Immune and artificial selection in the haemagglutinin (H) glycoprotein of measles virus

Christopher H. Woelk1, Li Jin2, Edward C. Holmes1 and David W. G. Brown2

Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK1
Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, London NW9 5HT, UK2

Author for correspondence: Christopher Woelk. Fax +44 1865 310447. e-mail Christopher.Woelk{at}zoo.ox.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
We present a maximum likelihood (ML) analysis of the selection pressures that have shaped the evolution of the large (L) protein and the haemagglutinin (H) glycoprotein of measles virus (MV). A number of amino acid sites that have potentially been subject to adaptive evolution were identified in the H protein using sequences from every known genotype of MV. All but one of these putative positively selected sites reside within the ectodomain of the H protein, where they often show an association with positions of potential B-cell epitopes and sites known to interact with the CD46 receptor. This suggests that MV may be under pressure from the immune system, albeit relatively weakly, to alter sites within epitopes and hence evade the humoral immune response. The positive selection identified at amino acid 546 was shown to correlate with the passage history of MV isolates in Vero cells. We reveal that Vero cell passaging has the potential to introduce an artificial signal of adaptive evolution through selection for changes that increase affinity for the CD46 receptor.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Measles virus (MV) is an enveloped virus (genus Morbillivirus, family Paramyxoviridae) containing a negative-sense (-) RNA genome of 15894 nucleotides. MV is one of the current eradication targets established by the World Health Organization (WHO) and widespread vaccination programmes have greatly reduced the incidence of measles in the Western hemisphere. Unfortunately, close to 1 million deaths a year still result from MV infection in the developing world and reintroductions into Western countries make MV a significant public health problem. Although MV is considered to be serologically monotypic, measles infections due to the wild-type virus can be classified into several genotypes, which may have distinct geographical origins (World Health Organization, 1998 ). The biological significance of this diversity is not well understood but since the haemagglutinin (H) and fusion (F) surface glycoproteins induce neutralizing antibody responses, it is possible that sequence differences may reflect immunological pressure (Griffin & Bellini, 1996 ). A rare complication of measles infection results in subacute sclerosing panencephalitis (SSPE; 1–5 per million cases), which is similar to measles inclusion body encephalitis (MIBE) in that it develops due to a persistent infection of neural cells. SSPE is a fatal neuro-degenerative disorder whose pathogenesis remains poorly understood.

The L protein is thought to be the viral polymerase due to its low abundance, large size and localization to transcriptionally active viral cores. The centre of this protein comprises five regions of high homology, which form an ancestral polymerase fold that is conserved in the RNA-dependent RNA polymerases of other virus families (Lamb & Kolakofsky, 1996 ). The H protein is thought to interact with two different receptors, CD46 and SLAM (Manchester et al., 2000 ; Tatsuo et al., 2000 ), and together with the F glycoprotein facilitates MV entry into host cells (Lamb, 1993 ). The H protein can be divided into three domains; a cytoplasmic domain, a transmembrane domain and a large ectodomain (Muller et al., 1993 ). The ectodomain consists of a {beta}-propeller structure projected from the cell surface by two helix-rich stem regions. Six antiparallel {beta}-sheets of four strands each form the propeller such that the fourth strand of each sheet is connected by a loop to the first strand of the next sheet. Two such loops connecting sheets 4 to 5 and 5 to 6 are thought to delineate the CD46 receptor-binding domain (Langedijk et al., 1997 ). Cysteine interactions allow pairing of amino acids 386 to 394 and 381 to 494, and the mature 78 kDa form of the H protein is produced by N-glycosylation of Asn residues 168, 187, 200 and 215, in the second stem region (Hu et al., 1994 ; Langedijk et al., 1997 ).

