Complete Genome Sequencing And Analysis Of The First Imported Middle East Respiratory Syndrome Coronavirus In China

1.Background and ObjectiveMiddle East Respiratory Syndrome Coronavirus is the pathogen of Middle East Respiratory Syndrome.The illness has an incubation period of 9?12 days.The usual symptoms are fever,cough,anhelation,dyspnea and acute renal failure,and sometimes diarrhoea in the population of imnune suppression.According to report,the infection of MERS-CoV will cause serve acute respiratory symptoms and led to atypical pneumonia and even death.Hence,MERS is a type of respiratory infectious disease with a high fatality rate of 40%.It has been a outbreak of this type of disease in Arab since MERS-CoV had been found in 2012.With the expansion of epidemic situation of MERS,more and more death cases had emerged.The new cases of MERS-Cov in Korea have appeared in April,2015 and then it turn into a epidemic outbreak.During the period of the MERS outbreak,the first imported case from Korea emerged in Guangdong,China.More and more researchers began to focus on MERS-Cov because of its high fatality rate.Most cases of MERS infection(more than 97%cases)were emerged in the area of Middle East on the current data of MERS patients.The infection of MERS-Cov has been reported in 20 countries or areas which has epidemic relationship with the area of Middle East.According to statistics of 402 MERS-Cov infectious cases in Saudi Arabia,27%cases belong to hospital staff but 57.8%of them released only mild or none symptoms and don't need medical treatment.All these factors show that MERS-Cov has developed the ability of human to human transmission but no evident shows it has the ability of continued human to human transmission.MERS-Cov is a new type of coronavirus.This new type of virus was named as Middle East Respiratory Syndrome Coronavirus(MERS-CoV)by the International Committee for the classification of viruses.MERS-Cov belongs to type of ? in C group of Coronavirus of Coronaviridae according to its serological characteristics and genome sequence.Virion of MERS-Cov can be polymorphism and close to spherical in shape.There is a protein envelope surrounding the virus and its surface covered with fiber so that the virus looks like a corona crown.The gene of MERS-Cov is single strand RNA with a total length of 30.1kb,so it is the longest genome of RNA virus.The gene of MERS-Cov contains a least 10 Open Reading Frane(ORF)which encode separately a large replication enzyme poly protein(ORF1a/1b),surface spike glycoprotein(S),small envelope protein(E),membrane protein(M),nucleocappsid(N)and 5 non-structural protein(ORF3?4a?4b?5 and 8b)etc.Most of the complete genome sequences in GenBank are isolate from the area of Middle East and Separated from human,camel and bat.The first complete gene of reference strain has released in GenBank(GenBank acessionJX869059).Now the method of laboratory diagnosis is always using RT-PCR to detect the sample of air tube(throat swab,sputum and tracheal aspirate).The positive result of universal primer of Corona virus and the positive signal of primer of upstream E gene(method of upE)or ORF1b gene of Open Reading Frame(method of ORF1b)can prompt the infection of MERS-Cov.The method of UpE shows high sensitivity while the method of ORF1b has high specificity.2 methods have no cross reation with human coronavirus OC43,NL63 and SARS-Cov.So the upE method as screening method and ORF1B method as validation test are recommended.The first imported case from Korea has emerged in China.There has no probability of infection and outbreak but if the disease broke out,it would be catastrophic.So the sequencing of the imported MERS-Cov complete gene will help to improve the cognition of its genomic structural and biological characteristics.It can also help to improve the cognition of its origin,variation,evolution,virulence and the relationship in the genetic evolution analysis with other areas'MERS-Cov.In summary,our study can provide imformation about the prevention and control of MERS and provide theoretical support of specific drug's research and development.Currently,researches of coronavirus protein structure have specifically focused on S protein.Although further research of other protein structUre and function are needed,they also have come into focus.In recent years,the important information of the gene and protein sequence can be obtained by the prediction of bioinformatics and sequence analysis software.Obtaining MERS CoV genome sequence,we used a bioinformatics approach to analyze the RNA,ORF5 of MERS CoV and the physicochemical properties,secondary structure,structural domain,transmembrane domain of E protein,with the use of database,web server and bioinformatics software.Comprehensive structural feature of the closely protein,related to viral infection,virulence and immunity,may offer theoretical basis for immunological screening and identification in vitro.2.Methods(1)Sample and virus isolation:On 28 May 2015,the first importation of suspected cases of Middle East respiratory syndrome from Korean was admitted to the people'sHospital of Huizhou City,Guangdong Province,Clinical manifestations include dramatic fever and the symptoms of severe acute respiratory syndromes.Buccal swabs were collected by Guangdong Provincial Center for Disease Control and Prevention on different date during fever,and case was documented MERS by China Center for Disease Control and Prevention.Virus RNA was extracted after sample was collected.