The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

Assessing in vivo mutation frequencies and creating a high-resolution genome-wide map of fitness costs of Hepatitis C virus.

Like many viruses, Hepatitis C Virus (HCV) has a high mutation rate, which helps the virus adapt quickly, but mutations come with fitness costs. Fitness costs can be studied by different approaches, such as experimental or frequency-based approaches. The frequency-based approach is particularly useful to estimate in vivo fitness costs, but this approach works best with deep sequencing data from many hosts are. In this study, we applied the frequency-based approach to a large dataset of 195 patients and estimated the fitness costs of mutations at 7957 sites along the HCV genome. We used beta regression and random forest models to better understand how different factors influenced fitness costs. Our results revealed that costs of nonsynonymous mutations were three times higher than those of synonymous mutations, and mutations at nucleotides A or T had higher costs than those at C or G. Genome location had a modest effect, with lower costs for mutations in HVR1 and higher costs for mutations in Core and NS5B. Resistance mutations were, on average, costlier than other mutations. Our results show that in vivo fitness costs of mutations can be site and virus specific, reinforcing the utility of constructing in vivo fitness cost maps of viral genomes.

Inferring Human Immunodeficiency Virus 1 Proviral Integration Dates With Bayesian Inference.

Human immunodeficiency virus 1 (HIV) proviruses archived in the persistent reservoir currently pose the greatest obstacle to HIV cure due to their evasion of combined antiretroviral therapy and ability to reseed HIV infection. Understanding the dynamics of the HIV persistent reservoir is imperative for discovering a durable HIV cure. Here, we explore Bayesian methods using the software BEAST2 to estimate HIV proviral integration dates. We started with within-host longitudinal HIV sequences collected prior to therapy, along with sequences collected from the persistent reservoir during suppressive therapy. We built a BEAST2 model to estimate integration dates of proviral sequences collected during suppressive therapy, implementing a tip date random walker to adjust the sequence tip dates and a latency-specific prior to inform the dates. To validate our method, we implemented it on both simulated and empirical data sets. Consistent with previous studies, we found that proviral integration dates were spread throughout active infection. Path sampling to select an alternative prior for date estimation in place of the latency-specific prior produced unrealistic results in one empirical data set, whereas on another data set, the latency-specific prior was selected as best fitting. Our Bayesian method outperforms current date estimation techniques with a root mean squared error of 0.89 years on simulated data relative to 1.23-1.89 years with previously developed methods. Bayesian methods offer an adaptable framework for inferring proviral integration dates.

HIV proviral genetic diversity, compartmentalization and inferred dynamics in lung and blood during long-term suppressive antiretroviral therapy.

The lung is an understudied site of HIV persistence. We isolated 898 subgenomic proviral sequences (nef) by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 8% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harboured at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. The extent of blood proviral diversity on ART was also a strong indicator of diversity in lung (Spearman's ρ = 0.98, p<0.0001). For three participants, insufficient lung sequences were recovered to reliably investigate genetic compartmentalization. Of the remainder, only two participants showed statistically significant support for compartmentalization when analysis was restricted to distinct proviruses per site, and the extent of compartmentalization was modest in both cases. When all within-host sequences (including duplicates) were considered, the number of compartmentalized datasets increased to four. Thus, while a subset of individuals harbour somewhat distinctive proviral populations in blood and lung, this can simply be due to unequal distributions of clonally-expanded sequences. For two participants, on-ART proviruses were also phylogenetically analyzed in context of plasma HIV RNA populations sampled up to 18 years prior, including pre-ART and during previous treatment interruptions. In both participants, on-ART proviruses represented the most ancestral sequences sampled within-host, confirming that HIV sequences can persist in the body for decades. This analysis also revealed evidence of re-seeding of the reservoir during treatment interruptions. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individual's proviral composition. Personalized HIV remission and cure strategies may be needed to overcome these challenges.

Longitudinal Screening of Retail Milk from Canadian Provinces Reveals No Detections of Influenza A Virus RNA (April - July 2024): Leveraging a Newly-Established Pan-Canadian Network for Responding to Emerging Viruses.