It was our aim to determine whether the H gene of MV might be subject to positive selection, such as that mediated by the immune response. In the context of comparing DNA sequences, this is most commonly done by counting the number of synonymous and nonsynonymous substitutions per site, referred to as dS and dN respectively. Omega ({omega}) is the ratio of dN to dS (dN/dS) and an {omega}<1 is indicative of purifying selection, an {omega}=1 suggests complete neutrality, while an {omega}>1 is an unambiguous signal of positive selection since it means that the rate of fixation is higher than the rate of mutation, which cannot be the case under neutral evolution (Yang & Bielawski, 2000 ). Pairwise methods are commonly used for estimating {omega} ratios but they suffer from the over-representation of distances associated with deeper branches in the phylogeny, averaging over large regions of sequence, and the movement of sites between the synonymous and nonsynonymous categories (Muse, 1996 ; Nei & Gojobori, 1986 ). A recently developed maximum likelihood (ML) method (Yang et al., 2000 ) for calculating {omega} ratios that accounts for phylogenetic structure as well as biases in both codon usage and transition/transversion ratio has been found to be superior to early pairwise methods and is able to detect localized positive selection (Zanotto et al., 1999 ). This ML method provides more realistic models of sequence evolution, and allows the classification of individual amino acid sites into conserved, neutral or positively selected classes.

Although the current measles vaccine protects against all known genotypes of MV, antigenic differences have been detected between strains and it has been suggested that mutations in critical protective epitopes could lead to the generation of vaccine escape mutants (Jin et al., 1998 ; Tamin et al., 1994 ). Since the H protein is the major target of the immune response we decided to determine if there were any sites, or phylogenetic lineages, under positive selection in this protein and, if so, whether they correspond to positions of T-cell or B-cell epitopes, suggesting that immune selection is in operation. For comparison purposes, the conserved L gene of MV, which is not thought to be under immune selection, was also analysed (Komase et al., 1995 ). The effect of using passaged strains in selection analysis may introduce a false signal of positive selection (Woelk & Holmes, 2001 ). Since the propagation of MV isolates in Vero cells has been well documented (Komase et al., 1990 ; McChesney et al., 1997 ; Rima et al., 1997 ; Rota et al., 1992 , 1994 , 1996 ) the effect of such passaging on selection analysis was also investigated.


   Methods
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Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses and passaging history.
Sequences of the L and H genes from acute infections with MV have been determined from MV cultures in B95a, B95-8, human embryonic kidney, Hep-2, primary monkey kidney or Vero cells. Viruses from SSPE patients were sequenced directly from brain material using RT-PCR products or after cloning (Cattaneo et al., 1988 ; Hu et al., 1993 ; Komase et al., 1990 ; Rima et al., 1997 ; M. Watanabe, personal communication). Nucleic acid from the four SSPE strains sequenced in this study (UK44/80s, UK83/56, UK85/56 and UK88/55) was extracted from frozen brain specimens using the silica-guanidinium thiocyanate method (Boom et al., 1990 ) and the entire H gene was amplified by RT-PCR as described previously (Jin et al., 1996 , 1998 ). The GenBank accession numbers of these newly determined isolates are Af399848Af399851.

{blacksquare} Multiple alignments.
All of the complete gene sequences available in GenBank for the H and L genes of MV were collected and aligned using CLUSTALW (Thompson et al., 1994 ) after the addition of the SSPE sequences determined in this study. One further H gene sequence from an SSPE patient was obtained from Michiko Watanabe (personal communication). A full list of the sequences analysed in this paper is available at http://evolve.zoo.ox.ac.uk. After identical sequences and vaccine strains had been removed, the L gene alignment contained 12 sequences, but the H gene alignment contained a much larger number of sequences such that its size would make the maximum likelihood analysis of selection pressures computationally unfeasible. Yang (1998) proposed that the removal of sequences with a high level of similarity has insignificant effects on the results of selection analysis and thus H gene sequences with greater than 99% similarity were removed to produce a data set of 50 sequences. This H gene data set is referred to as the ‘Global’ data set because it contains isolates from all of the genotypes of MV found worldwide. A further two data sets were created from this Global data set. The ‘-Vero’ data set contains 25 H gene sequences and was created by removing MV isolates that had been passaged in Vero cells or had unknown passaging histories. The ‘Vero’ data set contains 16 H gene sequences and consists of isolates that had only been Vero passaged.

