Relax,Keep Walking — A Practical Guide to Continuous Phylogeographic Inference with BEAST

Spatially explicit phylogeographic analyses can be performed with an inference framework that employs relaxed random walks to reconstruct phylogenetic dispersal histories in continuous space. This core model was first implemented 10 years ago and has opened up new opportunities in the field of phylodynamics, allowing researchers to map and analyze the spatial dissemination of rapidly evolving pathogens. We here provide a detailed and step-by-step guide on how to set up, run, and interpret continuous phylogeographic analyses using the programs BEAUti, BEAST, Tracer, and TreeAnnotator.     

Infection Frequency of Hepatitis C Virus and IL28B Haplotypes in Papua New Guinea,Fiji, and Kiribati

It has been estimated that there are more than 60 million Hepatitis C virus (HCV) carriers in the World Health Organisation''s Western Pacific region (WHO-WPR), where liver cancer is among the top three causes of cancer death. WHO and the US Centres for Disease Control and Prevention report the prevalence of HCV in the South Pacific islands (countries within the WHO-WPR) to be high (5–10% and >2% respectively). However, since HCV is not tested for in many of these countries, there is sparse data available to support this assertion. We screened ∼2000 apparently healthy individuals from Papua New Guinea, Fiji and Kiribati and found a sero-prevalence of 2.0%, 0.1% and 0%, respectively. All sero-positive samples tested negative for HCV RNA. Curious as to why all the sero-positive individuals were negative for HCV-RNA, we also screened them for the HCV protective IL28B SNP markers rs12979860 and rs8099917. All antibody-positive participants bar one had HCV protective haplotypes. Our results suggest that HCV is present in these Pacific island countries, albeit at a prevalence lower than previous estimates. As none of our participants had undergone antiviral treatment, and therefore must have cleared infection naturally, we hypothesise that genotypes 1 and/or 4 are circulating in South Pacific Island people and that these peoples are genetically predisposed to be more likely to spontaneous resolve HCV infection than to become chronic carriers.     

Multiple origins of European populations of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), a liver parasite of ruminants

The giant liver fluke, Fascioloides magna, a liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384bp) and nicotinamide dehydrogenase subunit I (nad1; 405bp) were used. Phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European foci of infection (Italy, Czech Republic and Danube floodplain forests involving the territories of Slovakia, Hungary and Croatia) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (eight), while a more substantial level of genetic diversity and a greater number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes, supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities which indicates that introduction of F. magna to the European continent occurred more than once. In the Czech focus of infection, a south-eastern USA origin was revealed. Identical haplotypes, common to parasites from the Czech Republic and from an expanding focus in Danube floodplain forests, implies that the introduction of F. magna to the Danube region came from an already established Czech focus of infection.     

The genomic rate of molecular adaptation of the human influenza A virus

Quantifying adaptive evolution at the genomic scale is an essential yet challenging aspect of evolutionary biology. Here, we develop a method that extends and generalizes previous approaches to estimate the rate of genomic adaptation in rapidly evolving populations and apply it to a large data set of complete human influenza A virus genome sequences. In accord with previous studies, we observe particularly high rates of adaptive evolution in domain 1 of the viral hemagglutinin (HA1). However, our novel approach also reveals previously unseen adaptation in other viral genes. Notably, we find that the rate of adaptation (per codon per year) is higher in surface residues of the viral neuraminidase than in HA1, indicating strong antibody-mediated selection on the former. We also observed high rates of adaptive evolution in several nonstructural proteins, which may relate to viral evasion of T-cell and innate immune responses. Furthermore, our analysis provides strong quantitative support for the hypothesis that human H1N1 influenza experiences weaker antigenic selection than H3N2. As well as shedding new light on the dynamics and determinants of positive Darwinian selection in influenza viruses, the approach introduced here is applicable to other pathogens for which densely sampled genome sequences are available, and hence is ideally suited to the interpretation of next-generation genome sequencing data.     

Evolutionary Genetics of Human Enterovirus 71: Origin,Population Dynamics,Natural Selection,and Seasonal Periodicity of the VP1 Gene

