Roman Biek

A high-resolution genetic signature of demographic and spatial expansion in epizootic rabies virus

Emerging pathogens potentially undergo rapid evolution while expanding in population size and geographic range during the course of invasion, yet it is generally difficult to demonstrate how these processes interact. Our analysis of a 30-yr data set covering a large-scale rabies virus outbreak among North American raccoons reveals the long lasting effect of the initial infection wave in determining how viral populations are genetically structured in space. We further find that coalescent-based estimates derived from the genetic data yielded an amazingly accurate reconstruction of the known spatial and demographic dynamics of the virus over time. Our study demonstrates the combined evolutionary and population dynamic processes characterizing the spread of pathogen after its introduction into a fully susceptible host population. Furthermore, the results provide important insights regarding the spatial scale of rabies persistence and validate the use of coalescent approaches for uncovering even relatively complex population histories. Such approaches will be of increasing relevance for understanding the epidemiology of emerging zoonotic diseases in a landscape context.

Wave-like spread of Ebola Zaire

In the past decade the Zaire strain of Ebola virus (ZEBOV) has emerged repeatedly into human populations in central Africa and caused massive die-offs of gorillas and chimpanzees. We tested the view that emergence events are independent and caused by ZEBOV variants that have been long resident at each locality. Phylogenetic analyses place the earliest known outbreak at Yambuku, Democratic Republic of Congo, very near to the root of the ZEBOV tree, suggesting that viruses causing all other known outbreaks evolved from a Yambuku-like virus after 1976. The tendency for earlier outbreaks to be directly ancestral to later outbreaks suggests that outbreaks are epidemiologically linked and may have occurred at the front of an advancing wave. While the ladder-like phylogenetic structure could also bear the signature of positive selection, our statistical power is too weak to reach a conclusion in this regard. Distances among outbreaks indicate a spread rate of about 50 km per year that remains consistent across spatial scales. Viral evolution is clocklike, and sequences show a high level of small-scale spatial structure. Genetic similarity decays with distance at roughly the same rate at all spatial scales. Our analyses suggest that ZEBOV has recently spread across the region rather than being long persistent at each outbreak locality. Controlling the impact of Ebola on wild apes and human populations may be more feasible than previously recognized.

The landscape genetics of infectious disease emergence and spread

The spread of parasites is inherently a spatial process often embedded in physically complex landscapes. It is therefore not surprising that infectious disease researchers are increasingly taking a landscape genetics perspective to elucidate mechanisms underlying basic ecological processes driving infectious disease dynamics and to understand the linkage between spatially dependent population processes and the geographic distribution of genetic variation within both hosts and parasites. The increasing availability of genetic information on hosts and parasites when coupled to their ecological interactions can lead to insights for predicting patterns of disease emergence, spread and control. Here, we review research progress in this area based on four different motivations for the application of landscape genetics approaches: (i) assessing the spatial organization of genetic variation in parasites as a function of environmental variability, (ii) using host population genetic structure as a means to parameterize ecological dynamics that indirectly influence parasite populations, for example, gene flow and movement pathways across heterogeneous landscapes and the concurrent transport of infectious agents, (iii) elucidating the temporal and spatial scales of disease processes and (iv) reconstructing and understanding infectious disease invasion. Throughout this review, we emphasize that landscape genetic principles are relevant to infection dynamics across a range of scales from within host dynamics to global geographic patterns and that they can also be applied to unconventional ‘landscapes’ such as heterogeneous contact networks underlying the spread of human and livestock diseases. We conclude by discussing some general considerations and problems for inferring epidemiological processes from genetic data and try to identify possible future directions and applications for this rapidly expanding field.

Spatial dynamics and genetics of infectious diseases on heterogeneous landscapes

Explicit spatial analysis of infectious disease processes recognizes that host–pathogen interactions occur in specific locations at specific times and that often the nature, direction, intensity and outcome of these interactions depend upon the particular location and identity of both host and pathogen. Spatial context and geographical landscape contribute to the probability of initial disease establishment, direction and velocity of disease spread, the genetic organization of resistance and susceptibility, and the design of appropriate control and management strategies. In this paper, we review the manner in which the physical organization of the landscape has been shown to influence the population dynamics and spatial genetic structure of host–pathogen interactions, and how we might incorporate landscape architecture into spatially explicit population models of the infectious disease process to increase our ability to predict patterns of disease occurrence and optimally design vaccination and control policies.

