Evolutionary dynamics of RNA viruses with zoonotic potential

RNA viruses with zoonotic potential represent a public health threat throughout the world. High mutation rates, short generation times and large population sizes are three factors responsible for their high genetic variability and enormous adaptive capacity to new environmental conditions. Understanding the genetic properties and the evolutionary dynamics of RNA viruses with zoonotic potential is crucial to prevent, control, treat and lessen the damage to animal and human health. This thesis investigates the most essential aspects related to the evolution and epidemiology of two widespread zoonotic diseases caused by two RNA viruses: Avian Influenza and rabies. Through the application of bioinformatics tools, I analysed a large amount of sequence data, generated using both first and second generation sequencing technology, from viruses collected during four distinct epidemics: a fox-rabies virus epidemic occurred in north-eastern Italy between 2008 and 2011, highly pathogenic H5N1 avian influenza outbreaks reported in Egypt between 2006 and 2010, and two avian influenza epidemics caused by two distinct subtypes that took place in northern Italy from 1999 to 2001 and 2002 to 2004. Through phylogenetic and Bayesian phylogeographic analyses of viral sequences sampled over multiple discrete spatio-temporal scales, the studies in this thesis reveal the co-circulation of multiple viral lineages, explore the viral gene flows and investigate the evolutionary dynamics of viruses under different selection pressures. In addition, to better understand the pattern of transmission of viral subpopulations from host to host, the intra-host variability and the evolution of viral pathogenicity, I employed an ultra-deep sequencing approach to assess the diversity of viral populations. The data generated in this thesis provide important insights into the a) impact and efficacy of surveillance strategies and control measures implemented during an outbreak, b) differences in the evolutionary dynamics and spatial spread between distinct genetic groups, c) emergence of amino acid mutations that may increase viral fitness, d) inter-host transmission of viral variants and e) gain of virulence determinants. Finally, this thesis shows the great opportunity offered by next generation sequencing technology for dramatic advancement in our understanding of the complicated evolutionary dynamics of these pathogen

Unexpected interfarm transmission dynamics during a highly pathogenic avian influenza epidemic

Next-generation sequencing technology is now being increasingly applied to study the within- and between-host population dynamics of viruses. However, information on avian influenza virus evolution and transmission during a naturally occurring epidemic is still limited. Here, we use deep-sequencing data obtained from clinical samples collected from five industrial holdings and a backyard farm infected during the 2013 highly pathogenic avian influenza (HPAI) H7N7 epidemic in Italy to unravel (i) the epidemic virus population diversity, (ii) the evolution of virus pathogenicity, and (iii) the pathways of viral transmission between different holdings and sheds. We show a high level of genetic diversity of the HPAI H7N7 viruses within a single farm as a consequence of separate bottlenecks and founder effects. In particular, we identified the cocirculation in the index case of two viral strains showing a different insertion at the hemagglutinin cleavage site, as well as nine nucleotide differences at the consensus level and 92 minority variants. To assess interfarm transmission, we combined epidemiological and genetic data and identified the index case as the major source of the virus, suggesting the spread of different viral haplotypes from the index farm to the other industrial holdings, probably at different time points. Our results revealed interfarm transmission dynamics that the epidemiological data alone could not unravel and demonstrated that delay in the disease detection and stamping out was the major cause of the emergence and the spread of the HPAI strain

Spatiotemporal reconstruction and transmission dynamics during the 2016-17 H5N8 highly pathogenic avian influenza epidemic in Italy

Effective control of avian diseases in domestic populations requires understanding of the transmission dynamics facilitating viral emergence and spread. In 2016–17, Italy experienced a significant avian influenza epidemic caused by a highly pathogenic A(H5N8) virus, which affected domestic premises housing around 2.7 million birds, primarily in the north‐eastern regions with the highest density of poultry farms (Lombardy, Emilia‐Romagna and Veneto). We perform integrated analyses of genetic, spatiotemporal and host data within a Bayesian phylogenetic framework. Using continuous and discrete phylogeography, we estimate the locations of movements responsible for the spread and persistence of the epidemic. The information derived from these analyses on rates of transmission between regions through time can be used to assess the success of control measures. Using an approach based on phylogenetic–temporal distances between domestic cases, we infer the presence of cryptic wild bird‐mediated transmission, information that can be used to complement existing epidemiological methods for distinguishing transmission within the domestic population from incursions across the wildlife–domestic interface, a common challenge in veterinary epidemiology. Spatiotemporal reconstruction of the epidemic reveals a highly skewed distribution of virus movements with a high proportion of shorter distance local movements interspersed with occasional long‐distance dispersal events associated with wild birds. We also show how such inference be used to identify possible instances of human‐mediated movements where distances between phylogenetically linked domestic cases are unusually high

