Inference of genetic relatedness between viral quasispecies from sequencing data

Background

RNA viruses such as HCV and HIV mutate at extremely high rates, and as a result, they exist in infected hosts as populations of genetically related variants. Recent advances in sequencing technologies make possible to identify such populations at great depth. In particular, these technologies provide new opportunities for inference of relatedness between viral samples, identification of transmission clusters and sources of infection, which are crucial tasks for viral outbreaks investigations.

Results

We present (i) an evolutionary simulation algorithm Viral Outbreak InferenCE (VOICE) inferring genetic relatedness, (ii) an algorithm MinDistB detecting possible transmission using minimal distances between intra-host viral populations and sizes of their relative borders, and (iii) a non-parametric recursive clustering algorithm Relatedness Depth (ReD) analyzing clusters’ structure to infer possible transmissions and their directions. All proposed algorithms were validated using real sequencing data from HCV outbreaks.

Conclusions

All algorithms are applicable to the analysis of outbreaks of highly heterogeneous RNA viruses. Our experimental validation shows that they can successfully identify genetic relatedness between viral populations, as well as infer transmission clusters and outbreak sources.

     

Semen-specific genetic characteristics of human immunodeficiency virus type 1 env

Human immunodeficiency virus type 1 (HIV-1) in the male genital tract may comprise virus produced locally in addition to virus transported from the circulation. Virus produced in the male genital tract may be genetically distinct, due to tissue-specific cellular characteristics and immunological pressures. HIV-1 env sequences derived from paired blood and semen samples from the Los Alamos HIV Sequence Database were analyzed to ascertain a male genital tract-specific viral signature. Machine learning algorithms could predict seminal tropism based on env sequences with accuracies exceeding 90%, suggesting that a strong genetic signature does exist for virus replicating in the male genital tract. Additionally, semen-derived viral populations exhibited constrained diversity (P < 0.05), decreased levels of positive selection (P < 0.025), decreased CXCR4 coreceptor utilization, and altered glycosylation patterns. Our analysis suggests that the male genital tract represents a distinct selective environment that contributes to the apparent genetic bottlenecks associated with the sexual transmission of HIV-1.     

Variability of human immunodeficiency virus type 1 group O strains isolated from Cameroonian patients living in France.

Human immunodeficiency virus type 1 (HIV-1) nucleotide sequences encoding p24Gag and the Env C2V3 region were obtained from seven patients who were selected on the basis of having paradoxical seronegativity on a subset of HIV enzyme-linked immunosorbent assay detection kits and having atypical Western blot (immunoblot) reactivity. Sequence analyses showed that all of these strains were more closely related to the recently described Cameroonian HIV isolates of group O (HIV-1 outlier) than to group M (HIV-1 major). All seven patients had Cameroonian origins but were living in France at the time the blood samples were taken. Characterization of a large number of group M strains has to date revealed eight distinct genetic subtypes (A to H). Genetic distances between sequences from available group O isolates were generally comparable to those observed in M intersubtype sequence comparisons, showing that the group O viruses are genetically very diverse. Analysis of sequences from these seven new viral strains, combined with the three previously characterized group O strains, revealed few discernable phylogenetic clustering patterns among the 10 patients' viral sequences. The level of diversity among group O sequences suggests that they may have a comparable (or greater) age than the M group sequences, although for unknown reasons, the latter group dispersed first and is the dominant lineage in the pandemic.     

Transmission patterns of HIV and hepatitis C virus among networks of people who inject drugs

Background

The risk-related behaviours and practices associated with injection drug use remain a driver of HIV and hepatitis C virus (HCV) transmission throughout the world. Here we evaluated HIV and HCV transmission patterns in the context of social networks of injection drug users (IDU) recruited from a higher incidence region in order to better understand factors that contribute to ongoing transmission among IDU.

Methods

IDU recruited through a chain-referral method provided biological specimens for analysis. HIV and HCV positive specimens were sequenced and analyzed using phylogenetic methods (Neighbour-joining and Bayesian) and transmission patterns of HIV and HCV evaluated in the context of the recruitment networks.

