Different models, different trees: the geographic origin of PTLV-I

Using nucleotide sequences from three genomic regions of the human and simian T-cell lymphotropic virus type I (HTLV-I/STLV-I)-consisting of 69 sequences from a 140-bp segment of the pol region, 98 sequences from a 503-bp segment of the LTR, and 154 sequences from a 386-bp segment of the env region-we tested two hypotheses concerning the geographic origin and evolution of STLV-I and HTLV-I. First, we tested the assumption of equal rates of evolution along STLV-I and HTLV-I lineages using a likelihood ratio test to ascertain whether current levels of genomic diversity can be used to determine ancestry. We demonstrated that unequal rates of evolution along HTLV-I and STLV-I lineages have occurred throughout evolutionary time, thus calling into question the use of pairwise distances to assign ancestry. Second, we constructed phylogenetic trees using multiple phylogenetic techniques to test for the geographic origin of STLV-I and HTLV-I. Using the principle of likelihood, we chose a statistically justified model of evolution for each data set. We demonstrated the utility of the likelihood ratio test to determine which model of evolution should be chosen for phylogenetic analyses, revealing that using different models of evolution produces conflicting results, and neither the hypothesis of an African origin nor the hypothesis of an Asian origin can be rejected statistically. Our best estimates of phylogenetic relationships, however, support an African origin of PTLV for each gene region.     

Phylogenetic associations of human and simian T-cell leukemia/lymphotropic virus type I strains: evidence for interspecies transmission.

Homologous env sequences from 17 human T-leukemia/lymphotropic virus type I (HTLV-I) strains from throughout the world and from 25 simian T-leukemia/lymphotropic virus type I (STLV-I) strains from 12 simian species in Asia and Africa were analyzed in a phylogenetic context as an approach to resolving the natural history of these related retroviruses. STLV-I exhibited greater overall sequence variation between strains (1 to 18% compared with 0 to 9% for HTLV-I), supporting the simian origin of the modern viruses in all species. Three HTLV-I phylogenetic clusters or clades (cosmopolitan, Zaire, and Melanesia) were resolved with phenetic, parsimony, and likelihood analytical procedures. Seven phylogenetic clusters of STLV-I were resolved with the most primitive (deeply rooted) divergence involving several STLV-I strains from Asian primate species. Combined analysis of HTLV-I and STLV-I revealed that neither STLV-I clusters nor HTLV-I clusters recapitulated host species specificity; rather, multiple clades from the same species were closer to clades from other species than to each other. We interpret these evolutionary associations as support for the occurrence of multiple discrete interspecies transmissions of ancestral viruses between primate species (including human) that led to recognizable phylogenetic clades that persist in modern species. Geographic concordance of divergent host species that harbor closely related viruses reinforces that physical feasibility for hypothesized interspecies virus transmission in the past and in the present.     

Multiple interspecies transmissions of human and simian T-cell leukemia/lymphoma virus type I sequences

Using two sets of nucleotide sequences of the human and simian T-cell leukemia/lymphoma virus type I (HTLV-I/STLV-I), one consisting of 522 bp of the env gene from 70 viral strains and the other a 140-bp segment from the pol gene of 52 viral strains, I estimated cladograms based on a statistical parsimony procedure that was developed specifically to estimate within-species gene trees. An extension of a nesting procedure is offered for sequence data that forms nested clades used in hypothesis testing. The nested clades were used to test three hypotheses relating to transmission of HTLV/STLV sequences: (1) Have cross-species transmissions occurred and, if so, how many? (2) In what direction have they occurred? (3) What are the geographic relationships of these transmission events? The analyses support a range of 11-16 cross-species transmissions throughout the history of these sequences. Additionally, outgroup weights were assigned to haplotypes using arguments from coalescence theory to infer directionality of transmission events. Conclusions on geographic origins of transmission events and particular viral strains are inconclusive due to small samples and inadequate sampling design. Finally, this approach is compared directly to results obtained from a traditional maximum parsimony approach and found to be superior at establishing relationships and identifying instances of transmission.      

