Evolutionary diversification of protein-coding genes of hantaviruses

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.     

Phylogenetic Analysis Reveals Rapid Evolutionary Dynamics in the Plant RNA Virus Genus Tobamovirus

Early studies on the evolutionary dynamics of plant RNA viruses suggested that they may evolve more slowly than their animal counterparts, sometimes dramatically so. However, these estimates were often based on an assumption of virus–host codivergence over time-scales of many millions of years that is difficult to verify. An important example are viruses of the genus Tobamovirus, where the assumption of host–virus codivergence over 100 million years has led to rate estimates in the range of ~1 × 10⁻⁸ nucleotide substitutions per site, per year. Such a low evolutionary rate is in apparent contradiction with the ability of some tobamoviruses to quickly overcome inbred genetic resistance. To resolve how rapidly molecular evolution proceeds in the tobomaviruses, we estimated rates of nucleotide substitution, times to common ancestry, and the extent of congruence between virus and host phylogenies. Using Bayesian coalescent methods applied to time-stamped sequences, we estimated mean evolutionary rates at the nucleotide and amino acid levels of between 1 × 10⁻⁵ and 1.3 × 10⁻³ substitutions per site, per year, and hence similar to those seen in a broad range of animal and plant RNA viruses. Under these rates, a conservative estimate for the time of origin of the sampled tobamoviruses is within the last 100,000 years, and hence a far more recently than proposed assuming codivergence. This is supported by our cophylogeny analysis which revealed significantly discordant evolutionary histories between the tobamoviruses and the plant families they infect.     

Bayesian Coalescent Analysis Reveals a High Rate of Molecular Evolution in GB Virus C

GB virus C/hepatitis G (GBV-C) is an RNA virus of the family Flaviviridae. Despite replicating with an RNA-dependent RNA polymerase, some previous estimates of rates of evolutionary change in GBV-C suggest that it fixes mutations at the anomalously low rate of ∼10⁻⁷ nucleotide substitution per site, per year. However, these estimates were largely based on the assumption that GBV-C and its close relative GBV-A (New World monkey GB viruses) codiverged with their primate hosts over millions of years. Herein, we estimated the substitution rate of GBV-C using the largest set of dated GBV-C isolates compiled to date and a Bayesian coalescent approach that utilizes the year of sampling and so is independent of the assumption of codivergence. This revealed a rate of evolutionary change approximately four orders of magnitude higher than that estimated previously, in the range of 10⁻² to 10⁻³ sub/site/year, and hence in line with those previously determined for RNA viruses in general and the Flaviviridae in particular. In addition, we tested the assumption of host-virus codivergence in GBV-A by performing a reconciliation analysis of host and virus phylogenies. Strikingly, we found no statistical evidence for host-virus codivergence in GBV-A, indicating that substitution rates in the GB viruses should not be estimated from host divergence times.     

Phylogenetic evidence for rapid rates of molecular evolution in the single-stranded DNA begomovirus tomato yellow leaf curl virus

Geminiviruses are devastating viruses of plants that possess single-stranded DNA (ssDNA) DNA genomes. Despite the importance of this class of phytopathogen, there have been no estimates of the rate of nucleotide substitution in the geminiviruses. We report here the evolutionary rate of the tomato yellow leaf curl disease-causing viruses, an intensively studied group of monopartite begomoviruses. Sequences from GenBank, isolated from diseased plants between 1988 and 2006, were analyzed using Bayesian coalescent methods. The mean genomic substitution rate was estimated to be 2.88 x 10(-4) nucleotide substitutions per site per year (subs/site/year), although this rate could be confounded by frequent recombination within Tomato yellow leaf curl virus genomes. A recombinant-free data set comprising the coat protein (V1) gene in isolation yielded a similar mean rate (4.63 x 10(-4) subs/site/year), validating the order of magnitude of genomic substitution rate for protein-coding regions. The intergenic region, which is known to be more variable, was found to evolve even more rapidly, with a mean substitution rate of approximately 1.56 x 10(-3) subs/site/year. Notably, these substitution rates, the first reported for a plant DNA virus, are in line with those estimated previously for mammalian ssDNA viruses and RNA viruses. Our results therefore suggest that the high evolutionary rate of the geminiviruses is not primarily due to frequent recombination and may explain their ability to emerge in novel hosts.     