Human enterovirus 71 (EV-71) is one of the major etiologic causes of hand, foot, and mouth disease (HFMD) among young children worldwide, with fatal instances of neurological complications becoming increasingly common. Global VP1 capsid sequences (n = 628) sampled over 4 decades were collected and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. We estimated that the common ancestor of human EV-71 likely emerged around 1941 (95% confidence interval [CI], 1929 to 1952), subsequently diverging into three genogroups: B, C, and the now extinct genogroup A. Genealogical analysis revealed that diverse lineages of genogroup B and C (subgenogroups B1 to B5 and C1 to C5) have each circulated cryptically in the human population for up to 5 years before causing large HFMD outbreaks, indicating the quiescent persistence of EV-71 in human populations. Estimated phylogenies showed a complex pattern of spatial structure within well-sampled subgenogroups, suggesting endemicity with occasional lineage migration among locations, such that past HFMD epidemics are unlikely to be linked to continuous transmission of a single strain of virus. In addition, rises in genetic diversity are correlated with the onset of epidemics, driven in part by the emergence of novel EV-71 subgenogroups. Using subgenogroup C1 as a model, we observe temporal strain replacement through time, and we investigate the evidence for positive selection at VP1 immunogenic sites. We discuss the consequences of the evolutionary dynamics of EV-71 for vaccine design and compare its phylodynamic behavior with that of influenza virus.Enterovirus 71 (EV-71) is a member of the genus Enterovirus in the family Picornaviridae. Classified as human enterovirus species A (HEV-A) along with some group A coxsackieviruses (CV-A), EV-71 is a small, nonenveloped, positive-stranded RNA virus with a genome approximately 7,400 bases long and is genetically most related to CV-A16. EV-71 is divided into three major genogroups (denoted A, B, and C), and various subgenogroups within genogroups B and C.Since its first isolation in the United States in 1969 (71), EV-71 has been identified worldwide as a common cause of hand, foot, and mouth disease (HFMD) in young children and infants. Large EV-71-associated HFMD outbreaks have been reported in the United States, Europe, Australia, and Asia and constitute a significant and emerging threat to global public health (9, 50, 62, 63). Although EV-71 infection manifests most frequently as a mild, self-limited febrile illness characterized by papulovesicular lesions on the hands, feet, oropharyngeal mucosa, and buttocks, a small proportion of acute infections are associated with fatal neurological symptoms, including brain stem encephalitis, aseptic meningitis, and poliomyelitis-like paralysis (4, 28, 47). Such cases of neurological disease with a high case fatality rate were first reported in Bulgaria in 1975 (21) and Hungary in 1978 (52). However, large HFMD epidemics with high mortality rates resurfaced 2 decades later, in Malaysia in 1997 (2, 13, 16, 43) and Taiwan in 1998 (33, 42). Following these outbreaks, the Asia-Pacific region has experienced more frequent large-scale EV-71-associated HFMD epidemics—most with a high incidence of neurotropic infections and significant case fatality rates—and the virus has attracted global attention (3, 5, 14, 15, 18, 37, 46, 48, 55, 57, 74, 81, 82). Intriguingly, almost all outbreaks reported in the Asia-Pacific region during the last decade were caused by previously undefined EV-71 subgenogroups, raising questions about their origin, genetic complexity, and epidemiological behavior.The icosahedral particles of EV-71, which are structurally similar to those of other members of the Picornaviridae, consist of structural proteins (capsid proteins VP1 to VP4) assembled as pentameric subunits (66). The VP1 protein is highly exposed and usually targeted by host neutralizing antibodies, predisposing the VP1 gene to constant immune selective pressure. This selection may drive the adaptive evolution of the capsid region of many enteroviruses, possibly resulting in amino acid fixations in virus populations (19, 45, 79). Because the VP1 gene of enteroviruses is thought to play an important role in viral pathogenesis and virulence (10, 12, 30), understanding the tempo and mode of evolution of the capsid protein can provide new insights into the epidemiological dynamics of EV-71 that may be useful in predicting the genetic basis and periodicity of future EV-71 epidemics and in facilitating the development of an effective EV-71 vaccine candidate.In this study, we investigated the evolutionary dynamics and genetic history of EV-71. We estimate the dates of emergence of various subgenogroups identified in recent HFMD outbreaks. Using recently developed Bayesian methods of evolutionary analysis, we estimate the divergence time of EV-71 from its closely related ancestor CV-A16, thereby providing a date of origin for EV-71. We also reconstruct the global population dynamics of EV-71 over the past 40 years, revealing temporal trends in genetic diversity within and between major epidemics. Finally, despite finding little evidence of positive selection in the VP1 capsid protein, we observed a pattern of continuous strain and lineage replacement through time, with strong selective pressure detected at several potentially immunogenic sites. The impact of EV-71 evolution on the development of an EV-71 vaccine is also discussed.     

Phylogenetic evidence for deleterious mutation load in RNA viruses and its contribution to viral evolution

Populations of RNA viruses are often characterized by abundant genetic variation. However, the relative fitness of these mutations is largely unknown, although this information is central to our understanding of viral emergence, immune evasion, and drug resistance. Here we develop a phylogenetic method, based on the distribution of nonsynonymous and synonymous changes, to assess the relative fitness of polymorphisms in the structural genes of 143 RNA viruses. This reveals that a substantial proportion of the amino acid variation observed in natural populations of RNA viruses comprises transient deleterious mutations that are later purged by purifying selection, potentially limiting virus adaptability. We also demonstrate, for the first time, the existence of a relationship between amino acid variability and the phylogenetic distribution of polymorphisms. From this relationship, we propose an empirical threshold for the maximum viable deleterious mutation load in RNA viruses.