Isolates of Zaire ebolavirus from wild apes reveal genetic lineage and recombinants

Over the last 30 years, Zaire ebolavirus (ZEBOV), a virus highly pathogenic for humans and wild apes, has emerged repeatedly in Central Africa. Thus far, only a few virus isolates have been characterized genetically, all belonging to a single genetic lineage and originating exclusively from infected human patients. Here, we describe the first ZEBOV sequences isolated from great ape carcasses in the Gabon/Congo region that belong to a previously unrecognized genetic lineage. According to our estimates, this lineage, which we also encountered in the two most recent human outbreaks in the Republic of the Congo in 2003 and 2005, diverged from the previously known viruses around the time of the first documented human outbreak in 1976. These results suggest that virus spillover from the reservoir has occurred more than once, as predicted by the multiple emergence hypothesis. However, the young age of both ZEBOV lineages and the spatial and temporal sequence of outbreaks remain at odds with the idea that the virus simply emerged from a long-established and widespread reservoir population. Based on data from two ZEBOV genes, we also demonstrate, within the family Filoviridae, recombination between the two lineages. According to our estimates, this event took place between 1996 and 2001 and gave rise to a group of recombinant viruses that were responsible for a series of outbreaks in 2001–2003. The potential for recombination adds an additional level of complexity to unraveling and potentially controlling the emergence of ZEBOV in humans and wildlife species.

Assembling evidence for identifying reservoirs of infection

Many pathogens persist in multihost systems, making the identification of infection reservoirs crucial for devising effective interventions. Here, we present a conceptual framework for classifying patterns of incidence and prevalence, and review recent scientific advances that allow us to study and manage reservoirs simultaneously. We argue that interventions can have a crucial role in enriching our mechanistic understanding of how reservoirs function and should be embedded as quasi-experimental studies in adaptive management frameworks. Single approaches to the study of reservoirs are unlikely to generate conclusive insights whereas the formal integration of data and methodologies, involving interventions, pathogen genetics, and contemporary surveillance techniques, promises to open up new opportunities to advance understanding of complex multihost systems.

Whole Genome Sequencing Reveals Local Transmission Patterns of Mycobacterium bovis in Sympatric Cattle and Badger Populations

Whole genome sequencing (WGS) technology holds great promise as a tool for the forensic epidemiology of bacterial pathogens. It is likely to be particularly useful for studying the transmission dynamics of an observed epidemic involving a largely unsampled ‘reservoir’ host, as for bovine tuberculosis (bTB) in British and Irish cattle and badgers. BTB is caused by Mycobacterium bovis, a member of the M. tuberculosis complex that also includes the aetiological agent for human TB. In this study, we identified a spatio-temporally linked group of 26 cattle and 4 badgers infected with the same Variable Number Tandem Repeat (VNTR) type of M. bovis. Single-nucleotide polymorphisms (SNPs) between sequences identified differences that were consistent with bacterial lineages being persistent on or near farms for several years, despite multiple clear whole herd tests in the interim. Comparing WGS data to mathematical models showed good correlations between genetic divergence and spatial distance, but poor correspondence to the network of cattle movements or within-herd contacts. Badger isolates showed between zero and four SNP differences from the nearest cattle isolate, providing evidence for recent transmissions between the two hosts. This is the first direct genetic evidence of M. bovis persistence on farms over multiple outbreaks with a continued, ongoing interaction with local badgers. However, despite unprecedented resolution, directionality of transmission cannot be inferred at this stage. Despite the often notoriously long timescales between time of infection and time of sampling for TB, our results suggest that WGS data alone can provide insights into TB epidemiology even where detailed contact data are not available, and that more extensive sampling and analysis will allow for quantification of the extent and direction of transmission between cattle and badgers.

