Human immunodeficiency virus type 1 subtype B ancestral envelope protein is functional and elicits neutralizing antibodies in rabbits similar to those elicited by a circulating subtype B envelope.

Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted.

DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.

DNA Surveillance is a Web-based application that assists in the identification of the species and population of unknown specimens by aligning user-submitted DNA sequences with a validated and curated data set of reference sequences. Phylogenetic analyses are performed and results are returned in tree and table format summarizing the evolutionary distances between the query and reference sequences. DNA Surveillance is implemented with mitochondrial DNA (mtDNA) control region sequences representing the majority of recognized cetacean species. Extensions of the system to include other gene loci and taxa are planned. The service, including instructions and sample data, is available at http://www.dna-surveillance.auckland.ac.nz.

Substitution model of sequence evolution for the human immunodeficiency virus type 1 subtype B gp120 gene over the C2-V5 region.

Phylogenetic analyses frequently rely on models of sequence evolution that detail nucleotide substitution rates, nucleotide frequencies, and site-to-site rate heterogeneity. These models can influence hypothesis testing and can affect the accuracy of phylogenetic inferences. Maximum likelihood methods of simultaneously constructing phylogenetic tree topologies and estimating model parameters are computationally intensive, and are not feasible for sample sizes of 25 or greater using personal computers. Techniques that initially construct a tree topology and then use this non-maximized topology to estimate ML substitution rates, however, can quickly arrive at a model of sequence evolution. The accuracy of this two-step estimation technique was tested using simulated data sets with known model parameters. The results showed that for a star-like topology, as is often seen in human immunodeficiency virus type 1 (HIV-1) subtype B sequences, a random starting topology could produce nucleotide substitution rates that were not statistically different than the true rates. Samples were isolated from 100 HIV-1 subtype B infected individuals from the United States and a 620 nt region of the env gene was sequenced for each sample. The sequence data were used to obtain a substitution model of sequence evolution specific for HIV-1 subtype B env by estimating nucleotide substitution rates and the site-to-site heterogeneity in 100 individuals from the United States. The method of estimating the model should provide users of large data sets with a way to quickly compute a model of sequence evolution, while the nucleotide substitution model we identified should prove useful in the phylogenetic analysis of HIV-1 subtype B env sequences.

The inference of stepwise changes in substitution rates using serial sequence samples.

It is frequently true that molecular sequences do not evolve in a strictly clocklike manner. Instead, substitution rate may vary for a number of reasons, including changes in selection pressure and effective population size, as well as changes in mean generation time. Here we present two new methods for estimating stepwise changes in substitution rates when serially sampled molecular sequences are available. These methods are based on multiple rates with dated tips (MRDT) models and allow different rates to be estimated for different intervals of time. These intervals may correspond to the sampling intervals or to a priori--defined intervals that are not coincident with the times the serial samples are obtained. Two methods for obtaining estimates of multiple rates are described. The first is an extension of the phylogeny-based maximum-likelihood estimation procedure introduced by Rambaut. The second is a new parameterization of the pairwise distance least-squares procedure used by Drummond and Rodrigo. The utility of these methods is demonstrated on a genealogy of HIV sequences obtained at five different sampling times from a single patient over a period of 34 months.

Reconstructing genealogies of serial samples under the assumption of a molecular clock using serial-sample UPGMA.

Reconstruction of evolutionary relationships from noncontemporaneous molecular samples provides a new challenge for phylogenetic reconstruction methods. With recent biotechnological advances there has been an increase in molecular sequencing throughput, and the potential to obtain serial samples of sequences from populations, including rapidly evolving pathogens, is fast being realized. A new method called the serial-sample unweighted pair grouping method with arithmetic means (sUPGMA) is presented that reconstructs a genealogy or phylogeny of sequences sampled serially in time using a matrix of pairwise distances. The resulting tree depicts the terminal lineages of each sample ending at a different level consistent with the sample's temporal order. Since sUPGMA is a variant of UPGMA, it will perform best when sequences have evolved at a constant rate (i.e., according to a molecular clock). On simulated data, this new method performs better than standard cluster analysis under a variety of longitudinal sampling strategies. Serial-sample UPGMA is particularly useful for analysis of longitudinal samples of viruses and bacteria, as well as ancient DNA samples, with the minimal requirement that samples of sequences be ordered in time.

Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E.

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.

Likelihood-based tests of topologies in phylogenetics.

