Oliver G. Pybus

Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic

In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. During the first few weeks of surveillance, the virus spread worldwide to 30 countries (as of May 11) by human-to-human transmission, causing the World Health Organization to raise its pandemic alert to level 5 of 6. This virus has the potential to develop into the first influenza pandemic of the twenty-first century. Here we use evolutionary analysis to estimate the timescale of the origins and the early development of the S-OIV epidemic. We show that it was derived from several viruses circulating in swine, and that the initial transmission to humans occurred several months before recognition of the outbreak. A phylogenetic estimate of the gaps in genetic surveillance indicates a long period of unsampled ancestry before the S-OIV outbreak, suggesting that the reassortment of swine lineages may have occurred years before emergence in humans, and that the multiple genetic ancestry of S-OIV is not indicative of an artificial origin. Furthermore, the unsampled history of the epidemic means that the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic, despite the fact that we included a panel of closely related and previously unpublished swine influenza isolates. Our results highlight the need for systematic surveillance of influenza in swine, and provide evidence that the mixing of new genetic elements in swine can result in the emergence of viruses with pandemic potential in humans.

Pandemic Potential of a Strain of Influenza A (H1N1): Early Findings

Swine Flu Benchmark The World Health Organization (WHO) announced on 29 April 2009, a level-5 pandemic alert for a strain of H1N1 influenza originating in pigs in Mexico and transmitting from human to human in several countries. Fraser et al. (p. 1557, published online 11 May; see the cover) amassed a team of experts in Mexico and WHO to make an initial assessment of the outbreak with a view to guiding future policy. The outbreak appears to have originated in mid-February in the village of La Gloria, Veracruz, where over half the population suffered acute respiratory illness, affecting more than 61% of children under 15 years old in the community. The basic reproduction number (the number of people infected per patient) is in the range of 1.5—similar or less than that of the pandemics of 1918, 1957, and 1968. There remain significant uncertainties about the severity of this outbreak, which makes it difficult to compare the economic and societal costs of intervention with lives saved and the risks of generating antiviral resistance. An international collaborative effort has analyzed the initial dynamics of the swine flu outbreak. A novel influenza A (H1N1) virus has spread rapidly across the globe. Judging its pandemic potential is difficult with limited data, but nevertheless essential to inform appropriate health responses. By analyzing the outbreak in Mexico, early data on international spread, and viral genetic diversity, we make an early assessment of transmissibility and severity. Our estimates suggest that 23,000 (range 6000 to 32,000) individuals had been infected in Mexico by late April, giving an estimated case fatality ratio (CFR) of 0.4% (range: 0.3 to 1.8%) based on confirmed and suspected deaths reported to that time. In a community outbreak in the small community of La Gloria, Veracruz, no deaths were attributed to infection, giving an upper 95% bound on CFR of 0.6%. Thus, although substantial uncertainty remains, clinical severity appears less than that seen in the 1918 influenza pandemic but comparable with that seen in the 1957 pandemic. Clinical attack rates in children in La Gloria were twice that in adults (<15 years of age: 61%; ≥15 years: 29%). Three different epidemiological analyses gave basic reproduction number (R0) estimates in the range of 1.4 to 1.6, whereas a genetic analysis gave a central estimate of 1.2. This range of values is consistent with 14 to 73 generations of human-to-human transmission having occurred in Mexico to late April. Transmissibility is therefore substantially higher than that of seasonal flu, and comparable with lower estimates of R0 obtained from previous influenza pandemics.

