Multiple recombinant ELISA for the detection of bovine viral diarrhoea virus antibodies in cattle sera

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

b

Instituto de Biotecnolog´ıa, CICVyA, INTA, Castelar, CC25 (1712), Buenos Aires, Argentina 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 infection is often subclinical or causes mild and non-specific clinical symptoms. However, transplacental infection can lead to reproductive 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 constantly 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 positivestranded 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: [email protected] (S. Chimeno Zoth).

0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.07.025

Among viral proteins, the structural proteins E0 and E2, and the non-structural NS3 protein have been identified as the principal immunogens of BVDV, being E2 the only one that elicits high titers of neutralizing antibodies after infection or vaccination (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 clinical features, laboratory diagnosis plays an important role. Therefore, a sensitive and specific test for the detection of antibodies would be a valuable tool for the diagnosis of bovine viral diarrhoea 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

100

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

comparison with ELISA. Therefore, the latter is usually preferred when a large sample throughput is required (Lindberg, 2003). Conventional ELISAs based on BVDV infected cells usually are disadvantageous because BVDV produces low levels of proteins in tissue culture and it is difficult to purify the virus because it is cell-associated. Thus, obtaining adequate quantities of BVDV pure proteins for diagnosis is laborious (Justewicz et al., 1987). Recombinant antigens constitute an alternative source. Reddy et al. (1997) described the use of recombinant BVDV antigens, NS3 and E0 obtained in bacteria and the precursor region of E0 and E1 obtained in insect cells for the development of ELISA. This and other studies (Petric et al., 1992; Vanderheijden et al., 1993) demonstrated the ability of BVDV antigens expressed in the baculovirus system to provide the reliable detection of BVDV antibodies in bovine sera. Nevertheless, none of the published reports include a detailed analysis of a large number of bovine serum samples, evaluating the individual contribution of recombinant antigens to the characterization of the samples tested. The aim of the present study was to develop recombinant ELISAs based on the three main antigens of BVDV to detect specific antibodies in bovine sera, with a sensitivity and specificity comparable with the virus neutralization test. The complete coding regions for E0 and E2 glycoproteins and a partial sequence coding for NS3 protein of BVDV NADL strain (genotype 1) were introduced into the baculovirus genome and the recombinant products were recovered from Sf9-infected cell lysates. The ability of the recombinant antigens to detect anti-BVDV antibodies in cattle sera was evaluated and the results compared with those obtained by virus neutralization. The results demonstrated that the simultaneous application of the three recombinant antigens in the design of a multiplex ELISA allowed the detection of anti-BVDV antibodies efficiently. 2. Materials and methods 2.1. Viruses and cells The NADL strain of BVDV (passage 3, provided by the Virology Institute of the Instituto Nacional de Tecnolog´ıa Agropecuaria (INTA)) was propagated in Madin Darby bovine kidney (MDBK) cells, cultured with minimum essential medium (MEM) (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum, 0.5% antibiotic solution (penicillin–streptomycin; Sigma–Aldrich, St. Louis, MO) and incubated at 37 ◦ C. The Autographa californica nuclear polyhedrosis virus (AcNPV, Pharmingen, San Diego, CA) was used as a vector for the expression of E0, E2 and NS3 proteins, under the regulation of the polyhedrin promoter. AcNPV was grown in monolayers of the Spodoptera frugiperda (Sf9) cell line. Insect cells were grown at 27 ◦ C in TNM-FH medium (Sigma–Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% antibiotic–antimycotic solution (Sigma–Aldrich, St. Louis, MO). Baculovirus and insect cell

