| BackgroundEmerging infectious diseases (EID) has posed a serious threat to human.According to the report of WHO, there are at least40new pathogens have been foundsince1967. More than70%of which are viral pathogens, including HIV virus, Ebolavirus, SARS coronavirus, avian influenza viruses and pH1N1viruses. On May9,2014,WHO has reported437laboratory confirmed human cases of H7N9virusinfection since the emergence of first infection cases on late March of2013in easternChina. There were134deaths. The mortality rate was30.7%and the cases weredistributed in13provinces/municipalities of mainland, Hong Kong, Taiwan andMalaysia. Until now the infection and death cases are still increasing. However, thecases are sporadic. Early diagnosis and antiviral therapy is the key to improve theprognosis and reduce mortality.The rapid identification and detailed information of pathogens in the emerginginfectious diseases is important for us to take effective measures to controlunexpected event. The detection methods of viral pathogens mainly rely on antigendetection, nucleic acid detection and virus isolation. Colloidal gold technique whichdetect the hemoagglutinin can give result in less30minutes, but it may loss somepatient who was really infected with H7N9virus as low sensitivity. The traditionalvirus isolation method is time-consuming and the isolation must be done in thebiosafety level III laboratory. Switching the evolutionary selection pressure from invivo human respiratory tract to embryonated chicken eggs might introduce mutationsinto the final genome sequences during culture. Genomic information which obtainedfrom virus isolation technology is not real condition in response to the host and maybeprovide some wrong information to treatment. For such factors, it is difficult to be conducted as a routine test in clinical laboratories. Currently, most clinicallaboratories use real-time PCR technology with high sensitivity and specificity forscreening patients suspected with H7N9virus infection. However, this method isbased on the genome information of pathogens which is not suitable for diagnosis ofan unknown or a novel viral pathogen.H7N9influenza viruse is low pathogenic in poultry. Once it infects to humanswhich might cause serious illness. Currently, there found no evidence about thetransmission ability of H7N9virus between people. But we can not exclude thepossibility. Once the virus occured mutation or reassortment, it may gain the ability tobind with the human respiratory tract cells preferentially. It is likely to evolve a newvirus which can transmit from person to person. Studies have shown that H7N9virusHA gene had already occurred the Q226L mutations which can enhance bindingcapacity to human respiratory receptor. If the PB2gene E627K and D701N mutationsoccur simultaneously, it could lead to the spread of H7N9virus between person toperson. By detecting respiratory and other organs of ferrets infected with the H7N9virus, they could found the virus RNA in upper and lower respiratory, lymph nodesand the brain which demonstrated that under appropriate conditions, it is possible tospread from person to person. Detecting the sequences of H7N9virus genome in time,especially the genome of H7N9virus isolated from human. Viral evolution, mutationand other molecular characteristics which can provide a scientific basis for furthermonitor, prevention and control of H7N9virus.The appearance of high throughput sequencing technology(HTS) in2005, alsoknown as next generation sequencing(NGS)technology has facilitated the possibilityof sequencing millions DNA molecules in a parallel and determining analysis ofgenomes landscape of a pathogen. Compared with the sanger sequencing, NGS canproduce more data in a shorter time with a lower cost. Ion torrent PGM is based onthe new semiconductor technology. Sequences data are obtained by directly sensingthe ions produced by template-directed DNA polymerase synthesis using all-naturalnucleotides on this massively parallel semiconductor-sensing device or ion chip. Itallows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. It is very suitable for hospital using. In recent years,the virus metagenome methods based on NGS have been successfully applied for theidentification and discovery of new viruses, sequencing of the full genome of virusand have the ability to detect viral quasispeciesThe metagenomic approach based on NGS technology was used to detect H7N9virus in the respiratory specimens of patients with infected with H7N9virus andconstruct a platform for the identification of pathogens in emergency infectiousdiseases. And then the genome of H7N9virus was sequenced directly in clinicalspecimens with PCR and NGS. The date was analyzed to see whether there was viralquasispecies related with