25 samples contained EVA71,and 17 samples contained CVA16 virus,which is the main pathogen of hand,foot and mouth disease,and Enteroviruses are mainly pathogenic to children.If carried by adults,it will increase the possibility of infection in children.Therefore,101 individual samples and mixed samples were amplified by semi-nested PCR using Enterovirus VP1 universal primers to obtain 11 sets of positive samples,and separate samples from the mixed group were obtained.After amplification,sample No.3486 and No.3390 serum were added to the cells for culture.Sample No.3390 failed to isolate and culture virus,and Sample No.3486 was successfully cultured.After culture,viral nucleic acid was extracted to obtain a complete Enterovirus sequence.(5)Sample No.3390 was combined with Sanger sequencing based on the NGS results to obtain the complete VP1 gene nucleotide sequence.The nucleotide homology between the obtained VP1 segment and Coxsackievirus A6464-QJ-YN-CHN-2019-CV-A6 is as high as 99.78%.And the reference strain was sampled in Yunnan in 2019,which suggests that this serotype Enterovirus continues to circulate in Yunnan.The amino acid homology between the VP1 segment of the Enterovirus isolated and cultured from sample No.3486 and Echovirus E4 is 97.50%,and the nucleotide homology is between 80.63%and 89.9%.The amino acid homology between its P1 segment and Echovirus E4 is 97.64%,which is in line with the existing Enterovirus typing standards.Therefore,it can be judged that the sequence is Human echovirus 4 of the EVB group in Enteroviruses.Since its whole genome sequence does not have high homology with the obtained Echovirus 4,the obtained virus may be a new isolate of Human echovirus 4.After recombination analysis of the obtained Enterovirus sequence,signs of recombination were found in segments 2A,2B,2C,3A,3B,3C,and 3D:signs of recombination were found in segment 2A and Echovirus E30 4-MRS2013,and signs of recombination were found in segment 2B and Human Enterovirus 85 strain HTYT-ARL-AFP02FXJCHN2011 found signs of recombination,found recombination signs in 2C and Enterovirus B80 KOUAN67/XZ/CHN/2010 strains;found recombination signs in 3A and Echovirus E14 E14P9682013China;in 3C,3D and Human Echovirus 30 1167438 phMC strain found signs of recombination,which suggests that a recombination event may have occurred.Conclusion:This study conducted an in-depth study on the serum virus group of patients with fever in Yunnan,and understood the spectrum and prevalence of potential viral pathogens in the Yunnan port area.The population in the border area of Yunnan is highly mobile and carries complex types of viruses.It not only carries viruses that are pathogenic to humans,but also viruses that are pathogenic to animals and plants.Viruses that are pathogenic to humans include HBV,Herpes virus,Enterovirus,etc.Enterovirus may be a common epidemic pathogen in my country's Yunnan port area.focusing on Enterovirus research,through NGS technology combined with Sanger sequencing technology,a complete VP1 gene of CVA6 was obtained.Sequence;and a strain of Echovirus 4 was isolated by cell culture.Enterovirus has weak pathogenicity to adults and strong pathogenicity to children.However,adults carry Enterovirus and spread to children through intestinal routes,contact and droplets,which leads to the epidemic of Enterovirus in children.,Causing serious consequences;and enterovirus recombination is complicated,virus typing is more,and virus culture is complicated.Therefore,this study has new discoveries about the transmission route of Enteroviruses that are more likely to be prevalent in children.Enteroviruses may Latent infection from adults and then to children,causing disease in children.Therefore,we also need to focus on and prevent in advance. |
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