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Prevalence and phylogenetic analysis of OsHV-1 in wild populations of Pacific oyster Crassostrea gigas in Italy

Erika A. Burioli1, 2*, Maria C. Bona1, Paolo Pastorino1, Maria V. Riina1, Maria L. Fioravanti2, Maryline Houssin3 and Marino Prearo1
  • 1 Istituto Zooprofilattico Sperimentale Piemonte Liguria Valle d'Aosta, Italy
  • 2 Department of Veterinary Medical Sciences, Alma Mater Studiorum, Università di Bologna, Italy
  • 3 Laboratoire Frank Duncombe LABEO, France

Ostreid Herpesvirus type 1 (OsHV-1), and especially the µVar and related genotypes, is considered one of the most serious pathogens for young stages of cupped oysters Crassostrea gigas/angulata (OIE, 2013). Despite the heavy economical impact of virus-induced mortalities, effective measures to prevent and control the disease are nowadays unavailable, in part because of the scarce information on the real diffusion of the virus and its variants, and their relative pathogenicity. During the last five years, detection of OsHV-1 in young oyster stocks farmed in Italy but originating from French hatcheries, was signalled (Dundon et al., 2015; Rosani et al., 2014). However, the health status related to OsHV-1 of these individuals at their arrival has not been established, preventing from concluding on the infection origin. Considering the close contact between wild and reared populations and the excellent water capacities as vehicle of infectious agents, the present study aimed to obtain precious epidemiological information in order to assess the risk of contamination of surrounding wild oyster populations or, on the contrary, to evidence a remote introduction of the pathogen and wild beds acting as virus reservoir. For the first time in Europe, this work focused the study of OsHV-1 on wild stocks of cupped oysters. During spring and summer 2012 and 2014, a total of 600 individuals of Pacific oysters were collected in 12 sites, representative of different environment types (lagoon, gulf, open waters and harbour), along the Italian coasts. The sampling campaign conduced in 2012 consisted in two groups of animals, divided on the basis of their length (≤25mm and ≥80mm). COI sequencing confirmed that all sampled oyster specimens were C. gigas (Bucklin et al., 2011). Detection and quantification (viral genome units UG/50mg of oyster tissue) of OsHV-1 was carried out, for each individual, on a pool of minced organs (heart, gills, mantle and adductor muscle), by a TaqMan® real-time PCR as described by Martenot et al. (2010) and characterised by a detection limit of 6 UG/50mg. The occurrence of the virus was described in terms of prevalence, associated with the 95% confidence interval and a multiple logistic regression was used to test the association between virus infection and potential risk factor as length, environment type and year of sampling. The association was evaluated by calculating the odds ratio. In individuals tested positive for OsHV-1, three regions of the viral genome were explored by sequencing: C region (ORFs 4/5), IAP region (ORFs 42/43) and the third encompassing ORFs 35-38 (Renault & Arzul, 2001; Segarra et al., 2010; Renault et al., 2012). The C region and the concatemers of the three target loci sequences were used to conduce the phylogenetic analysis of the Italian genotypes. Phylogenetic trees were then constructed using the MEGA version 5 program, and included the sequences of OsHV-1 (Davison et al., 2005; Friedman et al., 2005; Grijalva-Chon et al., 2013; Hwang et al., 2013; Jenkins et al., 2013; Martenot et al., 2015; Renault et al., 2012; Segarra et al., 2010; Shimahara et al., 2012; Xia et al., 2015) and Acute Viral Necrosis Virus (AVNV) (Ren et al., 2013) retrieved from GenBank and isolated in C. gigas but also in other molluscs, all over the world. The prevalence rates of the different groups are reported in Figure 1 and were quite low, ranging from 0% to 26.7%. Interestingly, the prevalence observed in 2012 in small individuals (≤25mm) was lower than in large (≥80mm): 10% (95% CI 5.3-16.8%) and 20% (95% CI 13.3-28.3 %) respectively. No significant difference was revealed considering the type of environment but odds ratio analysis showed a statistical significance for the protective effect of year (2014 versus 2012) and length (small versus large individuals). The sequencing results evidenced a certain diversity within the OsHV-1 genome, with the description of 9 different genotypes and a geographical distribution of some variants. Several mutations are attempt to induce changes in the amino-acid sequence and to modify virus phenotype. A genotype very close to the µvar was present in all the sampling sites. The C region resulted the most variable area and the analysis of a high number of sequences present on GenBank revealed that the genotypes closely related to the µVar are all from Europe except two specimens isolated from larvae in a hatchery in South Korea. The OsHV-1 reference genotype and AVNV clustered together, with the majority of variants isolated in non Ostreidae bivalves and with two Italian specimens isolated in C. gigas, in open waters, but situated in individual branches (Figure 2). The detection of OsHV-1 in wild populations, also far from farming locations, and the evidence of different genotypes, suggest a remote introduction of the pathogen in Italian waters. The high virus diversity observed in natural environment in Italy, and reported to date only in East Asia (Mineur et al., 2015), respect to other studies conduced in Europe, could be related to the fact that these studies involved only farmed individuals. In response to a lower genetic variability of hosts, the culture conditions could be responsible of high virus specialisation degree, matching with a low genetic diversity.

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Keywords: Ostreid herpesvirus, wild oyster, Prevalence, genotypes, phylogeny

Conference: AquaEpi I - 2016, Oslo, Norway, 20 Sep - 22 Sep, 2016.

Presentation Type: Poster

Topic: Aquatic Animal Epidemiology

Citation: Burioli EA, Bona MC, Pastorino P, Riina MV, Fioravanti ML, Houssin M and Prearo M (2016). Prevalence and phylogenetic analysis of OsHV-1 in wild populations of Pacific oyster Crassostrea gigas in Italy. Front. Vet. Sci. Conference Abstract: AquaEpi I - 2016. doi: 10.3389/conf.FVETS.2016.02.00012

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Received: 30 May 2016; Published Online: 14 Sep 2016.

* Correspondence: Dr. Erika A Burioli, Istituto Zooprofilattico Sperimentale Piemonte Liguria Valle d'Aosta, Torino, Italy, erika.burioli@izsto.it