resistance or virulence and help to guide clinical treatment.For a comprehensive assessment of current situation of human H7N9virus infectionand for a better control of H7N9virus in case of pandemic. Total of63H7N9virusgenome sequences in current public database were statistical analyzed about theimportant mutation in the amino acid sites.Objective1. To detect H7N9virus directly in respiratory specimens with next generationsequencing technologies and provide solutions to identify pathogens in emerginginfectious diseases of respiratory in clinical laboratory.2. To establish a method which can obtain the full genome of H7N9directly from theoriginal clinical specimen using PCR combined with the NGS and analyze thesignificant gene mutation influencing the pathogenicity of the virus.3. To analyze the resistance gene locus, pathogenicity receptor binding sites andpotential glycosylation sites of the genome sequences of the novel H7N9viruscollected from the public database and provide scientific basis for furthermonitoring, prevention and control of H7N9virus.Methods1. Total virus DNA/RNA was extracted from throat swab specimen of a patient oflaboratory confirmed H7N9virus infection. Library was constructed with theSISPA method and was subjected to sequencing in Ion Torrent PGM platform with ion316chip. A stand-alone blast was analyzed to filter the reads belong to virus.2. The8segments of H7N9virus were amplified by one-step PCR and sequenced onthe Ion Torrent PGM platform with ion314chip. The genome was assembled withthe FluAtyping Plug-in, the isolate was designed asA/Guangdong03-A/H7N9(2013). The reported significant gene mutation locus ofH7N9virus was analyzed. A/Guangdong03-A/H7N9(2013)genome was comparedwith the genome sequences obtained through embryonated chicken eggs. Genesequences and amino acids differences were analyzed.3. All the available genome sequences of H7N9virus isolated from human host wereCollected. Phylogenetic, drug resistance site, virulence and transmissibility,receptor binding property and the glycosylation site were analyzed.Results1.453.31Mb was abtainted in this run. A total of2,561,122reads was abtainted.2,481,807reads belong to human,79,218reads belong to bacteria and eukaryotesand97reads belong to virus. There are13reads belong to H7N9virus.2. We got a total of53.86Mb data and391,805reads was analyzed. The full genomeof H7N9virus was obtained with a depth of1020. Further analysis of the obtainedgenome demostrated that this virus holded the important mutations sites, such asHA-Q226L, HA-T160A, M2-S31N, PB2-D701N and PB1-I368V. Homologyanalysis and phylogenetic tree of NA and HA gene showed that HA gene had ahighest sequence similarity with A/duck/Fujian/6380/2010(H7N3). NA gene hada highest sequence similarity with a/northern shoveler/Hong Kong/MPL133/2010(H2N9). The genome varies from that passaged in the embryonated chicken eggs.In the HA segment,7point mutations were found, of which4were synonymousand3induced amino acid changes. In the NA segment there was only one pointmutations and induced amino acid change. In the M segment6point mutationswere found, of which4were synonymous and2induced amino acid changes. Inthe nucleocapsid protein(NP) segment,53point mutations were found,47weresynonymous and6induced amino acid changes. In the nonstructural (NS) protein segment,33point mutations were found,24were synonymous and9inducedamino acid changes.3. HA cleavage site sequence analysis showed that the amino acid were "PEIPKGR↓G"(↓for the cleavage site). There are two basic amino acids (K and R) meetsthe standard of low virulence strains.96.8%(61/63)and87.3%(55/63)of theisolates occurs the HA G186Vand Q226L mutations respectively. All the isolateshold the M2gene S31N(100%)mutation.4.8%(3/63) of patients isolates hold theNA gene R294K mutation.66.1%(41/63),12.9%(8/63) and6.5%(4/63) of theH7N9patient isolates occur the PB2E627K, D701N and Q591K mutation,respectively.88.9%(56/63)of patients isolates occur the PB1-I368V mutation. Allthe isolates occur the NA gene69-73amino acids deletions,Conclusions1. A virus metagenomic analysis technique which can apply to identify unknownvirus pathogens of emerging infectious diseases was established.2. A method combined PCR with NGS was established to obtain depth genomesequence of H7N9virus directly from the clinical specimen and can providepathogenic and resistant information of H7N9virus in clinical timely.3. All the63isolates hold the M2-S31N mutation. Only some isolates occurred theresistantant of neuraminidase inhibitors. The emergence of resistantant ofneuraminidase inhibitors may arise during concomitant treatment. The interactionof them maybe the reason of high infection and high death rate. |
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