Evolutionary Diversification of Protein-Coding Genes of Hantaviruses

Austin L. Hughes2, and Robert Friedman

Department of Biological Sciences, University of South Carolina


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Phylogenetic analyses of the S, M, and L genes of the hantaviruses (Bunyaviridae: Hantavirus) revealed three well-differentiated clades corresponding to viruses parasitic on three subfamilies (Murinae, Arvicolinae, and Sigmodontinae) of the rodent family Muridae. In rooted trees of M and L genes, the viruses with hosts belonging to Murinae formed an outgroup to those with hosts in Arvicolinae and Sigmodontinae. This phylogeny corresponded with a phylogeny of the murid subfamilies based on mitochondrial cytochrome b sequences, supporting the hypothesis that hantaviruses have coevolved with their mammalian hosts at least since the common ancestor of these three subfamilies, which probably occurred about 50 MYA. The nucleocapsid protein (encoded by the S gene) differentiated among the viruses parasitic on the three subfamilies in such a way that a high frequency of amino acid residue charge changes occurred in a hypervariable (HV) portion of the molecule, and nonsynonymous nucleotide differences causing amino acid charge changes in the HV region occurred significantly more frequently than expected under random substitution. Along with evidence that at least in some hantaviruses the HV region is a target for host antibodies and the known importance of charged residues in determining antibody epitopes, these results suggest that changes in the HV region may represent adaptation to host-specific characteristics of the immune response.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The hantaviruses are classified as a genus (genus Hantavirus) of the viral family Bunyaviridae, which includes over 300 distinct viruses in five genera, all characterized by a tripartite, single-stranded RNA genome (Elliot, Schmaljohn, and Collet 1991Citation ; Pringle 1991Citation ; Bishop 1996Citation ; Plyusnin, Vapalahti, and Vaheri 1996Citation ). The first described species in the genus was the Hantaan virus, a rodent-borne virus discovered in Korea, which causes in humans a cluster of symptoms known as hemorrhagic fever with renal syndrome (HFRS) (Elliott, Schmaljohn, and Collet 1991Citation ; LeDuc et al. 1993Citation ). Subsequently, other members of the genus have been found in Asia, Europe, and North and South America (Schmaljohn 1996Citation ), and New World members of the genus have been implicated in severe pulmonary disease in humans, called hantavirus pulmonary syndrome (HPS) (Hughes et al. 1993Citation ). The natural hosts of most known hantaviruses are rodents in the family Muridae, in which these viruses cause a persistent but asymptomatic infection (Morzunov et al. 1998Citation ).

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 1996Citation ). 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. 1994Citation ; Lundkvist et al. 1995Citation ). In addition, cytotoxic T lymphocyte (CTL) epitopes have been identified in the nucleocapsid protein (Van Epps, Schmaljohn, and Ennis 1999Citation ).

Several authors have published phylogenetic analyses of selected S, M, or L segment sequences (Xiao et al. 1994Citation ; Hörling et al. 1996Citation ; Plyusnin, Vapalahti, and Vaheri 1996Citation ; Schmaljohn 1996Citation ; Nemirov et al. 1999Citation ). 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. 1998Citation ). On the other hand, both this and other studies (e.g., Scharninghausen et al. 1999Citation ) 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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Sequences Analyzed
Hantavirus sequences used in analyses are summarized in table 1 . Only complete coding sequences of the S, M, and L genes were used; certain sequences identical or nearly identical to others in the data set were not included. In order to root the phylogeny of M sequences, we used an M sequence from the Dugbe virus (GenBank accession number M94133), a member of the genus Nairovirus of the family Bunyaviridae. In order to root the phylogeny of L sequences, we used L sequences from the following species in the genus Tospovirus of the family Bunyaviridae: peanut bud necrosis virus (PBNV) (accession number AF025538) and tomato spotted wilt virus (TSWV) (accession number D10066).