Highly pathogenic avian influenza (HPAI) H5N1 has been associated with significant morbidity and mortality. Given recent detections of HPAI H5N1 in dairy cattle and RNA detections in pasteurized retail milk in the United States, we established the Pan-Canadian Milk Network in April 2024. Through this, retail milk was procured longitudinally and sent to a central laboratory to test for the presence of influenza A virus RNA. We tested 109 retail milk samples from all ten Canadian provinces and all samples tested negative. Our independent testing results have aligned with reporting from federal retail milk testing initiatives. Despite no known HPAI infections of dairy cattle in Canada to date, H5N1 poses a significant threat to the health of both humans and other animals. By performing routine surveillance of retail milk on a national scale, we have shown that academic networks and initiatives can rapidly establish nationwide emerging infectious disease surveillance that is cost-effective, standardized, scalable, and easily accessible. Our network can serve as an early detection system to help inform containment and mitigation activities if positive samples are identified and can be readily reactivated should H5N1 or other emerging zoonotic viruses be identified in agricultural or livestock settings.

Fundamental Identifiability Limits in Molecular Epidemiology.

Viral phylogenies provide crucial information on the spread of infectious diseases, and many studies fit mathematical models to phylogenetic data to estimate epidemiological parameters such as the effective reproduction ratio (Re) over time. Such phylodynamic inferences often complement or even substitute for conventional surveillance data, particularly when sampling is poor or delayed. It remains generally unknown, however, how robust phylodynamic epidemiological inferences are, especially when there is uncertainty regarding pathogen prevalence and sampling intensity. Here, we use recently developed mathematical techniques to fully characterize the information that can possibly be extracted from serially collected viral phylogenetic data, in the context of the commonly used birth-death-sampling model. We show that for any candidate epidemiological scenario, there exists a myriad of alternative, markedly different, and yet plausible "congruent" scenarios that cannot be distinguished using phylogenetic data alone, no matter how large the data set. In the absence of strong constraints or rate priors across the entire study period, neither maximum-likelihood fitting nor Bayesian inference can reliably reconstruct the true epidemiological dynamics from phylogenetic data alone; rather, estimators can only converge to the "congruence class" of the true dynamics. We propose concrete and feasible strategies for making more robust epidemiological inferences from viral phylogenetic data.

HIV-1 variants are archived throughout infection and persist in the reservoir.

The HIV-1 reservoir consists of latently infected cells that persist despite antiretroviral therapy (ART). Elucidating the proviral genetic composition of the reservoir, particularly in the context of pre-therapy viral diversity, is therefore important to understanding reservoir formation and the persistence of latently infected cells. Here we investigate reservoir proviral variants from 13 Zambian acutely-infected individuals with additional pre-therapy sampling for a unique comparison to the ART-naïve quasispecies. We identified complete transmitted/founder (TF) viruses from seroconversion plasma samples, and additionally amplified and sequenced HIV-1 from plasma obtained one year post-infection and just prior to ART initiation. While the majority of proviral variants in the reservoir were most closely related to viral variants from the latest pre-therapy time point, we also identified reservoir proviral variants dating to or near the time of infection, and to intermediate time points between infection and treatment initiation. Reservoir proviral variants differing by five or fewer nucleotide changes from the TF virus persisted during treatment in five individuals, including proviral variants that exactly matched the TF in two individuals, one of whom had remained ART-naïve for more than six years. Proviral variants during treatment were significantly less divergent from the TF virus than plasma variants present at the last ART-naïve time point. These findings indicate that reservoir proviral variants are archived throughout infection, recapitulating much of the viral diversity that arises throughout untreated HIV-1 infection, and strategies to target and reduce the reservoir must therefore permit for the clearance of proviruses encompassing this extensive diversity.

Unifying Phylogenetic Birth-Death Models in Epidemiology and Macroevolution.