Results

Among the 407 recruited IDU, HCV and HIV prevalence were 60.6% and 10.1%, respectively; 98% of HIV positive individuals were co-infected with HCV. Thirty-six percent of HCV sequences were associated with clusters, compared to 67% of HIV sequences. Four (16.7%) of the 24 HCV clusters contained membership separated by 2 or fewer recruitment cycles, compared to 10 (41.6%) derived from more than one recruitment component. Two (28.6%) of the 7 HIV clusters contained membership separated by 2 or fewer recruitment cycles while 6 (85.7%) were composed of inter component membership.

Conclusions

Few HIV and HCV transmissions coincided with the recruitment networks, suggesting that they occurred in a different social context or a context not captured by the recruitment network. However, among the complete cohort, a higher degree of HIV clustering indicates many are recent infections originating from within current social networks, whereas a larger proportion of HCV infections may have occurred earlier in injecting history and in the context of a different social environment.     

HIV-1 Transmission during Early Infection in Men Who Have Sex with Men: A Phylodynamic Analysis

Background

Conventional epidemiological surveillance of infectious diseases is focused on characterization of incident infections and estimation of the number of prevalent infections. Advances in methods for the analysis of the population-level genetic variation of viruses can potentially provide information about donors, not just recipients, of infection. Genetic sequences from many viruses are increasingly abundant, especially HIV, which is routinely sequenced for surveillance of drug resistance mutations. We conducted a phylodynamic analysis of HIV genetic sequence data and surveillance data from a US population of men who have sex with men (MSM) and estimated incidence and transmission rates by stage of infection.

Methods and Findings

We analyzed 662 HIV-1 subtype B sequences collected between October 14, 2004, and February 24, 2012, from MSM in the Detroit metropolitan area, Michigan. These sequences were cross-referenced with a database of 30,200 patients diagnosed with HIV infection in the state of Michigan, which includes clinical information that is informative about the recency of infection at the time of diagnosis. These data were analyzed using recently developed population genetic methods that have enabled the estimation of transmission rates from the population-level genetic diversity of the virus. We found that genetic data are highly informative about HIV donors in ways that standard surveillance data are not. Genetic data are especially informative about the stage of infection of donors at the point of transmission. We estimate that 44.7% (95% CI, 42.2%–46.4%) of transmissions occur during the first year of infection.

Conclusions

In this study, almost half of transmissions occurred within the first year of HIV infection in MSM. Our conclusions may be sensitive to un-modeled intra-host evolutionary dynamics, un-modeled sexual risk behavior, and uncertainty in the stage of infected hosts at the time of sampling. The intensity of transmission during early infection may have significance for public health interventions based on early treatment of newly diagnosed individuals. Please see later in the article for the Editors'' Summary     

Genetically Related Human Immunodeficiency Virus Type 1 in Three Adults of a Family with No Identified Risk Factor for Intrafamilial Transmission

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son’s virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.     

Viral Genetic Linkage Analysis in the Presence of Missing Data

Analyses of viral genetic linkage can provide insight into HIV transmission dynamics and the impact of prevention interventions. For example, such analyses have the potential to determine whether recently-infected individuals have acquired viruses circulating within or outside a given community. In addition, they have the potential to identify characteristics of chronically infected individuals that make their viruses likely to cluster with others circulating within a community. Such clustering can be related to the potential of such individuals to contribute to the spread of the virus, either directly through transmission to their partners or indirectly through further spread of HIV from those partners. Assessment of the extent to which individual (incident or prevalent) viruses are clustered within a community will be biased if only a subset of subjects are observed, especially if that subset is not representative of the entire HIV infected population. To address this concern, we develop a multiple imputation framework in which missing sequences are imputed based on a model for the diversification of viral genomes. The imputation method decreases the bias in clustering that arises from informative missingness. Data from a household survey conducted in a village in Botswana are used to illustrate these methods. We demonstrate that the multiple imputation approach reduces bias in the overall proportion of clustering due to the presence of missing observations.     