techniques were carried out as described by O’Reilly et al. (1994). 2.2. Antibodies Monoclonal antibodies (mAb) 15C5 (NADL-E0 specific) and 19f9fb (NADL-E2 specific) were kindly provided by Dr. Rub´en Donis of Nebraska University, USA. An anti-his tag monoclonal antibody (Amersham, Buckinghamshire, UK) was employed for the detection of NS3 protein. Cattle sera were provided by the Virology Institute (INTACastelar, Argentina) and by Dr. A. Ode´on (INTA-Balcarce, Argentina). Serum samples were obtained from collections of bovine sera evaluated by viral neutralization against BVDV (type 1) NADL strain, available in the Virology Institute, INTACastelar. 2.3. Construction of recombinant baculoviruses expressing E0, E2 and NS3 E0, E2 and NS3 coding regions were amplified from cDNA synthesized from genomic BVDV RNA (NADL strain), isolated by TRIzol method (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. The precipitated RNA was dissolved in DEPC-treated water and 500 ng of random hexamers (Promega, Madison, USA) were added. After 10 min at 70 ◦ C and 1 min in ice, 5× RT buffer (Promega, Madison, USA), 10 mM dNTPs and 200 U of M-MLV RT (Promega, Madison, USA) were added. The reaction was incubated for 1 h at 37 ◦ C. Thereafter, the enzyme was inactivated 5 min at 95 ◦ C. Amplification of E0 and E2 coding regions was carried out as described previously (Zoth et al., 2001). Briefly, the E2 coding region was amplified with E2f and E2r oligonucleotides (Table 1), which amplify a 1300 bp fragment (from position 2415 to 3716) and E0 was amplified with E0f and E0r oligonucleotides, which amplify a 745 bp fragment (from position 1130 to 1875) based on NADL nucleotide sequence reported by Collett et al. (1988b). Oligonucleotides included the BamHI and XbaI restriction sites for cloning into pVL1393 transfer plasmid (Pharmingen, San Diego, CA). Amplification of NS3 coding region was carried out with NS3-f and NS3-r oligonucleotides, which amplify a 1099 bp fragment (from position 6544 to 7643) based on NADL nucleotide sequence (Collett et al., 1988b) and include the NdeI

Table 1 Oligonucleotides employed to amplify E0, E2 and NS3 coding regions Oligonucleotide

Sequence

E0f E0r E2f E2r NS3f NS3r

5 -CGCGGATCCATGAAACTGGAAAAAGCATTG-3 5 -AGGTCTAGATTAAGCGTATGCTCCAAACCAC-3 5 -GGGATCCACCATGGTACAGGGCATTCTG-3 5 -GAAGCTTCTAGATTAGAGTAAGACCCACTT-3 5 -TTCATATGCAAAGGGGAGGATCTT-3 5 -AGTCTGCAGTTATGTCCCATCGGT-3

Underlined sequences correspond to restriction sites used for cloning into the transfer plasmid.

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

101

Fig. 1. Detection of recombinant proteins by Western blot. Total proteins of cellular extracts of Sf9 cells infected with the recombinant baculoviruses (A, AcNPV/E0; B, AcNPV/E2; C, AcNPV/NS3) were separated by SDS-PAGE and the recombinant proteins were detected by Western blot with monoclonal antibodies (A, anti E0; B, anti E2; C, anti his). Wt, Sf9 cells infected with wild type baculovirus AcNPV; Mi, Sf9 cells mock infected; MW, standards of molecular weight.

and PstI restriction sites for cloning into pAcGHLT-C transfer plasmid (Pharmingen, San Diego, CA). All the reaction mixes contained 10× Taq DNA polymerase buffer (Promega, Madison, USA), 25 mM MgCl2 , 10 mM dNTPs, 300 ng of each oligonucleotide, 2 U of Taq DNA polymerase (Promega, Madison, USA) and 5 ␮l of cDNA. Each of the 35 cycles consisted of 30 s at 94 ◦ C, 30 s at 55 ◦ C (for E2 and NS3) or 50 ◦ C (for E0) and 60 s at 72 ◦ C followed by 7 min at 72 ◦ C after the last cycle. PCR products were cloned in the corresponding transfer plasmids and recombinant plasmids were isolated following classical molecular biology protocols (Maniatis et al., 1987). Sf9 insect cells were co-transfected with each recombinant vector and A. californica nuclear polyhedrosis virus (AcNPV) DNA, following the BaculoGold kit instructions (Pharmingen, San Diego, CA). Recombinant baculoviruses were purified by two rounds of final end point dilution (O’Reilly et al., 1994) and selected clones were amplified by three passages in Sf9 insect cells. Finally, each viral stock (AcNPV/E0, AcNPV/E2 and AcNPV/NS3) was titrated and stored at 4 ◦ C. 2.4. Western blot assay Monolayers of Sf9 cells were infected with each viral inoculum at a multiplicity of infection (moi) of 5. After 72 h, cells were collected and centrifuged for 15 min at 200 × g. Then, cells were washed with phosphate buffer (PBS) pH 6.2 and resuspended in cracking buffer (Harlow and Lane, 1988). Cel-

