Department of Biological Sciences, University of South Carolina
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Abstract |
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Introduction |
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The three segments of the hantavirus genome are referred to as S (small), M (medium), and L (large). The S segment encodes the nucleocapsid protein; the M segment includes a single reading frame encoding two envelope glycoproteins, G1 and G2, in the order 5'-G1-G2-3' with respect to the coding RNA sequence complementary to viral RNA; and the L segment encodes a single protein (the L protein), which is believed to be an RNA-dependent RNA polymerase (Schmaljohn 1996
). There is evidence of antibody recognition by vertebrate hosts of the nucleocapsid protein and of the G1 and G2 proteins, and specific antibody epitopes have been mapped for the nucleocapsid protein and G1 (Jenison et al. 1994
; Lundkvist et al. 1995
). In addition, cytotoxic T lymphocyte (CTL) epitopes have been identified in the nucleocapsid protein (Van Epps, Schmaljohn, and Ennis 1999
).
Several authors have published phylogenetic analyses of selected S, M, or L segment sequences (Xiao et al. 1994
; Hörling et al. 1996
; Plyusnin, Vapalahti, and Vaheri 1996
; Schmaljohn 1996
; Nemirov et al. 1999
). Consistently, these analyses have revealed three major clusters of hantavirus sequences corresponding to species whose rodent hosts belong to the subfamilies Murinae, Sigmodontinae, and Arvicolinae. However, because only unrooted phylogenetic trees have been constructed, the relationship among these three major clusters has not been addressed by previous phylogenetic analyses. In addition, previous phylogenetic analyses have used only partial gene sequences or have been based on all nucleotide sites in coding regions. In many comparisons among hantavirus isolates, synonymous sites in coding regions are saturated with changes or are near saturation (see below); under these circumstances, use of all sites can lead to errors in phylogenetic reconstruction.
Phylogenetic analyses of North American hantaviruses and of populations of their host Peromyscus leucopus showed a correspondence between the phylogenies of viral and host populations; these results suggested that virus and host populations have coevolved, at least over the short evolutionary time since the origin of P. leucopus (Morzunov et al. 1998
). On the other hand, both this and other studies (e.g., Scharninghausen et al. 1999
) have provided evidence that host switching may sometimes have occurred earlier in hantavirus evolution. The existence of viral clades specific to the rodent subfamilies Murinae, Sigmodontinae, and Arvicolinae suggests that hantaviruses may have coevolved with rodent lineages over the much longer evolutionary time (probably 50 Myr or more) during which the radiation of the Muridae took place. However, in order to test this hypothesis, it is necessary to know the evolutionary relationship among hantavirus clades and to compare their phylogeny with that of the subfamilies of Muridae.
The present paper uses phylogenetic analysis of sequence data from S, M, and L genes to address the relationship of the major clades of hantaviruses and compare their phylogeny with that of their rodent hosts. In addition, we test for evidence of natural selection by the host immune system and its contribution to diversification of these genes among the major rodent subfamily-specific clades of hantaviruses. Note that antibody and CTL epitope regions have typically been described in only one hantavirus, and the same regions of other hantaviruses belonging to other clades may not function as epitopes. Nonetheless, comparison of such regions among different hantaviruses can provide evidence regarding the role of natural selection in their evolution, including the differentiation among hantaviral clades.
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Materials and Methods |
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In order to compare hantavirus phylogenies with those of their rodent hosts, the following sequences of the mitochondrial cytochrome b gene were used: ArvicolinaeMicrotus arvalis (U54488) and Microtus rossiaemeridionalis (U55472); SigmodontinaePhyllotis osilae (U86827), Auliscomys pictus (U03545), Calomys sorellus (U03543), Neacomys spinosus (U58391), Oligorhizomys longicaudatus (U03535), Irenomys tarsalis (U03534), Akodon boliviensis (M35691), Lenoxus apicalis (U03541), and Notiomys edwardsii (U03537); MurinaeArvicanthus niloticus (AF004570), Mus musculus (J01420), and Rattus norvegicus (J01436). The phylogeny was rooted with sequences from musk ox Ovibos moschatus (U90303) and Hawaiian monk seal Monachus schauinslandii (X72209).
Statistical Methods
Sequences were aligned at the amino acid level using the CLUSTAL W program (Thompson, Higgins, and Gibson 1994
); the alignments are available from the authors on request. When pairwise comparisons were made among a set of sequences, any codon position at which the alignment postulated a gap in one or more of the sequences was excluded from all pairwise comparisons so that comparable sets of sites were compared in each case.