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Table 1 Hantavirus Sequences Used in the Analyses

 
In order to understand the impact of selection by the host immune system on hantavirus genes, patterns of nucleotide substitution were analyzed separately in the case of known antigenic regions. In the case of the M gene, the antibody epitope (AbE) in G1 identified by Jenison et al. (1994)Citation was compared with the remainder of G1 and with G2. The AbE corresponds to residues 59–89 of G1 in the Sin Nombre virus. Both antibody and CTL epitopes have been found in both the and the C-terminal portions of the nucleocapsid protein. We dealt with the following distinct regions of the nucleocapsid protein:

  1. We analyzed CTL epitopes (CTLEs) corresponding to residues 12–20 and 421–429 of the nucleocapsid protein of the Hantaan virus (Van Epps, Schmaljohn, and Ennis 1999Citation ).
  2. An AbE was identified at residues 17–59 of the Sin Nombre virus nucleocapsid protein by Jenison et al. (1994)Citation . We analyzed this epitope excluding the four N-terminal residues, which overlap the CTLE.
  3. Several studies have identified a hypervariable (HV) region of the amino acid sequences of hantavirus nucleocapsid proteins (Plyusnin et al. 1994aCitation ; Hörling et al. 1996Citation ). We defined the HV region to correspond to residues 242–281 of our joint alignment (Plyusnin et al. 1994aCitation ). This region includes an epitope region for both monoclonal antibodies and human patient sera (Lundkvist et al. 1995Citation ).

In order to compare hantavirus phylogenies with those of their rodent hosts, the following sequences of the mitochondrial cytochrome b gene were used: Arvicolinae—Microtus arvalis (U54488) and Microtus rossiaemeridionalis (U55472); Sigmodontinae—Phyllotis 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); Murinae—Arvicanthus 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 1994Citation ); 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 1987Citation ) based on the uncorrected proportion of amino acid differences, and the maximum-parsimony (MP) method (Swofford 1990Citation ). 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 1985Citation ); 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)Citation 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)Citation method (Zhang, Rosenberg, and Nei 1998Citation ). 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 1993Citation ). In applying Zhang, Rosenberg, and Nei's (1998)Citation 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. 1–3 >). 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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Fig. 1.—Phylogenetic tree of hantavirus L gene sequences, based on the proportion of amino acid differences (p). The tree is rooted with PBNV and TSWV sequences. Numbers on branches are percentages of 1,000 bootstrap pseudosamples supporting the branch; only values >=60% are shown

 
Hughes, Ota, and Nei (1990)Citation proposed a method of testing whether nonsynonymous nucleotide differences occur at random with respect to some qualitative amino acid residue property of interest (e.g., charge, polarity, etc.). Briefly, this method categorizes nonsynonymous differences as conservative or radical (nonconservative) with respect to the property of interest and computes the proportion of conservative nonsynonymous differences per conservative site (pNC) and the proportion of radical nonsynonymous differences per radical nonsynoynmous site (pNR). If nonsynonymous differences occur at random with respect to the property of interest, it is expected that pNC = pNR. If nonsynonymous differences occur in such a way as to conserve the property of interest, pNC > pNR. On the other hand, if nonsynonymous differences occur in such a way that the property is changed to a greater extent than expected under random substitution, pNR > pNC. A pattern of pNR > pNC in a given gene region is suggestive of positive Darwinian selection acting to favor a change in residue properties in that region.

The original method of Hughes, Ota, and Nei (1990)Citation , like Nei and Gojobori's (1986)Citation 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. 2000Citation ). 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)Citation method. For comparisons between two sets of sequences, Nei and Jin (1989)Citation 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.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Phylogenetic Analyses
NJ trees of L, M, and S genes are shown in figures 1–3 , respectively. In each tree, there were separate clusters containing isolates whose rodent hosts are members of the subfamilies Arvicolinae, Sigmodontinae, and Murinae; these clusters are designated subfamilies Arv, Sig, and Mur, respectively, in figures 1–3 . In the rooted trees of L and M sequences, the Mur subfamily formed an outgroup to the Arv and Sig subfamilies; this pattern received highly significant (100%) bootstrap support in both cases (figs. 1 and 2 ). In the case of the S sequence tree, there was no outgroup available for rooting the tree. The tree was thus drawn with the root in the midpoint of the longest internal branch; this rooting again placed Mur as an outgroup to Arv and Sig (fig. 3 ).



