Rebecca Rose

Explaining the geographic spread of emerging epidemics: a framework for comparing viral phylogenies and environmental landscape data

BackgroundPhylogenetic analysis is now an important tool in the study of viral outbreaks. It can reconstruct epidemic history when surveillance epidemiology data are sparse, and can indicate transmission linkages among infections that may not otherwise be evident. However, a remaining challenge is to develop an analytical framework that can test hypotheses about the effect of environmental variables on pathogen spatial spread. Recent phylogeographic approaches can reconstruct the history of virus dispersal from sampled viral genomes and infer the locations of ancestral infections. Such methods provide a unique source of spatio-temporal information, and are exploited here.ResultsWe present and apply a new statistical framework that combines genomic and geographic data to test the impact of environmental variables on the mode and tempo of pathogen dispersal during emerging epidemics. First, the spatial history of an emerging pathogen is estimated using standard phylogeographic methods. The inferred dispersal path for each phylogenetic lineage is then assigned a “weight” using environmental data (e.g. altitude, land cover). Next, tests measure the association between each environmental variable and lineage movement. A randomisation procedure is used to assess statistical confidence and we validate this approach using simulated data. We apply our new framework to a set of gene sequences from an epidemic of rabies virus in North American raccoons. We test the impact of six different environmental variables on this epidemic and demonstrate that elevation is associated with a slower rabies spread in a natural population.ConclusionThis study shows that it is possible to integrate genomic and environmental data in order to test hypotheses concerning the mode and tempo of virus dispersal during emerging epidemics.

HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer.

ABSTRACT While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV+ cART-treated (cART+) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART+ and cART-naive (cART−) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA+ cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread. IMPORTANCE Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In this study, we isolated and sequenced HIV from postmortem tissues from three HIV+ cART+ individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve, and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV expansion, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.

Exceptional Heterogeneity in Viral Evolutionary Dynamics Characterises Chronic Hepatitis C Virus Infection.

The treatment of HCV infection has seen significant progress, particularly since the approval of new direct-acting antiviral drugs. However these clinical achievements have been made despite an incomplete understanding of HCV replication and within-host evolution, especially compared with HIV-1. Here, we undertake a comprehensive analysis of HCV within-host evolution during chronic infection by investigating over 4000 viral sequences sampled longitudinally from 15 HCV-infected patients. We compare our HCV results to those from a well-studied HIV-1 cohort, revealing key differences in the evolutionary behaviour of these two chronic-infecting pathogens. Notably, we find an exceptional level of heterogeneity in the molecular evolution of HCV, both within and among infected individuals. Furthermore, these patterns are associated with the long-term maintenance of viral lineages within patients, which fluctuate in relative frequency in peripheral blood. Together, our findings demonstrate that HCV replication behavior is complex and likely comprises multiple viral subpopulations with distinct evolutionary dynamics. The presence of a structured viral population can explain apparent paradoxes in chronic HCV infection, such as rapid fluctuations in viral diversity and the reappearance of viral strains years after their initial detection.

The meningeal lymphatic system: a route for HIV brain migration?

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4+ T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.

Using Viral Gene Sequences to Compare and Explain the Heterogeneous Spatial Dynamics of Virus Epidemics

Rabies is an important zoonotic disease distributed worldwide. A key question in rabies epidemiology is the identification of factors that impact virus dispersion. Here we apply new analytical methods, based on phylogeographic reconstructions of viral lineage movement, to undertake a comparative evolutionary-epidemiological study of the spatial dynamics of rabies virus (RABV) epidemics in different hosts and habitats. We compiled RABV data sets from skunk, raccoon, bat and domestic dog populations in order to investigate the viral diffusivity of different RABV epidemics, and to detect and compare the environmental factors that impact the velocity of viral spread in continuous spatial landscapes. We build on a recently developed statistical framework that uses spatially- and temporally-referenced phylogenies. We estimate several spatial statistics of virus spread, which reveal a higher diffusivity of RABV in domestic dogs compared with RABV in other mammals. This finding is explained by subsequent analyses of environmental heterogeneity, which indicate that factors relating to human geography play a significant role in RABV dispersion in domestic dogs. More generally, our results suggest that human-related factors are important worldwide in explaining RABV dispersion in terrestrial host species. Our study shows that phylogenetically informed viral movements can be used to elucidate the factors that impact virus dispersal, opening new opportunities for a better understanding of the impact of host species and environmental conditions on the spatial dynamics of rapidly evolving populations.

