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Kathrine Stene-Johansen NIPH

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1 Kathrine Stene-Johansen NIPH
Genotyping of hepatitis A virus (HAV) - a useful tool for outbreak investigations Kathrine Stene-Johansen NIPH My name is KSJ I 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) Hedmark Oslo HAV Marokko Oslo Kristiansand Østfold, Nølkeby skole (4) Sudan Oslo Picornavirus family ss RNA viral genome of 7.5 kB The capsid is composed of 3 structural proteins; VP1, VP2 and VP3 A single serotype Israel Hedmark Chile 1 Brasil Levanger Chile 2 IVDU-epidemi IVDU-Finland MSM-utbrudd 1997/98 Lørenskog HAV belongs to the picornavirus family The genome consists of a single-stranded RNA genome of 7.5 kilobases The capsid proteins as shown here is composed of 3 structural proteins VP1, VP2 and VP3, as well as a putative VP4 protein The structural proteins of HAV are very conserved and reflected by the presence of only a single serotype Bergen Hellas Meråker England MSM-utbrudd 0.1 Tyrkia USA

3 Genome organisation of HAV
This figure shows the genome organisation of HAV The genome consist of a long ORF divided into 3 regions; The structural proteins P1 And 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 identity A sub-genotype is defined as a group of viruses with sequence variability of less than 7.5% 3 human genotypes genotype I >90% genotype III < 10% genotype II 2 isolates 3 simian genotypes A genotype is defined as a group of viruses with >85% nt identity They are further divided into sub-genotypes with a sequence variability of less than 7.5% 3 human and 3 simian genotypes has been identified Genotype 1 are found world wide and consist of more than 90% of the isolates characterized to day Genotype III represent less than 10% of the isolates and are endemic in parts of Asia Genotype II are represented with only 2 isolates Also the simian strain are represented by one isolate each.

5 HAV genotypes Genotype IA Genotype IB Genotype IIIA Genotype IIA
FH3 LY6 AH3 Genotype IA LU38 AH2 FH2 FH1 GBM AH1 F.G. NCACG HM-175 HAF-203 1000 Genotype IB L-A-1 1000 PA21 GA76 MBB Genotype IIIA P27 1000 1000 NOR21 1000 989 612 Genotype IIA CF53 Genotype IV simian strain Genotype IIB SLF88 This 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 V simian strain CY-145 AGM27 0.1

6 HAV strains High endemic regions Low endemic regions
Endemic HAV populations with closely related strains and geographic relatedness Low endemic regions Imported strains from high endemic regions with diverse origin and relatedness In 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
Sequencing Resent 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 strain Within outbreaks HAV sequences are identical or very closely related so that epidemiological defined cases are confirmed or precluded In 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 region We 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 period The blue part of the bars show the epidemic among IVDUs from The read part show 2 outbreaks among MSM in and 2004 Yellow bars indicate unknown transmission route

10 Genotyping used for outbreak investigation in Norway (1995-2004)
Epidemic among drug users Outbreak among homosexual men and 2004 Imported cases Small local outbreaks Outbreak among hemophiliacs We have used molecular epidemiology as a tool in investigation of the IVDU endemic in the period 2 MSM outbreaks inn and 2004 respectively imported cases small local outbreaks an outbreak among hemaphiliacs

11 Notified cases from January 1995 - July 1998
Genotyping 581 drug users 46 723 cases associated with the epidemic among drug users 65 54 genotype IA (IVDU-strain I) cases 142 secondary cases 19 11 genotype IIIA (IVDU-strain II) 519 cases not associated with the epidemic among drug users 19 drug users (IVDU- strain I) 49 30 other variants In 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. A IVDU= Intravenous drug abusers

12 Nosocomial outbreak faecal-oral transmission
A hospitalised alcoholic transmitted the virus to 14 secondary cases 8 nurses 4 other patients 2 relatives Outbreak 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 casers These 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 transmission 2 batches of coagulation factor VIII and serum from 2 hemophiliacs were analysed Identical virus were detected among the hemophiliacs and in the batches with coagulation factor This 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 98 18/23 PCR-positive Genotyping revealed 2 strains 12 MSM-strain I (genotype IA) 5 MSM-strain II (genotype IA) Outbreak strains distinct from the IVDU-strains MSM-strain II was also detected in a local family outbreak MSM= men with sexual contact with men

15 Outbreak among homosexual men
Family Outbreak Grandfather Grandmother IgM- +3d/PCR- Outbreak among homosexual men Father Mother Uncle Aunt +32d/PCR- 0d/PCR+ IgM- IgM-/PCR- IgM+ 18/23 PCR+ Sister Sister Sister - 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 2004 HAV RNA detected in 67/79 Identical sequences of genotype IA were detected The 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
Chile Genotype IA MSM outbreak Bergen IVDU outbreak Finland USA IVDU epidemic Turkey Chile Brazil Marocco Kristiansand Sudan Israel Genotype IIIA Genotype IB IVDU epidemic IVDU 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 Malmø Samarbeidsprosjekt med Eastbourn i England - større utbrudd blant IVDU påviste samme hAV-variant Genotype III B Indonesia 0.1

18 Conclusion By 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.

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