Characterization of a major neutralization domain of Ross river virus using anti-viral and anti-peptide antibodies.

The E2 glycoprotein of the alphavirus Ross River virus (RRV) contains three defined neutralization epitopes (a, b1 and b2) with determinants located between amino acids 216 and 251 in the linear sequence (Vrati et al., 1988, Virology 162, 346-353). The antigenic structure of this region has been examined using hyperimmune mouse antiserum against RRV and antiserum against four synthetic peptides representing linear amino acid sequences in the neutralization region of E2. In plaque reduction neutralization tests using hyperimmune antiserum to RRV, an RRV mutant altered at all three neutralization epitopes was markedly more resistant than the parental virus; variants altered at single epitopes could not be distinguished in these tests. Sera from mice immunized with synthetic RRV E2 peptides conjugated to keyhole limpet haemocyanin reacted, in a direct ELISA, with the specific region of RRV represented by the peptide. The same sera did not neutralize or immunoprecipitate RRV in solution or bind to RRV in a capture ELISA. The RRV peptides did not prime mice to react to a subimmunogenic dose of RRV; they did not bind monoclonal or polyclonal antibodies to RRV. We conclude that a significant proportion of the neutralizing antibody response in mice is elicited by epitopes a, b1, and b2 of RRV E2 and that the sites to which neutralizing antibodies bind are formed by complex folding.     

Epitope mapping of the gp53 envelope protein of bovine viral diarrhea virus

Epitopes recognized by nine monoclonal antibodies (mAbs) on the envelope protein, gp53, of two strains of bovine viral diarrhoea virus (NADL and Oregon C24V) were mapped by competitive binding assays and by the characterization and sequence analyses of mAb neutralization escape mutants. This defined an antigenic domain on gp53 that was shared by many BVDV strains, while other less conserved epitopes were possibly distinct. Sequencing of escape mutant viruses revealed that a cluster of three amino acids in the N-terminal half of gp53 were involved in the main antigenic domain shared by both NADL and Oregon C24V viruses, while an amino acid 31 residues further toward the N-terminus was involved in a second site present only on the NADL strain. Since other amino acids defining these epitopes were located at distant positions within the gp53 protein, it is likely that a major domain [corrected] on gp53 consist of composite, conformation-dependent epitopes.     

Fine mapping of a peptide sequence containing an antigenic site conserved among arenaviruses

The lymphocytic choriomeningitis virus (LCMV) structural glycoproteins GP-1 (Mr 44K) and GP-2 (Mr 35K) are encoded on a single intracellular proteolytic cleavage precursor glycoprotein, GP-C (Mr 76K). We have used a series of synthetic peptides derived from the deduced amino acid sequence of LCMV GP-C to define an antigenic site containing two topographically overlapping epitopes. Three mouse monoclonal antibodies directed against two epitopes on GP-2 were assayed for binding in solution phase blocking and solid-phase enzyme-linked immunoadsorbant assays to a series of peptides representing the sequence of the intracellular precursor glycopeptide GP-C. Both epitopes were initially localized to a single peptide GP-C 370-382 (Cys-Asn-Tyr-Ser-Lys-Phe-Trp-Tyr-Leu-Glu-His-Ala-Lys) in the GP-2 segment of GP-C. Further analysis demonstrated that both epitopes were contained within a nine amino acid segment, GP-C 370-378, which contains five residues conserved among LCMV, Lassa, Pichinde, and Tacaribe viruses. Assays with N-terminal deletions from this sequence suggested that the minimal epitope recognized by the broadly cross-reactive monoclonal 33.6 (epitope GP-2a) consisted of five amino acids, GP-C 374-378 (Lys-Phe-Trp-Tyr-Leu). Reactivity of a second monoclonal, 9-7.9 (epitope GP-2B) but not 33.6, was abolished when substitution of tyrosine for phenylalanine was made at position 375 in the antigenic sequence corresponding to a naturally occurring sequence difference between LCM and Lassa viruses. Polyclonal sera from human cases and from animals experimentally infected with Junin, LCM, and Lassa viruses, respectively, bound to the antigenic peptide GP-C 370-382 but not to control peptides. As was the case with the monoclonals, this binding activity was abrogated by blocking with the antigenic peptide but not with control peptides in solution.     

Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins

The complete sequence of the 26 S RNA of Ross River virus (T48 strain) has been obtained and from this the amino acid sequences of the encoded structural proteins have been deduced. These include a basic capsid protein and two envelope glycoproteins. The nucleotide sequence was obtained by chemical sequence analysis of both single-stranded and double-stranded cDNA made to RNA and the sequence data so obtained was rapidly aligned by making use of the protein homology found among the alphaviruses. The polyprotein precursor encoded by the 26 S RNA of Ross River virus is 75% homologous to that of Semliki Forest virus and 48% homologous to that of Sindbis virus. The extent of homology is not uniform within a protein or between proteins and this is discussed with respect to the possible function of the various polypeptide domains in the virus life cycle. In each case the putative attachment site of the amino proximal carbohydrate chains of the three glycoproteins is conserved, whereas the attachment site of a second chain, if present, is not conserved. The 3'-untranslated region of Ross River virus RNA is 524 nucleotides long. It contains a sequence of about 50 nucleotides in length which is present in four copies but which is not shared with other alphaviruses examined.     

A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein

Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.     

Classical swine fever virus: Antigenic architecture of the E2 glycoprotein elucidated by epitope mapping using synthetic peptides

Detailed epitope characterization of the major envelope glycoprotein E2 of the classical swine fever virus (CSFV) is important for our understanding of interactions between the virus and the host immune system, as well as for the development of CSFV-specific diagnostic assays and epitope- or peptide-based marker vaccines. As was shown previously by competitive binding assay, monoclonal antibodies obtained by our group recognize eight epitopes on the E2 protein. Here, we report mapping of five linear, nonoverlapping B-cell epitopes that use a set of synthetic peptides, which encompass the full sequence of the CSFV E2 protein (Shimen strain). Two of the epitopes are located in the antigenic domain A of the E2, while another three belong to a highly structured region of this glycoprotein. The alignment of the identified gene sequences was performed for 12 CSFV strains, three strains of bovine viral diarrhea virus (BVDV), and two strains of the border disease virus (BDV). The data obtained could be used to improve CSFV diagnostic assays, as well as to investigate the effects of aminoacid substitutions in E2 on its antigenic properties.     

Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector

Deletion mutants and chimeras of the glycoprotein (G) genes of vesicular stomatitis virus serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were cloned in plasmids and vaccinia virus vectors under control of the bacteriophage T7 polymerase promoter for expression in CV-1 cells co-infected with a T7 polymerase-expressing vaccinia virus recombinant. Truncated and chimeric G proteins expressed by these vectors were tested for their capacity to react with VSV-Ind and VSV-NJ epitope-specific monoclonal antibodies (MAbs) by Western blot analysis for those antigenic determinants not affected by disulfide-bond reducing conditions or by immuno dotblot analysis for those that are. These experiments allowed us to create putative epitope maps for glycoproteins of both serotypes based on binding affinity and cross-reactivity of VSV-Ind and VSV-NJ MAbs for truncated or chimeric G proteins of known amino acid sequences. Seven of the 9 VSV-NJ G epitopes, including all 4 epitopes involved in virus neutralization by MAbs, mapped to the center (amino acid sequence 193-289) of the 517 amino acid VSV-NJ G protein. Four of the 11 VSV-Ind G epitopes, including 2 neutralizable epitopes, mapped to the cysteine-rich amino-terminal domain (amino acid sequence 80-183) of the 511 amino acid VSV-Ind G protein; the remaining 7 VSV-Ind G epitopes, including 2 involved in virus neutralization, were clustered in the cysteine-poor carboxy-terminal domain (amino acid sequence 286-428). In site-specific mutants of the VSV-Ind G gene defective in one or both glycosylation sites, only the amino-terminal epitopes of the VSV-Ind G protein were affected by deletion of the carbohydrate chain at residue 179; deletion of the carbohydrate chain at residue 336 did not alter reactivity of the G protein with any of the relevant monoclonal antibodies. These results are discussed in relation to earlier attempts to map the antigenic determinants of VSV-NJ and VSV-Ind G proteins by proteolysis of the G protein and by sequencing the G genes of mutant viruses selected for their resistance to neutralization by epitope-specific monoclonal antibodies.     

