Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands1
Amsterdam Institute of Viral Genomics, 1105 BA Amsterdam, The Netherlands2
Author for correspondence: Vladimir Lukashov. Fax +31 20 691 6531. e-mail v.lukashov{at}amc.uva.nl
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Abstract |
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Introduction |
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The family Astroviridae contains a single genus, Astrovirus, which comprises all astroviruses. The antigenic relationships among astroviruses have been studied extensively. Based on serological data, eight serotypes of human astroviruses (HAstV serotypes 18) have been distinguished. Multiple studies have found no evidence for cross-reactivity between any two astroviruses from the many host species tested.
Over the last few years, the amount of sequence information on various astroviruses has grown rapidly. The entire genome has been sequenced for HAstV serotypes 1 (Willcocks & Carter, 1993 ; Lewis et al., 1994
), 2 (Jiang et al., 1993
), 3 (Oh & Schreier, 2001
) and 8 (Mendez-Toss et al., 2000
), astroviruses from sheep [OAstV (Jonassen et al., 2001
)] and turkey [TAstV-1 (Jonassen et al., 1998
) and 2 (Koci et al., 2000
)], as well as avian nephritis virus [ANV (Imada et al., 2000
)]. In addition, 3050% of the genome has been sequenced for several other members of the Astroviridae family, including HAstV serotypes 4 (Willcocks et al., 1995
; M. Hachiya, M. Matsui, M. Oseto, K. Morroka and H. Ushijima, unpublished data), 5 (Wang et al., 2001
; S. S. Monroe, S. E. Stine and R. I. Glass, unpublished data), 6 (M. M. Willcocks, J. B. Kurtz, T. W. Lee and M. J. Carter, unpublished data; I. Oishi, K. Yamazaki, T. Kase, T. Kimoto, N. Sakon, E. Utagawa, Y. Okuno, T. Ando and R. Glass, unpublished data) and 7 (Jonassen et al., 2001
; Walter et al., 2001
) as well as astroviruses from cat [FAstV (M. M. Wilcocks, M. J. Carter and D. A. Harbour, unpublished data)], pig [PAstV (Jonassen et al., 2001
; Wang et al., 2001
)] and ANV serotype 2 (T. Imada, S. Yamaguchi, M. Mase, K. Tsukamoto, M. Kubo and A. Morooka, unpublished data).
Sequence information has been used in several earlier studies to analyse the amino acid similarity among a group of astroviruses based on relatively short sequences (Monceyron et al., 1997 ; Mendez-Toss et al., 2000
; Belliot et al., 1997
; Wang et al., 2001
; Walter et al., 2001
). In the largest study so far, in terms of the number of virus species included, the amino acid sequences of ORF2 of mammalian astroviruses were shown to be more similar to each other than to avian astroviruses (Jonassen et al., 2001
). Based on similarities between ORF2 amino acid sequences of HAstV, FAstV and PAstV, a suggestion of relatively recent cross-species transmission among their host species has been put forward (Jonassen et al., 2001
). In another study, which was based on HAstV sequences of approximately 300 nt in length, relationships among HAstV serotypes were found to differ in the three ORFs (Belliot et al., 1997
). In ORF1a, the authors distinguished two phylogenetic groups of HAstV, designated genogroups A and B and comprising HAstV-1 to HAstV-5 and HAstV-6 and 7, respectively (Belliot et al., 1997
). These genogroups were not seen in ORFs 1b and 2. In ORF2, four clusters of HAstV were distinguished, comprising HAstV-1 and 6, 2 and 3, 4 and 8 and 5 and 7 (Belliot et al., 1997
). This latter observation contrasts with two more recent sets of data that, in turn, contradict each other (Wang et al., 2001
; Walter et al., 2001
). In one study, which was based on full ORF2 amino acid sequences, three bootstrap-supported phylogenetic clusters of HAstV were observed: (i) HAstV-1, 7 and 3; (ii) 5 and 6; and (iii) 4 and 8, with HAstV-2 being more related to the third cluster (Wang et al., 2001
). In another study based on nucleotide sequences of 363 nt in length, the only close relationships were seen between HAstV-2, 3 and 7, with HAstV-1, 5 and 6 being virtually equidistant from this cluster and from each other (Walter et al., 2001
). Moreover, PAstV appeared to be more related to other HAstV serotypes than HAstV-4 was (Walter et al., 2001
), which contrasts with the results of the amino acid sequence analysis of ORF2 (Jonassen et al., 2001
). Since the three studies aimed at characterization of the phylogenetic relationships among HAstV (Belliot et al., 1997
; Wang et al., 2001
; Walter et al., 2001
) differed in the genetic region and the type of sequences (nucleotide or amino acid) analysed as well as the phylogenetic method used, the reasons for the discrepancies between their results are uncertain. No systematic and explicit study on evolutionary relationships among members of the family Astroviridae has been performed yet.
