Spatial and temporal phylogeny of border disease virus in pyrenean chamois (Rupicapra p. Pyrenaica)

Border disease virus (BDV) affects a wide range of ruminants worldwide, mainly domestic sheep and goat. Since 2001 several outbreaks of disease associated to BDV infection have been described in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain, France and Andorra. In order to reconstruct the most probable places of origin and pathways of dispersion of BDV among Pyrenean chamois, a phylogenetic analysis of 95 BDV 5'untranslated sequences has been performed on chamois and domestic ungulates, including novel sequences and retrieved from public databases, using a Bayesian Markov Chain Monte Carlo method. Discrete and continuous space phylogeography have been applied on chamois sequences dataset, using centroid positions and latitude and longitude coordinates of the animals, respectively. The estimated mean evolutionary rate of BDV sequences was 2.9x10(-3) subs/site/year (95% HPD: 1.5-4.6x10(-3)). All the Pyrenean chamois isolates clustered in a unique highly significant clade, that originated from BDV-4a ovine clade. The introduction from sheep (dated back to the early 90s) generated a founder effect on the chamois population and the most probable place of origin of Pyrenean chamois BDV was estimated at coordinates 42.42 N and 1.9 E. The pathways of virus dispersion showed two main routes: the first started on the early 90s of the past century with a westward direction and the second arise in Central Pyrenees. The virus spread westward for more than 125 km and southward for about 50km and the estimated epidemic diffusion rate was about 13.1 km/year (95% HPD 5.2-21.4 km/year). The strong spatial structure, with strains from a single locality segregating together in homogeneous groups, and the significant pathways of viral dispersion among the areas, allowed to reconstruct both events of infection in a single area and of migrations, occurring between neighboring areas

Reconstructing the recent West Nile virus lineage 2 epidemic in Europe and Italy using discrete and continuous phylogeography

West Nile virus lineage 2 (WNV-2) was mainly confined to sub-Saharan Africa until the early 2000s, when it was identified for the first time in Central Europe causing outbreaks of human and animal infection. The aim of this study was to reconstruct the origin and dispersion of WNV-2 in Central Europe and Italy on a phylodynamic and phylogeographical basis. To this aim, discrete and continuous space phylogeographical models were applied to a total of 33 newly characterised full-length viral genomes obtained from mosquitoes, birds and humans in Northern Italy in the years 2013-2015 aligned with 64 complete sequences isolated mainly in Europe. The European isolates segregated into two highly significant clades: a small one including three sequences and a large clade including the majority of isolates obtained in Central Europe since 2004. Discrete phylogeographical analysis showed that the most probable location of the root of the largest European clade was in Hungary a mean 12.78 years ago. The European clade bifurcated into two highly supported subclades: one including most of the Central/East European isolates and the other encompassing all of the isolates obtained in Greece. The continuous space phylogeographical analysis of the Italian clade showed that WNV-2 entered Italy in about 2008, probably by crossing the Adriatic sea and reaching a central area of the Po Valley. The epidemic then spread simultaneously eastward, to reach the region of the Po delta in 2013, and westward to the border area between Lombardy and Piedmont in 2014; later, the western strain changed direction southward, and reached the central area of the Po valley once again in 2015. Over a period of about seven years, the virus spread all over an area of northern Italy by following the Po river and its main tributaries

Within-Host Dynamics of the Hepatitis C Virus Quasispecies Population in HIV-1/HCV Coinfected Patients

HIV/HCV coinfected individuals under highly active antiretroviral therapy (HAART) represent an interesting model for the investigation of the role played by the immune system in driving the evolution of the HCV quasispecies. We prospectively studied the intra-host evolution of the HCV heterogeneity in 8 coinfected subjects, selected from a cohort of 32 patients initiating HAART: 5 immunological responders (group A) and 3 immunological non-responders (group B), and in two HCV singly infected controls not assuming drugs (group C). For all these subjects at least two serial samples obtained at the first observation (before HAART) and more than 1 year later, underwent clonal sequence analysis of partial E1/E2 sequences, encompassing the whole HVR1. Evolutionary rates, dated phylogenies and population dynamics were co-estimated by using a Bayesian Markov Chain Monte Carlo approach, and site specific selection pressures were estimated by maximum likelihood-based methods. The intra-host evolutionary rates of HCV quasispecies was 10 times higher in subjects treated with HAART than in controls without immunodeficiency (1.9 and 2.3×10−3 sub/site/month in group A and B and 0.29×10−3 sub/site/month in group C individuals). The within-host Bayesian Skyline plot analysis showed an exponential growth of the quasispecies populations in immunological responders, coinciding with a peak in CD4 cell counts. On the contrary, quasispecies population remained constant in group B and in group C controls. A significant positive selection pressure was detected in a half of the patients under HAART and in none of the group C controls. Several sites under significant positive selection were described, mainly included in the HVR1. Our data indicate that different forces, in addition to the selection pressure, drive an exceptionally fast evolution of HCV during HAART immune restoration. We hypothesize that an important role is played by the enlargement of the viral replicative space