Molecular evolution of poxviruses

Previous restriction fragment length polymorphism analysis divided variola virus (VARV) strains into two subtypes, one of which included West African and South American isolates. This allowed a dating to be introduced for the first time in estimation of the VARV evolution rate. The results were used to analyze the molecular evolution of the total family Poxviridae. Comparisons of the known nucleotide sequences were performed for the extended conserved central genome region in 42 orthopoxvirus strains and for the eight genes of multisubunit RNA polymerase in 65 viruses belonging to various genera of the family Poxviridae. Using the Bayesian dating method, the mutation accumulation rate of poxviruses was estimated at (1.7–8.8) × 10^?6 nucleotide substitutions per site per year. Computations showed that the modern poxvirus genera started diverging from an ancestral virus more than 200 thousand years ago and that an ancestor of the genus Orthopoxvirus emerged 131 ± 45 thousand years ago. The other genera of mammalian poxviruses with a low GC content diverged approximately 110–90 thousand years ago. The independent evolution of VARV started 3.4 ± 0.8 thousand years ago. It was shown with the example of VARV and the monkeypox virus (MPXV) that divergent evolution of these orthopoxviruses started and the West African subtypes of VARV and MPXV were formed as geographical conditions changed to allow isolation of West African animals from other African regions.     

Tempo and mode of human and simian T-lymphotropic virus (HTLV/STLV) evolution revealed by analyses of full-genome sequences

We investigated the tempo and mode of evolution of the primate T-lymphotropic viruses (PTLVs). Several different models of nucleotide substitution were tested on a general phylogenetic tree obtained using the 20 full-genome HTLV/STLV sequences available. The likelihood ratio test showed that the Tamura and Nei model with discrete gamma-distributed rates among sites is the best-fitting substitution model. The heterogeneity of nucleotide substitution rates along the PTLV genome was further investigated for different genes and at different codon positions (cdp's). Tests of rate constancy showed that different PTLV lineages evolve at different rates when first and second cdp's are considered, but the molecular-clock hypothesis holds for some PTLV lineages when the third cdp is used. Negative selection was evident throughout the genome. However, in the gp46 region, a small fragment subjected to positive selection was identified using a Monte Carlo simulation based on a likelihood method. Employing correlations of the virus divergence times with anthropologically documented migrations of their host, a possible timescale was estimated for each important node of the PTLV tree. The obtained results on these slow-evolving viruses could be used to fill gaps in the historical records of some of the host species. In particular, the HTLV-I/STLV-I history might suggest a simian migration from Asia to Africa not much earlier than 19,500-60,000 years ago.     

Molecular evolution of poxviruses

Previous restriction fragment length polymorphism analysis divided variola virus (VARV) strains into two subtypes, one of which included West African and South American isolates. This allowed a dating to be introduced for the first time in estimation of the VARV evolution rate. The results were used to analyze the molecular evolution of the total family Poxviridae. Comparisons of the known nucleotide sequences were performed for the extended conserved central genome region in 42 orthopoxvirus strains and for the eight genes of multisubunit RNA polymerase in 65 viruses belonging to various genera of the family Poxviridae. Using the Bayesian dating method, the mutation accumulation rate of poxviruses was estimated at (1.7-8.8) x 10(-6) nucleotide substitutions per site per year. Computations showed that the modem poxvirus genera started diverging from an ancestral virus more than 200 thousand years ago and that an ancestor of the genus Orthopoxvirus emerged 131 +/- 45 thousand years ago. The other genera of mammalian poxviruses with a low GC content diverged approximately 110-90 thousand years ago. The independent evolution of VARV started 3.4 +/- 0.8 thousand years ago. It was shown with the example of VARV and the monkeypox virus (MPXV) that divergent evolution of these orthopoxviruses started and the West African subtypes of VARV and MPXV were formed as geographical conditions changed to allow isolation of West African animals from other African regions.     