lular extracts were analysed by SDS-PAGE and the presence of recombinant proteins was confirmed by Western blot (Fig. 1), following standard procedures (Harlow and Lane, 1988). Briefly, nitrocellulose membranes were blocked for 1 h at room temperature with a solution containing 5 mM Tris–HCl pH8, 15 mM NaCl, 0.05% Tween-20 (TBS-T) and 5% non-fatty powder milk. Recombinant E2 was detected with 19f9fb mAb (NADL-E2 specific, diluted 1:10000), recombinant E0 with 15C5 mAb (NADL-E0 specific, diluted 1:5000) and recombinant NS3 with anti-his mAb diluted 1:5000. After three washes with TBS-T, membranes were incubated with a solution containing 0.25 ␮g/ml of alkaline phosphatase conjugate anti-mouse IgG (Sigma–Aldrich, St. Louis, MO), washed, and finally stained with 33 ␮l of nitro blue tetrazolium (NBT, Promega, Madison, USA) and 16.5 ␮l of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, Promega, Madison, USA) in 5 ml of alkaline phosphatase buffer (100 mM Tris–HCl pH9, 150 mM NaCl, 1 mM MgCl2 ). 2.5. Production of ELISA antigen 2 × 108 Sf9 cells were infected with AcNPV/E0 or AcNPV/E2 at a moi of 5 for the production of E0 and E2 antigens. The infected cells were collected 72 h after infection and pelleted by centrifugation for 15 min at 200 × g. After one wash with PBS pH 6.2, cells were resuspended in 15 ml of lysis buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% IGEPAL CA-630 (Sigma–Aldrich, St. Louis, MO)). Following an incubation of 45 min on ice and centrifugation (9300 × g,

102

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

20 min at 4 ◦ C), the supernatant (soluble fraction) was collected, aliquoted and stored at −70 ◦ C to be used as ELISA antigen. The same procedure was carried out with mock-infected Sf9 cells, to obtain antigen for the negative control. 1.8 × 108 Sf9 cells were infected with AcNPV/NS3 at a moi of 5 for the production of NS3 antigen. The infected cells were collected 72 h after infection and pelleted by centrifugation for 15 min at 200 × g. After one wash with PBS pH 6.2, cells were resuspended in 2.5 ml of distilled water and 0.5 ml of 6× cracking buffer. Proteins present in cellular extracts were separated in SDS-PAGE and stained with Coomassie blue. The band corresponding to the NS3 protein was cut and electroeluted in 9 ml of Tris–glycine–SDS buffer (Harlow and Lane, 1988). The recovered fraction was aliquoted and stored at −70 ◦ C to be used as an ELISA antigen. The same procedure was repeated with mockinfected Sf9 cells, to obtain antigen for the negative control. 2.6. Indirect ELISA Independent ELISA tests based on E0, E2 or NS3 antigens were developed. Optimal dilutions of each antigen as well as bovine sera and anti-bovine conjugated with horseradish peroxidase (KPL, Gaithersburg, MD) were determined by checkerboard titration. Positive and negative samples were employed in order to establish the optimal conditions of each step of the assays. They consisted of a pool of bovine sera previously tested by viral neutralization assay. Microtitration plates (Maxisorp, Nunc, Rochester, NY) were coated with an optimal dilution of each antigen (1:729 for E0 and E2, 1:81 for NS3) containing approximately 100 ng/well for E0 and E2, and 300 ng/well for NS3 (as estimated by Western blot), in carbonate buffer pH 9.6, by overnight incubation at 4 ◦ C. After each step, plates were washed five times with washing buffer (PBS, 0.05% Tween-20). After removing the excess of the unbound antigen, a blocking step was performed for 1 h at 37 ◦ C with PBS, 0.05% Tween-20, 5% horse serum (PBS-T-HS), 5% unfatty powder milk. A 100 ␮l volume of an optimal dilution of bovine sera (1:200 for E0 and E2, 1:100 for NS3) in PBS-T-HS was prepared and, before adding it to the plate, it was incubated for 1 h in another microplate coated with an extract of Sf9 cells (the same used as negative control, diluted 1:200) in order to decrease the background. After this incubation, sera dilutions were transferred to the ELISA plate and incubated for 1 h at 37 ◦ C. Finally, 100 ␮l of a 1:2000 dilution (25 ng/well) of horseradish peroxidase conjugated antibody (KPL, Guildford UK GU2 5GN) in PBS-T-HS were applied for 1 h at 37 ◦ C. A 100 ␮l volume of the substrate solution (ABTS 0.5 mg/ml (Sigma–Aldrich, St. Louis, MO) in 0.1 M citrate buffer, pH 4.2 containing 0.03% hydrogen peroxide) was added, and absorbance readings at 405 nm were determined after incubation at room temperature (45–60 min for E0 and E2, 15–20 min for NS3). Each sample was tested in duplicate and the corresponding result was expressed as the difference between the OD405nm value obtained with the recombinant antigen (E0, E2, NS3) and the one obtained with the negative control (Sf9 antigen) for each serum sample.