Birth-death stochastic processes are the foundations of many phylogenetic models and are widely used to make inferences about epidemiological and macroevolutionary dynamics. There are a large number of birth-death model variants that have been developed; these impose different assumptions about the temporal dynamics of the parameters and about the sampling process. As each of these variants was individually derived, it has been difficult to understand the relationships between them as well as their precise biological and mathematical assumptions. Without a common mathematical foundation, deriving new models is nontrivial. Here, we unify these models into a single framework, prove that many previously developed epidemiological and macroevolutionary models are all special cases of a more general model, and illustrate the connections between these variants. This unification includes both models where the process is the same for all lineages and those in which it varies across types. We also outline a straightforward procedure for deriving likelihood functions for arbitrarily complex birth-death(-sampling) models that will hopefully allow researchers to explore a wider array of scenarios than was previously possible. By rederiving existing single-type birth-death sampling models, we clarify and synthesize the range of explicit and implicit assumptions made by these models. [Birth-death processes; epidemiology; macroevolution; phylogenetics; statistical inference.].

Genetic Diversity, Compartmentalization, and Age of HIV Proviruses Persisting in CD4+ T Cell Subsets during Long-Term Combination Antiretroviral Therapy.

The HIV reservoir, which comprises diverse proviruses integrated into the genomes of infected, primarily CD4+ T cells, is the main barrier to developing an effective HIV cure. Our understanding of the genetics and dynamics of proviruses persisting within distinct CD4+ T cell subsets, however, remains incomplete. Using single-genome amplification, we characterized subgenomic proviral sequences (nef region) from naive, central memory, transitional memory, and effector memory CD4+ T cells from five HIV-infected individuals on long-term combination antiretroviral therapy (cART) and compared these to HIV RNA sequences isolated longitudinally from archived plasma collected prior to cART initiation, yielding HIV data sets spanning a median of 19.5 years (range, 10 to 20 years) per participant. We inferred a distribution of within-host phylogenies for each participant, from which we characterized proviral ages, phylogenetic diversity, and genetic compartmentalization between CD4+ T cell subsets. While three of five participants exhibited some degree of proviral compartmentalization between CD4+ T cell subsets, combined analyses revealed no evidence that any particular CD4+ T cell subset harbored the longest persisting, most genetically diverse, and/or most genetically distinctive HIV reservoir. In one participant, diverse proviruses archived within naive T cells were significantly younger than those in memory subsets, while for three other participants we observed no significant differences in proviral ages between subsets. In one participant, "old" proviruses were recovered from all subsets, and included one sequence, estimated to be 21.5 years old, that dominated (>93%) their effector memory subset. HIV eradication strategies will need to overcome within- and between-host genetic complexity of proviral landscapes, possibly via personalized approaches.IMPORTANCE The main barrier to HIV cure is the ability of a genetically diverse pool of proviruses, integrated into the genomes of infected CD4+ T cells, to persist despite long-term suppressive combination antiretroviral therapy (cART). CD4+ T cells, however, constitute a heterogeneous population due to their maturation across a developmental continuum, and the genetic "landscapes" of latent proviruses archived within them remains incompletely understood. We applied phylogenetic techniques, largely novel to HIV persistence research, to reconstruct within-host HIV evolutionary history and characterize proviral diversity in CD4+ T cell subsets in five individuals on long-term cART. Participants varied widely in terms of proviral burden, genetic diversity, and age distribution between CD4+ T cell subsets, revealing that proviral landscapes can differ between individuals and between infected cell types within an individual. Our findings expose each within-host latent reservoir as unique in its genetic complexity and support personalized strategies for HIV eradication.

Variable routes to genomic and host adaptation among coronaviruses.

Natural selection operating on the genomes of viral pathogens in different host species strongly contributes to adaptation facilitating host colonization. Here, we analyse, quantify and compare viral adaptation in genomic sequence data derived from seven zoonotic events in the Coronaviridae family among primary, intermediate and human hosts. Rates of nonsynonymous (dN ) and synonymous (dS ) changes on specific amino acid positions were quantified for each open reading frame (ORF). Purifying selection accounted for 77% of all sites under selection. Diversifying selection was most frequently observed in viruses infecting the primary hosts of each virus and predominantly occurred in the orf1ab genomic region. Within all four intermediate hosts, diversifying selection on the spike gene was observed either solitarily or in combination with orf1ab and other genes. Consistent with previous evidence, pervasive diversifying selection on coronavirus spike genes corroborates the role this protein plays in host cellular entry, adaptation to new hosts and evasion of host cellular immune responses. Structural modelling of spike proteins identified a significantly higher proportion of sites for SARS-CoV-2 under positive selection in close proximity to sites of glycosylation relative to the other coronaviruses. Among human coronaviruses, there was a significant inverse correlation between the number of sites under positive selection and the estimated years since the virus was introduced into the human population. Abundant diversifying selection observed in SARS-CoV-2 suggests the virus remains in the adaptive phase of the host switch, typical of recent host switches. A mechanistic understanding of where, when and how genomic adaptation occurs in coronaviruses following a host shift is crucial for vaccine design, public health responses and predicting future pandemics.