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.     

Stone age diseases and modern AIDS

Background

Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ~10^-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered.

Results

We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 × 10^-4 and 7.9 × 10^-4 subs/site/year.

Conclusion

These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules.     

The phylogenetic position of "Acomyinae" (Rodentia, Mammalia) as sister group of a Murinae + Gerbillinae clade: evidence from the nuclear ribonuclease gene.

The phylogenetic relationships of Acomys and Uranomys within Muridae were investigated using nuclear pancreatic ribonuclease A gene sequences. The various kinds of substitutions in the data matrix (15 taxa x 375 nucleotides) were examined for saturation, in order to apply a weighted parsimony approach. Phylogenies were derived by maximum parsimony (weighted and unweighted) and maximum likelihood procedures, using a dormouse (Gliridae) as outgroup. Maximum likelihood gave the most robust results. All analyses cluster some traditional taxa with a strong robustness, such as three species of the genus Mus, two South-East Asian rats, and two genera in each of the gerbil and vole families. When analyzed with those of other murid rodents representing Murinae, Gerbillinae, Arvicolinae, Cricetinae, and Sigmodontinae, sequences of the ribonuclease gene suggest that Acomys and Uranomys constitute a monophyletic clade at the subfamily level, denoted "Acomyinae." The relationships between the six subfamilies of Muridae appear poorly resolved, except for a clade uniting Murinae, Acomyinae, and Gerbillinae. Within this clade, the sister group of Acomyinae could not be identified, as the branch length defining a Gerbillinae + Murinae cluster is extremely short. The poor resolution of our phylogenetic inferences is probably the result of two confounding factors, namely the limited size of the pancreatic ribonuclease sequence and the probable short time intervals during the radiation of the six murid subfamilies involved in this study.     

Molecular evolution and phylogeny of the human AIDS viruses LAV,HTLV-III,and ARV

Summary A phylogenetic tree for the human lymphadenopathy-associated virus (LAV), the human T-cell lymphotrophic virus type III (HTLV-III), and the acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV) has been constructed from comparisons of the amino acid sequences of their gag proteins. A method is proposed for estimating the divergence times among these AIDS viruses and the rates of nucleotide substitution for their RNA genomes. The analysis indicates that the LAV and HTLV-III strains diverged from one another after 1977 and that their common ancestor diverged from the ARV virus no more than 10 years earlier. Hence, the evolutionary diversity among strains of the AIDS viruses apparently has been generated within the last 20 years. It is estimated that the genome of the AIDS virus has a nucleotide substitution rate on the order of 10^–3 per site per year, with the rate in the second half of the genome being double that in the first half.     

Evolutionary history of the most speciose mammals: molecular phylogeny of muroid rodents

Phylogenetic relationships between 32 species of rodents representing 14 subfamilies of Muridae and four subfamilies of Dipodidae were studied using sequences of the nuclear protein-coding genes Lecithin Cholesterol Acyl Transferase (LCAT) and von Willebrand Factor (vWF). An examination of some evolutionary properties of each data matrix indicates that the two genes are rather complementary, with lower rates of nonsynonymous substitutions for LCAT. Both markers exhibit a wide range of GC3 percentages (55%-89%), with several taxa above 70% GC3 for vWF, which indicates that those exonic regions might belong to the richest class of isochores. The primary sequence data apparently harbor few saturations, except for transitions on third codon positions for vWF, as indicated by comparisons of observed and expected pairwise values of substitutions. Phylogenetic trees based on 1,962 nucleotidic sites from the two genes indicate that the 14 Muridae subfamilies are organized into five major lineages. An early isolation leads to the clade uniting the fossorial Spalacinae and semifossorial Rhizomyinae with a strong robustness. The second lineage includes a series of African taxa representing nesomyines, dendromurines, cricetomyines, and the sole living member of mystromyines. The third one comprises only the mouselike hamster CALOMYSCUS: The fourth clade represents the cricetines, myospalacines, sigmodontines, and arvicolines, whereas the fifth one comprises four "traditional" subfamilies (Gerbillinae, Murinae, Otomyinae, and Acomyinae). Within these groups, we confirm the monophyly of almost all studied subfamilies, namely, Spalacinae, Rhizomyinae, Nesomyinae, Cricetomyinae, Arvicolinae, Sigmodontinae, Cricetinae, Gerbillinae, Acomyinae, and Murinae. Finally, we present evidence that the sister group of Acomyinae is Gerbillinae, and we confirm a nested position of Myospalacinae within Cricetinae and Otomyinae within Murinae. From a biogeographical point of view, the five main lineages spread and radiated from Asia with different degrees of success: the first three groups are now represented by a limited number of species and genera localized in some regions, whereas the last two groups radiated in a large variety of species and genera dispersed all over the world.     