PAQ: Partition Analysis of Quasispecies

MOTIVATION: The complexities of genetic data may not be accurately described by any single analytical tool. Phylogenetic analysis is often used to study the genetic relationship among different sequences. Evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. Genetic databases are rapidly accumulating large amounts of sequences. Newly acquired sequences, which have not yet been characterized, may require preliminary genetic exploration in order to build models describing the evolutionary relationship among sequences. There are clustering techniques that rely less on models of evolution, and thus may provide nice exploratory tools for identifying genetic similarities. Some of the more commonly used clustering methods perform better when data can be grouped into mutually exclusive groups. Genetic data from viral quasispecies, which consist of closely related variants that differ by small changes, however, may best be partitioned by overlapping groups. RESULTS: We have developed an intuitive exploratory program, Partition Analysis of Quasispecies (PAQ), which utilizes a non-hierarchical technique to partition sequences that are genetically similar. PAQ was used to analyze a data set of human immunodeficiency virus type 1 (HIV-1) envelope sequences isolated from different regions of the brain and another data set consisting of the equine infectious anemia virus (EIAV) regulatory gene rev. Analysis of the HIV-1 data set by PAQ was consistent with phylogenetic analysis of the same data, and the EIAV rev variants were partitioned into two overlapping groups. PAQ provides an additional tool which can be used to glean information from genetic data and can be used in conjunction with other tools to study genetic similarities and genetic evolution of viral quasispecies.     

The Role of Viral Introductions in Sustaining Community-Based HIV Epidemics in Rural Uganda: Evidence from Spatial Clustering,Phylogenetics, and Egocentric Transmission Models

Background

It is often assumed that local sexual networks play a dominant role in HIV spread in sub-Saharan Africa. The aim of this study was to determine the extent to which continued HIV transmission in rural communities—home to two-thirds of the African population—is driven by intra-community sexual networks versus viral introductions from outside of communities.

Methods and Findings

We analyzed the spatial dynamics of HIV transmission in rural Rakai District, Uganda, using data from a cohort of 14,594 individuals within 46 communities. We applied spatial clustering statistics, viral phylogenetics, and probabilistic transmission models to quantify the relative contribution of viral introductions into communities versus community- and household-based transmission to HIV incidence. Individuals living in households with HIV-incident (n = 189) or HIV-prevalent (n = 1,597) persons were 3.2 (95% CI: 2.7–3.7) times more likely to be HIV infected themselves compared to the population in general, but spatial clustering outside of households was relatively weak and was confined to distances <500 m. Phylogenetic analyses of gag and env genes suggest that chains of transmission frequently cross community boundaries. A total of 95 phylogenetic clusters were identified, of which 44% (42/95) were two individuals sharing a household. Among the remaining clusters, 72% (38/53) crossed community boundaries. Using the locations of self-reported sexual partners, we estimate that 39% (95% CI: 34%–42%) of new viral transmissions occur within stable household partnerships, and that among those infected by extra-household sexual partners, 62% (95% CI: 55%–70%) are infected by sexual partners from outside their community. These results rely on the representativeness of the sample and the quality of self-reported partnership data and may not reflect HIV transmission patterns outside of Rakai.

Conclusions

Our findings suggest that HIV introductions into communities are common and account for a significant proportion of new HIV infections acquired outside of households in rural Uganda, though the extent to which this is true elsewhere in Africa remains unknown. Our results also suggest that HIV prevention efforts should be implemented at spatial scales broader than the community and should target key populations likely responsible for introductions into communities. Please see later in the article for the Editors'' Summary     

Viral linkage in HIV-1 seroconverters and their partners in an HIV-1 prevention clinical trial

Background

Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #{"type":"clinical-trial","attrs":{"text":"NCT00194519","term_id":"NCT00194519"}}NCT00194519) was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners.

Methodology/Principal Findings

We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners'' sequences and a Bayesian posterior probability of ≥50%. Adjudicators classified each seroconversion, finding 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%). Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters.

Conclusions/Significance

In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage determination process.     

