Presentation on theme: "Kathrine Stene-Johansen NIPH"— Presentation transcript:
1 Kathrine Stene-Johansen NIPH Genotyping of hepatitis A virus (HAV) - a useful tool for outbreak investigationsKathrine Stene-JohansenNIPHMy name is KSJI am a researcher at the department of blood and sexually transmitted diseases at NIPH. The last 10 years I have been working with molecular epidemiology of HAV and I will present our main findings in this talk on Genotyping of HAV – a tool for outbreak investigation.
2 HAV Picornavirus family ss RNA viral genome of 7.5 kB Hedmark (2)HedmarkOsloHAVMarokkoOsloKristiansandØstfold, Nølkeby skole (4)SudanOsloPicornavirus familyss RNA viral genome of 7.5 kBThe capsid is composed of 3 structural proteins; VP1, VP2 and VP3A single serotypeIsraelHedmarkChile 1BrasilLevangerChile 2IVDU-epidemiIVDU-FinlandMSM-utbrudd 1997/98LørenskogHAV belongs to the picornavirus familyThe genome consists of a single-stranded RNA genome of 7.5 kilobasesThe capsid proteins as shown here is composed of 3 structural proteins VP1, VP2 and VP3, as well as a putative VP4 proteinThe structural proteins of HAV are very conserved and reflected by the presence of only a single serotypeBergenHellasMeråkerEnglandMSM-utbrudd0.1TyrkiaUSA
3 Genome organisation of HAV This figure shows the genome organisation of HAVThe genome consist of a long ORF divided into 3 regions;The structural proteins P1And the non-structural proteins P2 and P3.The C-terminal part of VP3, the N-terminal part of VP1, as well as the VP1-2PA junction are the most variable regions and has therefore been used in molecular epidemiological studies.Based upon sequencing 168 bp within the VP1-2PA Betty Roberston and workers mange to distinguish geographical distributed strains and classified them into genotypes.Costa-Mattioli M et al. J Gen Virol 84 (2003),
4 HAV genotype classification A genotype is defined as a group of viruses with > 85% nucleotide identityA sub-genotype is defined as a group of viruses with sequence variability of less than 7.5%3 human genotypesgenotype I >90%genotype III < 10%genotype II 2 isolates3 simian genotypesA genotype is defined as a group of viruses with >85% nt identityThey are further divided into sub-genotypes with a sequence variability of less than 7.5%3 human and 3 simian genotypes has been identifiedGenotype 1 are found world wide and consist of more than 90% of the isolates characterized to dayGenotype III represent less than 10% of the isolates and are endemic in parts of AsiaGenotype II are represented with only 2 isolatesAlso the simian strain are represented by one isolate each.
5 HAV genotypes Genotype IA Genotype IB Genotype IIIA Genotype IIA FH3LY6AH3Genotype IALU38AH2FH2FH1GBMAH1F.G.NCACGHM-175HAF-2031000Genotype IBL-A-11000PA21GA76MBBGenotype IIIAP2710001000NOR211000989612Genotype IIACF53Genotype IVsimian strainGenotype IIBSLF88This is a phylogenetic tree based upon the nt sequence of the structural proteins, that show the 3 different genotypes with human isolates and subtypes, as well as 2 of the simian isolates..Genotype Vsimian strainCY-145AGM270.1
6 HAV strains High endemic regions Low endemic regions Endemic HAV populations with closely related strains and geographic relatednessLow endemic regionsImported strains from high endemic regions with diverse origin and relatednessIn high endemic regions most of the population are infected during childhood and the circulating virus population are closely related.Distinction between strains in an endemic situation is therefore difficult nor necessary.In low endemic regions strains are imported from different high endemic regions and therefore represent different relatedness. We can therefore distinguish between different outbreak strains.Imported strains may be linked by phylogenetic analysis to geographical regions that correspond to the patient’s destination of travel or the other way around that the patient has aquired the infection at home and not abroad as indicated in the notifications.
7 Genotyping HAV RNA isolation from serum Reverse transcriptase (rt) PCR SequencingResent studies has shown an extensive viremia of HAV. Virus is present in blood during the incubation period and for several week after clinical onset.The viral load may be very low but by PCR we can in theory detect a single virus genome. We have successfully been using serum for isolation of HAV RNA. Serum is also very accessible as all notified cases of HAV are confirmed by detection of HAV-IgM. Isolation from serum is also much less laborious than isolation from stools.HAV RNA is transcribed into cDNA by reverse transcriptase, followed by a semi-nested PCR. The amplified product is then sequenced.