(2)Molecular biology:We use RNA isolation kit and then adopt real-time PCR and Nest PCR amplification,with the use of sequence information of Genbank,we designed 24 primers,using primer premier5.0,to amplify the genome of MERS CoV separately.We use NEST PCR for the fragments which were unable to amplify in real-time PCR.After the fragment was isolated and eluted from a gel,gel purified and cloned,the sequences of these constructs were confirmed by the dideoxy chain termination method.(3)Bioinformatics analysis:We spliced the sequence by NDAstar V7.0 and submitted the assembly of viral genomic sequence to Genbank.Similarity,variation,evolution and restructuring events of Viral genomic sequence was analyzed by BLAST,Bioedit 7,MEGA 6.0,SIMPLOT and RDP4.Secondary structure of RNA was analyzed by RNAfold.Domains of ?-helieal,?-folding,T-turn and irregular coiled-coil of E protein were analyzed by Chou.Fasman and Gamier-Robson in Protean of DNAstar.Transmembrane helix of fused protein and restriction enzyme cutting site of signal peptide of fuesd protein were predicted by TMHMM and PHYRE server2 separately.3.ResultMERS CoV China GD01 has the basic characteristics of coronavirus genomes,genome length is measured as 29,928 nucleotides(NT),5' end for 134nt 5,UTR(untranslated region).Terminate the password from No.29663 to 3' end,266nt 3'UTR(untranslated region).Measured nearly full length sequences of the genome BLAST,discovery and Korea recently reported series most closely.In sequence alignments in the process,discover a different sequencing methods and sample sources,time may be corresponding impact on the accuracy of the sequencing structure.Using Genbank published in different areas of isolated strains were constructed with time genome evolutionary tree,s genes,ORFla,results show that the three did not differ significantly from the results of the evolutionary tree,viral nucleotide sequences and sequences of strains isolated from Arab countries in the Middle East in the near future similar up to 99.53%?99.92%;Than found in the poly has 3 new mutations in the protein ORFlab,s,e,m,n variation of protein has not yet been found;RDP4 and simplot software restructuring analysis found that fragments of the nuclear genome sequence of the virus has no obvious restructuring.On the third day of fever(samples collected on May 28)amplification,22nd primer product of size except for access purposes,there are two amplified fragment,after purification,cloning,sequencing and Assembly week,other than wild-type MERS CoV are found in samples,there are also two variants missing fragment sizes 29514bp and 29509bp,respectively.From the third day of fever,throat swab samples in patients with back three days,variant on the fifth day of fever which cannot be detected in a sample,second week of fever(the tenth and 13th day)cannot be amplified in samples of variant sequences.In the comparative analysis of genome sequences,missing virus found two sequences,which lack base and 419bp bases in 414bp,this may result in some changes in protein structure,partial integration or truncated.Application of bioinformatics to gene function analysis found that may exist in the body of the Middle East and respiratory syndrome corona virus defective interfering RNA.Analysis of the amino acid sequence of nucleic acid sequence translation,find mainly ORF5 and e proteins.To predict the nature of the RNA secondary structure and protein analysis,found the site for protein hydrophobic transmembrane domains.4.Conclusion(1)The study successfully amplified from the first Korea imported case has the Middle East nearly all of the respiratory syndrome coronavirus genome sequence,MERS CoV infection in patients with confirmed once again,and was found in samples of natural addition to the wild-type sequence MERS CoV,while there are two defective RNA variants.Sequences have been submitted to GenBank databases,access log,divided into:KT036372,KT036373,KT036374,named ChinaGDOl,ChinaGDO1,Variant 1,China GD01 Variant 2.(2)Similarity comparison and genome,s genes,phylogenetic analysis of ORF1a,confirmed China GD01 Korea similarity between isolates from the highest,wild strain genome sequence also found no restructuring phenomenon.The evolution analysis also suggested that this strain comes from Saudi Arabia.Three evolutionary tree structure shows that genomic evolutionary tree it is difficult to construct reality,ORF1a better suited to study its evolution.(3)The bioinformatic methods were used to analyze the MERS CoV RNA secondary structure,ORF5 protein and E protein structure.Found that fusion protein of ORF5 and the E protein could affect the hydrophobicity and the transmembrane structure of the virus.The mutations located in N terminal of E protein may result in virulence reduced.We detected the content of the variants during the period when patients with fever.While the variants existed in the early stage of the fever,it could not be checked out in the later stages of the illness development.This phenomenon also prompt the varients of MERS CoV may be associated with disease progression.Which were also provided support for the protein related functions study of MERS CoV.These were further validated in fol-low study.