Phylogenetic approach to recover integration dates of latent HIV sequences within-host.

Given that HIV evolution and latent reservoir establishment occur continually within-host, and that latently infected cells can persist long-term, the HIV reservoir should comprise a genetically heterogeneous archive recapitulating within-host HIV evolution. However, this has yet to be conclusively demonstrated, in part due to the challenges of reconstructing within-host reservoir establishment dynamics over long timescales. We developed a phylogenetic framework to reconstruct the integration dates of individual latent HIV lineages. The framework first involves inference and rooting of a maximum-likelihood phylogeny relating plasma HIV RNA sequences serially sampled before the initiation of suppressive antiretroviral therapy, along with putative latent sequences sampled thereafter. A linear model relating root-to-tip distances of plasma HIV RNA sequences to their sampling dates is used to convert root-to-tip distances of putative latent lineages to their establishment (integration) dates. Reconstruction of the ages of putative latent sequences sampled from chronically HIV-infected individuals up to 10 y following initiation of suppressive therapy revealed a genetically heterogeneous reservoir that recapitulated HIV's within-host evolutionary history. Reservoir sequences were interspersed throughout multiple within-host lineages, with the oldest dating to >20 y before sampling; historic genetic bottleneck events were also recorded therein. Notably, plasma HIV RNA sequences isolated from a viremia blip in an individual receiving otherwise suppressive therapy were highly genetically diverse and spanned a 20-y age range, suggestive of spontaneous in vivo HIV reactivation from a large latently infected cell pool. Our framework for reservoir dating provides a potentially powerful addition to the HIV persistence research toolkit.

Longitudinal within-host evolution of HIV Nef-mediated CD4, HLA and SERINC5 downregulation activity: a case study.

The HIV accessory protein Nef downregulates the viral entry receptor CD4, the Human Leukocyte Antigen (HLA)-A and -B molecules, the Serine incorporator 5 (SERINC5) protein and other molecules from the infected cell surface, thereby promoting viral infectivity, replication and immune evasion. The nef locus also represents one of the most genetically variable regions in the HIV genome, and nef sequences undergo substantial evolution within a single individual over the course of infection. Few studies however have simultaneously characterized the impact of within-host nef sequence evolution on Nef protein function over prolonged timescales. Here, we isolated 50 unique Nef clones by single-genome amplification over an 11-year period from the plasma of an individual who was largely naïve to antiretroviral treatment during this time. Together, these clones harbored nonsynonymous substitutions at 13% of nef's codons. We assessed their ability to downregulate cell-surface CD4, HLA and SERINC5 and observed that all three Nef functions declined modestly over time, where the reductions in CD4 and HLA downregulation (an average of 0.6% and 2.0% per year, respectively) achieved statistical significance. The results from this case study support all three Nef activities as being important to maintain throughout untreated HIV infection, but nevertheless suggest that, despite nef's mutational plasticity, within-host viral evolution can compromise Nef function, albeit modestly, over prolonged periods.

HIV Diversity and Genetic Compartmentalization in Blood and Testes during Suppressive Antiretroviral Therapy.