Characterization of monoclonal antibodies to bovine enteric coronavirus and antigenic variability among Quebec isolates

Summary Twenty monoclonal antibodies (MAbs) were prepared against the Mebus strain of bovine enteric coronavirus, 14 of them reacting with the peplomeric S (gp 100) glycoprotein. Competition binding assays allowed the definition of at least 4 distinct antigenic domains for the S glycoprotein, designated as A, B, C, and D; epitopes associated to neutralizing activity being located in sites A, B, and C. One MAb directed to the hemagglutinin HE (gp 140/gp 65) glycoprotein inhibited the hemagglutinating activity of the virus, but had no neutralizing activity. Comparison of Quebec enteropathogenic BCV isolates using polyclonal antiserum and MAbs directed to the S glycoprotein confirmed their close antigenic relationship, but also revealed the occurrence of at least three distinct antigenic subgroups. Antigenic domain D was highly conserved among BCV isolates, as well as non-neutralizing epitopes assigned to antigenic domains A and C. The Minnesota strain of turkey enteric coronavirus could be distinguished from BCV isolates by MAbs directed to epitopes of antigenic domain C, whereas human coronavirus HCV-OC 43 could be distinguished by MAbs directed to epitopes of antigenic domain A. The porcine hemagglutinating encephalomyelitis virus could be distinguished from the other hemagglutinating coronaviruses by neutralizing epitopes located on antigenic domains A, B, and C.     

Location of a neutralization determinant in the E protein of yellow fever virus (17D vaccine strain)

The location of a major antigenic determinant involved in the neutralization of a flavivirus, yellow fever virus (YF), has been defined in terms of its position in the amino acid sequence of the E protein. Neutralization escape variants of the 17D vaccine strain of YF were selected with two neutralizing monoclonal antibodies. Nucleotide sequencing of the envelope protein genes (E and M) of the variants showed that in each variant there was a single nucleotide change in the E gene leading to a nonconservative amino acid substitution in the E protein at position 71 or 72. The changes are in a region of the E protein which is hydrophilic, rich in cysteine residues, and not conserved between flavivirus subgroups. Since the selecting monoclonal antibodies neutralize attenuated 17D and virulent Asibi strains of YF with equal efficiency (J. J. Schlesinger, M. W. Brandriss, and T. P. Monath, 1983, Virology 125, 8-17), it can be concluded that the neutralization determinant defined for 17D YF is also present in Asibi YF.     

Reduced mouse neurovirulence of poliovirus type 2 Lansing antigenic variants selected with monoclonal antibodies

The Lansing strain of poliovirus type 2 is a mouse-adapted virus that induces a fatal paralytic disease in mice after intracerebral inoculation. Our previous results indicated that the mouse-adapted phenotype maps to the Lansing viral capsid. To further define regions of the capsid that are specifically involved in the infection of mice, antigenic variants resistant to neutralization with monoclonal antibodies were selected, and their mouse neurovirulence was studied. The monoclonal antibodies used were directed against antigenic site 1, an immunodominant loop of capsid polypeptide VP1 located on the virion surface. Ten of twenty-two variants selected had lower intracerebral neurovirulence in mice when compared to the parental virus. Four of the ten antigenic variants with reduced neurovirulence were temperature sensitive (ts) for replication in HeLa cells, while the remaining six variants replicated in HeLa cells as well as the parent virus. Two ts+ variants that were studied had a reduced ability to replicate in the mouse brain. There was no difference in the histopathology and pattern of involvement in the central nervous system of one variant compared to the parent virus. In three variants, reduction of neurovirulence correlated with specific amino acid substitutions at positions 100 and 101 of VP1, located within antigenic site 1. The ts phenotype in three variants was associated with a single amino acid deletion at position 105. Virus recovered from the brain of paralyzed mice that had been inoculated with the antigenic variants was characterized to identify the virus causing disease. In most cases, brain isolates resembled the inoculated virus in neurovirulence and amino acid sequence at the antigenic site. Virus recovered from brains of paralyzed mice that had been inoculated with the ts variants was either ts+ or cold sensitive, and had become more neurovirulent. These results suggest that specific amino acid changes within an antigenic site on the virion surface may result in reduction of mouse neurovirulence without affecting viral replication in cultured cells.     

Subtyping of human immunodeficiency virus isolates with a panel of monoclonal antibodies: identification of conserved and divergent epitopes on p17 and p25 core proteins.