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Fig. 3.—Unrooted phylogenetic tree of hantavirus S gene sequences, based on the proportion of amino acid differences (p). Numbers on branches are percentages of 1,000 bootstrap pseudosamples supporting the branch; only values >=60% are shown

 
In the case of the L genes, there was a single MP tree, the topology of which was identical to that of the NJ tree (fig. 1 ). In the case of M genes, there were 132 equally parsimonious trees; the consensus tree (not shown) was similar to the NJ tree (fig. 2 ) and, like the latter, showed the Mur subfamily as an outgroup to the Arv and Sig subfamilies. In the case of S genes, there were eight equally parsimonious trees; again, the consensus tree (not shown) closely resembled the NJ tree (fig. 3 ).



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Fig. 2.—Phylogenetic tree of hantavirus M gene sequences, based on the proportion of amino acid differences (p). The tree is rooted with a Dugbe virus sequence. Numbers on branches are percentages of 1,000 bootstrap pseudosamples supporting the branch; only values >=60% are shown

 
While the natural distribution of the Murinae is restricted to the Old World and that of Sigmodontinae is restricted to the New World, Arvicolinae are naturally found in both the New World and the Old World (Hoffmann and Koeppl 1985Citation ). In the NJ tree of S genes, there were two major subclusters within the Arv cluster: (1) a cluster whose hosts are in the genus Microtus, inhabiting both the Old World and the New World (Tula, Isla Vista, Prairie Vole, and Prospect Hill viruses), and (2) a cluster of exclusively Old World viruses, including Puumala, with hosts in the genus Clethrionomys, and Khabarovsk, whose host is in the genus Microtus (table 1 and fig. 3 ). The former subcluster represents the only known group of closely related hantaviruses with both New World and Old World representatives.

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 1981Citation ). 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)Citation ; in those trees also, Arvicolinae and Sigmodontinae were sister taxa, although the statistical support was low.



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Fig. 4.—Phylogenetic tree of cyt b sequences of Muridae, based on the number of nucleotide substitutions per site (d). The tree is rooted with sequences from an artiodactyl and a carnivore. Numbers on branches are percentages of 1,000 bootstrap pseudosamples supporting the branch; only values >=60% are shown

 
Nucleotide Substitution
Table 2 shows mean dN values in regions of M genes, computed within and between the Arv, Sig, and Mur subfamilies. Generally, dN was found to be higher in the AbE and in the remainder of G1 than in G2, and dN in the AbE tended to be somewhat higher than that in the remainder of G1 (table 3 ). For S genes, the mean dN was generally much lower in the CTLE than in the remainder of the gene for the Arv subfamily but not for other subfamilies, including Mur, for which these epitopes were identified (Van Epps, Schmaljohn, and Ennis 1999Citation ) (table 3 ). dN in the AbE was generally higher than that in the remainder of the gene in between-subfamilies comparisons but not in within-subfamily comparisons (table 4 ). In contrast, dN in the HV region was consistently and significantly greater than that in the remainder of the gene in all comparisons.