HIV's ability to persist during suppressive antiretroviral therapy is the main barrier to cure. Immune-privileged tissues, such as the testes, may constitute distinctive sites of HIV persistence, but this has been challenging to study in humans. We analyzed the proviral burden and genetics in the blood and testes of 10 individuals on suppressive therapy who underwent elective gender-affirming surgery. HIV DNA levels in matched blood and testes were quantified by quantitative PCR, and subgenomic proviral sequences (nef region) were characterized from single templates. HIV diversity, compartmentalization, and immune escape burden were assessed using genetic and phylogenetic approaches. Diverse proviruses were recovered from the blood (396 sequences; 354 nef-intact sequences) and testes (326 sequences; 309 nef-intact sequences) of all participants. Notably, the frequency of identical HIV sequences varied markedly between and within individuals. Nevertheless, proviral loads, within-host unique HIV sequence diversity, and the immune escape burden correlated positively between blood and testes. When all intact nef sequences were evaluated, 60% of participants exhibited significant blood-testis genetic compartmentalization, but none did so when the evaluation was restricted to unique sequences per site, suggesting that compartmentalization, when present, is attributable to the clonal expansion of HIV-infected cells. Our observations confirm the testes as a site of HIV persistence and suggest that individuals with larger and more diverse blood reservoirs will have larger and more diverse testis reservoirs. Furthermore, while the testis microenvironment may not be sufficiently unique to facilitate the seeding of unique viral populations therein, differential clonal expansion dynamics may be at play, which may complicate HIV eradication.IMPORTANCE Two key questions in HIV reservoir biology are whether immune-privileged tissues, such as the testes, harbor distinctive proviral populations during suppressive therapy and, if so, by what mechanism. While our results indicated that blood-testis HIV genetic compartmentalization was reasonably common (60%), it was always attributable to differential frequencies of identical HIV sequences between sites. No blood-tissue data set retained evidence of compartmentalization when only unique HIV sequences per site were considered; moreover, HIV immune escape mutation burdens were highly concordant between sites. We conclude that the principal mechanism by which blood and testis reservoirs differ is not via seeding of divergent HIV sequences therein but, rather, via differential clonal expansion of latently infected cells. Thus, while viral diversity and escape-related barriers to HIV eradication are of a broadly similar magnitude across the blood and testes, clonal expansion represents a challenge. The results support individualized analysis of within-host reservoir diversity to inform curative approaches.

Phylogenetic Tools for Generalized HIV-1 Epidemics: Findings from the PANGEA-HIV Methods Comparison.

Viral phylogenetic methods contribute to understanding how HIV spreads in populations, and thereby help guide the design of prevention interventions. So far, most analyses have been applied to well-sampled concentrated HIV-1 epidemics in wealthy countries. To direct the use of phylogenetic tools to where the impact of HIV-1 is greatest, the Phylogenetics And Networks for Generalized HIV Epidemics in Africa (PANGEA-HIV) consortium generates full-genome viral sequences from across sub-Saharan Africa. Analyzing these data presents new challenges, since epidemics are principally driven by heterosexual transmission and a smaller fraction of cases is sampled. Here, we show that viral phylogenetic tools can be adapted and used to estimate epidemiological quantities of central importance to HIV-1 prevention in sub-Saharan Africa. We used a community-wide methods comparison exercise on simulated data, where participants were blinded to the true dynamics they were inferring. Two distinct simulations captured generalized HIV-1 epidemics, before and after a large community-level intervention that reduced infection levels. Five research groups participated. Structured coalescent modeling approaches were most successful: phylogenetic estimates of HIV-1 incidence, incidence reductions, and the proportion of transmissions from individuals in their first 3 months of infection correlated with the true values (Pearson correlation > 90%), with small bias. However, on some simulations, true values were markedly outside reported confidence or credibility intervals. The blinded comparison revealed current limits and strengths in using HIV phylogenetics in challenging settings, provided benchmarks for future methods' development, and supports using the latest generation of phylogenetic tools to advance HIV surveillance and prevention.

Transmission of hepatitis C virus infection among younger and older people who inject drugs in Vancouver, Canada.