2.7. Virus neutralization test All bovine sera employed in these assays were tested for the presence of virus neutralizing antibodies against BVDV using standard microtitration procedures (Maisonnave and Rossi, 1982). Briefly, volumes of 100 ␮l of serial two-fold dilutions of heat inactivated sera were mixed with 100 TCID50 of BVDV (cytopathic NADL strain) in 50 ␮l and incubated for 1 h at 37 ◦ C in a humidified CO2 incubator. The mix was added to monolayers of MDBK cells seeded in microtitration plates with Minimum Eagle Medium (Life Technologies, Grand Island, NY), supplemented with 2% fetal bovine serum (Natocor, Argentina), 0.5% antibiotic solution (penicillin–streptomycin; Sigma–Aldrich, St. Louis, MO). After 72 h of incubation at 37 ◦ C in a humidified CO2 incubator, the test was read microscopically. Neutralizing titers were determined according to Reed and Muench method (Reed and Muench, 1938). Virus neutralization against BVDV type 2 was performed with 80613 CP isolate, (Weber, personal communication). 2.8. Determination of cut-off values In order to determine the cut-off value of each ELISA, a total of 141 negative and 60 positive sera (tested previously by virus neutralization) were evaluated. Obtained values were then analysed by Box and Whisker test (Statistix 1.0 Version, © 1996 Analytical Software), which identified the “probable outliers”, data that were eliminated of the analysis. Different values that are applied usually as cut-off values (negative mean + 2s, negative mean + 3s, negative mean + 4s), where the negative mean corresponds to the mean of the values showed by the negative sera, and s corresponds to the standard deviations, were calculated (Sutula et al., 1986). With the purpose of selecting the optimal cut-off value, each of these points was employed to estimate the sensitivity (Se) and specificity (Sp) of the three assays (Jacobson, 1998), where: Se =

number of positives by the developed assay (ELISA) number of positives by the virus neutralization reference test

Sp =

number of negatives by the developed assay (ELISA) number of negatives by the virus neutralization reference test

The Se and Sp values were analysed by a Scatter Plot test (Statistix 1.0 Version, © 1996 Analytical Software), which assessed the diagnostic performance of the system in terms of Se and (1 − Sp) for each possible cut-off value of the test. Therefore, the value that achieved the best relationship between diagnostic sensitivity and specificity was selected as the cut-off value of each ELISA. 2.9. Determination of variation coefficients Intra- and inter-assay variation coefficients (VC) were estimated according to the Jacobson’s procedure (Jacobson, 1998), where: standard deviation of replicates VC = mean of replicates

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

Intra- and inter-assay VC were estimated by evaluating results from six replicates of control samples in each plate (intraplate variation), and interplate variation using the same samples run in different plates. 3. Results 3.1. Synthesis of E0, E2 and NS3 proteins in Sf9 cells In order to determine whether recombinant baculoviruses expressed the proteins of interest, Sf9 cells were infected individually with AcNPV/E0, AcNPV/E2 and AcNPV/NS3. Cells were collected 72 h post infection and the presence of recombinant proteins was evaluated by SDS-PAGE and Western blot with monoclonal antibodies (Fig. 1). Cells infected with AcNPV/E0 showed a double band close to 47.4 kDa band of the molecular weight marker (Fig. 1A). This double band pattern has been described previously for E0 glycoprotein (Silva-Krott et al., 1994; Hulst et al., 1994). The presence of these two proteins of different migration would be the result of glycosylation heterogeneity, as has been reported for several glycoproteins synthesized in the baculovirus system (O’Reilly et al., 1994). Cells infected with AcNPV/E2 showed a major band between 47.4 and 60.7 kDa bands of the molecular weight marker (Fig. 1B). An additional band of around 100 kDa was identified and attributed to the homodimeric form of E2 (Weiland et al., 1990). The apparent molecular weight of recombinant E0 and E2 proteins suggested that they were subjected to glycosylation as happens during natural infection with BVDV. The presence of N-glycosylation was confirmed by treatment with tunycamicin, which showed an alteration of the molecular weight similar to that described by Collett et al. (1988a) for E0 and E2 in MDBK cells infected with BVDV (data not shown). Cells infected with AcNPV/NS3 (Fig. 1C) showed a major band of about 60 kDa, in agreement with the expected molecular weight, taking into account that the expressed protein fragment was synthesized as a fusion protein with GST and a stretch of poly-histidines coded by the transfer vector pAcGHLT-C (Pharmingen, San Diego, CA). The recombinant NS3 protein showed high level of expression (8% of total proteins), which allowed its visualization in gels stained with Coomassie blue (data not shown). 3.2. Development of the ELISA tests based on the E0, E2 and NS3 proteins Different methods were evaluated in order to determine the best strategy to produce the antigens (data not shown). E0 and E2 recombinant proteins were extracted by incubation of the infected cells with lysis buffer containing IGEPAL CA-630 (Sigma). On the other hand, it was not possible to purify NS3 by this methodology. Since neither of the detergents evaluated could extract successfully the recombinant protein and considering the high level of expression, NS3 was obtained directly from