Although amino acid sequences can be used to study phylogenetic relationships among species, this approach does not allow a number of basic evolutionary issues to be addressed, like, for example, the detection of the driving forces of virus evolution or the distinction between the common evolutionary origin and convergent evolution as it ignores synonymous mutations. In particular, it remained unknown how and to what degree the evolution of astroviruses was influenced by positive versus negative selection, recombinations, host-dependent evolution, cross-species transmissions and the independent evolution of genomic regions. In the present study, we collected all available sequence information on astroviruses and applied powerful phylogenetic methods to address these basic evolutionary issues.
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Methods |
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Phylogenetic analysis was performed using several methods. For all methods, positions containing an alignment gap were excluded from pairwise sequence comparisons. Bootstrap resampling was performed for each analysis (100 replications). Nucleotide distances were analysed using the neighbour-joining (NJ) algorithm as implemented in PHYLIP (NEIGHBOR), based on the Kimura two-parameter distance estimation method or the proportion of differences (p-distance). For ORFs, analyses of non-synonymous and synonymous nucleotide substitutions (those which change or do not change the amino acid, respectively) were performed using MEGA (Kumar et al., 1993 ). Estimation of both synonymous (Ds) and non-synonymous (Da) distances was based on the NeiGojobori method (Kumar et al., 1993
). The ratios of synonymous to non-synonymous substitutions (Ds:Da) were calculated (Lukashov et al., 1995
). Additionally, analyses of Ds and Da were performed using the JukesCantor correction for the NeiGojobori method. Bootscanning was performed as implemented in SIMPLOT (http://www.med.jhu.edu/deptmed/sray/).
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Results |
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To study evolutionary forces that are operational among astroviruses, we subsequently analysed nucleotide, synonymous and non-synonymous substitutions separately for the three ORFs.
Phylogenetic analysis of ORF1a found higher numbers of synonymous substitutions than non-synonymous substitutions in this genetic region, with the Ds:Da ratios being above 1 for all pairwise sequence comparisons (Fig. 2). Whereas analyses of ORF1a based on nucleotide and non-synonymous substitutions resulted in trees with the same topology, as based on full-length genomes (compare Fig. 2A
, C
with Fig. 1
), OAstV did not cluster with HAstV when synonymous substitutions were analysed, nor did TAstV-1 and 2 (Fig. 2B
). In the tree based on synonymous substitutions, OAstV, ANV and TastV-1 and 2 were equidistant from each other, with the Ds being between 0·70 and 0·80 for their pairwise comparisons, which is the level of saturation (Fig. 2B
). In comparisons within and among HAstV serotypes, the Da between any two sequences did not exceed 0·02 but the Ds varied from 0·25 to 0·35. For the two sequences of HAstV-1, a Ds:Da ratio of 9·86 (0·069:0·007) was observed. Ds:Da ratios among different serotypes of HAstV were stable, varying from 15·00 to 20·06. In comparisons among the avian viruses and OAstV, Ds:Da ratios varied from 1 to 2. Similar results were obtained using the JukesCantor correction for the NeiGojobori method: the Ds between HAstV, OAstV, ANV and TAstV varied from 2·00 to 2·95, while the Da were between 0·90 and 1·44.