BACKGROUND & AIMS: Understanding HCV transmission among people who inject drugs (PWID) is important for designing prevention strategies. This study investigated whether HCV infection among younger injectors occurs from few or many transmission events from older injectors to younger injectors among PWID in Vancouver, Canada. METHODS: HCV antibody positive participants at enrolment or follow-up (1996-2012) were tested for HCV RNA and sequenced (Core-E2). Time-stamped phylogenetic trees were inferred using Bayesian Evolutionary Analysis Sampling Trees (BEAST). Association of age with phylogeny was tested using statistics implemented in the software Bayesian Tip Significance (BaTS) testing. Factors associated with clustering (maximum cluster age: five years) were identified using logistic regression. RESULTS: Among 699 participants with HCV subtype 1a, 1b, 2b and 3a infection (26% female, 24% HIV+): 21% were younger (<27years), and 10% had recent HCV seroconversion. When inferred cluster age was limited to <5years, 15% (n=108) were in clusters/pairs. Although a moderate degree of segregation was observed between younger and older participants, there was also transmission between age groups. Younger age (<27 vs. >40, AOR: 3.14; 95% CI: 1.54, 6.39), HIV (AOR: 1.97; 95% CI: 1.22, 3.18) and subtype 3a (AOR: 2.12; 95% CI: 1.33, 3.38) were independently associated with clustering. CONCLUSIONS: In this population of PWID from Vancouver, HCV among young injectors was seeded from many transmission events between HCV-infected older and younger injectors. Phylogenetic clustering was associated with younger age and HIV. These data suggest that HCV transmission among PWID is complex, with transmission occurring between and among older and younger PWID.

Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation.

The immunoglobulin heavy-chain locus (IGH) encodes variable (IGHV), diversity (IGHD), joining (IGHJ), and constant (IGHC) genes and is responsible for antibody heavy-chain biosynthesis, which is vital to the adaptive immune response. Programmed V-(D)-J somatic rearrangement and the complex duplicated nature of the locus have impeded attempts to reconcile its genomic organization based on traditional B-lymphocyte derived genetic material. As a result, sequence descriptions of germline variation within IGHV are lacking, haplotype inference using traditional linkage disequilibrium methods has been difficult, and the human genome reference assembly is missing several expressed IGHV genes. By using a hydatidiform mole BAC clone resource, we present the most complete haplotype of IGHV, IGHD, and IGHJ gene regions derived from a single chromosome, representing an alternate assembly of ∼1 Mbp of high-quality finished sequence. From this we add 101 kbp of previously uncharacterized sequence, including functional IGHV genes, and characterize four large germline copy-number variants (CNVs). In addition to this germline reference, we identify and characterize eight CNV-containing haplotypes from a panel of nine diploid genomes of diverse ethnic origin, discovering previously unmapped IGHV genes and an additional 121 kbp of insertion sequence. We genotype four of these CNVs by using PCR in 425 individuals from nine human populations. We find that all four are highly polymorphic and show considerable evidence of stratification (Fst = 0.3-0.5), with the greatest differences observed between African and Asian populations. These CNVs exhibit weak linkage disequilibrium with SNPs from two commercial arrays in most of the populations tested.

Theoretical and experimental assessment of degenerate primer tagging in ultra-deep applications of next-generation sequencing.

Primer IDs (pIDs) are random oligonucleotide tags used in next-generation sequencing to identify sequences that originate from the same template. These tags are produced by degenerate primers during the reverse transcription of RNA molecules into cDNA. The use of pIDs helps to track the number of RNA molecules carried through amplification and sequencing, and allows resolution of inconsistencies between reads sharing a pID. Three potential issues complicate the above applications. First, multiple cDNAs may share a pID by chance; we found that while preventing any cDNAs from sharing a pID may be unfeasible, it is still practical to limit the number of these collisions. Secondly, a pID must be observed in at least three sequences to allow error correction; as such, pIDs observed only one or two times must be rejected. If the sequencing product contains copies from a high number of RT templates but produces few reads, our findings indicate that rejecting such pIDs will discard a great deal of data. Thirdly, the use of pIDs could influence amplification and sequencing. We examined the effects of several intrinsic and extrinsic factors on sequencing reads at both the individual and ensemble level.

The impact of clinical, demographic and risk factors on rates of HIV transmission: a population-based phylogenetic analysis in British Columbia, Canada.

