Appendix 26 Genetic and antigenic analysis of Italian 1993 FMDV isolates a
J. I. Núñeza,b , P. Fusic, B. Borregoc, E. Brocchic, M.L. Pacciarinic and F. Sobrinoa,b,* Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Cantoblanco 28049, Madrid, Spain b Centro de Investigación en Sanidad Animal, INIA, Valdeolmos 28130, Madrid, Spain c Istituto Zooprofilatico Sperimentale di la Lombardia e la Emilia. Brescia, Italy *Corresponding author: F. Sobrino. Tfn. 34-91-4978246; Fax: 34-91-4978087; e-mail: fsobrino@cbm.uam.es
Abstract: The origin and evolution of foot-and-mouth disease type O viruses that caused the outbreak occurred in Italy in 1993, the first episode of the disease in the EU after adoption of a non-vaccination policy that started in 1991, have been studied by the analysis of sequences encoding three main antigenic sites on viral capsid proteins. The phylogenetic tree derived from sequences spanning the carboxyterminal end of VP1 showed that the Italian viruses were closely related to previous Middle East isolates. For some of the isolates analyzed values of fixation of nucleotide substitutions per year were observed in the three regions analyzed, ranging from 1.7 to 2 x 10-2. The use of a panel of new monoclonal antibodies rose against an isolate from this outbreak, as well as monoclonal antibodies raised against FMDV O1-Switz, showed differences in the reactivity pattern among some of the Italian isolates analyzed. These results support the potential for viral diversification in a limited period of time and under epidemiological conditions in which no vaccination campaigns were being implemented. Introduction: Foot-and-mouth disease (FMD) was eradicated in the EU in 1991, after more than two decades of regular vaccination with chemically inactivated viruses (Barteling and Vreeswijk, 1992). Since then, a non-vaccination, total stamping out policy was adopted, following demonstration of the vaccine origin of the most of the latest outbreaks (Beck and Stromaier, 1987; Carrillo et al., 1990). However, there is an evident risk of reintroduction of the disease not only from border areas but also from far locations as took place in U.K. and other European countries in 2001 (Knowles 2001), associated to the growing globalisation of livestock trade (Sobrino and Domingo, 2001). Protection against FMD is associated to the induction of FMDV neutralizing antibodies in vaccinated and convalescent animals (Van Bekkum, 1969; Martin and Chapman 1961). FMDV capsid proteins contain B cell antigenic sites that constitute the targets of the neutralizing antibodies elicited in natural hosts (reviewed in Brown, 1995; Mateu et al., 1995). The main B cell antigenic site in capsid proteins has been identified for serotypes A, O and C (reviewed in Brown, 1995; Mateu, 1995). The high potential for genetic and antigenic variation is one of the most important FMDV features (Domingo et al., 1990; 2003), which derives from the cuasispecies structure of its viral populations (Domingo et al., 1992). This structure poses important implications on its biology and control: among them, the high antigenic diversity of viral populations that is reflected in seven serotypes and a multitude of variants (Arrowsmith 1977; Pereira, 1981). This antigenic variability affects vaccination strategies because of the need to adequate the vaccines to the antigenic properties of field viruses (Kitching, 1989). As complement of classical serological methods, nucleotide sequencing and phylogenetic analyses have become valuable tools for the characterization and epidemiological tracing of FMD outbreaks (Armstrong et al., 1994; Kitching et al., 1989, Samuel et al., 1988). Phylogenetic analyses have been mostly based on sequences from the VP1-coding region (Beck and Strohmaier, 1987; Dopazo et al., 1988; Martínez et al., 1992; Sangare et al., 2003), particularly on its 3’end 250 nt (Pattnaik et al., 1998; Samuel et al., 1999; Vosloo et al., 1992), which contains one of the main B cell antigenic site, corresponding to VP1 residues 140-160 (Bittle et al., 1982; Strohmaier et al., 1992). Phylogenetic trees constructed from these 3’ end region reproduce those derived from the whole VP1 sequence (Núñez et al, 2001). The use of panels of monoclonal antibodies (MAbs) has proven useful to study the antigenic variation of type C viruses in the field (reviewed in Mateu, 1996). Thus, selection of antigenic variants, with amino acid replacements at neutralizing antigenic sites, has been reported during type C FMDV epizootics in epidemiological situations in which part of the susceptible animals were vaccinated (Mateu et al., 1987; 1988). For type O FMDV, a main neutralizing antigenic site has been identified on the capsid protein VP1 (site 1), being composed by residues 140-160 located at the G-H loop and residues 200-213 at the carboxyterminal end (Kitson et al., 1990). In addition, residues 70 to 77 and position 131 in VP2 and 56 to 58 in VP3 have been reported to contribute to the discontinuous neutralizing antigenic sites 2 and 4, respectively (Kitson et al., 1990).
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