Based on the analysis of ORF1b, phylogenetic relations among all viruses except HAstV-3 (Fig. 3) were similar to those observed for ORF1a. Again, analysis of synonymous substitutions resulted in the loss of tree structure, with TAstV-1 and 2, ANV and OAstV being virtually equidistant from each other and from HAstV (Fig. 3B
). Compared to ORF1a, analysis of ORF1b demonstrated a major difference in the position of HAstV-3 among other human astroviruses. HAstV-1, 2 and 8 clustered together whether synonymous or non-synonymous substitutions were analysed, with HAstV-3 branching out before the main group of human astroviruses (bootstrap values of 100, Fig. 3
). Whereas pairwise Da among HAstV-1, 2 and 8 varied in a narrow range from 0·006 to 0·012, their non-synonymous distances to HAstV-3 were 0·0330·036. For the synonymous substitutions, the distance ranges were 0·2250·312 and 0·5380·624, respectively. The latter range was close to the distances of HAstV-1, 2 and 8 to non-human astroviruses (for instance, the Ds between HAstV-1 and TAstV-1 was 0·664, Fig. 3
). Ds:Da ratios among human astroviruses were even greater in ORF1b than in ORF1a. They were 46·00 for the comparison of the two HAstV-1 sequences, 22·5039·00 for the comparisons among HAstV-1, 2 and 8, and 15·3718·39 for their comparisons with HAstV-3.
In addition to the analysis of the full-length sequences of ORF1a and 1b, we performed a phylogenetic analysis for all available partial sequences. Since many of them were derived from non-overlapping genetic regions, a single tree could not include all of them. Therefore, we performed a separate analysis for each partial sequence, which was analysed with the consensus sequences of all HAstV serotypes. All partial sequences clustered together with the consensus sequence of their serotype (data not shown).
Compared to ORFs 1a and 1b, many more full-length sequences were available for the analysis of ORF2, allowing the study of FAstV, PAstV and ANV-2, for which no full-genome sequences are currently available. We also had an opportunity to analyse evolutionary relationships among viruses of the same HAstV serotype, finding that each serotype represents a monophyletic group supported by bootstrap values of 100, whether the analysis was based on synonymous or non-synonymous substitutions (Fig. 4). Extensive intraserotype sequence heterogeneity was observed for each HAstV serotype, except HAstV-2, for which only two closely related sequences were available. HAstV-4 appeared to be the most heterogeneous serotype (perhaps only due to the larger number of sequences available), with Ds among the individual sequences being up to 0·322 and Da being up to 0·032. For the interserotype comparisons, our analysis of non-synonymous substitution revealed that some HAstV serotypes are more closely related than others. Astroviruses belonging to HAstV-3 clustered with those of HAstV-7 and together with HAstV-1. This group of three HAstV serotypes clustered together with another group, which included HAstV-5 and 6, and separately from the third group of HAstV-4 and 8, with bootstrap values of those clusters being between 87 and 100 (Fig. 4C
). These associations, except for that between HAstV-4 and 8, were lost when synonymous substitutions were analysed, with various HAstV serotypes being almost equidistant from each other (Fig. 4B
). All the mean interserotype Ds were close to the level of saturation and varied from 0·541 (between HAstV-4 and 8) to 0·736 (HAstV-1 to 4 or HastV-7). The mean interserotype Da was much lower, varying from just 0·097 (between HAstV-3 and 7) to 0·322 (between HAstV-1 and 4). As the result, the Ds:Da ratios for the interserotype comparisons varied from 2·29 to 6·43.
The phylogenetic tree of mammalian astroviruses had a cactus-like topology, when non-synonymous substitutions were analysed, with OAstV branching out first (the mean Da to different HAstV serotypes was 0·5680·619), PAstV branching out second (0·3800·451) and FAstV branching out third and being the closest to HAstV (0·2580·330). This tree structure was preserved when JukesCantor correction for the NeiGojobori method was used (data not shown). The mean Da of FAstV to HAstV serotypes varied from 0·258 to 0·330, being lower than many of the mean Da among HAstV serotypes. Nevertheless, in the phylogenetic tree based on non-synonymous substitutions, all HAstV clustered together and separately from FAstV, with a bootstrap support of 100 (Fig. 4C). This cactus-like topology of the tree of mammalian astroviruses was completely lost when we analysed synonymous substitutions and found that all virus species and HAstV serotypes, except for HAstV-4 and 8, branched out virtually from a single phylogenetic node (Fig. 4B
). This finding was a reflection of the high Ds among virus species, which were at least 0·7. Still, viruses belonging to the same HAstV serotype clustered together, with a bootstrap support of 100 for each serotype (Fig. 4B
). Similar results were obtained using the JukesCantor correction for the NeiGojobori method: the Ds between HAstV, FAstV, PAstV, OAstV, ANV and TAstV varied from 2·01 to 3·00, while the Da were between 0·39 (HAstV versus FAstV) and 1·38.