{blacksquare} Phylogenetic analyses.
The PAUP* package was used to construct maximum likelihood (ML) trees from sequences of L and H gene (Global, -Vero and Vero) data sets using the HKY85+{Gamma} model of nucleotide substitution (Swofford, 2000 ). Maximum likelihood trees for the L gene and Global data sets are presented in Fig. 1(a) and Fig. 1(b), respectively. Values for both the transition/transversion (TS/TV) ratio and the shape parameter ({alpha}) of a gamma distribution of rate variation among sites (with eight categories) were estimated from the data (Table 1).



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Fig. 1. (a) Maximum likelihood (ML) tree for 12 L gene sequences of MV and (b) an ML tree of the 50 H gene sequences from the Global data set. The unrooted phylogenies were midpoint rooted for purposes of clarity and bootstrap values (1000 replicates) are indicated at the nodes when over 75%. All horizontal branches are drawn to scale and genotypes are indicated on the right-hand side. Isolates in bold were sampled from SSPE cases and the isolate in italics (Brx/Germany/1971) was from a patient with a form of measles encephalitis that had different properties to SSPE. Isolate names are in the format: traditional name (when applicable)/country of isolation/year of isolation/isolate number (when applicable). In terms of the SSPE cases, dates in parentheses refer to the instance of initial MV infection (IU, information unknown). For more information on the isolates used in this study please refer to http://evolve.zoo.ox.ac.uk.

 

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Table 1. Maximum likelihood parameters for the phylogenetic trees of the L and H gene data sets of MV

 
{blacksquare} Selection analyses.
The ML method for determining selection pressures developed by Yang et al. (2000) incorporates a variety of models that use statistical distributions to account for variable {omega} (dN/dS) ratios among codon sites and applies them to a phylogenetic tree of protein-coding DNA sequences. Yang et al. (2000) originally used a total of 13 models (FR and M0 through M13) to investigate selection pressures in gene sequences. From their research it became evident that a subset of these models (FR, M0, M1, M2, M3, M7, M8) is sufficient for accurate selection analysis. The identification of amino acid/codon sites under positive selection involves two major steps, the first of which is to test whether sites exist where {omega}>1. This is done through the comparison of nested models that accommodate positive selection with those that do not, using a likelihood ratio test (LRT). Models unable to account for positively selected sites include M0, M1 and M7. M0 calculates a single {omega} parameter averaged over all sites between the bounds 0 and 1, whereas M7 uses a discrete beta distribution (with 10 categories) to model different {omega} ratios among sites between the same bounds. The shape of this beta distribution is governed by the two parameters p and q. The M1 model accounts for neutral evolution by estimating the proportion of sites that are conserved ({omega}=0) and neutral ({omega}=1). In contrast, the M2, M3 and M8 models are able to account for positive selection since they all have parameters that can estimate {omega}>1. M2 and M8 extend the capabilities of M1 and M7, respectively, through the addition of two parameters that have the potential to estimate an {omega}>1 for a further class of sites. The M3 model provides the most sensitive test for positive selection by estimating a {omega} ratio for a predetermined number of classes (in this case three). M2 and M3 are both nested with M0 and M1, while M7 is nested with M8, and nested models can be compared in LRTs. Once positively selected sites have been shown to exist the second step involves using Bayesian methods to locate their position. Sites belonging to a site class with {omega}>1 and having high posterior probabilities are likely candidates for positive selection. Finally, by making use of the free ratio (FR) model it is possible to detect individual branches that have undergone positive selection (Yang, 1998 ). Instead of analysing sites, the FR model estimates a single {omega} ratio for the entire gene along each branch in a phylogeny and can be compared to the M0 model, which assumes the same {omega} ratio for the entire tree. It should be noted that this latter test for positive selection is very conservative since the average {omega} ratio along the entire gene must be >1 for a branch to be deemed positively selected. The methods and models described were implemented using the CODEML program of the PAML package, version 3.0prior (Yang, 1997 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Parameter estimates resulting from selection analyses are given in Table 2 and a summary of LRTs appears in Table 3. Although the FR model significantly outperformed the M0 model in the L gene, Global and -Vero data sets (Table 3), there was no instance of an {omega} ratio significantly greater than 1 for any lineage. Hence, there is no evidence for positive selection on any particular branch of the MV phylogenies derived from either the L or H sequences used in this study. Further, none of the models able to identify a positively selected class of sites (M2, M3 and M8) did so for the L gene data set. Although the M2 and M3 models are able to reject the simpler M0 and M1 models, both suggest that the majority of sites in the L gene are strongly conserved with a small proportion (4·7%) of neutrally evolving sites. This small neutral class was also estimated by M8 but, since M8 could not reject M7, it could not be confirmed significantly in this case.