Recent Common Ancestry of Ebola Zaire Virus Found in a Bat Reservoir

Identifying a natural reservoir for Ebola virus has eluded researchers for decades [1,2]. Recently, Leroy et al. presented the most compelling evidence to date that three species of fruit bats (Hypsignathus monstrosus, Epomops franqueti, and Myonycteris torquata) may constitute a long-missing wildlife reservoir for Ebola virus Zaire (EBOVZ) [3]. These bats, caught near affected villages at the Gabon–Congo border, appear to have been asymptomatically infected and, in seven cases, yielded virus sequences that closely matched those found in the human outbreaks happening about the same time. Leroy et al.s phylogenetic analysis of the partial sequences of the viral polymerase (L) gene derived from humans and bats emphasized the interspecific relationships to related filoviruses. Here, we show that (1) despite their short length (265 bp), these sequences also provide critical information about the intraspecific history of EBOVZ, and (2) based on the genetic data available so far, the association of the virus with fruit bats in the sampled area can only be traced back a few years. Consistent with previous analysis using glycoprotein (GP) gene sequences [4], results for the L gene show that viruses amplified from more recently collected samples appear to be direct descendents of viruses seen during previous outbreaks. This relationship is not only apparent for viruses found in 1976–1995 compared with those found in 2001–2003, but also within the latter group (Figure 1). In essence, this means that all genetic variation seen thus far in EBOVZ, including virus amplified from fruit bats, appears to be the product of mutations that have accumulated within the last 30 years. Finding such strong evidence for temporal structure by chance seems highly unlikely, especially given the concordance with our earlier results from the GP gene [4]. Although the lack of any mutational differences between the sequences Mayinga 1976 and Kikwit 1995 is perplexing in this context, it is most likely a stochastic artifact due to the short length of the sequence considered. Full-length sequences are available for both these isolates, which over the entire genome are 1.2% different. Over 19 years this yields an ad hoc evolutionary rate estimate of 6.2 × 10−4 substitutions per site per year, close to the rate we had previously estimated for the GP gene (~8.0 × 10−4) [4] and to the point estimate for all partial L sequences in the current analysis (1.1 × 10−3; 95% highest posterior density interval: 6.3 × 10−7 to 2.4 × 10−3). Thus, even though the L sequences are rather short, they yield evolutionary rate estimates similar to the longer GP sequences. Figure 1 Maximum Likelihood Tree from Partial L Sequences of Ebola Virus Zaire The temporal structure visible in the L gene genealogy implies that all viruses sampled from both humans and bats between 2001 and 2003 can be traced back to a very recent common ancestor, by which we mean a recent coalescence of genetic lineages, not an ancestral alternative reservoir species. In fact, according to our phylogenetic estimate, the partial L sequence of this genetic ancestor would have looked identical to that sampled from infected humans during outbreaks in late 2001 and early 2002 (Entsiami and Mendemba, Figure 1), suggesting that this ancestor could not be much older. This is in agreement with the previous analysis of the GP gene, which indicated that all viruses sampled from outbreaks since 2001 had a most recent common ancestor in 1999 (confidence region: 1998–2000) [4]. While these findings do not question whether fruit bats may represent a wild reservoir for EBOVZ, they do raise important issues. If the three identified fruit bat hosts were the natural reservoir for EBOVZ, the recent common ancestry of all sequences derived from them so far is surprising because, at least at first sight, it seems to contradict the idea of a long-established association of bat and virus. The most reasonable explanation for this result is that the virus experienced a recent genetic bottleneck. We present three alternative scenarios of what could have caused such a bottleneck. One possibility is that somewhere around 1999, the total number of infected bats within the sampled area became extremely small (likely much less than the peak 23% incidence determined by Leroy et al.). Under such a scenario, the most recent common ancestor could not be traced back further into the past because an extremely small, effective viral population size has caused the descendents of all but one of the previously existing viral lineages to be lost. Since no trapping study on bats was undertaken before 2001, we could not directly address this issue. However, some epidemiological and virological observations may account for this situation. The need to apply the very sensitive nested PCR to detect viral RNA suggests a very low viral load in organs of infected bats. Furthermore, the presence of a high prevalence rate of seropositive bats (16.7%, 4/24) compared with only 3.2% (2/63) that were PCR positive (but seronegative) just three months after the appearance of the first human cases in Mendemba, Gabon, indicates that viral replication within bats may be highly restricted and possibly only taking place prior to the onset of the host immune response. Especially if infections are synchronized, for example, by some environmental trigger, this may lead to periods with an extremely small number of productively infected bats, repeatedly forcing the virus population through a genetic bottleneck. Alternatively, the recent common ancestor could be explained by infected bats introducing the virus into the EBOVZ-affected area of Gabon and Congo around 1999. Previous results for the GP gene actually support this hypothesis by revealing a consistent signature of geographic spread within the spatial, temporal, and genetic data for EBOVZ over the last 30 years [4]. Similar genetic patterns associated with local founder events followed by spatial spread have also been documented from rabies virus in wildlife host populations [5]. Some observed epidemiological changes in sampled bat populations over time may also support this hypothesis. Leroy et al. found that during the first visit to one of their sampling locations, 23% (7/31) of the bats were PCR positive, whereas 0% (0/10) were seropositive. At a second visit five months later, these numbers had changed to 2% (4/184) and 8% (12/160) [3]. Again, no bats were positive by both PCR and serology. Though other factors may also explain these opposing trends, the observed temporal pattern is consistent with an infection wave moving through the sampled population, resulting in a high proportion of infectious individuals at first, followed by an increased proportion of seropositive animals. Given that the three implicated fruit bat species may not be the only reservoir for EBOVZ, as Leroy et al. were already careful to point out [3], another possible explanation for the existence of a common viral ancestor in the recent past is that the virus was introduced to these fruit bats around the same time it affected other wildlife populations and emerged in humans. It is important to note that this scenario does not rule out bats as reservoir species, a hypothesis for which there is additional independent support [6]. Instead, it would imply that the primary reservoir of EBOVZ, whether it involves additional bat species or representatives of other taxonomic groups, has yet to be found. We expect that distinguishing between these possible scenarios will become increasingly easier as more temporal, spatial, and genetic data are generated. Additional viral sequences from infected fruit bats and large-scale serological prevalence in bat populations both within and outside the affected area should give some much needed answers regarding the dynamics of the virus in its wild reservoir. Together with other viral sequences in human cases and vulnerable animal species, and a better understanding of the factors associated with its emergence in human and wildlife populations, these combined approaches will hopefully lead to new and more successful strategies for preventing and controlling outbreaks of EBOVZ in the near future.