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Discussion |
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Our analyses demonstrated that all astrovirus species and HAstV serotypes currently recognized do represent individual lineages within the phylogenetic tree of the family Astroviridae. All individual virus sequences belonging to the same species or serotype clustered together, irrespective of the phylogenetic model used and genomic region analysed. Moreover, all serotypes of HAstV consistently clustered together and separately from non-human viruses, in spite of the fact that some of the mean interserotype Da in ORF2 was higher than the Da between HAstV and FAstV. This observation is in agreement with an earlier analysis of ORF2 amino acid sequences (Wang et al., 2001 ) and supports the common evolutionary origin of all HAstV.
Evolutionary relationships among HAstV serotypes differed in the three ORFs. In ORF1a, HAstV-1 branched out significantly earlier than other HAstV serotypes (Fig. 2), whereas in ORF1b, HAstV-3 was an outlier (Fig. 3
). In ORF2, the serotypes of HAstV formed several bootstrap-supported phylogenetic groups (Fig. 4
). Earlier, three contradictory studies aimed at characterization of the phylogenetic relationships among HAstV within ORF2 have been published (Belliot et al., 1997
; Wang et al., 2001
; Walter et al., 2001
). Evolutionary relationships among HAstV serotypes, as demonstrated in our study based on full-length ORFs, disagreed in several principle points with the results of two earlier studies, which were based on analyses of short genetic regions of about 300 nt in length (Belliot et al., 1997
; Walter et al., 2001
), and were generally in agreement with the analysis of full ORF2 amino acid sequences (Wang et al., 2001
). These discrepancies, together with an observation of tree incongruence among HAstV serotypes in different regions of ORF2 (Mendez-Toss et al., 2000
), indicate that the relationships within short genetic regions may not represent the true relationships among astroviruses. We demonstrated the close evolutionary relationships between HAstV-3 and 7, HAstV-5 and 6 and among HAstV-1, 3 and 7 in ORF2. Together with observations of higher variability of ORF2 compared to ORFs 1a and 1b, these data point to the independent evolution of genes and even gene regions of astroviruses, suggesting different selection pressures and/or evolutionary constraints for different genomic regions.
To study operational evolutionary forces, we analysed synonymous and non-synonymous nucleotide substitutions separately in the three ORFs. Non-synonymous substitutions, since they change the amino acids, are generally subjected to strong positive or negative selection pressure. In contrast, synonymous substitutions, which preserve amino acids, are considered to be subjected to a weaker or no selection pressure. Because the mutation rates of synonymous and non-synonymous sites should be the same, the Ds and Da, as well as their ratio, indicate the direction and intensity of selection during the evolutionary history of a group of species. For other viruses, it is known that the Ds:Da ratio may vary for different genetic regions. For instance, Ds:Da ratios within the human immunodeficiency virus type 1 (HIV-1) polymerase gene are well above 1, reflecting the deleterious character of most non-synonymous substitutions within this gene (Cornelissen et al., 1997 ), which is in contrast to the short-term evolution of its envelope gene, where the mean Ds:Da ratios is equal to 0·4: amino acid changes in this immunogenic region are generally advantageous (Lukashov et al., 1995
). In addition, the evolutionary time-scale must be considered, since, for long-term evolution, as in the separation among HIV-1 subtypes, the Ds:Da ratios within the env gene are generally above 1 (Lukashov & Goudsmit, 1997
), reflecting the accumulation of synonymous substitutions with time (Goudsmit & Lukashov, 1999
).