The origin and diversity of the HIV-1 pandemic

This review examines the enormous progress that has been made in the past decade in understanding the origin of HIV, HIV genetic variability, and the impact of global HIV diversity on the pandemic. Multiple zoonotic transmissions of simian immunodeficiency virus (SIV) have resulted in different HIV lineages in humans. In addition, the high mutation and recombination rates during viral replication result in a great genetic variability of HIV within individuals, as well as within populations, upon which evolutionary selection pressures act. The global HIV pandemic is examined in the context of HIV evolution, and the global diversity of HIV subtypes and recombinants is discussed in detail. Finally, the impact of HIV diversity on pathogenesis, transmission, diagnosis, treatment, the immune response, and vaccine development is reviewed.     

Simple epidemiological dynamics explain phylogenetic clustering of HIV from patients with recent infection

Phylogenies of highly genetically variable viruses such as HIV-1 are potentially informative of epidemiological dynamics. Several studies have demonstrated the presence of clusters of highly related HIV-1 sequences, particularly among recently HIV-infected individuals, which have been used to argue for a high transmission rate during acute infection. Using a large set of HIV-1 subtype B pol sequences collected from men who have sex with men, we demonstrate that virus from recent infections tend to be phylogenetically clustered at a greater rate than virus from patients with chronic infection ('excess clustering') and also tend to cluster with other recent HIV infections rather than chronic, established infections ('excess co-clustering'), consistent with previous reports. To determine the role that a higher infectivity during acute infection may play in excess clustering and co-clustering, we developed a simple model of HIV infection that incorporates an early period of intensified transmission, and explicitly considers the dynamics of phylogenetic clusters alongside the dynamics of acute and chronic infected cases. We explored the potential for clustering statistics to be used for inference of acute stage transmission rates and found that no single statistic explains very much variance in parameters controlling acute stage transmission rates. We demonstrate that high transmission rates during the acute stage is not the main cause of excess clustering of virus from patients with early/acute infection compared to chronic infection, which may simply reflect the shorter time since transmission in acute infection. Higher transmission during acute infection can result in excess co-clustering of sequences, while the extent of clustering observed is most sensitive to the fraction of infections sampled.     

Phylogenetic surveillance of viral genetic diversity and the evolving molecular epidemiology of human immunodeficiency virus type 1

With ongoing generation of viral genetic diversity and increasing levels of migration, the global human immunodeficiency virus type 1 (HIV-1) epidemic is becoming increasingly heterogeneous. In this study, we investigate the epidemiological characteristics of 5,675 HIV-1 pol gene sequences sampled from distinct infections in the United Kingdom. These sequences were phylogenetically analyzed in conjunction with 976 complete-genome and 3,201 pol gene reference sequences sampled globally and representing the broad range of HIV-1 genetic diversity, allowing us to estimate the probable geographic origins of the various strains present in the United Kingdom. A statistical analysis of phylogenetic clustering in this data set identified several independent transmission chains within the United Kingdom involving recently introduced strains and indicated that strains more commonly associated with infections acquired heterosexually in East Africa are spreading among men who have sex with men. Coalescent approaches were also used and indicated that the transmission chains that we identify originated in the late 1980s to early 1990s. Similar changes in the epidemiological structuring of HIV epidemics are likely to be taking in place in other industrialized nations with large immigrant populations. The framework implemented here takes advantage of the vast amount of routinely generated HIV-1 sequence data and can provide epidemiological insights not readily obtainable through standard surveillance methods.     

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.      