HIV-1 Evolutionary Patterns Associated with Metastatic Kaposi’s Sarcoma during AIDS

Kaposis sarcoma (KS) in HIV-infected individuals can have a wide range of clinical outcomes, from indolent skin tumors to a life-threatening visceral cancer. KS tumors contain endothelial-related cells and inflammatory cells that may be HIV-infected. In this study we tested if HIV evolutionary patterns distinguish KS tumor relatedness and progression. Multisite autopsies from participants who died from HIV-AIDS with KS prior to the availability of antiretroviral therapy were identified at the AIDS and Cancer Specimen Resource (ACSR). Two patients (KS1 and KS2) died predominantly from non-KS-associated disease and KS3 died due to aggressive and metastatic KS within one month of diagnosis. Skin and visceral tumor and nontumor autopsy tissues were obtained (n = 12). Single genome sequencing was used to amplify HIV RNA and DNA, which was present in all tumors. Independent HIV tumor clades in phylogenies differentiated KS1 and KS2 from KS3, whose sequences were interrelated by both phylogeny and selection. HIV compartmentalization was confirmed in KS1 and KS2 tumors; however, in KS3, no compartmentalization was observed among sampled tissues. While the sample size is small, the HIV evolutionary patterns observed in all patients suggest an interplay between tumor cells and HIV-infected cells which provides a selective advantage and could promote KS progression.

Complex patterns of Hepatitis-C virus longitudinal clustering in a high-risk population

We investigated longitudinal viral clustering among and within subjects in a highly networked cohort of people who inject drugs (PWID). All subjects had estimated dates of infection and two or more E1 sequences (bp 943-1288 relative to H77) with 1 to 14years of follow up. Two methods (HIV-TRACE and PhyloPart) were used to determine clusters. Genetic distance thresholds were determined by comparing intra-and inter-host distances. Additional phylogenetic analysis was performed on subjects with complicated viral histories. At the optimal threshold of 3.9%, HIV-TRACE found 77 clusters and PhyloPart found 63 clusters, of which 27 and 32 contained multiple subjects, respectively. Furthermore, 1/3 of the subjects had sequences in different clusters over the course of the study, including some cases in which a later-sampled sequence matched a cluster detected much earlier in the infection, despite being separated by RNA-negative lab visit and detection of sequences in different clusters. A detailed phylogenetic analysis of four subjects with such patterns showed that in all four cases, the earlier and later variants grouped closely on the tree, and did not group with concurrent sequences from any other subject. These observations suggest that subjects are either experiencing rapid and recurring infection-clearance-reinfection cycles from the same source, or a single transmission event produces a chronic infection that may go undetected and/or co-circulate with different viruses from separate transmission events. Furthermore, our results show the utility of using longitudinal sampling to obtain a more comprehensive view of the viral linkages in high-risk populations.

Predicted coreceptor usage at end-stage HIV disease in tissues derived from subjects on antiretroviral therapy with an undetectable plasma viral load

HIV cure research is increasingly focused on anatomical tissues as sites for residual HIV replication during combined antiretroviral therapy (cART). Tissue-based HIV could contribute to low-level immune activation and viral rebound over the course of infection and could also influence the development of diseases, such as atherosclerosis, neurological disorders and cancers. cART-treated subjects have a decreased and irregular presence of HIV among tissues, which has resulted in a paucity of actual evidence concerning how or if HIV persists, replicates and evolves in various anatomical sites during therapy. In this study, we pooled 1806 HIV envelope V3 loop sequences from twenty-six tissue types (seventy-one total tissues) of six pre-cART subjects, four subjects with an unknown cART history who died with profound AIDS, and five subjects who died while on cART with an undetectable plasma viral load. A computational approach was used to assess sequences for their ability to utilize specific cellular coreceptors (R5, R5 and X4, or X4). We found that autopsied tissues obtained from virally suppressed cART+ subjects harbored both integrated and expressed viruses with similar coreceptor usage profiles to subjects with no or ineffective cART therapy (i.e., significant plasma viral load at death). The study suggests that tissue microenvironments provide a sanctuary for the continued evolution of HIV despite cART.