Observation of positive selection within hypervariable regions of a newly identified DNA virus (SEN virus)

To elucidate the evolution of SEN virus (SEN-V), serial sequences of chronically SEN-V-infected patients were analyzed. In the hypervariable regions, non-synonymous substitutions significantly predominated. This could be attributed to positive selection in evading immune surveillance of the hosts and to establish a persistent infection. On the basis of the sequences in the two open reading frames of SEN-V DNA, the rate of synonymous substitutions was 7.32×10⁻⁴ per site per year. Since this rate is close to RNA viruses and higher than other DNA viruses, the SEN-V might be replicated by machinery with poor or no proofreading function.     

Phylogeny of muroid rodents: relationships within and among major lineages as determined by IRBP gene sequences

The rodent family Muridae is the single most diverse family of mammals with over 1300 recognized species. We used DNA sequences from the first exon ( approximately 1200bp) of the IRBP gene to infer phylogenetic relationships within and among the major lineages of muroid rodents. We included sequences from every recognized muroid subfamily except Platacanthomyinae and from all genera within the endemic Malagasy subfamily Nesomyinae, all recognized tribes of Sigmodontinae, and a broad sample of genera in Murinae. Phylogenetic analysis of the IRBP data suggest that muroid rodents can be sorted into five major lineages: (1) a basal clade containing the fossorial rodents in the subfamilies Spalacinae, Myospalacinae, and Rhizomyinae, (2) a clade of African and Malagasy genera comprising the subfamilies Petromyscinae, Mystromyinae, Cricetomyinae, Nesomyinae, and core dendromurines, (3) a clade of Old World taxa belonging to Murinae, Otomyinae, Gerbillinae, Acomyinae, and Lophiomyinae, (4) a clade uniting the subfamilies Sigmodontinae, Arvicolinae, and Cricetinae, and (5) a unique lineage containing the monotypic Calomyscinae. Although relationships among the latter four clades cannot be resolved, several well-supported supergeneric groupings within each are identified. A preliminary examination of molar tooth morphology on the resulting phylogeny suggests the triserial murid molar pattern as conceived by evolved at least three times during the course of muroid evolution.     

A large variation in the rates of synonymous substitution for RNA viruses and its relationship to a diversity of viral infection and transmission modes

RNA viruses successfully adapt to various environments by repeatedly producing new mutants, often through generating a number of nucleotide substitutions. To estimate the degree of variation in mutation rates of RNA viruses and to understand the source of such variation, we studied the synonymous substitution rate because synonymous substitution is exempt from functional constraints at the protein level, and its rate reflects the mutation rate to a great extent. We estimated the synonymous substitution rates for a total of 49 different species of RNA viruses, and we found that the rates had tremendous variation by 5 orders of magnitude (from 1.3 x 10(-7) to 6.2 x 10(-2) /synonymous site/year). Comparing the synonymous substitution rates with the replication frequencies and replication error rates for the RNA viruses, we found that the main source of the rate variation was differences in the replication frequency because the rates of replication error were roughly constant over different RNA viruses. Moreover, we examined a relationship between viral life strategies and synonymous substitution rates to understand which viral life strategies affect replication frequencies. The results show that the variation of synonymous substitution rates has been influenced most by either the difference in the infection modes or the differences in the transmission modes. In conclusion, the variation of mutation rates for RNA viruses is caused by different replication frequencies, which are affected strongly by the infection and transmission modes.     