H5N1 Virus Evolution in Europe—An Updated Overview

Since its emergence in South East Asia in 2003, Highly Pathogenic Avian Influenza (HPAI) A/H5N1 has reportedly caused outbreaks in poultry and/or wild birds in 62 countries, of which 24 were in Europe. Interestingly, out of the many genetic clades circulating in Asia, the westward spread of HPAI A/H5N1 to Central Asia, the Middle East, Europe and Africa was dominated by one single clade, namely clade 2.2. In this paper, we review and update through phylogenetic and gene migrational analysis the information concerning the evolution and the molecular epidemiology of HPAI A/H5N1 on the European continent

Molecular Evolution and Phylogeography of Co-circulating IHNV and VHSV in Italy

Infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) are the most important viral pathogens impacting rainbow trout farming. These viruses are persistent in Italy, where they are responsible for severe disease outbreaks (epizootics) that affect the profitability of the trout industry. Despite the importance of IHNV and VHSV, little is known about their evolution at a local scale, although this is likely to be important for virus eradication and control. To address this issue we performed a detailed molecular evolutionary and epidemiological analysis of IHNV and VHSV in trout farms from northern Italy. Full-length glycoprotein gene sequences of a selection of VHSV (n=108) and IHNV (n=89) strains were obtained. This revealed that Italian VHSV strains belong to sublineages Ia1 and Ia2 of genotype Ia and are distributed into 7 genetic clusters. In contrast, all Italian IHNV isolates fell within genogroup E, for which only a single genetic cluster was identified. More striking was that IHNV has evolved more rapidly than VHSV (mean rates of 11 and 7.3 × 10-4 nucleotide substitutions per site, per year, respectively), indicating that these viruses exhibit fundamentally different evolutionary dynamics. The time to the most recent common ancestor of both IHNV and VHSV was consistent with the first reports of these pathogens in Italy. By combining sequence data with epidemiological information it was possible to identify different patterns of virus spread among trout farms, in which adjacent facilities can be infected by either genetically similar or different viruses, and farms located in different water catchments can be infected by identical strains. Overall, these findings highlight the importance of combining molecular and epidemiological information to identify the determinants of IHN and VHS spread, and to provide data that is central to future surveillance strategies and possibly control

Different environmental gradients associated to the spatiotemporal and genetic pattern of the H5N8 highly pathogenic avian influenza outbreaks in poultry in Italy

Comprehensive understanding of the patterns and drivers of avian influenza outbreaks is pivotal to inform surveillance systems and heighten nations’ ability to quickly detect and respond to the emergence of novel viruses. Starting in early 2017, the Italian poultry sector has been involved in the massive H5N8 highly pathogenic avian influenza epidemic that spread in the majority of the European countries in 2016/2017. Eighty‐three outbreaks were recorded in north‐eastern Italy, where a densely populated poultry area stretches along the Lombardy, Emilia‐Romagna and Veneto regions. The confirmed cases, affecting both the rural and industrial sectors, depicted two distinct epidemic waves. We adopted a combination of multivariate statistics techniques and multi‐model regression selection and inference, to investigate how environmental factors relate to the pattern of outbreaks diversity with respect to their spatiotemporal and genetic diversity. Results showed that a combination of eco‐climatic and host density predictors were associated with the outbreaks pattern, and variation along gradients was noticeable among genetically and geographically distinct groups of avian influenza cases. These regional contrasts may be indicative of a different mechanism driving the introduction and spreading routes of the influenza virus in the domestic poultry population. This methodological approach may be extended to different spatiotemporal scale to foster site‐specific, ecologically informed risk mitigating strategies

Introduction into Nigeria of a Distinct Genotype of Avian Influenza Virus (H5N1)

Genetic characterization of highly pathogenic avian influenza viruses (H5N1) isolated in July 2008 in Nigeria indicates that a distinct genotype, never before detected in Africa, reached the continent. Phylogenetic analysis showed that the viruses are genetically closely related to European and Middle Eastern influenza A (H5N1) isolates detected in 2007

Modelling the impact of co-circulating low pathogenic avian influenza viruses on epidemics of highly pathogenic avian influenza in poultry

It is well known that highly pathogenic avian influenza (HPAI) viruses emerge through mutation of precursor low pathogenic avian influenza (LPAI) viruses in domestic poultry populations. The potential for immunological cross-protection between these pathogenic variants is recognised but the epidemiological impact during co-circulation is not well understood. Here we use mathematical models to investigate whether altered flock infection parameters consequent to primary LPAI infections can impact on the spread of HPAI at the population level. First we used mechanistic models reflecting the co-circulatory dynamics of LPAI and HPAI within a single commercial poultry flock. We found that primary infections with LPAI led to HPAI prevalence being maximised under a scenario of high but partial cross-protection. We then tested the population impact in spatially-explicit simulations motivated by a major avian influenza A(H7N1) epidemic that afflicted the Italian poultry industry in 1999-2001. We found that partial cross-protection can lead to a prolongation of HPAI epidemic duration. Our findings have implications for the control of HPAI in poultry particularly for settings in which LPAI and HPAI frequently co-circulate

Reassortant Avian Influenza Virus (H5N1) in Poultry, Nigeria, 2007

Reassortant Influenza Virus (H5N1) in Poultry, Nigeria, 200