viruses Article Retrospective Characterization of the 2006–2007 Swine Vesicular Disease Epidemic in Northern Italy by Whole Genome Sequence Analysis Giulia Pezzoni 1 , Arianna Bregoli 1, Chiara Chiapponi 1 , Santina Grazioli 1, Antonello Di Nardo 2,* and Emiliana Brocchi 1 1 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, 25124 Brescia, Italy; [email protected] (G.P.); [email protected] (A.B.); [email protected] (C.C.); [email protected] (S.G.); [email protected] (E.B.) 2 The Pirbright Institute, Pirbright, Woking, Surrey GU24 0NF, UK * Correspondence: [email protected] Abstract: Advances in the epidemiological tracing of pathogen transmission have been largely driven by the increasing characterisation of whole-genome sequence data obtained at a finer resolution from infectious disease outbreaks. Dynamic models that integrate genomic and epidemiological data further enhance inference on the evolutionary history and transmission dynamics of epidemic outbreaks by reconstructing the network of ‘who-infected-whom’. Swine Vesicular Disease (SVD) was present in Italy from 1966 until 2015, and since the mid-1990s, it has mainly been circulating Citation: Pezzoni, G.; Bregoli, A.; within Italy’s central-southern regions with sporadic incursions to the north of the country. However, Chiapponi, C.; Grazioli, S.; Di Nardo, a recrudescence of SVD in northern Italy was recorded between November 2006 and October 2007, A.; Brocchi, E. Retrospective leading to a large-scale epidemic that significantly affected the intensive pig industry of the Lombardy Characterization of the 2006–2007 region. In this study, by using whole-genome sequence data in combination with epidemiological Swine Vesicular Disease Epidemic in information on disease occurrences, we report a retrospective epidemiological investigation of the Northern Italy by Whole Genome 2006–2007 SVD epidemic, providing new insights into the transmission dynamics and evolutionary Sequence Analysis. Viruses 2021, 13, mode of the two phases that characterised the epidemic event. Our analyses support evidence of 1186. https://doi.org/10.3390/ v13071186 undetected premises likely missed in the chain of observed infections, of which the role as the link between the two phases is reinforced by the tempo of SVD virus evolution. These silent transmissions, Academic Editors: Douglas Gladue, likely resulting from the gradual loss of a clear SVD clinical manifestation linked to sub-clinical Fernando Rodriguez and infections, may pose a risk of failure in the early detection of new cases. This study emphasises the Manuel Borca power of joint inference schemes based on genomic and epidemiological data integration to inform the transmission dynamics of disease epidemics, ultimately aimed at better disease control. Received: 21 May 2021 Accepted: 18 June 2021 Keywords: swine vesicular disease; epidemiological tracing; outbreak investigation; phylodynamics; Published: 22 June 2021 transmission tree; Italy Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Swine vesicular disease (SVD) is a viral infectious disease in pigs caused by a member of the Enterovirus genus (Enterovirus B species) of the family Picornaviridae [1]. Vesicular lesions are usually found on the coronary bands of infected animals, but vesicles might also appear on their snout, lips, tongue, and teats. Differential laboratory diagnosis with Copyright: © 2021 by the authors. foot-and-mouth disease (FMD) is critical due to the indistinguishable features of the clinical Licensee MDPI, Basel, Switzerland. manifestations of the disease. For this reason, SVD was included in the World Organisation This article is an open access article for Animal Health (OIE)’s list of notifiable diseases until 2015. However, compared to FMD, distributed under the terms and SVD is moderately contagious and is characterised by lower morbidity, milder clinical signs, conditions of the Creative Commons Attribution (CC BY) license (https:// and may progress subclinically in pigs. Pathogenesis varies according to the infecting virus creativecommons.org/licenses/by/ strain, age of the host, route and dose of infection, and the farming conditions [2]. Disease 4.0/). transmission between farms is mainly linked to the movements of infected animals and/or Viruses 2021, 13, 1186. https://doi.org/10.3390/v13071186 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 1186 2 of 17 via fomites, despite a limited tendency for the virus to diffuse even at the within-farm level [2–4]. The SVD virus (SVDV) is a single-stranded, non-enveloped, positive-sense RNA virus of ~7.4 kb in length. All of the viral lineages belong to a single serotype, which is further characterised