A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes

A new method is proposed for estimating the number of synonymous and nonsynonymous nucleotide substitutions between homologous genes. In this method, a nucleotide site is classified as nondegenerate, twofold degenerate, or fourfold degenerate, depending on how often nucleotide substitutions will result in amino acid replacement; nucleotide changes are classified as either transitional or transversional, and changes between codons are assumed to occur with different probabilities, which are determined by their relative frequencies among more than 3,000 changes in mammalian genes. The method is applied to a large number of mammalian genes. The rate of nonsynonymous substitution is extremely variable among genes; it ranges from 0.004 X 10(-9) (histone H4) to 2.80 X 10(-9) (interferon gamma), with a mean of 0.88 X 10(-9) substitutions per nonsynonymous site per year. The rate of synonymous substitution is also variable among genes; the highest rate is three to four times higher than the lowest one, with a mean of 4.7 X 10(-9) substitutions per synonymous site per year. The rate of nucleotide substitution is lowest at nondegenerate sites (the average being 0.94 X 10(-9), intermediate at twofold degenerate sites (2.26 X 10(-9)). and highest at fourfold degenerate sites (4.2 X 10(-9)). The implication of our results for the mechanisms of DNA evolution and that of the relative likelihood of codon interchanges in parsimonious phylogenetic reconstruction are discussed.     

The Evolutionary Dynamics of Bluetongue Virus

Bluetongue virus (BTV) is a midge-borne member of the genus Orbivirus that causes an eponymous debilitating livestock disease of great agricultural impact and which has expanded into Europe in recent decades. Reassortment among the ten segments comprising the double-stranded (ds) RNA genome of BTV has played an important role in generating the epidemic strains of this virus in Europe. In this study, we investigated the dynamics of BTV genome segment evolution utilizing time-structured data sets of complete sequences from four segments, totalling 290 sequences largely sampled from ruminant hosts. Our analysis revealed that BTV genome segments generally evolve under strong purifying selection and at substitution rates that are generally lower (mean rates of ~0.5–7 × 10⁻⁴ nucleotide substitutions per site, per year) than vector-borne positive-sense viruses with single-strand (ss) RNA genomes. These also represent the most robust estimates of the nucleotide substitution rate in a dsRNA virus generated to date. Additionally, we determined that patterns of geographic structure and times to most recent common ancestor differ substantially between each segment, including a relatively recent origin for the diversity of segment 10 within the past millennium. Together, these findings demonstrate the effect of reassortment to decouple the evolutionary dynamics of BTV genome segments.     

Phylogenetic evidence for the rapid evolution of human B19 erythrovirus

Human B19 erythrovirus is a ubiquitous viral pathogen, commonly infecting individuals before adulthood. As with all autonomous parvoviruses, its small single-stranded DNA genome is replicated with host cell machinery. While the mechanism of parvovirus genome replication has been studied in detail, the rate at which B19 virus evolves is unknown. By inferring the phylogenetic history and evolutionary dynamics of temporally sampled B19 sequences, we observed a surprisingly high rate of evolutionary change, at approximately 10(-4) nucleotide substitutions per site per year. This rate is more typical of RNA viruses and suggests that high mutation rates are characteristic of the Parvoviridae.     

Intrahost human immunodeficiency virus type 1 evolution is related to length of the immunocompetent period.

The antigenic diversity threshold theory predicts that antigenic sites of human immunodeficiency virus type 1, such as the V3 region of the external glycoprotein gp120, evolve more rapidly during the symptom-free period in individuals progressing to AIDS than in those who remain asymptomatic for a long time. To test this hypothesis, genomic RNA sequences were obtained from the sera of 44 individuals at seroconversion and 5 years later. The mean number of nonsynonymous nucleotide substitutions in the V3 region of the viruses circulating in 31 nonprogressors (1.1 x 10(-2) +/- 0.1 x 10(-2) per site per year) was higher than the corresponding value for 13 progressors (0.66 x 10(-2) +/- 0.1 x 10(-2) per site per year) (P < 0.01), while no difference between the mean numbers of synonymous substitutions in the two groups was seen (0.37 x 10(-2) +/- 0.1 x 10(-2) and 0.51 x 10(-2) +/- 0.2 x 10(-2) per site per year for nonprogressors and progressors, respectively; P > 0.1). The mean ratios of synonymous nucleotide p distance to nonsynonymous p distance were 0.35 for nonprogressors and 0.62 for progressors. The number of nonsynonymous substitutions was not associated with virus load or virus phenotype, which are established predictors of disease progression, but correlated strongly with the duration of the immunocompetent period (r2 = 0.41; P = 0.001). This indicates that there is no causative relationship between intrahost evolution and CD4+ cell decline. Our data suggest that intrahost evolution in human immunodeficiency virus type 1 infection is driven by selective forces, the strength of which is related to the duration of the immunocompetent period.     

