Division of Infectious Disease Control, Norwegian Institute of Public Health, PO Box 4404, Nydalen, NO-0403 Oslo, Norway
Correspondence
Kathrine Stene-Johansen
kasj{at}fhi.no
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence of NOR-21 reported in this paper is AJ299464.
Primer sequences and PCR conditions are available as supplementary material in JGV Online.
Present address: Department of Microbiology, Akershus University Hospital, Akershus, Norway.
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INTRODUCTION |
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We have presented here characterization of outbreaks caused by genotype IIIA and the first complete genome sequence of HAV genotype IIIA (NOR-21). In this work, we have compared these HAV genotype IIIA isolates with the other human and simian genotypes to elucidate the characteristics and origin of this HAV genotype.
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METHODS |
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Molecular epidemiology of HAV.
HAV RNA was isolated and a 350 bp region within the VP12PA junction of the HAV genome was amplified by RT-PCR and subsequently sequenced as described previously (Stene-Johansen et al., 1998). Epidemiological data were obtained from MSIS, the notification system for infectious diseases in Norway, at the Norwegian Institute of Public Health (http://www.fhi.no).
RT-PCR and sequencing.
Viral RNA was extracted from 140 µl anti-HAV IgM-positive serum by using a QIAamp viral RNA kit (Qiagen). RNAgard (0·5 U µl1; Pharmacia) and dithiothreitol (10 mM) were added to the isolated RNA (50 µl). cDNA was generated with avian myeloblastosis virus reverse transcriptase (Promega) or Superscript II (Invitrogen) following the manufacturers' recommendations. PCR amplifications were performed with AmpliTaq Gold DNA polymerase or AmpliTaq DNA polymerase according to Applied Biosystem's recommendations, with 0·5 µM dNTPs and 0·5 µM each primer. Primer sequences and amplification conditions are given in the Supplementary Table, available in JGV Online.
Rapid amplification of cDNA ends (RACE).
The 5' end of the HAV genome was amplified by using 5' RACE (Invitrogen). First-strand cDNA synthesis was carried out with 0·1 µM HAV3-59M primer. Subsequent PCR amplifications of tailed cDNAs were generated with the 5' RACE abridged anchor primer (AAP) and abridged universal amplification primer (AUAP) and different combinations of sequence-specific primers (see Supplementary Table) according to Invitrogen's recommendations. The sequence of the 3' end of HAV RNA was obtained by 3' RACE using the adapter primer (AP) and AUAP (Invitrogen). cDNA was synthesized at 70 °C according to Invitrogen's recommendations. An Advantage 2 PCR kit (Clontech) was used with 200 µM dNTPs, 200 nM AUAP primer and 500 nM sequence-specific primers. In the first PCR, using primers HAV6 and AUAP, the first ten cycles included touchdown annealing from 60 to 50 °C and then 20 cycles with annealing at 50 °C (denaturation at 95 °C for 30 s, annealing for 30 s, extension at 68 °C for 5 min). Semi-nested PCR products (4·5 kb), generated with primers HAV8 and AUAP, were extracted from the gel (QIAquick gel extraction kit; Qiagen) and sequenced. HAV sequence was only obtained with primer HAV8. A 1000-fold dilution of the AUAP/HAV8 PCR product, purified on MicroSpin S400 columns (Amersham Biosciences), was used as template in several PCRs with other primers. Sequencing of these products was not successful. PCR on the diluted product with primer pair Hav3368+/AUAP, Havgt3-3156+/Havgt3-4806M and HAV8/Hav1-3'm was used successfully in a hybrid PCRsequencing method (Berg & Olaisen, 1994). PCR products, digested with restriction enzymes EcoRI, Sau3A, XbaI and AccI, were ligated to the restriction enzyme-digested cassettes and amplified successfully with the M13 sequencing primers and sequence-specific primers. Bands of the estimated size were extracted from gels as described above and sequenced. All fragments were sequenced in both directions on an ABI PRISM 310 Genetic Analyser (Applied Biosystems) with BigDye Terminator sequencing reagents, using Applied Biosystems' recommendations.
Sequence analysis.
Sequences were aligned by using the BioEdit software (http://www.mbio.ncsu.edu/BioEdit). Genetic distances were calculated by using the DNADIST (kimura two-parameter model) and PROTDIST (dayhoff pam matrix) programs in the PHYLIP package (Felsenstein, 1993) and BioEdit. Phylogenetic trees were conducted by using the Kimura two-parameter model for nucleotide sequences and the Poisson correction method for amino acid sequences, and the neighbour-joining method in MEGA version 3 (Kumar et al., 2004
). The reliability of the trees was confirmed by bootstrap sampling of 950 replicas, also in MEGA.
Nucleotide sequences and accession numbers.
The following strain sequences from GenBank were used: AH1 (AB020564), AH2 (AB020565), AH3 (AB020566), FH1 (AB020567), FH2 (AB020568), FH3 (AB020569), HAF-203 (AF268396), L-A-1 (AF314208), LU38 (AF357222), LY6 (AF485328), SLF88 (AY032861), F.G. (X83302), GBM (X75215), NOR-21 (AJ299464), HM-175 (M14707), MBB (M20273), NCACG (K02990), PA21 (M34084), AGM27 (D00924), CY-145 (M59286), P27 (AJ519486), GA76 (M66695), CF53 (AY644676), HMH (AY644337), NOR-18 (AJ296172), NOR-19NOR-24 (AJ299462AJ299467), NOR-27 (AJ968415), Eastbourne-1 and -2 (AJ968416AJ968417).