Likelihood-based statistical tests of competing evolutionary hypotheses (tree topologies) have been available for approximately a decade. By far the most commonly used is the Kishino-Hasegawa test. However, the assumptions that have to be made to ensure the validity of the Kishino-Hasegawa test place important restrictions on its applicability. In particular, it is only valid when the topologies being compared are specified a priori. Unfortunately, this means that the Kishino-Hasegawa test may be severely biased in many cases in which it is now commonly used: for example, in any case in which one of the competing topologies has been selected for testing because it is the maximum likelihood topology for the data set at hand. We review the theory of the Kishino-Hasegawa test and contend that for the majority of popular applications this test should not be used. Previously published results from invalid applications of the Kishino-Hasegawa test should be treated extremely cautiously, and future applications should use appropriate alternative tests instead. We review such alternative tests, both nonparametric and parametric, and give two examples which illustrate the importance of our contentions.

Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection.

To understand the high variability of the asymptomatic interval between primary human immunodeficiency virus type 1 (HIV-1) infection and the development of AIDS, we studied the evolution of the C2-V5 region of the HIV-1 env gene and of T-cell subsets in nine men with a moderate or slow rate of disease progression. They were monitored from the time of seroconversion for a period of 6 to 12 years until the development of advanced disease in seven men. Based on the analysis of viral divergence from the founder strain, viral population diversity within sequential time points, and the outgrowth of viruses capable of utilizing the CXCR4 receptor (X4 viruses), the existence of three distinct phases within the asymptomatic interval is suggested: an early phase of variable duration during which linear increases ( approximately 1% per year) in both divergence and diversity were observed; an intermediate phase lasting an average of 1.8 years, characterized by a continued increase in divergence but with stabilization or decline in diversity; and a late phase characterized by a slowdown or stabilization of divergence and continued stability or decline in diversity. X4 variants emerged around the time of the early- to intermediate-phase transition and then achieved peak representation and began a decline around the transition between the intermediate and late phases. The late-phase transition was also associated with failure of T-cell homeostasis (defined by a downward inflection in CD3(+) T cells) and decline of CD4(+) T cells to

HIV-1 dynamics in children.

HIV-1-infected children have higher plasma viral loads and progress to disease more quickly than infected adults. To gain insight into the accelerated pathogenesis of HIV-1 in children, viral dynamics were measured following the initiation of highly active antiretroviral therapy (HAART) and compared with those reported for adults. A biphasic decline in plasma HIV-1 RNA was observed, with a rapid decrease during the first 1 to 2 weeks of therapy (phase I) followed by a slower decline (phase II). The phase I and II decay rates were not significantly different among children of different ages, pretherapy plasma HIV-1 RNA levels, or CD4 cell counts. Estimated phase I decay rates were similar to those previously reported in adults with a mean of 0.43 days(-1) and a half-life of 1.6 days. The phase II decay rates were slower in children compared with adults with a mean of 0.016 days(-1) versus 0.066 days(-1), and a half-life of 43.3 versus 14.1 days, respectively (p < .05). The mean time required to reach viral levels below detection thresholds was also longer in these children compared with that in adults. These data suggest that HIV-1 dynamics may be different in children, and that these differences may necessitate different treatment strategies.

Coalescent estimates of HIV-1 generation time in vivo.

The generation time of HIV Type 1 (HIV-1) in vivo has previously been estimated using a mathematical model of viral dynamics and was found to be on the order of one to two days per generation. Here, we describe a new method based on coalescence theory that allows the estimate of generation times to be derived by using nucleotide sequence data and a reconstructed genealogy of sequences obtained over time. The method is applied to sequences obtained from a long-term nonprogressing individual at five sampling occasions. The estimate of viral generation time using the coalescent method is 1.2 days per generation and is close to that obtained by mathematical modeling (1.8 days per generation), thus strengthening confidence in estimates of a short viral generation time. Apart from the estimation of relevant parameters relating to viral dynamics, coalescent modeling also allows us to simulate the evolutionary behavior of samples of sequences obtained over time.

Evolution of envelope sequences from the genital tract and peripheral blood of women infected with clade A human immunodeficiency virus type 1.

