Revealing the evolutionary history and epidemiological dynamics of emerging RNA viral pathogens

Fast-evolving RNA viruses are a leading cause of morbidity and mortality among human and animal populations, contributing significantly to both global health and economic burden. The advent and revolution of high-throughput sequencing has empowered phylogenetic analyses with increasing amounts of temporally and spatially sampled viral data. Moreover, the parallel advancement in molecular evolution and phylogenetic methods has provided investigators with a unique opportunity to gain detailed insight into the evolutionary and epidemiological dynamics of emerging viral pathogens. Using state-of-the-art statistical approaches, this thesis addresses some of the important but controversial questions in viral emergence. Chapter 2 introduces a new framework to quantify and investigate reassortment events in influenza A viruses. By developing a computationally efficient algorithm to calculate the largest common subtree for a pair of tree sets, which are estimated from diffe rent parts of the genome for the same taxa set, the level of phylogenetic incongruency due to reassortment can be appropriately ascertained. Chapters 3, 4 and 5 investigate the evolutionary origins of three diff erent viruses: the novel emergence and cross-species transmission of SARSCoV, the genesis and dissemination of the unique HCV circulating recombinant form, and the ancient divergence of all influenza viruses, respectively. Moreover, Chapter 4 presents an improved statistical framework, which provides more precise evolutionary estimates, by utilizing the hierarchical bayes approach to investigate recombination events in emerging RNA viruses. The last empirical study, presented in Chapter 6, applies the recently developed Bayesian phylogeography models to a large viral sequence dataset sampled from southern Viet Nam to examine the fine-scale spatiotemporal dynamics of endemic dengue in Southeast Asia. The work presented here reflects both the advancements made in sequencing technology and statistical phylogenetics, along with some of the challenges that remain in studying the emergence of fast-evolving RNA viruses. This thesis proposes new and improved solutions to these evolutionary problems, such as incorporating non-vertical evolution (i.e. homologous recombination and reassortment) into the phylodynamic framework, with the aim of facilitating future investigations of emerging viral diseases

Pathogen genomics and One Health: a scoping review of current practices in zoonotic disease research

Objectives: Whole-genome sequencing has revolutionised the field of infectious disease surveillance, enabling near real-time detection of pathogens and tracking how infections may spread. Our study aimed to characterise genomic applications to cross-domain zoonotic pathogen transmission at the human-animal and/or human-environment interfaces. Methods: We performed a scoping review of studies that have applied genomic epidemiology to zoonotic disease transmission across One Health domains (human, animal, and environment). We identified 114 records published between 2005 and 2022 which reported multi-domain genomic data of zoonotic pathogens integrated into phylogenetic models. Results: Most studies investigated bacterial pathogens, highlighting key knowledge gaps for other zoonotic agents, particularly arboviruses. Sampling and sequencing efforts varied greatly across domains: the median number and range of pathogen genomes analysed were highest for humans (23; 1-29,586) and lowest for the environment domain (13; 1-956). Genomics was used to track zoonotic disease outbreaks and cross-domain transmission, to improve pathogen surveillance, and to disentangle evolutionary dynamics driving lineage diversification and virulence. Conclusions: Our study highlights current practices and knowledge gaps to guide future study designs and genomic applications to multi-domain and cross-species transmission of zoonoses, with the potential to identify key infection sources and inform interventions for local and global health security

Utilizing citizen science data to rapidly assess changing associations between wild birds and avian influenza outbreaks in poultry

High pathogenicity avian influenza virus (HPAIV) is a rapidly evolving virus causing significant economic and environmental harm. Wild birds are a key viral reservoir and an important source of viral incursions into animal populations, including poultry. However, we lack a thorough understanding of which species drive incursions and whether this changes over time. We explored associations between the abundances of 152 avian species and outbreaks of highly pathogenic avian influenza (HPAI) in poultry premises across Great Britain between October 2021 and January 2023. Spatial generalized additive models were used, with species abundance distributions sourced from eBird. Associations were investigated at the species-specific level and across species aggregations. During autumn/winter, associations were generally strongest with waterbirds such as ducks and geese; however, we also found significant associations in groups such as non-native gamebirds and rapid change in species-specific associations over time. Our results demonstrate the value of citizen science to rapidly explore wild species as potential facilitators of disease incursions into well-monitored populations, especially in regions where viral surveillance in wild species is limited. This can be a critical step towards prioritizing targeted surveillance that could inform species-specific biosecurity measures; particularly for HPAIV, which has undergone sudden shifts in host range and continues to rapidly evolve

A de novo approach to inferring within-host fitness effects during untreated HIV-1 infection

