Although parasite-host co-speciation is a long-held hypothesis, convincing evidence for long-term co-speciation remains elusive, largely because of small numbers of hosts and parasites studied and uncertainty over rates of evolutionary change. Co-speciation is especially rare in RNA viruses, in which cross-species transfer is the dominant mode of evolution. Simian foamy viruses (SFVs) are ubiquitous, non-pathogenic retroviruses that infect all primates. Here we test the co-speciation hypothesis in SFVs and their primate hosts by comparing the phylogenies of SFV polymerase and mitochondrial cytochrome oxidase subunit II from African and Asian monkeys and apes. The phylogenetic trees were remarkably congruent in both branching order and divergence times, strongly supporting co-speciation. Molecular clock calibrations revealed an extremely low rate of SFV evolution, 1.7 x 10(-8) substitutions per site per year, making it the slowest-evolving RNA virus documented so far. These results indicate that SFVs might have co-speciated with Old World primates for at least 30 million years, making them the oldest known vertebrate RNA viruses.

It is generally accepted that the fitness cost of resistance mutations plays a role in the persistence of transmitted drug-resistant human immunodeficiency virus type 1 and that mutations that confer a high fitness cost are less able to persist in the absence of drug pressure. Here, we show that the fitness cost of reverse transcriptase (RT) mutations can vary within a 72-fold range. We also demonstrate that the fitness cost of M184V and K70R can be decreased or enhanced by other resistance mutations such as D67N and K219Q. We conclude that the persistence of transmitted RT mutants might range widely on the basis of fitness and that the modulation of fitness cost by mutational interactions will be a critical determinant of persistence.Antiretroviral drug resistance is an important cause of treatment failure in human immunodeficiency virus type 1-infected persons treated with reverse transcriptase (RT) and protease inhibitors. Emergence of resistance during treatment usually involves the initial selection of deleterious mutations that reduce drug susceptibility and decrease replicative capacity. Compensatory evolution through the acquisition of additional mutations generally results in partial restorations of viral fitness (19). Despite the accumulation of compensatory mutations, drug-resistant viruses tend to replicate less efficiently than wild-type viruses.The transmission of drug-resistant mutants with diminished fitness and the evolution of these viruses in the absence of drug are typically associated with the reversion and loss of resistance mutations (3,9,22). Different rates of persistence and reversion of mutations have been documented in vivo and have usually been explained by the impact of mutations on viral fitness. For instance, less-fit zidovudine-resistant mutants carrying T215Y/F are generally replaced by more-fit 215D/C/S revertants within less than a year, while more-fit M41L or D67N mutants tend to revert more slowly (1-3, 5, 6, 8, 13, 17, 20, 25). However, in vivo observations do not always show the expected relationship between fitness cost and persistence, and a range of persistence for the same mutation has been noted among patients infected with multidrug-resistant viruses. For instance, M184V mutants have been found to persist between 4 and 16 months after primary infection, while persistence of K103N can range between 1 and 3 years (2,3,5,17). We hypothesize that mutational interactions in multidrug-resistant viruses might modulate the fitness cost of resistance mutations and might influence the rate of reversion and persistence of mutations.

BackgroundThe success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.MethodologyWe developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.SignificanceCombined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.