The core 2 beta-1,6-N-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus 4 was acquired from an ancestor of the African buffalo

The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only viral gene known to date that encodes a homologue of the cellular core 2 beta-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M). To investigate the origin and evolution of the Bo17 gene, we analyzed its distribution among BoHV-4 strains and determined the sequences of Bo17 from nine representative strains and of the C2GnT-M gene from six species of ruminants expected to encompass the group within which the gene acquisition occurred. Of 34 strains of BoHV-4, isolated from four different continents, all were found to contain the Bo17 gene. Phylogenetic analyses indicated that Bo17 was acquired from a recent ancestor of the African buffalo, implying that cattle subsequently acquired BoHV-4 by cross-species transmission. The rate of synonymous nucleotide substitution in Bo17 was estimated at 5 x 10(-8) to 6 x 10(-8) substitutions/site/year, consistent with previous estimates made under the assumption that herpesviruses have cospeciated with their hosts. The Bo17 gene acquisition was dated to around 1.5 million years ago. Bo17 sequences from BoHV-4 strains from African buffalo and from cattle formed two separate clades, estimated to have split about 700,000 years ago. Analysis of the ratio of nonsynonymous to synonymous nucleotide substitutions revealed a burst of amino acid replacements subsequent to the transfer of the cellular gene to the viral genome, followed by a return to a strong constraint on nonsynonymous changes during the divergence of contemporary BoHV-4 strains. The Bo17 gene represents the most recent of the known herpesvirus gene acquisitions and provides the best opportunity for learning more about this important process of viral evolution.     

Molecular evolution of the tick-borne encephalitis and Powassan viruses

Issues associated with newly emerging viruses, their genetic diversity, and viral evolution in modern environments are currently attracting growing attention. In this study, a phylogenetic analysis was performed and the evolution rate was evaluated for such pathogenic flaviviruses endemic to Russia as tick-borne encephalitis virus (TBEV) and Powassan virus (PV). The analysis involved 47 nucleotide sequences of the TBEV genome region encoding protein E and 17 sequences of the PV NS5-encoding region. The nucleotide substitution rate was estimated as 1.4 × 10⁻⁴ and 5.4 × 10⁻⁵ substitutions per site per year for the E protein-encoding region of the TBEV genome and for the NS5 genome region of PV, respectively. The ratio of non-synonymous to synonymous nucleotide substitutions (dN/dS) in viral sequences was calculated as 0.049 for TBEV and 0.098 for PV. The highest dN/dS values of 0.201–0.220 were found in the subcluster of Russian and Canadian PV strains, and the lowest value of 0.024 was observed in the cluster of Russian and Chinese strains of the Far Eastern TBEV genotype. Evaluation of time intervals between the events of viral evolution showed that the European subtype of TBEV diverged from the common TBEV ancestor approximately 2750 years ago, while the Siberian and Far Eastern subtypes emerged approximately 2250 years ago. The PV was introduced into its natural foci of the Russian Primorskii krai only approximately 70 years ago; these strains were very close to Canadian PV strains. The pattern of PV evolution in North America was similar to the evolution of the Siberian and Far Eastern TBEV subtypes in Asia. The moments of divergence between major genetic groups of TBEV and PV coincide with historical periods of climate warming and cooling, suggesting that climate change was a key factor in the evolution of flaviviruses in past millennia.     