Characterization of HIV Transmission in South-East Austria

To gain deeper insight into the epidemiology of HIV-1 transmission in South-East Austria we performed a retrospective analysis of 259 HIV-1 partial pol sequences obtained from unique individuals newly diagnosed with HIV infection in South-East Austria from 2008 through 2014. After quality filtering, putative transmission linkages were inferred when two sequences were ≤1.5% genetically different. Multiple linkages were resolved into putative transmission clusters. Further phylogenetic analyses were performed using BEAST v1.8.1. Finally, we investigated putative links between the 259 sequences from South-East Austria and all publicly available HIV polymerase sequences in the Los Alamos National Laboratory HIV sequence database. We found that 45.6% (118/259) of the sampled sequences were genetically linked with at least one other sequence from South-East Austria forming putative transmission clusters. Clustering individuals were more likely to be men who have sex with men (MSM; p<0.001), infected with subtype B (p<0.001) or subtype F (p = 0.02). Among clustered males who reported only heterosexual (HSX) sex as an HIV risk, 47% clustered closely with MSM (either as pairs or within larger MSM clusters). One hundred and seven of the 259 sequences (41.3%) from South-East Austria had at least one putative inferred linkage with sequences from a total of 69 other countries. In conclusion, analysis of HIV-1 sequences from newly diagnosed individuals residing in South-East Austria revealed a high degree of national and international clustering mainly within MSM. Interestingly, we found that a high number of heterosexual males clustered within MSM networks, suggesting either linkage between risk groups or misrepresentation of sexual risk behaviors by subjects.     

Transmission of Equine Influenza Virus during an Outbreak Is Characterized by Frequent Mixed Infections and Loose Transmission Bottlenecks

The ability of influenza A viruses (IAVs) to cross species barriers and evade host immunity is a major public health concern. Studies on the phylodynamics of IAVs across different scales – from the individual to the population – are essential for devising effective measures to predict, prevent or contain influenza emergence. Understanding how IAVs spread and evolve during outbreaks is critical for the management of epidemics. Reconstructing the transmission network during a single outbreak by sampling viral genetic data in time and space can generate insights about these processes. Here, we obtained intra-host viral sequence data from horses infected with equine influenza virus (EIV) to reconstruct the spread of EIV during a large outbreak. To this end, we analyzed within-host viral populations from sequences covering 90% of the infected yards. By combining gene sequence analyses with epidemiological data, we inferred a plausible transmission network, in turn enabling the comparison of transmission patterns during the course of the outbreak and revealing important epidemiological features that were not apparent using either approach alone. The EIV populations displayed high levels of genetic diversity, and in many cases we observed distinct viral populations containing a dominant variant and a number of related minor variants that were transmitted between infectious horses. In addition, we found evidence of frequent mixed infections and loose transmission bottlenecks in these naturally occurring populations. These frequent mixed infections likely influence the size of epidemics.     

Genetic and stochastic influences on the interaction of human immunodeficiency virus type 1 and cytotoxic T lymphocytes in identical twins

Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8+ T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.     

Frequent transmission of immunodeficiency viruses among bobcats and pumas

With the exception of human immunodeficiency virus (HIV), which emerged in humans after cross-species transmissions of simian immunodeficiency viruses from nonhuman primates, immunodeficiency viruses of the family Lentiviridae represent species-specific viruses that rarely cross species barriers to infect new hosts. Among the Felidae, numerous immunodeficiency-like lentiviruses have been documented, but only a few cross-species transmissions have been recorded, and these have not been perpetuated in the recipient species. Lentivirus seroprevalence was determined for 79 bobcats (Lynx rufus) and 31 pumas (Puma concolor) from well-defined populations in Southern California. Partial genomic sequences were subsequently obtained from 18 and 12 seropositive bobcats and pumas, respectively. Genotypes were analyzed for phylogenic relatedness and genotypic composition among the study set and archived feline lentivirus sequences. This investigation of feline immunodeficiency virus infection in bobcats and pumas of Southern California provides evidence that cross-species infection has occurred frequently among these animals. The data suggest that transmission has occurred in multiple locations and are most consistent with the spread of the virus from bobcats to pumas. Although the ultimate causes remain unknown, these transmission events may occur as a result of puma predation on bobcats, a situation similar to that which fostered transmission of HIV to humans, and likely represent the emergence of a lentivirus with relaxed barriers to cross-species transmission. This unusual observation provides a valuable opportunity to evaluate the ecological, behavioral, and molecular conditions that favor repeated transmissions and persistence of lentivirus between species.