Spatial and Temporal Dynamics of Hepatitis B Virus D Genotype in Europe and the Mediterranean Basin

Hepatitis B virus genotype D can be found in many parts of the world and is the most prevalent strain in south-eastern Europe, the Mediterranean Basin, the Middle East, and the Indian sub-continent. The epidemiological history of the D genotype and its subgenotypes is still obscure because of the scarcity of appropriate studies. We retrieved from public databases a total of 312 gene P sequences of HBV genotype D isolated in various countries throughout the world, and reconstructed the spatio-temporal evolutionary dynamics of the HBV-D epidemic using a Bayesian framework

Time and Mode of Epidemic HCV-2 Subtypes Spreading in Europe: Phylodynamics in Italy and Albania

Hepatitis C virus (HCV) genotype 2 causes about 10% of global infections and has the most variable circulation profile in Europe. The history of “endemic” HCV-2 subtypes has been satisfactorily reconstructed, instead there is little information about the recent spread of the “epidemic” subtypes, including HCV-2c. To investigate the origin and dispersion pathways of HCV-2c, 245 newly characterized Italian and Albanian HCV-2 NS5B sequences were aligned with 247 publicly available sequences and included in phylogeographic and phylodynamic analyses using the Bayesian framework. Our findings show that HCV-2c was the most prevalent subtype in Italy and Albania. The phylogeographic analysis suggested an African origin of HCV-2c before it reached Italy about in the 1940s. Phylodynamic analysis revealed an exponential increase in the effective number of infections and Re in Italy between the 1940s and 1960s, and in Albania between the 1990s and the early 2000s. It seems very likely that HCV-2c reached Italy from Africa at the time of the second Italian colonization but did not reach Albania until the period of dramatic migration to Italy in the 1990s. This study contributes to reconstructing the history of the spread of epidemic HCV-2 subtypes to Europe

Compartmentalization of Hepatitis C Virus Quasispecies in Blood Mononuclear Cells of Patients with Mixed Cryoglobulinemic Syndrome

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance

Prevalenza ed epidemiologia molecolare di HBV in soggetti HIV-1 positivi e in coppie mamma/bambino viventi in Per\uf9

Tra le principali infezioni a trasmissione sessuale, il virus dell\u2019epatite B (HBV) e quello dell\u2019immunodeficienza umana (HIV) rappresentano un\u2019 importante minaccia alla salute pubblica in molti Paesi dell\u2019America Latina. Secondo i dati UNAIDS 2003, attualmente vivono in Per\uf9 82.000 persone infette da HIV-1 (c.ca 0.5% della popolazione), di cui 27.000 donne e 2.000 bambini. Un\u2019elevata endemia di infezione da HBV \ue8 riportata sia in popolazioni native dell\u2019Amazzonia che nei pi\uf9 importanti centri urbani (prevalenza di portatori di HBsAg intorno al 10%). Pazienti e metodi Sono stati inclusi nello studio 395 soggetti, di cui 150 bambini figli di madre HIV-1 positiva e 245 individui adulti (in prevalenza donne) tutti HIV positivi, afferenti ad una casa famiglia di Lima. La determinazione dell\u2019HBV-DNA \ue8 stata eseguita mediante amplificazione del gene S di HBV utilizzando un protocollo di nested-PCR. La tipizzazione molecolare \ue8 stata ottenuta mediante sequenziamento e analisi filogenetica dei prodotti di amplificazione. Risultati In totale 57 soggetti (14.4%) sono risultati HBV-DNA positivi. Tra gli adulti la prevalenza di HBV-DNA \ue8 risultata del 12%, mentre tra i bambini \ue8 pari al 19.3%. In particolare, 41 (27.3%) dei 150 bambini inclusi nello studio, erano infettati da HIV, mentre i rimanenti erano HIV negativi: la prevalenza di HBV-DNA \ue8 risultata simile nei due gruppi (15% vs. 21%, rispettivamente-p=0.4). In 6 casi abbiamo identificato la contemporanea positivit\ue0 per HBV-DNA della mamma e del bambino. L\u2019 analisi filogenetica ha mostrato la presenza di 4 genotipi: F, A, G e D. Il genotipo F \ue8 risultato il pi\uf9 rappresentato (80.7% di prevalenza) seguito dai genotipi A e G (5 individui ciascuno) e dal D (1 individuo). Analizzando le coppie mamma/bambino, abbiamo potuto osservare che in 2 dei 6 casi di contemporanea positivit\ue0 il genotipo presente nella mamma non corrispondeva a quello del bambino. Questi dati indicano una larga diffusione dell\u2019infezione da HBV tra i soggetti HIV positivi e tra i bambini figli di mamme HIV positive viventi in Per\uf9. L\u2019elevata prevalenza di infezione da HBV evidenziata tra i bambini sembra essere attribuibile solo in parte a trasmissione verticale. Questi dati indicano l\u2019esigenza di una pi\uf9 attenta sorveglianza dell\u2019infezione da HBV e l\u2019urgenza dell\u2019adozione di programmi di immunizzazione per la prevenzione dell\u2019infezione in et\ue0 infantile