Global distribution and prevalence of hepatitis C virus genotypes

Hepatitis C virus (HCV) exhibits high genetic diversity, characterized by regional variations in genotype prevalence. This poses a challenge to the improved development of vaccines and pan‐genotypic treatments, which require the consideration of global trends in HCV genotype prevalence. Here we provide the first comprehensive survey of these trends. To approximate national HCV genotype prevalence, studies published between 1989 and 2013 reporting HCV genotypes are reviewed and combined with overall HCV prevalence estimates from the Global Burden of Disease (GBD) project. We also generate regional and global genotype prevalence estimates, inferring data for countries lacking genotype information. We include 1,217 studies in our analysis, representing 117 countries and 90% of the global population. We calculate that HCV genotype 1 is the most prevalent worldwide, comprising 83.4 million cases (46.2% of all HCV cases), approximately one‐third of which are in East Asia. Genotype 3 is the next most prevalent globally (54.3 million, 30.1%); genotypes 2, 4, and 6 are responsible for a total 22.8% of all cases; genotype 5 comprises the remaining <1%. While genotypes 1 and 3 dominate in most countries irrespective of economic status, the largest proportions of genotypes 4 and 5 are in lower‐income countries. Conclusion: Although genotype 1 is most common worldwide, nongenotype 1 HCV cases—which are less well served by advances in vaccine and drug development—still comprise over half of all HCV cases. Relative genotype proportions are needed to inform healthcare models, which must be geographically tailored to specific countries or regions in order to improve access to new treatments. Genotype surveillance data are needed from many countries to improve estimates of unmet need. (Hepatology 2015;61:77–87)

The genomic and epidemiological dynamics of human influenza A virus.

The evolutionary interaction between influenza A virus and the human immune system, manifest as ‘antigenic drift’ of the viral haemagglutinin, is one of the best described patterns in molecular evolution. However, little is known about the genome-scale evolutionary dynamics of this pathogen. Similarly, how genomic processes relate to global influenza epidemiology, in which the A/H3N2 and A/H1N1 subtypes co-circulate, is poorly understood. Here through an analysis of 1,302 complete viral genomes sampled from temperate populations in both hemispheres, we show that the genomic evolution of influenza A virus is characterized by a complex interplay between frequent reassortment and periodic selective sweeps. The A/H3N2 and A/H1N1 subtypes exhibit different evolutionary dynamics, with diverse lineages circulating in A/H1N1, indicative of weaker antigenic drift. These results suggest a sink–source model of viral ecology in which new lineages are seeded from a persistent influenza reservoir, which we hypothesize to be located in the tropics, to sink populations in temperate regions.

Testing macro–evolutionary models using incomplete molecular phylogenies

Phylogenies reconstructed from gene sequences can be used to investigate the tempo and mode of species diversification. Here we develop and use new statistical methods to infer past patterns of speciation and extinction from molecular phylogenies. Specifically, we test the null hypothesis that per–lineage speciation and extinction rates have remained constant through time. Rejection of this hypothesis may provide evidence for evolutionary events such as adaptive radiations or key adaptations. In contrast to previous approaches, our methods are robust to incomplete taxon sampling and are conservative with respect to extinction. Using simulation we investigate, first, the adverse effects of failing to take incomplete sampling into account and, second, the power and reliability of our tests. When applied to published phylogenies our tests suggest that, in some cases, speciation rates have decreased through time.

Rates of molecular evolution in RNA viruses: a quantitative phylogenetic analysis.

The study of rates of nucleotide substitution in RNA viruses is central to our understanding of their evolution. Herein we report a comprehensive analysis of substitution rates in 50 RNA viruses using a recently developed maximum likelihood phylogenetic method. This analysis revealed a significant relationship between genetic divergence and isolation time for an extensive array of RNA viruses, although more rate variation was usually present among lineages than would be expected under the constraints of a molecular clock. Despite the lack of a molecular clock, the range of statistically significant variation in overall substitution rates was surprisingly narrow for those viruses where a significant relationship between genetic divergence and time was found, as was the case when synonymous sites were considered alone, where the molecular clock was rejected less frequently. An analysis of the ecological and genetic factors that might explain this rate variation revealed some evidence of significantly lower substitution rates in vector-borne viruses, as well as a weak correlation between rate and genome length. Finally, a simulation study revealed that our maximum likelihood estimates of substitution rates are valid, even if the molecular clock is rejected, provided that sufficiently large data sets are analyzed.