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Table 2. Likelihood and parameter estimates for selection analysis of the L and H gene data sets of MV

 

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Table 3. Likelihood ratio tests (LRTs) between models of codon evolution for the L and H gene data sets of MV

 
Selection analysis of H gene isolates from worldwide MV genotypes
For the analysis of the Global data set all the models which can account for positive selection (M2, M3 and M8) significantly rejected those that do not (M0, M1 and M7) (Table 3). The M3 and M8 models produced the best likelihood score and they both suggest that a small proportion of codon sites in the H gene are under weak positive selection. M3 estimates 1·9% of sites to have a positive selection pressure of 2·319 whereas M8 suggests a larger number of sites (6·1%) to have a slightly weaker selection pressure of 1·658. M3 further suggests that the majority of sites (88·8%) are fairly conserved ({omega}1=0·146) with 9·3% effectively neutral ({omega}0=0·984). M3 and M8 were also significantly favoured in LRTs indicating that they provide the best representations of the data. Using Bayesian methods we identified 11 amino acid sites belonging to the positively selected class under the M8 model. These were positions 12 and 348 at the 90% significance level; positions 62, 303, 423 and 476 at the 95% significance level; and positions 211, 481, 546, 562 and 575 at the 99% significance level (Table 4). The M3 model could only confirm that site 575 was under positive selection at the 90% level. Since M3 and M8 cannot be compared with an LRT it is unclear which provides the more accurate description of the positive selection pressure affecting the H gene. Therefore, we will discuss the positively selected sites identified by both M3 and M8.


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Table 4. Correlation of positively selected sites in the H protein of MV with known biological features

 
Selection analysis of Vero-passaged H gene isolates
The effect of including Vero-passaged isolates in selection analysis was investigated by (i) removing them from the Global data set and (ii) analysing them as a separate data set. Exclusion of Vero-passaged isolates from the Global data set did not inhibit the ability of models M2, M3 and M8 to reject M0, M1 and M7 (Table 3). In fact, parameter estimates under most of the models were remarkably similar to those obtained when Vero-passaged isolates were included (Table 2). The exception was M3, which in this case estimated a larger proportion of sites (6·7%) under a weaker selection pressure ({omega}2=1·549). The majority of sites (93·3%) were again fairly conserved ({omega}0 and {omega}1=0·162). Under M3 and M8, Bayes theory significantly confirmed positive selection at sites 12, 211, 303, 348, 423, 476, 562 and 575, although in some cases with marginally different significance levels than before. However, this -Vero data set failed to re-identify positive selection at sites 62, 481 and 546. The M3 model additionally identified sites 302, 451 and 560 to be positively selected at the 90% level (Table 4) of which sites 302 and 560 were reconfirmed by the M8 model. Although these newly identified sites all had posterior probabilities below the 90% level when the Global data set was analysed, they were all in the 80th percentile.

When Vero-passaged isolates were analysed separately the parameter estimates differed extensively from results of the Global and -Vero data sets (Table 2). Although the significance of positive selection under M3 and M8 was still confirmed by LRTs (Table 3), both these models estimated a smaller proportion of positively selected sites (0·6%) with a relatively high {omega} ratio (>5 in both cases). M3 again had the best likelihood score and divided the remainder of sites such that 53·2% were moderately conserved ({omega}0=0·387) and 46·2 % were strongly conserved ({omega}1=0·001). For both M3 and M8, posterior probabilities could only assign two sites at positions 546 and 562 to the positively selected class above the 90% level (Table 4).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Our analysis was unable to identify amino acid sites under positive selection in the L protein of MV but did suggest that a number of amino acid residues within the H protein may be subject to adaptive evolution (Fig. 2). Current thinking dictates that substitutions in T-cell epitopes (TCEs) and B-cell epitopes (BCEs) of the H gene are under immune selection because T-cell and B-cell immunity provide the major defence against viral infections (El Kasmi et al., 2000 ; Jaye et al., 1998 ). Several studies have investigated the antigenic profile of the H protein through testing the reactivity of synthetic peptides with human sera (Muller et al., 1993 ; Obeid et al., 1994 ). The peptides shown to react with human antibodies in these studies, therefore representing potential BCEs, are labelled in Fig. 2. Obeid et al. (1994) showed that peptides 29 (343–357) and 41 (463–477) were recognized by 20% of serum samples and these regions correspond to positively selected sites 348 and 476, respectively. Muller et al. (1993) showed that peptides H21 (201–215), H42 (411–425) and H57 (561–575) reacted with 40, 48 and 83% of serum samples, respectively. Putative positively selected sites 211, 423, 562 and 575 fall within these peptides.