Synonymous sites in the S, M, and L genes were saturated or nearly saturated in many comparisons among hantavirus isolates. Therefore, phylogenetic analyses of these genes were based on amino acid sequences. Two methods were used: the neighbor-joining (NJ) method (Saitou and Nei 1987
) based on the uncorrected proportion of amino acid differences, and the maximum-parsimony (MP) method (Swofford 1990
). Since these methods gave similar results, only NJ trees are illustrated here. The reliability of clustering patterns in NJ trees was tested by bootstrapping (Felsenstein 1985
); 1,000 bootstrap pseudosamples were used. In the case of mammalian mitochondrial cytochrome b (cyt b) genes, synonymous sites were not saturated. The NJ tree of these genes was therefore constructed on the basis of the number of nucleotide substitutions per site, estimated by Kimura's (1980)
two-parameter method, as well as on the basis of the proportion of amino acid difference.
To examine the pattern of nucleotide substitution in different gene regions, the numbers of synonymous substitutions per synonymous site (dS) and the number of nonsynonymous substitutions per nonsynonymous site (dN) were estimated using a modified version of Nei and Gojobori's (1986)
method (Zhang, Rosenberg, and Nei 1998
). Nei and Gojobori's original method can underestimate the number of synonymous sites, because it assumes equal frequencies of transitions and transversions at twofold-degenerate sites (Li 1993
). In applying Zhang, Rosenberg, and Nei's (1998)
modified version, we used an independent estimate of the transition : transversion ratio (R) to count synonymous and nonsynonymous sites. This estimate was obtained by counting transitional and transversional differences at fourfold-degenerate sites between phylogenetically independent pairs of closely related sequences. (Note that because the effects of purifying selection and transitional bias are confounded at twofold-degenerate sites, methods that estimate R from such sites are unreliable.)
These phylogenetically independent pairs of sequences were chosen on the basis of the phylogenetic trees (figs. 13 >). In the case of the S gene, 11 phylogenetically independent pairs were used: Tula/Koziky/5276Ma/94 and Tula/Koziky/5247Ma/94; Tula/Kozice 144/Ma/94 and Tula/Kozice 667/Ma/95; Isla Vista MC-SB-1 and Isla Vista PC-SB-77; Prospect Hill and Prospect Hill PH-NY1; Puumala Kamiiso-8Cv-95 and Puumala Tobetsu-60Cv-93; Puumala Sotkamo and Puumala Evo/13Cg/93; Puumala Udmartia/894Cg/91 and Puumala Puu/Kazan; Puumala P360 and Puumala K27; New York H-NY1 and New York RI-1; Hantaan C1-1 and Hantaan CFC94-2; and Dobrava Saarema/160V and Dobrava Saar/90Aa/97. In the case of the M gene, eight phylogenetically independent pairs were used: Puumala PUU/Cg-Erft and Puumala CG13891; New York-1 and New York RI-1; Convict Creek 74 and Convict Creek 107; Hantaan C1-1 and Hantaan CVMC-B11; Lee and HoJo; Hanavirus Z10 and Hantaan HV114; Seoul 80-39 and Sapporo SR-11; and Hantavirus HB55 and Hantavirus R22. For the S gene, the R value obtained was 4.6, while for the M gene it was 2.7.
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The original method of Hughes, Ota, and Nei (1990)
, like Nei and Gojobori's (1986)
method, did not take into account the possibility of transitional bias. In the present paper, we used a modified version of that method which does take into account transitional bias (Hughes et al. 2000
). A computer program implementing this method is available on request from the authors. In applying this method, we used the estimates of R obtained as described above.
Standard errors of mean dS, dN, pNC, and pNR were estimated by Nei and Jin's (1989)
method. For comparisons between two sets of sequences, Nei and Jin (1989)
considered two types of mean distance: (1) an overall distance, which is the mean of all pairwise distances between the two sets of sequences, and (2) a net distance, from which distances within each set of sequences are subtracted out. In comparisons among major clusters of hantavirus sequences, established on the basis of phylogenetic analysis, we computed distances (and their standard errors) of both types. When the groups involved are phylogenetically distinct clusters, the net distance estimates nucleotide substitutions on the internal branch of the tree between the clusters. We also reconstructed patterns of amino acid change in phylogenies by the MP method.
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Results |
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In phylogenetic trees of rodent mitochodrial cyt b genes, Arvicolinae and Sigmodontinae clustered together as sister taxa, with Murinae outside. In the NJ tree based on DNA sequences, the branch supporting this topology received significant (95%) bootstrap support (fig. 4 ). In the NJ tree based on amino acid sequences (not shown), the bootstrap support for the corresponding branch was lower (73%). In MP analyses, a single most-parsimonious tree was found for the DNA sequences (not shown), and in it, Arvicolinae and Sigmodontinae were sister taxa (i.e., the most closely related pair of monophyletic groups; Wiley 1981
). Three equally parsimonious trees were found for amino acid sequences (not shown), and in all three, Arvicolinae and Sigmodontinae were sister taxa. These results agreed with those of MP and maximum-likelihood analyses of pancreatic ribonuclease genes by Dubois, Catzeflis, and Beintema (1999)
; in those trees also, Arvicolinae and Sigmodontinae were sister taxa, although the statistical support was low.