Funder: Isaac Newton Trust; funder-id: http://dx.doi.org/10.13039/501100004815Funder: Li Ka Shing Foundation; funder-id: http://dx.doi.org/10.13039/100007421Funder: Division of Intramural Research, National Institute of Allergy and Infectious Diseases; funder-id: http://dx.doi.org/10.13039/100006492Funder: Helsingin Yliopisto; funder-id: http://dx.doi.org/10.13039/100007797In the absence of effective antiviral therapy, HIV-1 evolves in response to the within-host environment, of which the immune system is an important aspect. During the earliest stages of infection, this process of evolution is very rapid, driven by a small number of CTL escape mutations. As the infection progresses, immune escape variants evolve under reduced magnitudes of selection, while competition between an increasing number of polymorphic alleles (i.e., clonal interference) makes it difficult to quantify the magnitude of selection acting upon specific variant alleles. To tackle this complex problem, we developed a novel multi-locus inference method to evaluate the role of selection during the chronic stage of within-host infection. We applied this method to targeted sequence data from the p24 and gp41 regions of HIV-1 collected from 34 patients with long-term untreated HIV-1 infection. We identify a broad distribution of beneficial fitness effects during infection, with a small number of variants evolving under strong selection and very many variants evolving under weaker selection. The uniquely large number of infections analysed granted a previously unparalleled statistical power to identify loci at which selection could be inferred to act with statistical confidence. Our model makes no prior assumptions about the nature of alleles under selection, such that any synonymous or non-synonymous variant may be inferred to evolve under selection. However, the majority of variants inferred with confidence to be under selection were non-synonymous in nature, and in most cases were have previously been associated with either CTL escape in p24 or neutralising antibody escape in gp41. We also identified a putative new CTL escape site (residue 286 in gag), and a region of gp41 (including residues 644, 648, 655 in env) likely to be associated with immune escape. Sites inferred to be under selection in multiple hosts have high within-host and between-host diversity although not all sites with high between-host diversity were inferred to be under selection at the within-host level. Our identification of selection at sites associated with resistance to broadly neutralising antibodies (bNAbs) highlights the need to fully understand the role of selection in untreated individuals when designing bNAb based therapies

A Molecular-Level Account of the Antigenic Hantaviral Surface

Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.Peer reviewe

A Molecular-Level Account of the Antigenic Hantaviral Surface

Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.Peer reviewe

Structure of the Lassa virus glycan shield provides a model for immunological resistance

Lassa virus is an Old World arenavirus endemic to West Africa that causes severe hemorrhagic fever. Vaccine development has focused on the envelope glycoprotein complex (GPC) that extends from the virion envelope. The often inadequate antibody immune response elicited by both vaccine and natural infection has been, in part, attributed to the abundance of N-linked glycosylation on the GPC. Here, using a virus-like-particle system that presents Lassa virus GPC in a native-like context, we determine the composite population of each of the N-linked glycosylation sites presented on the trimeric GPC spike. Our analysis reveals the presence of underprocessed oligomannose-type glycans, which form punctuated clusters that obscure the proteinous surface of both the GP1 attachment and GP2 fusion glycoprotein subunits of the Lassa virus GPC. These oligomannose clusters are seemingly derived as a result of sterically reduced accessibility to glycan processing enzymes, and limited amino acid diversification around these sites supports their role protecting against the humoral immune response. Combined, our data provide a structure-based blueprint for understanding how glycans render the glycoprotein spikes of Lassa virus and other Old World arenaviruses immunologically resistant targets.Peer reviewe

Seasonal dynamics of the wild rodent faecal virome

Viral discovery studies in wild animals often rely on cross-sectional surveys at a single time point. As a result, our understanding of the temporal stability of wild animal viromes remains poorly resolved. While studies of single host–virus systems indicate that host and environmental factors influence seasonal virus transmission dynamics, comparable insights for whole viral communities in multiple hosts are lacking. Utilizing noninvasive faecal samples from a long-term wild rodent study, we characterized viral communities of three common European rodent species (Apodemus sylvaticus, A. flavicollis and Myodes glareolus) living in temperate woodland over a single year. Our findings indicate that a substantial fraction of the rodent virome is seasonally transient and associated with vertebrate or bacteria hosts. Further analyses of one of the most common virus families, Picornaviridae, show pronounced temporal changes in viral richness and evenness, which were associated with concurrent and up to ~3-month lags in host density, ambient temperature, rainfall and humidity, suggesting complex feedbacks from the host and environmental factors on virus transmission and shedding in seasonal habitats. Overall, this study emphasizes the importance of understanding the seasonal dynamics of wild animal viromes in order to better predict and mitigate zoonotic risks

A Protective Monoclonal Antibody Targets a Site of Vulnerability on the Surface of Rift Valley Fever Virus

Summary: The Gn subcomponent of the Gn-Gc assembly that envelopes the human and animal pathogen, Rift Valley fever virus (RVFV), is a primary target of the neutralizing antibody response. To better understand the molecular basis for immune recognition, we raised a class of neutralizing monoclonal antibodies (nAbs) against RVFV Gn, which exhibited protective efficacy in a mouse infection model. Structural characterization revealed that these nAbs were directed to the membrane-distal domain of RVFV Gn and likely prevented virus entry into a host cell by blocking fusogenic rearrangements of the Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability on the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection and a model for understanding Ab-mediated neutralization of bunyaviruses more generally. : Allen et al. reveal a molecular basis of antibody-mediated neutralization of Rift Valley fever virus, an important human and animal pathogen. They isolate and demonstrate the protective efficacy of a monoclonal antibody in a murine model of virus infection, providing a blueprint for rational therapeutic and vaccine design. Keywords: phlebovirus, Rift Valley fever virus, antibody, structure, bunyavirus, virus-host interactions, immune response, vaccine, antiviral, neutralizatio