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Fig. 2. Amino acid sequence of the H protein from the Edmonston strain of MV. Notation above the sequence divides the protein into the cytoplasmic (1–34) and transmembrane domains (35–58) (Muller et al., 1993 ), and the stem and {beta}-propeller regions of the ectodomain (59–617). Amino acids represented by lower-case letters form strands in {beta}-sheets from which the propeller structure is constructed (Langedijk et al., 1997 ). Circled amino acids indicate those identified to be under positive selection with a posterior probability above the 90% level. Circles that are shaded were lost, and grey circles gained, when Vero-passaged isolates were removed from the selection analysis. Boxed sequences indicate 15-mer peptides that reacted with human sera, and when in the ectodomain are thought to be potential BCEs, or TCEs when present in the cytoplasmic and transmembrane domains (Muller et al., 1993 ; Obeid et al., 1994 ). The dotted underlined region refers to the CD46 receptor-binding domains (Langedijk et al., 1997 ) and amino acids in bold refer to residues thought to be involved in CD46 interaction (Bartz et al., 1996 ; Lecouturier et al., 1996 ; Patterson et al., 1999 ). Cysteine residues believed to form pairings are labelled C1 and C2 (Langedijk et al., 1997 ). The {Delta} symbol indicates both conserved and nonconserved N-glycosylation sites.

 
Positively selected sites 62, 302, 303 and 560 have been shown to correlate with BCEs recognized by MAbs or with peptides that react with mouse sera (Hu et al., 1993 ; Jin et al., 1998 ; Obeid et al., 1994 ; Rota et al., 1992 ; Tamin et al., 1994 ). However, Muller et al. (1993) noticed discrepancies in the reactivity patterns of peptides between different animal sera so they are probably not relevant to the immune situation in humans. Further, virus-infected cells have been shown to express two types of H protein with different extents of glycosylation (Ogura et al., 2000 ) and it has been suggested that N-glycosylation can contribute to conformational epitopes (Hu et al., 1994 ). However, the positively selected sites we identified in the H protein did not correspond to Asn positions of N-glycosylation (Fig. 2), suggesting that MV does not modulate the extent of glycosylation of the H protein to mask antigenic sites or abolish carbohydrate epitopes.

Extensive work has been done on the cellular immune response to MV (Jacobson et al., 1984 , 1989 ; Kreth et al., 1979 ; Lucas et al., 1982 ; Richert et al., 1985 ; Sethi et al., 1982 ; van Binnendijk et al., 1990 ), the most recent of which showed specific cytotoxic T-lymphocyte (CTL) responses in Caucasian and West African adults to MV fusion (F) and H proteins and to a lesser extent to the nucleoprotein (NP) (Jaye et al., 1998 ). They concluded that different MHC class I and class II molecules bind to different sets of peptides scattered along the entire sequence of F, H and NP, but that the main CTL response was mediated through CD8 HLA class I-restricted cells that were largely directed to the F and H proteins. Synthetic peptides comprising amino acids 29–37, 31–45 and 41–55 of the H protein (Fig. 2) have been shown to induce T-cell proliferation when exposed to either peripheral blood mononuclear cells (PBMC) or peripheral blood leukocytes (PBL) (Muller et al., 1993 ; Nanan et al., 1995 ). These peptide regions did not correlate to any putative sites of positive selection identified in our study. Obeid et al. (1993) did distinguish peptides bearing TCEs that corresponded to positively selected sites, namely peptides 423–437 (423), 443–457 (451), 473–487 (476 and 481) and 543–557 (546) (Table 4). However, these peptides were identified by testing responses of spleen cells from peptide-primed mice to MV in vitro and this is unlikely to be relevant to the immune response in humans.