Epidemiology, Genetic Diversity, and Evolution of Endemic Feline Immunodeficiency Virus in a Population of Wild Cougars

ABSTRACT Within the large body of research on retroviruses, the distribution and evolution of endemic retroviruses in natural host populations have so far received little attention. In this study, the epidemiology, genetic diversity, and molecular evolution of feline immunodeficiency virus specific to cougars (FIVpco) was examined using blood samples collected over several years from a free-ranging cougar population in the western United States. The virus prevalence was 58% in this population (n = 52) and increased significantly with host age. Based on phylogenetic analysis of fragments of envelope (env) and polymerase (pol) genes, two genetically distinct lineages of FIVpco were found to cooccur in the population but not in the same individuals. Within each of the virus lineages, geographically nearby isolates formed monophyletic clusters of closely related viruses. Sequence diversity for env within a host rarely exceeded 1%, and the evolution of this gene was dominated by purifying selection. For both pol and env, our data indicate mean rates of molecular evolution of 1 to 3% per 10 years. These results support the premise that FIVpco is well adapted to its cougar host and provide a basis for comparing lentivirus evolution in endemic and epidemic infections in natural hosts.

Emergence of divergent Zaire ebola virus strains in Democratic Republic of the Congo in 2007 and 2008.

BACKGROUND Zaire ebolavirus was responsible for 2 outbreaks in Democratic Republic of the Congo (DRC), in 1976 and 1995. The virus reemerged in DRC 12 years later, causing 2 successive outbreaks in the Luebo region, Kasai Occidental province, in 2007 and 2008. METHODS Viruses of each outbreak were isolated and the full-length genomes were characterized. Phylogenetic analysis was then undertaken to characterize the relationships with previously described viruses. RESULTS The 2 Luebo viruses are nearly identical but are not related to lineage A viruses known in DRC or to descendants of the lineage B viruses encountered in the Gabon-Republic of the Congo area, with which they do, however, share a common ancestor. CONCLUSIONS Our findings strongly suggest that the Luebo 2007 outbreak did not result from viral spread from previously identified foci but from an independent viral emergence. The previously identified epidemiological link with migratory bat species known to carry Zaire ebolavirus RNA support the hypothesis of viral spillover from this widely dispersed reservoir. The high level of similarity between the Luebo2007 and Luebo2008 viruses suggests that local wildlife populations (most likely bats) became infected and allowed local viral persistence and reemergence from year to year.