103

polyacrylamide gels stained with Coomassie blue followed by electroelution. Once the source of the antigens was established, different dilutions of each antigen were assayed. Dilutions of serum and conjugate, as well as composition of blocking solutions were also evaluated by a checkerboard titration protocol. During the development of the assays, it appeared that the specificity could be improved by including a pre-adsorption step. It consisted in the incubation of the sera samples for 1 h at 37 ◦ C in another plate coated with extract of Sf9 cells diluted 1:200. This additional step was incorporated to the protocol of the three assays, before adding the sera to the ELISA plate and found to be efficient for reducing non-specific interactions. The final setup of the assays has been described in Section 2. 3.3. Validation of the ELISAE0 , ELISAE2 and ELISANS3 Once the final protocol for the assays had been determined, specificity and sensitivity were evaluated in comparison with the reference test, by using 141 negative sera and 60 positive sera tested previously by viral neutralization (Fig. 2). Values obtained with the negative sera were evaluated by the Box and Whisker statistic method (Statistix 1.0 Version, © 1996 Analytical Software), which allowed the identification of probable outlier values. Considering that the probability of these values belonging to the data group is nearly zero, they were excluded from the analysis. It was possible to identify three probable outliers by the assay based on E0, and five by the assay based on NS3. Nevertheless, Fig. 2A and C includes these values to demonstrate the ability of these assays of detecting specific anti-BVDV antibodies in cattle sera classified as “negative” by the viral neutralization test. Different methods were applied in order to determine the cut-off value of each assay. The negative mean plus three standard deviations (Xneg + 3σ) was the selected value that achieved the best relationship between sensitivity and specificity, estimated by using the viral neutralization as reference test (Table 2). Using the cut-off value selected for the ELISAE2 , seven of the 60 positive samples evaluated were found negative by this method, indicating that its sensitivity was 88.3%. On the other hand, only two of the 141 negative samples evaluated were positive by this assay, achieving a specificity value of 98.6%. In the case of ELISAE0 , the sensitivity was 76.7%, as 14 of the 60 positive samples showed ELISA values lower than the established cut-off point. When evaluating the 139 negative sera samples, all of them were negative by this ELISA. For this reason, the specificity of the ELISAE0 was 100%. Finally, the analysis of sera samples with the ELISA based on Table 2 Sensitivity (Se) and specificity (Sp) values estimated for the ELISA tests based on E2, E0 and NS3 recombinant antigens, using VNT as reference test

Cut-off value Se (%) Sp (%)

E2

E0

NS3

0.104 88.3 98.6

0.113 76.7 100

0.083 93.8 100

104

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

Fig. 2. ELISA tests developed with the recombinant antigens: E0 (A), E2 (B) and NS3 (C). Each assay was applied to 141 negative sera (VNT −) and 60 positive sera (VNT +), previously characterized by the viral neutralization test. Each sample was tested in duplicate and the corresponding result was expressed as the difference between the OD405nm value obtained with the recombinant antigen (E0, E2, NS3) and the one obtained with the negative control (Sf9 antigen) for each serum sample. The horizontal line indicates the cut-off value of each assay. E0 (A) and NS3 (C) graphs remark samples that were excluded from the analysis because they were identified as “probable outliers” by the Box and Whisker test (Statistix 1.0 Version, © 1996 Analytical Software).

NS3 showed a sensitivity value of 93.8%, as four of the 64 positive samples resulted negative by this method. The specificity was 100%, as there was total coincidence between the reference test and the ELISANS3 , when evaluating the negative samples.