Zika virus in the Americas: Early epidemiological and genetic findings

Zika virus genomes from Brazil The Zika virus outbreak is a major cause for concern in Brazil, where it has been linked with increased reports of otherwise rare birth defects and neuropathology. In a phylogenetic analysis, Faria et al. infer a single introduction of Zika to the Americas and estimated the introduction date to be about May to December 2013—some 12 months earlier than the virus was reported. This timing correlates with major events in the Brazilian cultural calendar associated with increased traveler numbers from areas where Zika virus has been circulating. A correlation was also observed between incidences of microcephaly and week 17 of pregnancy. Science, this issue p. 345 Virus sequencing indicates that Zika arrived in Brazil during the middle of 2013, coincident with a surge in air travelers. Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

Adaptation of HIV-1 to human leukocyte antigen class I

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host–pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8+ T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8+ T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128–135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8+ T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Recent epidemic of acute hepatitis C Virus in HIV-positive men who have sex with men linked to high-risk sexual behaviours.

Objective:To characterize the mode of hepatitis C virus (HCV) transmission in a recent epidemic of acute HCV in HIV-infected individuals using linked molecular and clinical epidemiological studies. Design:Individuals diagnosed with acute HCV between 1999 and 2005 at three urban HIV units in the UK were enrolled into a phylogenetic and case–control study. Phylogenetic trees were constructed from the amplified sequences of the E1/E2 region of the HCV genome and were used to compare cases with unrelated sequences. A questionnaire-based, case–control study using matched controls recruited from each HIV unit identified putative transmission factors. Results:One hundred and eleven HIV-positive men who have sex with men with acute HCV (genotype 1: 84%) were enrolled. Phylogenetic analysis of 93 E1/E2 sequences revealed seven monophyletic clusters signifying multiple independent HCV lineages co-circulating in the HIV-positive population. Permucosal rather than percutaneous transmission factors were associated with case/control status. Cases (n = 60) had more sexual partners, increased levels of high-risk sexual behaviour and were more likely to have shared drugs via a nasal or anal route in the preceding year in comparison with controls (n = 130). Sex in a group of more than two people was the strongest predictor of case/control status; odds ratios associated with participation in two or at least three types of high-risk sexual behaviour in a group were 9.16 (95% confidence interval, 3.51–23.90) and 23.50 (95% confidence interval, 9.47–58.33), respectively. Conclusion:The identified co-circulating HCV lineages belong to different subtypes and genotypes, implying that rather than viral change, the epidemic is due to permucosal transmission factors that should be the focus of public health interventions.

Tracing the origin and history of the HIV-2 epidemic

In this study we date the introduction of HIV-2 into the human population and estimate the epidemic history of HIV-2 subtype A in Guinea-Bissau, the putative geographic origin of HIV-2. The evolutionary history of the simian immunodeficiency virussooty mangabey/HIV-2 lineage was reconstructed by using available database sequences with known sampling dates, and a timescale for this history was calculated by using maximum likelihood methods. The date of the most recent common ancestor of HIV-2 subtype A strains was estimated to be 1940 ± 16 and that of B strains was estimated to be 1945 ± 14. In addition we used coalescent theory to estimate the past population dynamics of HIV-2 subtype A in a rural population of Guinea-Bissau. Parametric and nonparametric estimates of the effective number of infections through time were obtained for an equal sample of gag, pol, and env sequences. Our estimates of the epidemic history of HIV-2 subtype A in Guinea-Bissau show a transition from constant size to rapid exponential growth around 1955–1970. Our analysis provides evidence for a zoonotic transfer of HIV-2 during the first half of the 20th century and an epidemic initiation in Guinea-Bissau that coincides with the independence war (1963–1974), suggesting that war-related changes in sociocultural patterns had a major impact on the HIV-2 epidemic.