8 Molecular epidemiology of HAV By sequencing bp in the VP1-2PA region we can distinguish between outbreak strainWithin outbreaks HAV sequences are identical or very closely related so that epidemiological defined cases are confirmed or precludedIn our molecular epidemiological studies we have chosen a region within the VP1-2PA junction of either 350 or 450 bases that is suitable for the distinction of strains.Our experience is that HAV from the same outbreak are identical or closely related, but may vary with a nt or two within this regionWe can thereby either confirm or preclude cases from an outbreak, as well as distinguish between outbreaks and follow the dissemination of the virus in the population. We have therefore used genotyping as a tool in outbreak investigation
9 Notified cases of HAV infected in Norway 1994-2004 This figure shows the number of notified cases transmitted in Norway in the periodThe blue part of the bars show the epidemic among IVDUs fromThe read part show 2 outbreaks among MSM in and 2004Yellow bars indicate unknown transmission route
10 Genotyping used for outbreak investigation in Norway (1995-2004) Epidemic among drug usersOutbreak among homosexual men and 2004Imported casesSmall local outbreaksOutbreak among hemophiliacsWe have used molecular epidemiology as a tool in investigation ofthe IVDU endemic in the period2 MSM outbreaks inn and 2004 respectivelyimported casessmall local outbreaksan outbreak among hemaphiliacs
11 Notified cases from January 1995 - July 1998 Genotyping581 drug users46723 cases associated with the epidemic among drug users6554 genotype IA (IVDU-strain I)cases142 secondary cases1911 genotype IIIA (IVDU-strain II)519 cases not associated with the epidemic among drug users19 drug users (IVDU- strain I)4930 other variantsIn the period January 1995-July 1998, 1242 cases of HAV was notified in Norway.Of these 723 cases were associated with the epidemic, 581 IVDUs and 142 secondary cases.We selected cases for genotyping that represented different geographical region and the whole time period.We found an identical strain of genotype IA in 54 cases. This strain was found from the beginning of the epidemic in Oslo and spread to most parts of Norway throughout the 3.5 years we studied the epidemic. At the autumn of 1997 From Oslo this strains spread to regions around Oslo and further along the southern- and western coast finally to the northerne part of Norway. AIVDU= Intravenous drug abusers
12 Nosocomial outbreak faecal-oral transmission A hospitalised alcoholic transmitted the virus to 14 secondary cases8 nurses4 other patients2 relativesOutbreak strain of genotype IA identical to outbreak strain circulating among drug users at this time ( IVDU- strain I)At the beginning of the epidemic among IVDUs there was a nosocomial outbreak due to fecal-oral transmission.An hospitalized alcoholic was the source to 14 secondary casersThese included 8 nurses of witch 6 were on sick-leave for an average of 22 days, and 3 of the fellow patient was re-hospitalised because of HAV infection.We obtained serum from these patients and found that they were infected with the same outbreak strain, the time of onset of infections suggested that the alcoholic was the source of transmission.The outbreak strain was identical to the outbreak strain circulating among IVDUs at this time. This show spread from the IVDU community to the main population.One cases was believed to have acquired the infection abroad, but molecular characterization revealed identical strains indicating that she had been infected at the hospital at work.
13 Outbreak among hemophiliacs Parenteral transmission In hemophiliacs were transmitted with HAV, where coagulation factor VIII was the possible source of transmission2 batches of coagulation factor VIII and serum from 2 hemophiliacs were analysedIdentical virus were detected among the hemophiliacs and in the batches with coagulation factorThis outbreak strain was identical to the IVDU-strain II (genotype IIIA)In 1994 there was an outbreak of HAV among hemophiliacs.This outbreak show spread from the IVDU community to blood donors in the main population.
14 Outbreak among homosexual men 1997-1998 26 notified cases of HAV infection among homosexual men from October 97- March 9818/23 PCR-positiveGenotyping revealed 2 strains12 MSM-strain I (genotype IA)5 MSM-strain II (genotype IA)Outbreak strains distinct from the IVDU-strainsMSM-strain II was also detected in a local family outbreakMSM= men with sexual contact with men
15 Outbreak among homosexual men Family OutbreakGrandfatherGrandmotherIgM-+3d/PCR-Outbreak among homosexual menFatherMotherUncleAunt+32d/PCR-0d/PCR+IgM-IgM-/PCR-IgM+18/23 PCR+SisterSisterSister- 67d+11d+11d+11d+11d
16 Outbreak among homosexual men (2004) 79 notified cases of HAV associated with homosexual men in the period May - November 2004HAV RNA detected in 67/79Identical sequences of genotype IA were detectedThe same outbreak strain caused concurrent outbreaks among homosexual men in Denmark, Sweden and the Netherlands
17 England Greece Genotype IA MSM outbreak 1997-98 Bergen USA ChileGenotype IAMSM outbreakBergenIVDU outbreak FinlandUSAIVDU epidemicTurkeyChileBrazilMaroccoKristiansandSudanIsraelGenotype IIIAGenotype IBIVDU epidemicIVDU variant I sirkulerte i perioden og omfattet til slutt hele Norge. Identifiserte de aller første tilfellene i januar 1995 i Østfold. Den samme varianten påviste hos en stoffmisbrukere fra Karlstad i1997.IVDU-variant 2 tilhører genogruppe III, mindre vanlig variant påvist i sør-øst og sentral Asia (India, Nepal, Sri Lanka, Malaysia),Spain,IVDU utbrudd i MalmøSamarbeidsprosjekt med Eastbourn i England - større utbrudd blant IVDU påviste samme hAV-variantGenotype III BIndonesia0.1
18 ConclusionBy molecular epidemiology we have been able to characterise and distinguish outbreaks of HAV, as well as studied the dissemination of outbreak strains in the population.These studies have shown that sequencing of outbreak strains is essential in outbreak investigation, and that molecular epidemiology is an excellent tool for the surveillance of HAV.