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Table 2 Mean Numbers of Nonsynonymous Substitutions per 100 Sites (dN ± SE) in Comparisons Among Hanavirus M Gene Regions

 

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Table 3 Mean Numbers of Nonsynonymous Substitutions per 100 Sites (dN ± SE) in Comparisons Among Hanavirus S Gene Regions

 

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Table 4 Mean Numbers of Synonymous (dS) and Nonsynonymous (dN) Nucleotide Substitutions per Site (±SE) for 11 Phylogenetically Independent Comparisons of Closely Related Paris of Hantavirus S Genes

 
When the rate of nonsynonymous substitution is unusually high in one region of a gene, this suggests either that the region is one of low functional constraint at the amino acid level or that positive selection has acted to favor amino acid replacements in the region. One way of deciding between these hypotheses is to compare dS and dN; the latter is expected to exceed the former in cases of positive selection (Hughes and Nei 1988Citation ; Hughes 1999Citation ). However, in the case of hantaviruses isolates, synonymous sites are saturated in comparisons between distantly related isolates, for example, in comparisons between the Arv, Sig, and Mur subfamilies (data not shown). Therefore, we compared dS and dN in 11 phylogenetically independent comparisons of closely related S sequences (see Materials and Methods). Mean dS was significantly greater than mean dN in all regions analyzed except the AbE (table 4 ). The lack of a significant difference in the AbE was not, however, caused by an elevated dN in this region, but by a lower than usual dS (table 4 ). Thus, comparisons of closely related S gene sequences showed no evidence of positive selection on the AbE or any other region.

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)Citation 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 1981Citation ). 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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Table 5 Mean Percentages of Conservative (pNC) and Radical (pNR) Nonsynonymous Difference (±SE) Among Hantavirus S Gene Regions

 
In order to further examine the pattern of amino acid change in the HV region, we reconstructed amino acid changes in the nucleocapsid protein using the MP method. Among changes reconstructed to have occurred on the major internal branches of the phylogeny (i.e., the branches connecting the Arv, Sig, and Mur clusters), 15 of 24 (62.5%) changes in the HV region involved a charge change, whereas only 41 of 136 (30.1%) changes occurring in the remainder of the protein involved a charge change. The difference between these proportions was significant ({chi}2 = 7.01, df = 1; P < 0.01).Thus, this analysis also showed a significant bias toward charge changes in the HV region among hantavirus subfamilies.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Phylogenetic analyses of the L, M, and S genes of hantaviruses (figs. 1–3 ) supported the hypothesis that hantaviruses form three major clades (subfamilies Arv, Sig, and Mur) and that subfamily Mur (whose hosts are Murinae) forms an outgroup to subfamilies Arv (host Arvicolinae) and Sig (host Sigmodontinae). This relationship among the hantavirus subfamilies implies that the phylogeny of the virus is congruent with that of its rodent hosts (fig. 4 ). This, in turn, suggests that the hantaviruses have coevolved with their hosts since at least the common ancestor of the subfamilies Arvicolinae, Sigmodontinae, and Murinae. Molecular data have provided strong evidence that the genera Mus and Rattus, both in Murinae, diverged about 40 MYA (Kumar and Hedges 1998Citation ). Given the phylogeny of figure 4 , it seems likely that the last common ancestor of Arvicolinae, Sigmodontinae, and Murinae occurred at least 50–60 MYA. Thus, it appears that the hantaviruses have been coevolving with their rodent hosts for 50–60 Myr.

Thottapalayam (TPM) virus is a virus related to hantaviruses which was isolated from a shrew (Insectivora: Soricidae: Suncus murinus) in India (Xiao et al. 1994Citation ). Plyusnin, Vapalahti, and Vaheri (1996)Citation 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)Citation . 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 1997Citation ), 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, 1.8–2.0 MYA (Hoffman and Koeppel 1985Citation ). 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 (2.41–2.68) 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 1997Citation ). 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 1990Citation ). It is of interest that this region includes epitopes for antibodies, particularly for antibodies that discriminate between different hantavirus serotypes (Lundkvist et al. 1995Citation ). 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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Table 1 Continued

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
This research was supported by grant GM34940 from the National Institutes of Health to A.L.H. and by a grant from the South Carolina Commission on Higher Education.


    Footnotes
 
Claudia Kappen, Reviewing Editor

1 Keywords: adaptive evolution Bunyaviridae Hantavirus host-parasite coevolution viral evolution Back

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 Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 

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Accepted for publication July 4, 2000.