Journal of Virological Methods 138 (2006) 99–108 Multiple recombinant ELISA for the detection of bovine viral diarrhoea virus antibodies in cattle sera S. Chimeno Zoth a,b,∗ , Oscar Taboga a a Instituto de Biotecnolog´ ıa, CICVyA, INTA, Castelar, CC25 (1712), Buenos Aires, Argentina b Consejo Nacional de Investigaciones Cient´ ıficas y Tecnol´ ogicas (CONICET), Rivadavia 1917 (1033), Ciudad de Buenos Aires, Argentina Received 18 April 2006; received in revised form 26 July 2006; accepted 31 July 2006 Available online 8 September 2006 Abstract The most immunogenic proteins (E0, E2 and NS3) of bovine viral diarrhoea virus (BVDV) (NADL strain) were expressed in the baculovirus/insect cells system. Recombinant antigens were applied to the design of enzyme immunoabsorbent assays (ELISAs) for the detection of specific antibodies in cattle sera. The assays developed were shown to be highly sensitive and specific in comparison with the viral neutralization test, which is the reference test for the serological diagnosis of BVDV. The present results demonstrate the contribution of each recombinant antigen to determine clearly the pattern of anti-BVDV antibodies in bovine serum samples. © 2006 Elsevier B.V. All rights reserved. Keywords: BVDV; Recombinant antigens; ELISA 1. Introduction Bovine viral diarrhoea virus (BVDV) infection is distributed worldwide. Although the prevalence of the infection varies, the infection tends to be endemic in many countries, reaching a maximum level of 1–2% of cattle persistently infected (PI) and 60–85% of antibody-positive cattle (Houe, 1999). BVDV infec- tion is often subclinical or causes mild and non-specific clinical symptoms. However, transplacental infection can lead to repro- ductive disorders, teratogenic defects or birth of immunotolerant persistently infected calves (Baker, 1995). These calves have either no or low levels of specific antibodies, but shed virus con- stantly and, therefore, contribute to maintain the infection in the herd. BVDV is a member of the Pestivirus genus in the Flaviviridae family. Classical swine fever virus (CSFV) and Border disease virus (BDV) are the two other members of this group of positive- stranded RNA viruses. The genome of BVDV has a single open reading frame that is translated to a polyprotein of about 4000 amino acids, which is processed further by viral and cellular proteases into the final components (Collett et al., 1991). ∗ Corresponding author. Tel.: +54 11 4621 1278; fax: +54 11 4621 0199. E-mail address: schimeno@cicv.inta.gov.ar (S. Chimeno Zoth). Among viral proteins, the structural proteins E0 and E2, and the non-structural NS3 protein have been identified as the prin- cipal immunogens of BVDV, being E2 the only one that elicits high titers of neutralizing antibodies after infection or vaccina- tion (Donis et al., 1988; Bolin, 1993). In Argentina, the prevalence of BVDV antibodies in adult cattle is around 70% (Rweyemamu et al., 1990; Pacheco and Lager, 2000); 20% of normal fetuses are detected as infected by viral isolation (Mu˜ noz et al., 1996), and 2% are seropositive (Pinto et al., 1993). Jones et al. (2001) reported the presence of BVDV type 1 and BVDV type 2 in Argentina. The genetic typing of 29 BVDV isolates indicated that 90% belonged to BVDV type 1 and 10%, to BVDV type 2. Ode´ on et al. (2003) described the association between Argentinean strains of genotype 1 and 2 and different clinical manifestations, such as acute enteritis, generalized dermatitis and mucosal disease. Considering the complexity of BVDV pathogenesis and clini- cal features, laboratory diagnosis plays an important role. There- fore, a sensitive and specific test for the detection of antibodies would be a valuable tool for the diagnosis of bovine viral diar- rhoea and to monitor the infection status of individual animals and herds. The “gold standard” for antibody detection against BVDV is the virus neutralization test (Edwards, 1990). It is a sensitive and specific assay but cell culture-dependent and labour-intensive in 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.07.025