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On the other hand, it is possible that positive selection has occurred in the HV region in the past, particularly in the differentiation on the nucleocapsid protein among hantavirus subfamilies. In order to test this hypothesis, we used the modified Hughes, Ota, and Nei (1990)
method to compare pNC and pNR with respect to amino acid residue charge. We chose to consider residue charge because of evidence that the pattern of residue charges is important in determining epitopes for antibodies (Hopp and Woods 1981
). Residues were categorized as positively charged (H, K, R), negatively charged (D, E), and neutral, with any mutation causing a change in category being counted as radical.
In the CTLE, the AbE, and the remainder of the S gene, pNC generally exceeded pNR or the two quantities were about equal. For all pairwise comparisons, pNC was significantly greater than pNR in both the AbE and the remainder (table 5 ). In contrast, in the HV region, the mean pNR for all pairwise comparisons was significantly greater than pNC (table 5 ). This pattern appeared to be mainly explained by comparisons between subfamilies rather than within subfamilies. There was no significant difference between mean pNC and mean pNR for within-subfamily comparisons (table 5 ). However, mean pNR was significantly greater than mean pNC in net comparisons between subfamilies Arv and Sig and between these two subfamilies and subfamily Mur (table 5 ). These results thus indicate that nonsynonymous differences between the subfamilies of hantaviruses have occurred in such a way as to cause charge changes in the HV region to a greater extent than expected under random substitution.
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Discussion |
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Thottapalayam (TPM) virus is a virus related to hantaviruses which was isolated from a shrew (Insectivora: Soricidae: Suncus murinus) in India (Xiao et al. 1994
). Plyusnin, Vapalahti, and Vaheri (1996)
suggested that the existence of a hantavirus relative in insectivores suggests that these viruses have coevolved with mammals since before the divergence of insectivores and rodents. A small section (80 codons) of the S gene of TPM was sequenced by Xiao et al. (1994)
. The mean dN between this sequence and the corresponding region of hantavirus S genes was 0.373 ± 0.052; synonymous sites were saturated in the same comparison. The mean dN in the corresponding region between Mur subfamily hantaviruses and Arv and Sig subfamily hantaviruses was 0.137 ± 0.022. Assuming that the Mur subfamily diverged about 50 MYA and assuming a constant rate of nonsynonymous substitution, we used these dN values to estimate the divergence of TPM from rodent hantaviruses at about 136 MYA. This is probably close to the divergence of insectivores and rodents, supporting the hypothesis of coevolution.
So far, no estimates of the mutation rate in hantaviruses have been available. Because synonymous sites are subject to little if any purifying selection in most genomes (Li 1997
), the rate of synonymous substitution can provide an estimate of the mutation rate. Synonymous sites are saturated in the comparisons between Arv, Sig, and Mur subfamilies but not in some comparisons within subfamilies. Although certain species of the genus Microtus have more recently crossed the Bering Land Bridge from Asia to North America, it is believed that the first separation of Old World and New World Microtus occurred in the early Pleistocene, 1.82.0 MYA (Hoffman and Koeppel 1985
). The mean dS between the S genes from the Old World and New World clusters of viruses with hosts in the genus Microtus was 0.964 ± 0.052 (fig. 3 ). Assuming that these viruses coevolved with their hosts, this would correspond to a substitution rate of (2.412.68) x 10-7 substitutions per site per year. Although considerably lower than synonymous substitution rates seen in such viruses as influenza or HIV-1, this rate is about two orders of magnitude faster than typical mammalian mutation rates (Li 1997
). Given such a mutation rate difference between hantaviruses and their hosts, it is not surprising to find saturation of synonymous sites in the former occurring much more rapidly than in the latter.
The present analyses did not reveal any evidence of positive selection, as might occur in the case of adaptation to host immune recognition, in the CTL epitopes of the nucleocapsid protein, in the antibody epitope region of G1, or in the N-terminal antibody epitope region of the nucleocapsid protein. However, the divergence of the three hantavirus subfamilies has been accompanied by a high level of amino acid residue charge changes in the nucleocapsid protein, particularly in the HV region. Our results suggest that between subfamilies, charge changes have occurred in the HV region to a greater extent than expected under random substitution. Such a pattern is suggestive of positive Darwinian selection (Hughes, Ota, and Nei 1990
). It is of interest that this region includes epitopes for antibodies, particularly for antibodies that discriminate between different hantavirus serotypes (Lundkvist et al. 1995
). Thus, changes in the pattern of amino acid residue charges in the HV region of the nucleocapsid protein may have occurred as an adaptation to features of the immune response that are specific to each host subfamily.
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Acknowledgements |
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Footnotes |
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1 Keywords: adaptive evolution
Bunyaviridae
Hantavirus
host-parasite coevolution
viral evolution
2 Address for correspondence and reprints: Austin L. Hughes, Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208. E-mail: austin{at}biol.sc.edu
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