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RESULTS |
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DISCUSSION |
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Phylogeny based on the nucleotide sequences clearly distinguished the different genotypes, regardless of the region used for analysis (Fig. 2). The distinction between HAV genotypes I and II was less clear when the amino acid sequences were analysed. When the polymerase gene was used for analysis, the clustering of subgenotypes was less obvious, and for the capsid region, even the separation into genotype I and II was vague. The inferred phylogeny based on the amino acid sequence of the capsid region also showed that the human HAV strains were related more closely to each other than to the simian strains. Similar clustering of the human HAV strains has also been obtained by phylogenetic analysis of non-synonymous sites (Ching et al., 2002
; Lu et al., 2004
).
The nucleotide comparisons in Table 1 showed a slightly higher variability in the VP1 region than in the polymerase 3D gene. However, phylogenetic analysis of the nucleotide sequences clearly distinguished the different genotypes, although the distinction was better for the polymerase gene than for the VP1 gene. Unique amino acid substitutions in the polymerase gene showed great variability among genotypes, whereas VP1 was highly conserved, particularly among the human genotypes. In addition, molecular epidemiological studies showed that VP1 is conserved. The conserved amino acid sequence in VP1 is subjected to negative selection and this will necessarily be reflected in the nucleotide sequence. Therefore, it will not reflect the genetic relatedness of HAV strains as much as regions that are not subjected to such structural constraints. Summarizing these observations, the polymerase gene might be a better candidate for the distinction of outbreak strains. So far, regions within VP1 have been useful for distinction of strains but, in light of our findings, the polymerase gene might be more suitable.
The origin of HAV genotype IIIA strains has been debated due to their diverse origin, but close sequence similarity (Brown et al., 1989; Robertson, 2001
). PA21, isolated in Panama in 1980 from a feral owl monkey, was the first isolate of genotype III and the only isolate of simian origin (Brown et al., 1989
). HAV genotype III has since been detected among humans in geographically diverse regions. The subgenotype IIIA seems to be endemic in South-East and Central Asia (Robertson et al., 1992
). The best-characterized genotype III isolates are the GA76 isolate from an outbreak in Georgia in 1976 (Khanna et al., 1992
), and the P27 strain (Costa-Mattioli et al., 2002
), the NOR-21 strain and the HMH strain isolated recently in Europe. The NOR-21 strain shared 24 unique amino acid substitutions, as well as an insertion at the C terminus of the 3C protein, with the simian strains and showed considerably more amino acid changes than the two human isolates of genotype II relative to HM-175 (Fig. 3
). Phylogenetic analysis and nucleotide and amino acid comparisons showed that genotype IIIA was more or less equally distant from the other human and simian genotypes. In contrast, analysis of the amino acid sequence of the capsid region suggested a closer relationship between the human strains, which may be explained by adaptation to the human host. The widespread and increasing distribution of this genotype among humans, and their close relatedness to PA21, suggest strongly that genotype III is of human origin.
We wondered whether the genetic variability was higher for genotype IIIA than genotype IA. During the outbreak of hepatitis A in the IVDU community in Norway in the period 19951999, we found two circulating clades of HAV. In the genotype IA outbreak, starting in 1995, the virus (NOR-17-like isolates) was highly conserved throughout a 4·5 year period (data not shown), whereas the genotype IIIA outbreak, starting in 1997, showed up to 4 % nucleotide variability within a much shorter period. The general conservation of outbreak strains of genotype IA was not seen for HAV genotype IIIA. The difference in sequence variability could be the result of different selective pressures, as well as the mode of transmission. Virus that is transmitted by the faecaloral route must cross several barriers and is therefore subjected to strong selection. By parenteral transmission, there is a direct transfer from blood to blood, with limited barriers, so we assume that the selection pressure is lower than via the faecaloral route. HAV genotype IIIA has been associated with parenteral transmission through blood products. This genotype also caused outbreaks in Sweden, the UK, Estonia and Norway in IVDU communities, where parenteral transmission of virus infections through needle-sharing practices is common. The sequence variability seen for HAV genotype IIIA might therefore be a result of parenteral transmission.
HAV genotype IIIA has been much less prevalent than genotype I and has not been detected in Norway before. We therefore assumed that the genotype IIIA strains had evolved from the same outbreak strain and not from parallel introductions of different HAV genotype IIIA strains to the same community. However, phylogenetic analysis, including PA21 and HMH as reference strains, clustered the outbreak strains into two groups, suggesting at least two introductions of genotype IIIA strains. Increased immunity in the IVDU community at the time of introduction of HAV genotype III may also suggest the occurrence of several small, limited outbreaks due to the lack of susceptible IVDU to HAV. HAV genotype IIIA seems to be endemic in parts of Asia and might therefore have been imported from these countries by transmission through travellers (IVDU or drug dealers) who import drugs illegally into Europe. If this is the case, it might explain why this genotype is so frequently associated with IVDU. The close relationship between the isolates from IVDU in the UK and Norway may support this theory by import from the same high-endemic regions on different occasions. Alternatively, the close relationship may indicate a more global spread of HAV among IVDU communities in Europe. The incidence of HAV is declining in many parts of the world (Jacobsen & Koopman, 2004). HAV genotype IIIA is emerging in Europe, especially among IVDU. Transmission of HAV in association with illegal import of drugs from endemic regions might cause an increase in the incidence of infection with HAV genotype IIIA strains. Whether the variability observed in the genotype IIIA outbreak in Norway during 19971998 was due to the nature of this genotype, its mode of transmission or frequent import from endemic regions needs to be further studied. Close surveillance by molecular epidemiology is important to understand better the pattern of transmission of this emerging genotype in Europe.
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ACKNOWLEDGEMENTS |
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Received 29 April 2005;
accepted 17 June 2005.
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