BACKGROUND: The diversification of human immunodeficiency virus (HIV) is shaped by its transmission history. We therefore used a population based province wide HIV drug resistance database in British Columbia (BC), Canada, to evaluate the impact of clinical, demographic, and behavioral factors on rates of HIV transmission. METHODS: We reconstructed molecular phylogenies from 27,296 anonymized bulk HIV pol sequences representing 7747 individuals in BC-about half the estimated HIV prevalence in BC. Infections were grouped into clusters based on phylogenetic distances, as a proxy for variation in transmission rates. Rates of cluster expansion were reconstructed from estimated dates of HIV seroconversion. RESULTS: Our criteria grouped 4431 individuals into 744 clusters largely separated with respect to risk factors, including large established clusters predominated by injection drug users and more-recently emerging clusters comprising men who have sex with men. The mean log10 viral load of an individual's phylogenetic neighborhood (composed of 5 other individuals with shortest phylogenetic distances) increased their odds of appearing in a cluster by >2-fold per log10 viruses per milliliter. CONCLUSIONS: Hotspots of ongoing HIV transmission can be characterized in near real time by the secondary analysis of HIV resistance genotypes, providing an important potential resource for targeting public health initiatives for HIV prevention.

Global origin and transmission of hepatitis C virus nonstructural protein 3 Q80K polymorphism.

Hepatitis C virus (HCV) has a naturally occurring polymorphism, Q80K, in the nonstructural protein 3 (NS3) gene encoding the viral protease, which has been associated with reduced susceptibility to the direct-acting antiviral inhibitor simeprevir. Q80K is observed predominantly in HCV genotype 1a and seldom in other HCV genotypes; moreover, it has a markedly high prevalence in the United States. Here, we reconstruct the evolutionary history of this polymorphism to investigate why it is so highly localized in prevalence and whether it is stably transmitted between hosts. We found that the majority (96%) of HCV infections carrying Q80K were descended from a single lineage in which a Q80K substitution occurred around the 1940s in the United States, which implies that this polymorphism is likely highly transmissible. Furthermore, we identified 2 other substitutions in NS3 that may interact with Q80K and contribute to its stability. Our results imply that the current distribution and prevalence of Q80K are unlikely to change significantly in the short term.

Longitudinal evolution of the HIV effective reproduction number following sequential expansion of treatment as prevention and pre-exposure prophylaxis in British Columbia, Canada: a population-level programme evaluation.

BACKGROUND: Treatment as prevention and pre-exposure prophylaxis (PrEP) are key strategies in the control of HIV/AIDS. We aimed to characterise the longitudinal effects of antiretroviral therapy (ART), followed by treatment as prevention and the addition of PrEP, on the HIV effective reproduction number (Re) in British Columbia, Canada. METHODS: This population-level programme evaluation used data from the Drug Treatment Program of the British Columbia Centre for Excellence in HIV/AIDS (Vancouver, British Columbia, Canada). We also used estimates of HIV incidence and prevalence from the Public Health Agency of Canada, data on the number of new HIV diagnoses per year from the British Columbia Centre for Disease Control, and mortality data from the British Columbia Vital Statistics Agency. Data were obtained from 1985 until 2022, depending on the database source. Outcomes were the annual HIV prevalence, HIV incidence, number of new HIV diagnoses, number of people living with HIV on ART, HIV/AIDS-related and all-cause mortality rates, the HIV incidence-to-all-cause-mortality ratio, and Re. We calculated the modified effective reproduction number (Rme) using two thresholds of viral suppression and compared these values with Re. FINDINGS: We found a 95% decline in HIV/AIDS-related mortality and a 91% decrease in HIV incidence over the study period. The Re progressively declined from 1996 to 2022; however, from 1996 to 2017, Rme remained stable (>1) when calculated for people living with HIV with unsuppressed viraemia, suggesting that treatment as prevention reduces HIV incidence by decreasing the pool of individuals who are potentially able to transmit the virus. From 2018 to 2022, a decline in the estimated Re and Rme (<1) was observed regardless of whether we considered all people living with HIV or only those who were virologically unsuppressed. This finding suggests that PrEP decreases HIV incidence by reducing the number of susceptible individuals in the community, independently of viral suppression. INTERPRETATION: Our results show the synergy between generalised treatment as prevention and targeted PrEP in terms of decreasing HIV incidence. These findings support the incorporation of longitudinal monitoring of Re at a programmatic level to identify opportunities for the optimisation of treatment-as-prevention and PrEP programmes. FUNDING: British Columbia Ministry of Health, Health Canada, Public Health Agency of Canada, Vancouver Coastal Health, Vancouver General Hospital Foundation, Genome British Columbia, and the Canadian Institutes of Health Research.