Lack of tax diversity for tropical spastic paraparesis/human T-cell lymphotropic virus type 1 (HTLV-I) associated myelopathy development in HTLV-I-infected subjects in São Paulo, Brazil

The product of human T-cell lymphotropic virus type 1 (HTLV-1) tax gene has a transactivating effect of the viral and cellular gene expression. Genetic variations in this gene have been correlated with differences in clinical outcomes. Based upon its diversity, two closely related substrains, namely tax A and tax B, have been described. The tax A substrain has been found at a higher frequency among human T-cell leukemia virus type 1 (TSP/HAM) patients than among healthy HTLV-I-infected asymptomatic subjects in Japan. In this study, we determined the distribution of tax substrains in HTLV-I-infected subjects in the city of S?o Paulo, Brazil. Using the ACCII restriction enzyme site, we detected only tax A substrain from 48 TSP/HAM patients and 28 healthy HTLV-I carriers. The sequenced tax genes from nine TSP/HAM patients and five asymptomatic HTLV-I carriers showed a similar pattern of mutation, which characterizes tax A. Our results indicate that HTLV-I tax subtypes have no significant influences on TSP/HAM disease progression. Furthermore, monophyletic introduction of HTLV-I to Brazil probably occurred during the African slave trade many years ago.     

Molecular evolution of the tick-borne encephalitis and Powassan viruses

The problem of emerging viruses, their genetic diversity and viral evolution in nature are attracting more attention. The phylogenetic analysis and evaluationary rate estimation were made for pathogenic flaviviruses such as tick-borne encephalitis virus (TBEV) and Powassan (PV) circulated in natural foci in Russia. 47 nucleotide sequences of encoded protein E of the TBEV and 17 sequences of NS5 genome region of the PV have been used. It was found that the rate of accumulation of nucleotide substitutions for E genome region of TBEV was approximately 1.4 x 10(-4) and 5.4 x 10(-5) substitutions per site per year for NS5 genome region of PV. The ratio of non-synonymous nucleotide substitutions to synonymous substitution (dN/dS) for viral sequences were estimated of 0.049 for TBEV and 0.098 for PV. Maximum value dN/dS was 0.201-0.220 for sub-cluster of Russian and Canadian strains of PV and the minimum - 0.024 for cluster of Russian and Chinese strains of Far Eastern genotype TBEV. Evaluation of time intervals of evolutionary events associated with these viruses showed that European subtype TBEV are diverged from all-TBEV ancestor within approximately 2750 years and the Siberian and Far Eastern subtypes are emerged about 2250 years ago. The PV was introduced into natural foci of the Primorsky Krai of Russia only about 70 years ago and PV is a very close to Canadian strains of PV. Evolutionary picture for PV in North America is similar to evolution of Siberian and Far Eastern subtypes TBEV in Asia. The divergence time for main genetic groups of TBEV and PV are correlated with historical periods of warming and cooling. These allow to propose a hypothesis that climate changes were essential to the evolution of the flaviviruses in the past millenniums.     

Origin and evolution of influenza virus hemagglutinin genes

Influenza A, B, and C viruses are the etiological agents of influenza. Hemagglutinin (HA) is the major envelope glycoprotein of influenza A and B viruses, and hemagglutinin-esterase (HE) in influenza C viruses is a protein homologous to HA. Because influenza A virus pandemics in humans appear to occur when new subtypes of HA genes are introduced from aquatic birds that are known to be the natural reservoir of the viruses, an understanding of the origin and evolution of HA genes is of particular importance. We therefore conducted a phylogenetic analysis of HA and HE genes and showed that the influenza A and B virus HA genes diverged much earlier than the divergence between different subtypes of influenza A virus HA genes. The rate of amino acid substitution for A virus HAs from duck, a natural reservoir, was estimated to be 3.19 x 10(-4) per site per year, which was slower than that for human and swine A virus HAs but similar to that for influenza B and C virus HAs (HEs). Using this substitution rate from the duck, we estimated that the divergences between different subtypes of A virus HA genes occurred from several thousand to several hundred years ago. In particular, the earliest divergence time was estimated to be about 2,000 years ago. Also, the A virus HA gene diverged from the B virus HA gene about 4,000 years ago and from the C virus HE gene about 8,000 years ago. These time estimates are much earlier than the previous ones.     