Spatial and temporal reconstruction of bovine viral diarrhea virus genotype 1 dispersion in Italy

Bovine viral diarrhea virus (BVDV) is a widespread and economically important pathogen of cattle; genetic typing of BVDV isolates distinguished two species, namely BVDV-1 and BVDV-2. BVDV-1 is the most widespread worldwide and it includes at least 11 subtypes. With the aim of clarifying the routes of circulation of BVDV-1 subtypes in an endemic area and in order to investigate the relationships between the genetic diversity of BVDV and its geographic distribution, a phylogenetic analysis of 5' untranslated region of Italian sequences was performed using a new Bayesian framework allowing the spatial-temporal reconstruction of the evolutionary dynamics of highly variable viruses. Our analyses suggested that different BVDV subtypes entered the North-Eastern part of Italy at different times within a time span between 23 and 7 years ago. The largest virus dispersion occurred between the mid 1990s and the early 2000s. A possible gravity-like dynamic of the infection, originating in larger animal population then following patterns of national commercial-flow, should be hypothesized

Whole-genome sequencing and comparative genomic analysis of Salmonella enterica serovar Napoli strains isolated from human cases occurred in Lombardy and Emilia-Romagna, Northern Italy

Background: Salmonella enterica serovar Napoli (S. Napoli) is uncommon in Europe except in Italy, where it ranks among the top five serovars causing human infection. However, information about its epidemiology, ecology and virulence is poor, and no foodborne or environmental factor has been identified so far. Importantly, recent studies conducted by our group have revealed that S. Napoli and typhoid serovars are highly related, in terms of genomics and clinical characteristics. Material/methods: Forty-four S. Napoli strains isolated from human cases in Lombardy and Emilia-Romagna, Italy (2012-2014), were subjected to whole-genome sequencing (WGS) using the Illumina MiSeq platform. Bayesian SNP-based phylogeny was reconstructed using the kSNP software. A comparative genomic analysis was performed to detect the differences between the S. Napoli genomes, in terms of nucleotide variations. Results: Phylogenetic analysis revealed that S. Napoli isolates are grouped in two main clades that strongly correlate with their geographic origin (Figure 1). Clade A (n=16 isolates) included most of the isolates from Emilia-Romagna, and clade B (n=28 isolates) comprised most of the isolates from Lombardy, including the strain SMOUT310_MI, responsible for causing a recent outbreak in Milan (2014). Conclusions: In the present study, we compared S. Napoli isolates from two different Italian regions by SNP typing based on WGS. The results indicate that WGS coupled to SNP-based phylogeny seems an excellent approach to infer the genomic and geographic distribution of serovar Napoli isolates, and that our analysis can provide valuable supplemental information about this emerging public health concern in Italy. Future studies are needed to further improve the usefulness of WGS analysis, in particular regarding genetic changes over time and diversity within regions, in order to make hypothesis regarding the origin and the geographical dispersion of this poorly studied serovar