SPATIAL AND TEMPORAL DYNAMICS OF PESTIVIRUS, CRIMEAN-CONGO HEMORRHAGIC FEVER AND HEPATITIS E VIRUS: A PHYLOGENETIC APPROACH

The general aim of the PhD project was to apply phylogenetic analysis to viral sequences obtained in different geographic areas at different times, in order to reconstruct the most probable places of origin and pathways of dispersion of infections. Viral population growth and evolution leave a measurable imprint on the genome of viruses over the course of years, months or even days and occur simultaneously with geographic dispersal (Holmes, 2008; Pybus & Rambaut, 2009). This interaction characterizes a spatial phylodynamic process that can be recovered from genomic data using phylogeographic analyses (Faria et al., 2011). The research activity has been focused on Pestivirus genus, that includes pathogens of livestock (Bovine viral diarrhea virus - BDV) and wildlife (Border disease virus - BDV), and on zoonotic emerging diseases, involving in their epidemiological cycle both livestock and wildlife (Crimean-Congo hemorrhagic fever - CCHF, Hepatitis E virus- HEV). Concerning BDV, since 2001 several outbreaks of disease have been reported in Pyrenean chamois in Spain, France and Andorra. These outbreaks have decimated several Pyrenean chamois populations, with mortalities ranging from 40% to 85%. The infection has become endemic in the Central and Eastern Pyrenees. The aim of this study was to clarify the origin and dispersion of the Pyrenean chamois BDV genetic variant by reconstructing the spatial and temporal dynamics of BDV 5’ UTR sequences of Pyrenean chamois, 10 novel sequences and 41 retrieved from public databases and Sheep BDV sequences (n=44) from Spain and France were also retrieved. The phylogenetic analysis was performed using a Bayesian Markov chain Monte Carlo (MCMC) method implemented in the BEAST v.1.74 package. The chamois clade originated from sheep BDV genotype 4, generating a founder effect due to intra-species spread and spatial dispersion. The time of the most recent common ancestor estimates for the chamois clade dated back to a time span between 1974 and 1996, with a mean estimation falling in 1988. The pathway of dispersion of isolates suggests a complex exchange between neighboring Pyrenean chamois populations, still going on such as Western direction. Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5’ UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. Italy is one of the countries with the highest genetic diversity of BVDV worldwide. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction. CCHF is a zoonosis mainly transmitted by ticks that causes sporadic cases and severe hemorrhagic fever of acute human disease with a mortality rate of 5-60% and it has recently emerged in the Balkans and eastern Mediterranean areas. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach. CCHFV phylogeographic reconstruction suggests that the disease originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s and the second in the early 1900s. The most probable location for the origin of the European clade was Russia, but Turkey played a central role in spreading the virus throughout Europe. Our data suggest that the movement of wild and domestic ungulates from endemic areas probably represent the main cause of virus dissemination in Eastern Europe. Hepatitis E virus is classified into four genotypes that have different geographical and host distributions. The main cause of sporadic autochthonous type E acute hepatitis in developed countries is genotype 3, which has a worldwide distribution and widely infects pigs. The aim of this study was to make hypotheses concerning the origin and global dispersion routes of this genotype by reconstructing the spatial and temporal dynamics of 208 HEV genotype 3 ORF-2 sequences (retrieved from public databases) isolated in different geographical areas. The evolutionary rates, time of the most recent common ancestors (tMRCAs), epidemic growth and phylogeography of HEV-3 were co-estimated using a MCMC Bayesian method. On the basis of time-scaled phylogeographical reconstruction, we hypothesise that HEV-3, after originating in the early 1800s in Europe, reached Asia in the first decades of 1900, and then moved to America probably in the 1970s-1980s. Analysis of the skyline plot showed a sharp increase of the number of infections between the 1980s and 2005, suggesting the intervention of new and highly efficient routes of transmission, possibly related to changes in the pig industry.