For all pairwise sequence comparisons in our study, we found Ds:Da ratios above 1. This indicates that negative selection is generally dominating in the evolution of astroviruses. This conclusion does not rule out that positive selection is operational and certain amino acid changes are selected for (Wang et al., 2001 ). The structure of the phylogenetic trees indicates that separation between the HAstV and FAstV lineages is the most recent separation between two astrovirus species and an increase of non-synonymous substitutions during virus adaptation to a new host could be expected. Yet, even for the comparisons of HAstV and FAstV, the Ds:Da ratios were far above 1. In all three ORFs, Ds:Da ratios were, in general, inversely related to the overall divergence among the viruses, being the highest in comparisons of the most closely related viruses (e.g. sequences belonging to the same HAstV serotype), lower in comparisons among HAstV serotypes and only slightly above 1 in comparisons between mammalian and avian viruses. This finding was directly linked to the rapid accumulation of synonymous substitutions along virus lineages, which approached the saturation level for all interspecies comparisons, rather than to different evolutionary forces operational along the evolutionary scale of astroviruses. In all three ORFs, analysis of synonymous substitutions resulted in the loss of tree structure, with the lineages of distinct virus species and even HAstV serotypes in the most variable ORF2 branching out from effectively a single phylogenetic node and mammalian and avian astroviruses clustering with each other.
Recently, a hypothesis about the evolutionary history of HAstV has been put forward, according to which HAstV-5, 3 and 4 are the possible ancestors for HAstV-6, 7 and 8, respectively (Wang et al., 2001 ). Our results do not support such a hypothesis. All HAstV serotypes did represent monophyletic clusters within the tree, indicating that the separation among HAstV serotype lineages occurred before the contemporary HAstV serotypes have been formed. Moreover, HAstV-6 and 8 are actually closer to their common nodes with HAstV-5 and 4, respectively, based on the analysis of either Ds or Da (Fig. 4B
, C
), indicating the lower level of divergence from the most recent common ancestor. Neither are our data supportive of the hypothesis that diversification among HAstV serotype lineages started in the late 1970s and continued until the 1990s (Wang et al., 2001
). We demonstrated that the oldest available HAstV sequence (the HAstV-4 sample isolated in 1971, accession number Z33883) branches out far from the HAstV-4 common node (Fig. 4
), indicating that even intraserotype diversification has started (long) before 1971 and that the separations among HAstV serotype lineages are even older evolutionary events.
The phylogenetic tree of mammalian astroviruses did not correspond to the phylogenetic tree of the virus host species (Fig. 5). The human lineage was the first to branch out in the tree of mammals, followed by the separation of the cat lineage, with the separation between the pig and the sheep lineages being the most recent evolutionary event (Kumar & Hedges, 1998
). Yet the order of separation among mammalian astroviruses was exactly the opposite (Fig. 4
), with the separation of the OAstV lineage being the oldest evolutionary event and the separation of the human virus lineage being the most recent one. These data suggest that at least two cross-species transmissions occurred in the evolutionary history of astroviruses. The most likely scenario, based on the topology of the phylogenetic trees, would be that a PAstV-like virus passed to cats and then to humans, with both transmissions possibly involving (yet unidentified) intermediate hosts. The hypothesis of relatively recent cross-species transmissions involving humans, cats and pigs has been put forward based on the amino acid similarities among ORF2 sequences of HAstV, FAstV and PAstV (Jonassen et al., 2001
). However, the extremely high Ds:Da ratios demonstrated in our study indicate a strong selection against non-synonymous substitutions (or amino acid changes) during the evolution of astroviruses. Therefore, amino acid similarity does not necessarily reflect a recent separation between lineages. Our observation that the synonymous substitutions among mammalian astroviruses are saturated and the position of the 1971 HastV-4 sample (accession number Z33883) in the phylogenetic tree do not add weight to the hypothesis that these cross-species transmissions are recent evolutionary events. We conclude therefore that a single cross-species transmission introduced astroviruses into the human population, finding no evidence for ongoing animal-to-human transmissions.
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Acknowledgments |
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References |
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Received 19 October 2001;
accepted 25 January 2002.