by four antigenic/genomic variants [5]. Clinical cases of SVD were initially reported in October 1966 from two farms in the Lombardy region of Italy [1]. The virus was also isolated during late 1971 in Hong Kong [6]. Although the exact geographical origin of the SVDV still remains to be elucidated, its biological origin has been ascribed to co-infection, recombination, and a single anthroponotic transfer event which may have resulted from large viral meningitis epidemics recorded around 1960–1961 [7]. Enterovirus B serotype coxsackievirus B5 and A9 were regarded as the most likely viruses involved in this evolutionary process [7]. The SVDV introduction in Europe has allegedly been attributed to the importation of animals and/or animal products from the Far East, however the exact time of the introduction into Europe is uncertain since SVD cases might have been going undetected due to the magnitude of FMD cases reported in the 1960s [7]. Starting from the 1970s, the SVDV has been reported in countries in both Western and Eastern Europe, with outbreaks sequentially caused by the three different antigenic/genomic variants identified after the first one was detected in 1966 [5]. Since the early 1990sand after the latest outbreaks were reported in the Netherlands, Belgium, and Spain, SVD has been eliminated from most of the European territories, with the exception of Portugal and Italy. From then on, sporadic recurrences of the disease in Portugal were evidenced in 2003 and 2007 [8]. In Italy, the fourth SVDV antigenic variant [5] has been circulating for more than 20 years, mainly in Italy’s southern regions with sporadic incursions in the north of the country. During the last 20-year period, two different lineages have been detected in Italy: one evolved exclusively in Italy from viruses first introduced in 1992, and the second one was detected in2004 and is closely related to the viruses circulating in Portugal during 2003, 2004, and 2007 [8,9]. The last major epidemic of SVD occurring in Northern Italy was reported in 2006, causing a total of 53 outbreaks with the culling of ~150,000 animals and an estimated financial impact of ~EUR 30 million [3]. On the 2nd of October 2006, pigs were tested SVD seropositive at a slaughterhouse in the Italian province of Bergamo (within the Lombardy region) after 4 years of SVD being absent from northern Italy [3]. During the 11 months of the epidemic course recorded from November 2006 to October 2007, SVD spread widely in a densely populated pig area of Lombardy (Figure1). The epidemic event was characterised by two subsequent phases, with the recrudescence of infections detected at a 4 month from the last SVD case identified during the initial phase (Supplementary Figure S1). During the same time period, SVD outbreaks were also reported in both central and southern Italy (Supplementary Figure S1), but these were not found to be linked with the Lombardy cluster, according to field epidemiological tracing. In this study, 37 whole-genome sequences (WGS) generated from SVD isolates col- lected from farms affected during the entire timeframe of the epidemic and 6 further WGSs of contemporary viruses isolated from the SVD outbreaks reported in southern Italy (Figure1A) have been used to retrospectively retrace the transmission history of the epidemic by co-analysing the epidemiological and genetic data. We report a detailed reconstruction of the epidemic event, describing the space and rate of its epidemiological and evolutionary scales and providing evidence for undetected silent/sub-clinical virus transmissions. Knowledge of the risk factors for infectivity and transmission gained by a thorough understanding of the dynamics of infections can be translated into more effective interventions for better disease control. Viruses 2021, 13, 1186 3 of 17 Viruses 2021, 13, x FOR PEER REVIEW 3 of 18 FigureFigure 1.1. GeographicalGeographical distributiondistribution of of the the Italian Italian municipalities municipalities affected affected by by the the 2006–2007 2006–2007 SVD SVD epidemic epidemic investigated investigated in this in study.this study. Full geographicalFull geographical extent extent of the epidemicof the epid eventemic including event including the southern the southern Italian cluster Italian ( Acluster) and the(A) northern and the epidemicnorthern clusterepidemic only cluster (B). Geographicalonly (B). Geographical locations arelocations presented are aspresented region (firstas region admin-level), (first admin-level), province (second province admin-level), (second admin- and level), and municipalities (third admin-level). municipalities (third admin-level). In this study, 37 whole-genome