We have investigated the feasibility and significance of subtyping of human immunodeficiency virus (HIV) isolates with monoclonal antibodies (mAb) raised against the core proteins of HIV. A panel of 37 mAb tested for reactivity with HIV1 oligopeptides was used to analyse the antigenic relatedness among 14 HIV isolates which included 12 isolates of HIV1 from different geographical origins and 2 isolates of HIV2. Three out of these 37 mAb reacted with conserved epitopes expressed by all 14 HIV isolates tested. These reagents which included 2 mAb reacting with the 285-310 amino acid sequence of p25 and 1 mAb reacting with an epitope of p25 not mapped by the peptides' approach, also reacted with a non-human primate lentivirus. Five mAb reacting either with the 11-25 or 121-132 amino acid sequences of p17 or the 302-320 amino acid sequence of p25 reacted with strain-specific epitopes. The other 29 mAb reacted with polymorphic epitopes and thereby define subfamily and subtype-specific markers.     

Sequence of the genes encoding the structural proteins of the low-virulence tick-borne flaviviruses Langat TP21 and Yelantsev.

The structural protein coding regions of the genomes of Langat virus (strain TP21) and Yelantsev virus, which was originally described to be a low virulence natural isolate of tick-borne encephalitis virus, were cloned and sequenced. These viruses had both been used as experimental live vaccines against tick-borne encephalitis in Czechoslovakia and Russia, respectively. Peptide mapping and monoclonal antibody binding experiments yielded identical reaction patterns for Langat virus and Yelantsev virus which were distinct, however, from the pattern obtained with tick-borne encephalitis virus. Sequence analysis confirmed this distinctiveness and proved that the vaccine strain Yelantsev was also Langat virus. The envelope protein E of both viruses exhibits an 88% amino acid sequence homology with that of tick-borne encephalitis virus. Assessment of the antigenic reactivity and sequence comparison with the E protein of tick-borne encephalitis virus revealed several differences affecting epitopes involved in virus neutralization. These observations suggest that Langat-like virus-based vaccines may not represent the most effective means to achieve protection against tick-borne encephalitis virus.     

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22-58), and complex-specific (amino acids 304-332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60-97 and 298-397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60-135; amino acids 60-205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298-397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.     

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.     

Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein

Experiments with monoclonal antibodies raised against two laboratory strains of Sindbis virus, SB and SIN, suggested the existence of a strain-specific neutralizing antigenic site (E2-b) on the E2 glycoprotein. A comparison of monoclonal antibody binding patterns and E2 glycoprotein gene sequences of six laboratory strains distinguished three different configurations of E2-b that correlated with specific amino acid substitutions at position 216 of the E2 glycoprotein. Further study of neutralization escape mutants selected with E2-b-specific antibodies confirmed that amino acid 216 is a major determinant of the E2-b antigenic site. Eight of nine mutants showed a coding change at position 216. One neutralization escape mutation created a new glycosylation site at position 213 and resulted in an E2 protein with an altered migration rate in SDS-PAGE. The neutralization escape mutants studied included amino acid substitutions not found in the laboratory strains that revealed differing binding requirements for two E2-b-specific monoclonal antibodies. The E2-b site is contrasted with the E2-c neutralizing antigenic site described previously (R.A. Olmsted, W.J. Meyer, and R.E. Johnston, 1986, Virology 148, 245-254).     

Non-additive effects of multiple amino acid substitutions on antigen-antibody recognition.

Synthetic peptides have been used to mimic the main antigenic site of foot-and-mouth disease virus (FMDV) of serotype C and of several variant isolates. This region includes multiple continuous B cell epitopes. The effect of single amino acid replacements, individually or in combination, on antigen specificity has been evaluated using monoclonal antibodies. Quantitative enzyme immunodot assays have shown that both additive and non-additive effects of multiple replacements occur in continuous B cell epitopes, with regard to antibody recognition. Antigenically critical single replacements may be compensated by other, non-critical replacements. Thus, the role of a single amino acid on antibody recognition depends on the sequence context in the antigenic domain. The non-additive effects of multiple replacements may modulate the extent of antigenic diversification of highly variable RNA viruses, and keep viruses confined within antigenic groups by precluding linear antigenic divergence.     

