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.