MV infection is facilitated through the binding of the H protein to two different receptors. Vero-passaged isolates are thought to have an affinity for binding the CD46 receptor (Manchester et al., 2000 ) whereas isolates passaged in B-cells appear to prefer the SLAM receptor (Tatsuo et al., 2000 ). Binding of CD46 by the H protein leads to receptor down-regulation, syncytium formation, haemagglutination and haemadsorption (Lecouturier et al., 1996 ). The relevance of the CD46 receptor in natural measles infection has recently been confirmed (Manchester et al., 2000 ) and its down-regulation appears to be modular (Sakata et al., 1998 ). Low-affinity CD46-binding isolates can be converted to the high-affinity CD46-binding phenotype by a substitution of Asn for Tyr at position 481 (Hsu et al., 1998 ). Likewise, a substitution of Ser for Gly at site 546 has been shown to confer both the properties of CD46 binding and haemadsorption to isolates that were previously defective for these traits (Li et al., 1999 ). These are clearly selectable traits because they facilitate efficient spread of MV to other cells.

For positive selection at a particular site to be attributed to Vero passaging, the signal for positive selection should be lost at this particular site when Vero-passaged isolates are removed from the Global data set, and then this site should be positively selected when only Vero-passaged isolates are analysed. This pattern was only seen for the positive selection at site 546. This site was found to be under strong selection pressure ({omega}>5) when only Vero-passaged isolates were analysed, and this selection is therefore most likely the result of Vero passaging. Although positive selection at site 481 was lost in the -Vero data set, it could not be assigned to the positively selected class when only Vero isolates were analysed and cannot be confirmed to result from Vero passaging. Indeed, analysis of a larger number of MV strains than were used in this study suggests that isolates of several different passaging types contain a Tyr at position 481. Hence, it seems probable that the positive selection at site 481 results from natural selection for the CD46 receptor as opposed to artificial selection attributable to Vero passaging. This is further supported by the fact that site 481 was not found to correlate with any antigenic features discussed earlier. Site 562 was also deemed to be under strong positive selection when only Vero-passaged isolates were analysed. However, because positive selection at site 562 was not lost when Vero-passaged isolates were removed from the Global data set, it is also unlikely to result from Vero passaging.

Sites 211, 243, 451 and 476 have also been implicated in CD46 interaction (Bartz et al., 1996 ; Patterson et al., 1999 ) and our analysis of the -Vero data set suggested that sites 211, 451 and 476 are potentially under positive selection. As discussed previously, the positive selection at sites 211 and 476 could be the result of immune selection from antibodies but a possible selective force from the CD46 receptor cannot be ruled out entirely. Early studies indicated that Val and Tyr at sites 451 and 481, respectively, are critical for inducing CD46 down-regulation (Bartz et al., 1996 ; Lecouturier et al., 1996 ). A more recent study suggests that site 451 may be the more influential of this pair since it describes a strain generated by site-directed mutagenesis with a Tyr at position 481 that did not induce significant down-regulation (Xie et al., 1999 ). Down-regulation of CD46 is thought to confer an advantage to the virus because it disrupts antigen presentation of the H protein to T-cells (Rivailler et al., 1998 ). Interestingly, the majority of isolates have a Val at position 451, indicating that this is probably the ancestral state. On a number of occasions after the mid 1970s, the Val at position 451 appears to have been replaced by Ala, Glu, Lys or Met. This is unexpected since such replacements would increase the efficiency of H protein presentation to the immune system. Hence, it is likely that the replacement of Val at 451 and disruption of down-regulation may confer some other selective advantage upon the virus. For instance, the presence of CD46 upon the cell surface protects the cell from complement lysis and may allow for greater efficiency of virus generation (Schnorr et al., 1995 ).