Synthetic peptides of Venezuelan equine encephalomyelitis virus E2 glycoprotein. I. Immunogenic analysis and identification of a protective peptide

Fourteen peptides representing 67% of the extramembranal domain of the Venezuelan equine encephalomyelititis (VEE) virus E2 glycoprotein were synthesized and analyzed to determine their antigenic, immunogenic, and protective capacities. Thirteen of 14 peptides elicited antibody for the homologous peptide. Thirteen peptides elicited antiviral antibody that recognized either the Trinidad (TRD) strain of VEE virus or the TC-83 vaccine derivative, or both. Two peptides, VE2pep01(TC-83) and VE2pep01(TRD), protected significant numbers of mice from TRD virus challenge. The majority of the peptides were reactive with antisera from mice immunized with the various subtypes of VEE virus. A competition assay using antipeptide antibodies to block virus binding of anti-VEE virus monoclonal antibodies corroborated previous studies on the spatial relationship of E2 epitopes and provided evidence for a spatial overlap of the E2 amino terminus with a domain composed of residues 180-210.     

Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals

Two anti-glycoprotein E2 monoclonal (MC) antibodies, designated R6 and R13, prepared against an attenuated mutant (SB-RL) of Sindbis virus, preferentially neutralize attenuated, rapidly penetrating strains of Sindbis as opposed to virulent, more slowly penetrating strains. An antigenic variant of SB-RL which displayed reduced reactivity with R6 and R13, demonstrated virulence in suckling mice and slow penetration in baby hamster kidney cells. Anti-E2 MC antibodies to SB-RL and to an independently propagated wild-type Sindbis strain, SIN, were used to compare the topographical relationship of the R6 and R13 epitopes to other E2 antigenic sites. The R6 and R13 epitopes constituted a previously undescribed E2 site, E2-c. Antibody competition experiments using virions in solid phase and in suspension, as well as binding of MC antibody to antigenic variants, demonstrated that E2-c was a spatially distinct and independently mutable site at the surface of Sindbis virions. Of three E2 antigenic sites, E2-a and E2-c were conserved among the Sindbis strains examined, while E2-b was strain specific. Although attenuated strains were preferentially neutralized by E2-c specific MC antibodies, the critical mutation in these strains did not alter their ability to bind E2-c MC antibody. Rather, the mutation was responsible for the altered biological effect of antibody binding at E2-c; the in vitro rapid penetration phenotype; and the in vivo phenotype of attenuation in suckling mice.     

Two mechanisms of antigenic diversification of foot-and-mouth disease virus.

The amino acid replacements that underlay the diversification of the main antigenic site A (VP1 residues 138 to 150) of foot-and-mouth disease virus (FMDV) of serotype C have been identified. Sixteen new VP1 sequences of isolates from 1926 until 1989 belonging to subtypes C1, C2, C3, C4, C5, and unclassified are reported. The reactivities in enzyme-linked immunoelectrotransfer blot assays of capsid protein VP1 with a panel of neutralizing monoclonal antibodies that recognize sites A or C (the VP1 carboxy-terminus) have been correlated with the amino acid sequence at the relevant epitopes. The analyses involving the immunodominant site A reveal two mechanisms of antigenic change. One is a gradual increase in antigenic distance brought about by accumulation of amino acid replacements at two hypervariable segments within site A. A second mechanism consists of an abrupt antigenic change manifested by loss of many epitopes, caused by one replacement at a critical position (particularly Ala (145)----Val or His (146)----Gln). The identification of the amino acid substitutions responsible for such large antigenic changes provides new information for the design of synthetic anti-FMD vaccines. However, the screening of isolates from six decades suggests that the virus, even within the confines of a single serotype, has exploited a minimum of its potential for antigenic variation.     

Genetic stability of Ross River virus during epidemic spread in nonimmune humans

We have examined the rate of evolution of Ross River virus, a mosquito-borne RNA virus, during epidemic spread through tens of thousands of nonimmune humans over a period of 10 months. Two regions of the Ross River virus genome were sequenced: the E2 gene (1.2 kb in length), which encodes the major neutralization determinant of the virus, and 0.4 kb of the 3'-untranslated region. In the E2 gene, a single nucleotide change was selected which led to a predicted amino acid change at residue 219. No changes were selected in the 3'-untranslated region. By comparison with rates of evolution reported for non-arthropod-borne RNA viruses, the rate for Ross River virus is surprisingly low. We identify three features of the Ross River virus replication and transmission cycle which may limit the rate of evolution of arthropod-borne viruses in the field.