Homologous recombination and the pattern of nucleotide substitution in Ehrlichia ruminantium

Patterns of nucleotide substitution at orthologous loci were examined between three genomes of Ehrlichia ruminantium, the causative agent of heartwater disease of ruminants. The most recent common ancestor of two genomes (Erwe and Erwo) belonging to the Welgevonden strain was estimated to have occurred 26,500-57,000 years ago, while the most recent common ancestor of these two genomes and the Erga genome (Gardel strain) was estimated to have occurred 2.1-4.7 million years ago. The search for genes showing extremely high values of the number of synonymous substitutions per site was used to identify genes involved in past homologous recombination. The most striking case involved the map1 gene, encoding major antigenic protein-1; evidence for homologous recombination is consistent with previous phylogenetic analysis of map1 alleles. At this and certain other loci, homologous recombination may have contributed to the evolution of host-pathogen interactions. In addition, comparison of the patterns of synonymous and nonsynonymous substitution provided evidence for positive selection favoring a high level of amino acid change between the Welgevonden and Gardel strains at a locus of unknown function (designated Erum4340 in the Erwo genome).     

Evolutionary insights on the origin of human T-cell lymphoma/leukemia virus type I (HTLV-I) derived from sequence analysis of a new HTLV-I variant from Papua New Guinea.

Recent studies have established the presence of human T-cell lymphoma/leukemia virus type I (HTLV-I) in Melanesia. An HTLV-I strain, PNG-1, has now been isolated from a healthy member of the Hagahai, a remote, recently contacted group in Papua New Guinea. To further characterize PNG-1, we employed polymerase chain reaction amplification with subsequent cloning and sequencing of amplified products. Sequence analyses of amplified regions of pol, env, and pX genes of this variant indicate marked heterogeneity (approximately 7%) from prototype HTLV-I. Based on available sequence data, PNG-1 is distinct from all other known HTLV-I strains and diverges from the common ancestor of HTLV-I prior to prototype isolates. The data also suggest, therefore, that HTLV-I originated in the Indo-Malay region rather than Africa.     

Polymorphism and evolution of influenza A virus genes

The nucleotide sequences of four genes of the influenza A virus (nonstructural protein, matrix protein, and a few subtypes of hemagglutinin and neuraminidase) are compiled for a large number of strains isolated from various locations and years, and the evolutionary relationship of the sequences is investigated. It is shown that all of these genes or subtypes are highly polymorphic and that the polymorphic sequences (alleles) are subject to rapid turnover in the population, their average age being much less than that of higher organisms. Phylogenetic analysis suggests that most polymorphic sequences within a subtype or a gene appeared during the last 80 years and that the divergence among the subtypes of hemagglutinin genes might have occurred during the last 300 years. The high degree of polymorphism in this RNA virus is caused by an extremely high rate of mutation, estimated to be 0.01/nucleotide site/year. Despite the high rate of mutation, most influenza virus genes are apparently subject to purifying selection, and the rate of nucleotide substitution is substantially lower than the mutation rate. There is considerable variation in the substitution rate among different genes, and the rate seems to be lower in nonhuman viral strains than in human strains. The difference might be responsible for the so-called freezing effect in some viral strains.      

Low degree of human T-cell leukemia/lymphoma virus type I genetic drift in vivo as a means of monitoring viral transmission and movement of ancient human populations.