The origin and evolution of Ebola and Marburg viruses

Molecular evolutionary analyses for Ebola and Marburg viruses were conducted with the aim of elucidating evolutionary features of these viruses. In particular, the rate of nonsynonymous substitutions for the glycoprotein gene of Ebola virus was estimated to be, on the average, 3.6 x 10(-5) per site per year. Marburg virus was also suggested to be evolving at a similar rate. Those rates were a hundred times slower than those of retroviruses and human influenza A virus, but were of the same order of magnitude as that of the hepatitis B virus. When these rates were applied to the degree of sequence divergence, the divergence time between Ebola and Marburg viruses was estimated to be more than several thousand years ago. Moreover, most of the nucleotide substitutions were transitions and synonymous for Marburg virus. This suggests that purifying selection has operated on Marburg virus during evolution.      

Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses

European bat lyssaviruses types 1 and 2 (EBLV-1 and EBLV-2) are widespread in Europe, although little is known of their evolutionary history. We undertook a comprehensive sequence analysis to infer the selection pressures, rates of nucleotide substitution, age of genetic diversity, geographical origin, and population growth rates of EBLV-1. Our study encompassed data from 12 countries collected over a time span of 35 years and focused on the glycoprotein (G) and nucleoprotein (N) genes. We show that although the two subtypes of EBLV-1--EBLV-1a and EBLV-1b--have both grown at a low exponential rate since their introduction into Europe, they have differing population structures and dispersal patterns. Furthermore, there were strong constraints against amino acid change in both EBLV-1 and EBLV-2, as reflected in a low ratio of nonsynonymous to synonymous substitutions per site, particularly in EBLV-1b. Our inferred rate of nucleotide substitution in EBLV-1, approximately 5 x 10(-5) substitutions per site per year, was also one of the lowest recorded for RNA viruses and implied that the current genetic diversity in the virus arose 500 to 750 years ago. We propose that the slow evolution of EBLVs reflects their distinctive epidemiology in bats, where they occupy a relatively stable fitness peak.     

Relationships of the chromosomal species in the eurasian mole rats of theSpalax ehrenbergi group as determined by DNA-DNA hybridization,and an estimate of the spalacid-murid divergence time

Summary DNA-DNA hybridization was used to measure the average genomic divergence among the four chromosomal species of the Eurasian mole rats belonging to theSpalax ehrenbergi complex (Rodentia: Spalacidae). The percent nucleotide substitutions in the single-copy nuclear DNA among the species ranged from 0 to 5%, suggesting that speciation has occurred with minor genomic changes in these animals. The youngest chromosomal species appear to differ by 0.2–0.6% base pair mismatch, which is only between one and three base differences in a 500-bp fragment. The interspecific values of percent nucleotide differences permit the recognition of two well-separated speciation events in theS. ehrenbergi complex, the older (of Lower Pleistocene age) having isolated the chromosomal species 2n=54 before the divergence of the three other species.DNA-DNA hybridization was also used to compare the Spalacinae (Eurasian mole rats), Murinae (Old World rats and mice), and Arvicolinae (voles and lemmings). These data enabled us to estimate the time of divergence of the spalacids at ca. 19 million years ago. The dates of divergence among the other rodent lineages, as predicted by DNA hybridization results, agree well with paleontological data. These dates of divergence are obtained by the relation between geological time and single-copy nuclear DNA change, a relation that was calibrated by Catzeflis et al. (1987) through the use of fossil Arvicolinae and Murinae data.