The development of viral diversity during the course of human immunodeficiency virus type 1 (HIV-1) infection may significantly influence viral pathogenesis. The paradigm for HIV-1 evolution is based primarily on studies of male cohorts in which individuals were presumably infected with a single virus variant of subtype B HIV-1. In this study, we evaluated virus evolution based on sequence information of the V1, V2, and V3 portions of HIV-1 clade A envelope genes obtained from peripheral blood and cervical secretions of three women with genetically heterogeneous viral populations near seroconversion. At the first sample following seroconversion, the number of nonsynonymous substitutions per potential nonsynonymous site (dn) significantly exceeded substitutions at potential synonymous sites (ds) in plasma viral sequences from all individuals. Generally, values of dn remained higher than values of ds as sequences from blood or mucosa evolved. Mutations affected each of the three variable regions of the envelope gene differently; insertions and deletions dominated changes in V1, substitutions involving charged amino acids occurred in V2, and sequential replacement of amino acids over time at a small subset of positions distinguished V3. The relationship among envelope nucleotide sequences obtained from peripheral blood mononuclear cells, plasma, and cervical secretions was evaluated for each individual by both phylogenetic and phenetic analyses. In all subjects, sequences from within each tissue compartment were more closely related to each other than to sequences from other tissues (phylogenetic tissue compartmentalization). At time points after seroconversion in two individuals, there was also greater genetic identity among sequences from the same tissue compartment than among sequences from different tissue compartments (phenetic tissue compartmentalization). Over time, temporal phylogenetic and phenetic structure was detectable in mucosal and plasma viral samples from all three women, suggesting a continual process of migration of one or a few infected cells into each compartment followed by localized expansion and evolution of that population.

Evolution of human immunodeficiency virus type 1 envelope sequences in infected individuals with differing disease progression profiles.

Sequence variation displayed by the human immunodeficiency virus type 1 (HIV-1) has been proposed to be linked to the pathogenesis of acquired immunodeficiency syndrome (AIDS). To assess viral evolution during the course of infection, we evaluated sequence variability in the env variable domains in four HIV-1-infected individuals exhibiting differing profiles of CD4+ T cell decline when followed from seroconversion until the development of AIDS or loss of followup. Proviral sequences encoding the V3-V5 region of gp 120 were obtained following PCR amplification of peripheral blood mononuclear cell DNA and cloning. Virus in each patient was relatively homogeneous early in infection and then diverged with time, more consistently at its nonsynonymous sites. Just prior to or coincident with a rapid decline in CD4+ T cell numbers, sequences were found with basic amino acid substitutions clustered within and downstream of the gp 120 V3 domain. Within the constraints of the current data set, we conclude that the virus appears to continually accumulate changes in its amino acid sequences well into the time of marked CD4+ T cell decline.

Dynamics of syncytium-inducing and non-syncytium-inducing type 1 human immunodeficiency viruses during primary infection.

Syncytium-inducing (SI) type 1 human immunodeficiency viruses (HIV-1) replicate faster in vitro and are generally more cytopathic to T cells than non-syncytium-inducing (NSI) HIV-1. Early in infection, the virus population typically consists of NSI viruses, with SI viruses appearing later. This is true even when both SI and NSI viruses are transmitted, and when SI viruses dominate the virus population peak seen during primary infection. Here, Phillips's model of HIV dynamics during primary infection (Science 271:497-499) is modified to map the growth trajectories of SI and NSI subpopulations. The model predicts that with certain rate constants, SI viruses may show a more precipitous decline in their numbers during primary infection than NSI viruses, and this may account for the observed dominance of NSI viruses early in infection.

Quantitation of target molecules from polymerase chain reaction-based limiting dilution assays.

Polymerase chain reaction-based limiting dilution assays (PLDAs), commonly called end-point dilutions, are frequently used to quantify the copy numbers of human immunodeficiency virus (HIV) and other viruses in biological samples; however, the way in which these assays are done, and the mathematical method used to estimate copy numbers, vary from laboratory to laboratory. Here, we describe a statistical method for estimating the number of copies and the associated standard error of the estimate, using a PLDA. The copy number is estimated by the value that maximizes the goodness of fit between the observed numbers of negative reactions and the expected numbers of negative reactions (the latter estimated using a Poisson probability distribution) as measured by the chi2 statistic. The method described here also takes into account user-specified probabilities of obtaining a false-positive or a false-negative PCR result, a feature that is not generally available with other limiting dilution estimation procedures. QUALITY, a computer program that implements the estimation strategy, is also described. Simulations illustrate the efficiency of estimation with different numbers of PCR amplifications conducted at each dilution, and different dilution factors. Finally, a simple strategy for more efficient assays is proposed.

Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and distinct evolutionary rates among envelope sequence clades.

For the rapid genetic analysis of feline immunodeficiency virus (FIV), we developed a heteroduplex mobility assay (HMA) that utilizes a PCR-amplified fragment of the FIV envelope gene spanning the third and fourth variable regions of the envelope surface protein coding sequence. Viral sequences were successfully amplified from blood specimens from 98 naturally infected cats from Australia, Canada, Germany, Italy, South Africa, and the United States. Eighty were clearly assignable to the A or B envelope sequence subtypes. Three belonged to subtype C, one was dually infected with viruses harboring the A and B env subtypes, and several were categorized as outliers to any of the established subtypes or as probable intersubtype recombinants. Some geographic clustering was evident, with subtypes A and B found in greater frequency in the western and eastern regions of the United States, respectively. Subtypes A, B, and C were found on more than one continent, and countries with more than two samples analyzed contained at least two subtypes. The broadest representation of subtypes was found in Munich, Germany, where three subtypes and one virus that was not classifiable by HMA were found. Thirteen samples were selected for DNA sequence determination over the same region of env used for HMA. Analysis of all available FIV env sequences from this and previous studies revealed the existence of recombinant viruses generated from subtype A/B, B/D, and A/C envelope gene sequences. Subtype A env sequences were less diverse than subtype B sequences, although both groups had well-supported clusters. Furthermore, the mutational pattern giving rise to diversification in the two subtypes differed, with the subtype A viruses showing half as many synonymous site mutations compared to subtype B yet showing similar levels of nonsynonymous site changes. These results are consistent with the hypothesis that FIV-B is an older virus group and is possibly more host adapted than FIV-A.

Human immunodeficiency virus type 1 molecular evolution and the measure of selection.

Human immunodeficiency virus (HIV) envelope genes are highly variable between and often within individuals. Part of this variability is thought to be the result of immune-mediated positive selection for sequence diversity. To measure positive selection it has become customary in HIV research to calculate the ratio of the proportions of synonymous (ds) and nonsynonymous (dn) substitutions per potential synonymous or nonsynonymous site, respectively. However, another measure that can be used is the difference between ds and dn, delta d. We show, by example, that using the ratio, ds/dn, or the difference, delta d, may lead us to different conclusions regarding the existence of positive selection pressure. We conclude by noting that until we understand the processes that mediate nucleotide variation in a host selective environment, inferences based on summary statistics characterizing types of nucleotide substitutions should be made with caution.

Covariability of V3 loop amino acids.

We reanalyzed for covariability a set of 308 human immunodeficiency virus type 1 (HIV-1) V3 loop amino acid sequences from the B envelope sequence subtype previously analyzed by Korber et al.,1 as well as a new set of 440 sequences that also included substantial numbers of sequences from subtypes A, D, and E. We used the measure employed by Korber et al., essentially the likelihood ratio statistic for independence, plus two additional measures as well as clade information to examine the new set and both data sets simultaneously. We set forth the following conclusions and observations. The eight most highly connected sites identified through these statistical approaches included all of the six residues previously shown to have determining roles in structure, immunologic recognition, virus phenotype, and host range; each of the seven pairs of covariant sites found by Korber were signaled by our additional two measures in the set of 308 sequences, although 2 or 3 dropped out of the examination of the set of 440 when the requirement of stringent significance was applied for some or all of the three tests, respectively; using the same criteria, a total of 20 (including 5 Korber et al. pairs) or a total of 6 (including 4 Korber et al. pairs) were found when the set of 440 was added. Several limitations to statistical analysis of this type of HIV sequence data were also noted. For example, the data sets were, by historical necessity, collected haphazardly. For example, it was not possible to separate substantially sized groups out according to time of or since infection, disease status, antiviral treatment, geography, etc. There was also an enormous "wealth of significance" within the data. For example, for one measure the 440 data set showed 233 of the 465 pairs of sites with a likelihood ratio statistic of < 0.001. Last, most sites had consensus amino acids in 80% or more of the sequences; hence, there was an absence of data on many combinations of amino acids. Given the observed linkage between sites shown to be covariable and those known to have critical biological function, the statistical approaches we and Korber et al. have outlined may find use in predicting critical structural features of HIV proteins as targets for therapeutic intervention.