高通量测序与生物信息学在传染病疫情中的应用.pptx

1、高通量测序与生物信息学在传染病疫情中的应用Emerging infectious diseases are major world health threatsFrequent outbreaks in recent yearsMost of the emergingdisease comes from animals H7N9: Birds MERS: Camels Ebola: Bats Zika: Mosquitos SARS: BatsWe are more close toanimals We occupied more spaces of animals We eat more k

2、inds of animals We have more animal pets With rapid and intensive internationaltravels, infectious diseases have no countryboundaries.Bat: as an important source of highlylethal zoonotic viruses Severe Acute Respiratory Syndrome (SARS), 2003 Middle East respiratory syndrome (MERS), 2012- Ebola in We

3、st Africa, 2014 Other Human Diseases caused by CoV (229E, NL64, HKU1; OC43) Other Human Diseases caused by non-CoV: Rabies Virus;Hantaviruses; Filoviruses; Lassa virus; Henipavirus (Hendra &Nipah)SARS: Severe Acute Respiratory Syndrome2 0 0 3 8,000+ infection 774 deaths (10%) $ 59B lossesScience 200

4、5; Vol. 310: 676-679MERS: Middle East RespiratorySyndrome2 0 12mBio (2017) vol. 8 no. 2 e00373-172123 MERS-CoV cases, 740 deaths (35%),Ongoing in 27 CountriesMERS in South Korea,2 015186 infections, 36 deaths (20%)Why Bats ? The only mammal that can fly Longevity animals, can live for 40 years (Mous

5、e only 3 years) The second largest order of mammals, comprising about20% of all mammal species, with over 1,200 species. Present throughout the world, feeding on all kinds of insectsand fruits Flying leads to increased body temperature andincreased reactive oxygen species (ROS) , and enhancedDNA rep

6、air capacity Unique immune system: Constitutive low level expression of interferon (Persistent immunesurveillance, no cancer) Inflammatory genes deletion (allow viruses to coexist) Natural immunity comprimised (allow viruses to coexist)Ebola outbreaks in history1976-19771994-1996 Sudan, DRC SUDV, EB

7、OV DRC, Gabon EBOV, TAFV2001-20032007-20122014-present Gabon, Cango EBOV DRC, Uganda SUDV, EBOV,BDBV Guinea, Liberia,Sierra Leone EBOV2014 Outbreak in Sierra LeoneGeographical distribution of the EBOV outbreak in Sierra Leone（Gire et. al., Science, 2014)Initial studies on the genetics of 2014 EBOVOr

8、igin of 2014 EBOVIncreased mutation rate were proposedResults: 99 Sequences from 78 EVD Cases. Mutation rate doubled.Worries/Questions: More virulent? Higher contagious? Are vaccine/drug target mutated? Does PCR primers still work?（Gire et. al., Science, 2014)Initial studies on the genetics of 2014

9、EBOV2014 Outbreak in Sierra LeoneWith the situation worsen in West Africa,China urgently sent several medical teamssince August 2014ChinaSierra LeoneP3 Labs was deployed in Sierra Leone-China Friendship HospitalThe mobile P3 labThe fixed P3 labSample informationSamples werecollected primarilyin West

10、ern SierraLeone175 full-lengthEBOV genomesequences obtained：CMLTT (China Mobile Lab Test Team)（Tong et al., Nature, 2015)EBOV genome sequencing flow chartSuspected blood samples19 PCRs per sampleHeat inactivationSerum collectionDNA pool and purificationLibrary preparationIon Proton SequencingData an

11、alysisFurther inactivationwith Lysis bufferRNA Extraction with MagMaxRT-qPCRTo minimize the risk of infection, multiplex PCR was performed onlimited residual RNA sample to recover genome sequencesGenetic diversity of the 2014 EBOVCyan: synonymousMagenta: non-synGreen: UTRGrey: Inter-genic440 novel s

12、ubstitutions7 fixed substitutionsLineage-specific substitutionsBoxed are serial TC mutationsEvolutionary dynamics of the 2014 EBOVSeven novel sublineages identified.Posterior support is shown (MrBayes).EBOV genetic diversityEBOV genetic diversity kept increasing during early stage of the outbreakTra

13、nsmission routesMajor Transmission Routes ofEBOV in Western Sierra LeoneThree major locations played critical role in the spreading of EBOV in Sierra LeoneCorrection of EBOV evolution rate EBOV evolution rate is constant compared with previous outbreaks. Relieved worlds tension.Severe Acute Diarrhea

14、 Syndrome (SADS) Acute diarrhea / acute vomiting / rapid weight loss Died 2-6 days following onset High mortal rate (up to 90%) All known swine pathogens were excludedGuangdongProvince: SADS outbreak, 2016-10-28100 km: SARS index case, 2002-11-16Original farmThe other 3 farmsSerial outbreaks in 2017

15、A total of 24,963 piglets died as of 2017-05-0264% (4659/7268) piglets died in Feb. Farm AQuestions to be answered What is the pathogen? Where did it come from? How was it transmitted betweenthe 4 farms? What lessons can we learn fromthis outbreak?Pathogen Identification Pathogen screening using Nex

16、t-generation sequencing andbioinformatics tools Serum antibody verification in recoveredpigs using LIPS (LuciferaseImmunoPrecipitation System) Virus isolation and challenging with SPFanimals Immunohistochemical confirmation ofvirus infectionPathogen screening using Next-generation sequencingNext-gen

17、erationsequencingRaw ReadsHKU2-CoV related virus identifiedWhat is Bat coronavirus HKU2? Genus Alphacoronavirus First found in Guangdong Province in 2005 Host : Chinese horseshoe bats (中华菊头蝠)SADS-CoV complete genome sequencing Ampliseq primers designComplete genome 27,173 ntOverall identity with HKU

18、2 is 95%Envelop protein S1 shares only 86% identitySerological confirmationLuciferase Immunoprecipitation System (LIPS)S1 geneAntibody detected in recovered Sow: (+) 12/46Virus isolationnormalCPENP antibodyDAPImockSADS-CoVAnimal Challenge with isolated virusChallengeinoculatedwith virusinoculatedwit

19、h mockControlVirus challenge experimentsRed , anti- SADS-CoVN proteinBlue, DAPIControlChallengeHKU2 is not the direct ancestor of SADS-CoVIdentity95%86%HKU2What is the direct ancestor of SADS-CoVSADS-CoV source-trackingin wild bats in Guangdong Province7 locations4 yrs (2013-2016)596 bat samples71 S

20、ADS-CoV positive4 samples for NGSClose relatives in bats identifiedBat virusHomologyPhylogenetic analysis indicates batcoronavirus is the ancestorS1CompletegenomeTransmission AnalysisBat to Pig: single entryof multiple entry?FarmWGS strainsFarm AFarm BFarm CFarm D85119Phylogenetic analysis of SADS-C

21、oV in four farms Either the virus was introduced into pigs from batsmultiple times Or genetic recombination disturbed the molecularclock.Striking similarities between SADS and SARSGuangdong Province Time: Oct-Nov Location: Guangdong Host: Bats Virus: Coronaviruses Severity: Fatal diseaseSADS, 2016-1

22、0-28SARS, 2002-11-16100 kmAcknowledgement （Ebola Work） Prof. Wu-chun Cao, Corresponding author Prof Fuchu He, Co-corresponing author Prof. George F. Gao, Co-corresponing author Prof. Weifeng Shi Prof. Di Liu Chinese Mobile Testing Team in Sierra Leone Colleagues in State Key Laboratory of Pathogen and Biosecurity Department of Health, SL governmentTong Yigang, BIME 2015-12-22Thanks!