Prevalence and mapping of hepatitis C infections among men who have sex with men in New York City

Emerging sexually transmitted hepatitis C virus (HCV) epidemics among men who have sex with men (MSM) have been reported worldwide, with higher HCV infection rates among those who are HIV-infected. This study aims to determine prevalence of recent and chronic HCV infections among community-recruited MSM in New York City (NYC), map HCV infections by home, social, and sexual neighborhoods, and identify clusters of genetically linked HCV variants using phylogenetic analysis. The NYC M2M study recruited MSM via modified time-space, venue-based sampling and internet/mobile app-based recruitment during 2010–13. Participants completed a Google Earth map on neighborhoods of where they lived, socialized, and had sex in the last 3 months, an ACASI questionnaire, and a sexual network inventory about their sex partners. The men received HIV testing and provided serum samples. Testing on stored serum samples included HCV antibody and RNA viral load, HCV antibody avidity assay (avidity index <30% with positive viral load is considered recently infected), and HCV RNA extraction and amplification to generate a 432 base-pair region of Core/E1 for sequencing and phylogenetic analysis. Historic local controls were included in the phylogenetic analysis. Of 1,028 MSM, 79.7% were HIV-negative and 20.3% HIV-positive. Twenty nine MSM (2.8%) were HCV antibody-positive. MSM who were HCV antibody-positive reported a median of 2 male sex partners in last 3 months, with 6.9% aged 18–24, 17.2% 25–29, 13.8% 30–39, and 62.1% 40 and over. 8.1% of HIV-positive MSM were HCV antibody-positive vs. 1.5% of HIV-negative men (p<0.0001). Of 29 HCV-antibody positive MSM, 12 (41%) were HCV RNA-positive (11 subtype 1a and 1 subtype 1b). Two of 12 HCV RNA-positive participants had low antibody avidity values, suggesting recent HCV infection. HCV antibody seropositivity was significantly associated with older age >40 years, adjusted odds ratio (aOR) 3.56 (95% CI 1.57, 8.08), HIV-positive serostatus, aOR 3.18 (95% CI 1.40, 7.22), any sexually transmitted infection (STI) in the last 3 months, aOR 2.81 (95% CI 1.11, 7.13), and injection drug use (IDU) ever, aOR 4.34 (95% CI 1.69, 11.17). Mapping of HCV infections differed slightly by home, social, and sexual neighborhoods. Based on phylogenetic analysis from 12 HCV RNA-positive samples, no evidence of a clustered HCV epidemic was found. Overall HCV seroprevalence was 2.8% among community-recruited MSM in NYC, with higher prevalence among HIV-positive MSM compared to HIV-negative MSM. Only two participants were found to have recent HCV infection, with no evidence of a clustered HCV epidemic based on phylogenetic analysis. Our results support testing of HCV infection among HIV-negative MSM if they report having a recent STI and IDU in the past rather than universal HCV testing in all HIV-negative MSM.

Brain-specific HIV Nef identified in multiple patients with neurological disease

HIV-1 Nef is a flexible, multifunctional protein with several cellular targets that is required for pathogenicity of the virus. This protein maintains a high degree of genetic variation among intra- and inter-host isolates. HIV Nef is relevant to HIV-associated neurological diseases (HAND) in patients treated with combined antiretroviral therapy because of the protein’s role in promoting survival and migration of infected brain macrophages. In this study, we analyzed 2020 HIV Nef sequences derived from 22 different tissues and 31 subjects using a novel computational approach. This approach combines statistical regression and evolved neural networks (ENNs) to classify brain sequences based on the physical and chemical characteristics of functional Nef domains. Based on training, testing, and validation data, the method successfully classified brain Nef sequences at 84.5% and provided informative features for further examination. These included physicochemical features associated with the Src-homology-3 binding domain, the Nef loop (including the AP-2 Binding region), and a cytokine-binding domain. Non-brain sequences from patients with HIV-associated neurological disease were frequently classified as brain, suggesting that the approach could indicate neurological risk using blood-derived virus or for the development of biomarkers for use in assay systems aimed at drug efficacy studies for the treatment of HIV-associated neurological diseases.