Laboratorie de Virologie, Institut de Biologie, Centre Hospitalier Regional Universitaire de Nantes, Rue Quai Moncousu 9, 44093 Nantes, France1
Departamento de Técnicas Nucleares Aplicadas, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay2
Laboratorio de Biología Molecular, Asociación Española Primera de Socorros Mutuos, Boulevard Artigas 1465, 11200 Montevideo, Uruguay3
Instituto de Hematologia, Facultad de Medicina, Universidad Austral de Chile, Casilla 567, Valdivia, Chile4
Department of Cellular and Developmental Biology, University of Rome La Sapienza, Viale di Porta Tiburtina 28, 00185 Roma, Italy5
Author for correspondence: Juan Cristina. Fax +598 2 525 08 95. e-mail cristina{at}cin1.cin.edu.uy
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Abstract |
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Primers HAV2 and HAV3 differed by a single nucleotide that represents a deletion carried by some HAV strains (position 102, underlined in primer HAV3). An equimolecular mixture of the two primers was used in order to target all HAV strains encountered with the same efficiency. Twenty µl of the reaction mixture was added to PCR tubes containing 5 µl RNA from serum or stool samples. HAV RNA was reverse-transcribed into cDNA (40 min at 45 °C) and a 77 bp fragment was amplified by PCR (15 s at 94 °C and 1 min at 60 °C) for 45 cycles on an ABI Prism 7700 (Perkin Elmer). Samples testing positive in the RTPCR screening were used in order to analyse the VP1 amino-terminal and/or the VP1/2A regions of the genome. We used an RTPCR method based on primers described previously (Robertson et al., 1989 , 1992
). PCR products were purified and sequenced directly using a Big Dye DNA sequencing kit (Perkin Elmer) on a 373 DNA Sequencer apparatus (Perkin Elmer). Using this approach, 147 nt sequences in the VP1 amino terminus and 168 nt sequences in the VP1/2A region were obtained for all 22 South American isolates.
In order to determine the degree of genetic variability and the heterogeneity of the South American strains, evolutionary analysis was done by alignment of the VP1 amino-terminal region of 22 HAV strains recovered from the three different South American countries with three strains reported previously from Brazil and 21 other strains from different genotypes and geographical origins. The origins of the sequences and the strains used are listed in Table 1. Sequences were aligned using the CLUSTAL W program (Thompson et al., 1994
). A matrix of distances for Kimuras two-parameter model was then generated (Felsenstein, 1993
) and used to compute neighbour-joining phylogenetic trees. Their reliability was assessed by bootstrap resampling (1000 pseudo-replicas). These methods were implemented with software from the MEGA program (Kumar et al., 1994
).
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As can be seen in the figure, all the South American strains also clustered together in this region with all known IA strains. Strains belonging to other genotypes also clustered separately in this case. Nevertheless, genetic heterogeneity was also observed inside cluster IA among South American strains for this region of the genome and no geographically related cluster was found.
In order to study whether the phylogenetic relationships observed among the strains were appropriate using Kimuras two-parameter model (Felsenstein, 1993 ), we performed the same studies described above using the model of Tamura & Nei (1993)
. The results of these studies are shown in Fig. 2
.
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The results of this study suggest that the population of HAV that is circulating endemically in South America belongs to just one sub-genotype (IA). All of the 25 HAV strains isolated in four different South American countries belong this sub-genotype (Figs 1 and 2
; Table 1
). This is in contrast to what is observed in other regions of the world were HAV is endemic, like South Africa, where co-circulation of two different genotypes was observed (Taylor, 1997
). Moreover, in other regions of the world, like Europe and Japan, a more complex pattern is observed, since HAV isolates are derived from multiple genotypes, probably representing viruses imported from other regions (Robertson et al., 1992
; Apaire-Marchais et al., 1995
; Bruisten et al., 2001
). Genetic analysis of strains can therefore provide valuable information with regard to the source of the virus in both sporadic and epidemic infection (Apaire-Marchais et al., 1995
; Normann et al., 1995
; Kedda et al., 1995
).
The bootstrap values obtained in the phylogenetic trees allowed us to differentiate among the different genotypes and sub-types (Figs 1 and 2
). Nevertheless, bootstrap values within genotype IA did not allow us to establish definite relationships among strains situated in that cluster. However, the South American strains, isolated over a short period of time, even in the same country, were found not to be identical, and consequently more than one isolate was present and co-circulating in the same outbreak (Figs 1
and 2
). This was also unexpected, since the available data from other regions have shown that all isolates from the same outbreak were identical (Taylor, 1997
) or the majority of the cases were infected with the same strain (Robertson et al., 2000
). Moreover, strains isolated in North America (USA) show a very close genetic relationship among themselves and come from a country with low endemic rate for HAV, suggesting that they represent an almost exclusive endemic transmission of a predominant group of strains that continues to circulate in that region (Robertson et al., 1992
; see also Fig. 2
). In contrast to this view, our results suggest that strains isolated in South America, which came from a region with a medium to high endemic pattern of HAV (Robertson et al., 1992
), have a higher degree of genetic variability.
In order to gain insight into the genetic variability of the VP1 amino terminus and the VP1/2A junction regions studied, the sequences of South American strains were compared to the corresponding sequences of strain HM-175 (Cohen et al., 1987 ) (not shown). The percentage identity obtained for the VP1 amino terminus ranged from 87·1 to 91·2%, whereas the VP1/2A junction identity ranged from 88 to 92·3%. The sequences of both regions of the South American strains were then translated to amino acids and compared again with corresponding amino acid sequences from strain HM-175. This study revealed that there were more first- and second-base changes (7·1 and 14·3%, respectively) within the VP1 amino terminus (some of which resulted in non-homologous amino acid changes) than were found in the VP1/2A junction (6·7 and 10%). These results suggest that the VP1 amino terminus region of strains circulating in South America is slightly more variable than the VP1/2A junction region. This was also unexpected, since data reported previously from other regions of the world showed the VP1/2A region to be more variable (Robertson et al., 1991
, 1992
; Robertson & Nainan, 1997
). This might be due, at least in part, to the fact that few strains isolated in South America were available when these studies were performed.
Recent studies suggest a changing epidemiological pattern in HAV infection throughout South America, which may result in more clinical cases in teenagers and adults and a greater potential for outbreaks (Tapia-Conyer et al., 1999 ; Tanaka, 2000
). Whether this changing pattern is related to a higher genetic variability of HAV than previously expected, changes in hygiene conditions or a combination of these and other factors remains to be established.
Taking into account that partial sequencing of selected genome regions has been employed, a definitive picture of the biological meaning of these and other possible changes in the whole of the genome will emerge from more in-depth studies. Phylogenetic studies can provide important information for the design and evaluation of appropriate and suitable HAV vaccine candidate strain(s) for the South American region.
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Acknowledgments |
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Footnotes |
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References |
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Received 23 April 2001;
accepted 17 July 2001.