A similar analysis to that performed with Vero-passaged isolates was attempted with B95a-passaged isolates (results not shown) in order to determine if any of the positively selected sites were the result of selection for the SLAM receptor. However, selection analysis of 15 isolates from the Global data set that had only undergone B95a passaging did not estimate a positively selected class. If the positive selection pressure exerted by the SLAM receptor is weaker than that from the CD46 receptor, or if the SLAM receptor selects for a single site, then a larger data set of B95a-passaged isolates is needed to detect this. This work is ongoing.

Finally, we attempted to determine if any of the potential positively selected sites resulted from the presence of the SSPE strains in the Global data set. Removing the 13 SSPE strains from the analysis (results not shown) led to a loss of positive selection at sites 12, 62, 211, 303, 348, 423 and 476 in comparison to results from the Global data set. Unfortunately, positive selection at these sites could not be confirmed when the 13 SSPE strains were analysed as a separate data set, because the models that identified a positively selected class did not perform significantly well. Loss of positive selection at site 12 presents an interesting result because it lies in the cytoplasmic domain of the H protein (Fig. 2). The matrix (M) protein is thought to interact with the nucleocapsid protein and the cytoplasmic tails of the F and H glycoproteins to enable the morphogenesis of viral particles by a budding process (Peeples, 1991 ). Viral factors thought to abolish viral budding and, thus in part, facilitate the generation of SSPE cases, are defective M proteins and F proteins with truncated and/or distorted cytoplasmic domains (Schneider-Schaulies et al., 1995 ). It is possible that changes at site 12 may block interaction with the M protein and are selected so that viral budding is further impeded in SSPE-associated viruses. The serum of SSPE patients has been shown to have a high antibody titre directed against the H protein (Liebert, 1997 ). Consequently, even though positive selection at sites 211, 348, 423 and 476 was lost on removal of SSPE strains their discussion in terms of epitopes can still be considered valid. Selection analysis of a larger number of SSPE strains should confirm significant positive selection in these strains and further research will be concentrated in this area.

In summary, the conserved nature of the L protein was confirmed since we failed to predict any sites under positive selection. The putative positively selected sites identified in the H protein could have arisen for a variety of reasons (Table 4). Although positively selected sites were not shown to correlate with TCEs presently identified, sites 211, 348, 423, 476, 562 and 575 were shown to coincide with potential BCEs and may have been generated by immune selection. Further work is clearly needed to confirm whether these epitopes are protective. Some of the aforementioned sites are also associated with areas that are known to interact with the CD46 receptor and this may contribute to their selection. The selection at site 546 is probably generated by Vero passaging, which selects for more efficient binding of the CD46 receptor. Selection for limiting down-regulation of the CD46 receptor and for increased binding of this receptor probably led to the positive selection identified at sites 451 and 481, respectively. These two sites appear to have arisen naturally rather than by artificial selection from Vero cell passaging. Possible explanations for the positive selection at a number of sites could not be deduced (Table 4). These may have resulted from selection for the SLAM receptor during B95a passaging or coincide with some, as yet undetermined, antigenic feature. Further work is needed to confirm this.

Previous analyses of viral glycoproteins have estimated the selection pressure at positively selected sites to range from 2·433 (attachment glycoprotein of respiratory syncytial virus) to 6·898 (haemagglutinin gene of influenza virus) (Woelk & Holmes, 2001 ; Yang et al., 2000 ). In comparison, the positive selection pressure estimated for the H gene of MV is relatively weak (2·319 under M3 and 1·658 under M8). Although vaccines protect against all known strains of MV, substitutions at positively selected sites should still be monitored because they have the potential to generate escape mutants. Ideally, selection analysis in future studies should be performed using strains obtained directly from clinical material of acute cases, which does not suffer from passaging problems. This will provide the most accurate picture of the selection pressures affecting the H gene of MV during acute infections and determine the precise nature of adaptive evolution.


   Acknowledgments
 
We thank Dr Michiko Watanabe for providing sequence data for the Biken strain, and Dr Andrew Rambaut and Professor Alan Grafen for the provision of statistical support. We also thank two anonymous referees for useful suggestions and the BBSRC and The Royal Society for financial support.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 12 March 2001; accepted 9 July 2001.