We have studied the genetic variation of human T-cell leukemia/lymphoma virus type I (HTLV-I) isolates in the same individuals over time, as well as of HTLV-I isolates from various parts of the world. The viral DNA fragment studied encodes the carboxy terminus of gp46 and almost all of gp21, both of which are envelope glycoproteins. Samples were obtained from native inhabitants of five African countries, two South American countries, China, the French West Indies, and Haiti and included 14 patients with tropical spastic paraparesis/HTLV-I-associated myelopathy, 10 patients with adult T-cell leukemia, 1 patient with T-cell non-Hodgkin's lymphoma, and 3 healthy HTLV-I-seropositive individuals. DNA analyses of HTLV-I sequences demonstrated that (i) little or no genetic variation occurred in vivo in the same individual or in different hosts from the same region carrying the same virus, regardless of their clinical statuses; (ii) changes in nucleotide sequences in some regions of the HTLV-I genome were diagnostic of the geographical origin of the viruses; (iii) HTLV-I sequences from West African countries (Mauritania and Guinea Bissau) and some from the Ivory Coast and Central African Republic were virtually identical to those from the French West Indies, Haiti, French Guyana, and Peru, strongly suggesting that at least some HTLV-I strains were introduced into the New World through infected individuals during the slave trade events; and (iv) the Zairian HTLV-I isolates represent a separate HTLV-I cluster, in which intrastrain variability was also observed, and are more divergent from the other HTLV-I isolates. Because of the low genetic variability of HTLV-I in vivo, the study of the proviral DNA sequence in selected populations of infected individuals will increase our knowledge of the origin and evolution of HTLV-I and might be useful in anthropological studies.     

The simian origins of the pathogenic human T-cell lymphotropic virus type I

At least four, and possibly six, molecular subtypes of human T-cell lymphotropic virus type I (HTLV-I) exist: one is confined to Melanesia/Australia, one is ubiquitous, and the others are found only in Africa. Molecular epidemiology suggests that all subtypes arose from separate interspecies transmissions from simians to humans.     

Unequal evolutionary rates in the human immunodeficiency virus type 1 (HIV-1) pandemic: the evolutionary rate of HIV-1 slows down when the epidemic rate increases

HIV-1 sequences in intravenous drug user (IDU) networks are highly homogenous even after several years, while this is not observed in most sexual epidemics. To address this disparity, we examined the human immunodeficiency virus type 1 (HIV-1) evolutionary rate on the population level for IDU and heterosexual transmissions. All available HIV-1 env V3 sequences from IDU outbreaks and heterosexual epidemics with known sampling dates were collected from the Los Alamos HIV sequence database. Evolutionary rates were calculated using phylogenetic trees with a t test root optimization of dated samples. The evolutionary rate of HIV-1 subtype A1 was found to be 8.4 times lower in fast spread among IDUs in the former Soviet Union (FSU) than in slow spread among heterosexual individuals in Africa. Mixed epidemics (IDU and heterosexual) showed intermediate evolutionary rates, indicating a combination of fast- and slow-spread patterns. Hence, if transmissions occur repeatedly during the initial stage of host infection, before selective pressures of the immune system have much impact, the rate of HIV-1 evolution on the population level will decrease. Conversely, in slow spread, where HIV-1 evolves under the pressure of the immune system before a donor infects a recipient, the virus evolution at the population level will increase. Epidemiological modeling confirmed that the evolutionary rate of HIV-1 depends on the rate of spread and predicted that the HIV-1 evolutionary rate in a fast-spreading epidemic, e.g., for IDUs in the FSU, will increase as the population becomes saturated with infections and the virus starts to spread to other risk groups.     

Dating the time of viral subtype divergence

Precise dating of viral subtype divergence enables researchers to correlate divergence with geographic and demographic occurrences. When historical data are absent (that is, the overwhelming majority), viral sequence sampling on a time scale commensurate with the rate of substitution permits the inference of the times of subtype divergence. Currently, researchers use two strategies to approach this task, both requiring strong conditions on the molecular clock assumption of substitution rate. As the underlying structure of the substitution rate process at the time of subtype divergence is not understood and likely highly variable, we present a simple method that estimates rates of substitution, and from there, times of divergence, without use of an assumed molecular clock. We accomplish this by blending estimates of the substitution rate for triplets of dated sequences where each sequence draws from a distinct viral subtype, providing a zeroth-order approximation for the rate between subtypes. As an example, we calculate the time of divergence for three genes among influenza subtypes A-H3N2 and B using subtype C as an outgroup. We show a time of divergence approximately 100 years ago, substantially more recent than previous estimates which range from 250 to 3800 years ago.