Differentiation between field and vaccine strain of bluetongue virus serotype 16

Abstract

In August 2000, bluetongue virus (BTV) appeared for the first time in Sardinia and, since then, the infection spread across Sicily and into the mainland of Italy involving at the beginning serotypes 2 and 9 and then, from 2002, 4 and 16. To reduce direct losses due to disease and indirect losses due to new serotype circulation, the 2004 Italian vaccination campaign included the modified-live vaccines against BTV-4 and 16 produced by Onderstepoort Biological Product (OBP), South Africa. Few months after the end of the campaign, BTV-16 was reported broadly in the country and the need of differentiating field from the BTV-16 vaccine isolate became crucial. In this study, the gene segments 2, 5, 6 and 10 of both the Italian and vaccine BTV-16 strains were sequenced and their molecular relationship determined. As sequences of segment 5 were those showing the highest differences (17.3%), it was possible to develop a new diagnostic tool able to distinguish the Italian BTV-16 NS1 gene from that of the homologous vaccine strain. The procedure based on the use of a RT-PCR and the subsequent sequencing of the amplified product showed a high degree of sensitivity and specificity when samples from either BTV-16 vaccinated or infected sheep were tested.

Introduction

Bluetongue (BT) is an infectious, non-contagious disease caused by an arthropod-borne virus of the Orbivirus genus in the Reoviridae family; besides the Bluetongue virus (BTV), this genus includes other 20 serogroups as recently recognised by the International Committee for the Taxonomy of Viruses (Mertens et al., 2004). Within the BTV serogroup, 24 distinct serotypes have been identified by the virus-neutralisation test (VNT) and all of them used midges of the genus Culicoides as competent vectors (Mellor, 1992). The BTV genome consists of 10 double-stranded RNA segments distinguished from 1 to 10 on the basis of their molecular weight and electrophoretic mobility in 1% agarose gels. Each genome segment encodes for a viral protein for a total of seven structural (VP1–VP7) and four non-structural (NS1–NS3/A) proteins (Verwoerd et al., 1970, Roy et al., 1990a, Roy, 1992). The viral genome is surrounded by a double-layered protein capsid the outer part of which is composed by VP2 and VP5 (Roy, 1996). VP2, derived from segment 2, is the most variable protein (Roy, 1992), it is responsible for virus adsorption, entry into mammalian cells and might determine its virulence (Huismans et al., 1987). VP5, derived from segment 6, is not exposed on the virus surface. It serves as a scaffold for VP2 multimers (Huismans and Erasmus, 1981, Hassan and Roy, 1999) and though capable of binding to the cell surface, it cannot be internalised (Hassan et al., 2001). Both proteins are involved in the serotype specific antigenicity (Mertens et al., 1989). The NS1 protein is a non-structural protein encoded by segment 5. It has been found in BTV-infected cells to form tubular structures whose role is still to be determined (Hewat et al., 1992). The NS3/NS3A are also non-structural proteins differing for 13 amino acids only. Both proteins derived from segment 10, a very highly conserved gene among BTV strains and serotypes (Roy, 1996, Bonneau et al., 1999). The NS3/NS3A act as viral chaperones responsible for virus egression from either mammalian or insect cells (Bansal et al., 1998).

When infecting sheep, BTV might cause a severe clinical disease with mortality rates exceeding 10% (Mellor, 1994, Mellor, 1990, Koumbati et al., 1999). In cattle, goats and wild ruminants, the infection evolves mostly asymptomatically (Anderson et al., 1985). Due to its ability to spread rapidly under suitable circumstances, BT is classified by the Office International des Epizooties (OIE) as a notifiable disease (former List A). During outbreaks, affected countries are banned from trading in livestock and livestock products triggering serious socio-economic effects. Since 2000, Italy has experienced the most severe outbreaks of BT ever to be recorded involving at the beginning BTV serotypes 2 and 9, and then, from 2002, also 4 and 16. BTV has now been reported in Sardinia Sicily, Calabria, Basilicata, Puglia, Campania, Lazio, Tuscany, Abruzzo, Molise, Umbria, Emilia Romagna, Liguria and Marche (Anonymous, 2001, Anonymous, 2003). In an attempt to reduce direct losses due to disease and indirect losses due to virus circulation, since February 2002 the Italian government has been carrying out a compulsory BT vaccination campaign including all susceptible ruminants. Based on the serotype/s present in a given area, various monovalent serotype formulations of modified-live vaccine (MLV) produced by the Onderstepoort Biological Products (OBP), South Africa, were used. As a consequence of the new epidemiological scenario, in the first few months of the 2004 the monovalent BTV-16 modified-live vaccine either in combination with the monovalent BTV-2, BTV-4 and BTV-9 vaccines or with BTV-2 and BTV-4 was added in the composition and used in many Italian regions. Since May 2004, several BTV-16 seroconversions have been recorded in sentinel animals located either in vaccinated or unvaccinated areas supporting the hypothesis of vaccine strain circulation in the environment as already evidenced for BTV-2 (Ferrari et al., 2005). Although no scientific data were available for the BTV-16 vaccine strain, this event could not be discarded and a possible new scenario with both, vaccine and field BTV-16 isolates, contemporaneously circulating should be considered. In such a context, knowing exactly what kind of BTV-16 is circulating in a given area becomes crucial as such information will affect the livestock movement ban imposition with epidemiological and economical implications. This study compared the S2, 5, 6 and 10 sequences of the BTV-16 wild-type virus isolated in 2002 with those of the homologous vaccine strain, identified differences and developed a diagnostic method able to distinguish the two viruses.