In order to estimate the reproducibility of each assay, the intra- and inter-assay variation coefficients were determined, according to Jacobson’s procedure (Jacobson, 1998). Table 3 shows the results obtained, which indicate that both coefficients were lower than 20% for raw absorbance values denoting

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108 Table 3 Intra- and inter-assay variation coefficient (VC) estimated for the ELISA tests based on E2, E0 and NS3 recombinant antigens

Intra-assay CV Inter-assay CV

E2

E0

NS3

2.8% 4.1%

5.3% 9.7%

2.4% 17%

Variation coefficients (VC: standard deviation of replicates/mean of replicates) correspond to the raw absorbances of the positive control sample in six replicates run in the same plate and in different plates.

adequate repeatability at this stage of the development of the assay. 3.4. Comparison of the recombinant ELISAs and virus neutralization Table 4 shows the comparison of the recombinant ELISAs and the virus neutralization for the samples whose results disagreed with the reference test. When looking at ELISAE2 results, it was possible to appreciate that only two (FVC and 280MB) of the 141 negative sera found positive by this ELISA. However, the values observed were slightly higher than the cut-off value, and Western blot assays confirmed the absence of specific reactivity against E2 recombinant protein.

105

When studying the behaviour of the positive sera, seven out of 60 samples (453.4, 816.1, 815.1, 644.1, 531.1, 537.1 and 156.1) were negative by the ELISAE2 . These samples were also negative by the ELISAE0 and the absence of reactivity against both antigens (E0 and E2) was confirmed by Western blot. Nevertheless, all these samples were positive when evaluated against the NS3 recombinant antigen, regardless of the test used. The evaluation of the ELISAE0 showed that three out of 141 negative samples (49.11, 49.13 and 49.15) were positive by this assay. These three samples had been excluded from the analysis by the Box and Whisker test when it identified them as probable outliers. All the remaining sera were negative, in agreement with the reference test. Finally, when studying the performance of the ELISANS3 , all the negative samples (137 sera) were negative throughout this assay. Only five negative samples (705MB, 58.1VP, 58.2VP, 64.1VP, 48.13OD), which had been excluded from the analysis by the Box and Whisker test, were positive by the ELISANS3 . The reactivity of the samples against the NS3 antigen was confirmed by Western blot. When evaluating the behaviour of the positive samples, 93.8% were positive by the ELISANS3 . Only four samples (RS283MB, 601MB, 816.4MB, 452.3) showed values below the cut-off point. One sample (452.3) had been evaluated by the other ELISAs, and it was negative

Table 4 Comparison of the results obtained by viral neutralization test and the recombinant ELISA tests for the samples that revealed discrepancies between both methodologies Serum

VNT

ELISAE2 a , cut-off 0.104

WBE2

ELISAE0 a , cut-off 0.113

WBE0

ELISANS3 a , cut-off 0.083

WBNS3

FVC 280MB 453.4 816.1 815.1 644.1 531.1 537.1 156.1 49.11 49.13 49.15 452.3 153.aVC 153.bVC 153.cVC 155.aVC 453.aMB 453.bMB 705MB 58.1VP 58.2VP 64.1VP 48.13OD RS283MB 601MB 816.4MB

− − + + + + + + + − − − + + + + + + + − − − − − + + +

+ (0.132) + (0.112) − (0.059) − (0.063) − (0.039) − (0.009) − (0.059) − (0) − (0.044) − (0) − (0.022) − (0.018) + (0.274) + (0.214) + (0.261) + (0.244) + (0.146) + (0.184) + (0.113) − (0) − (0) − (0) − (0) − (0.004) ND ND ND

− − − − − − − − − − − − ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

− (0.048) − (0) − (0.064) − (0.061) − (0) − (0) − (0.012) − (0.033) − (0.018) + (0.243) + (0.378) + (0.305) − (0) − (0.007) − (0.032) − (0) − (0.069) − (0.231) − (0.118) − (0) − (0) − (0) − (0) − (0) ND ND ND

− − − − − − − − − + + + ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

− (0) − (0) + (0.287) + (0.343) + (1.132) + (1.445) + (1.548) + (0.848) + (0.282) ND ND ND − (0) ND ND ND ND ND ND + (0.209) + (0.544) + (0.179) + (0.293) + (0.2) − (0.038) − (0) − (0.029)

− − + + + + + + + ND ND ND − ND ND ND ND ND ND + + + + + − − −

Results (+ or −) are indicated for the different assays: viral neutralization test (VNT), ELISA based on E0, E2 or NS3 (ELISAE0 , ELISAE2 , ELISANS3 ) and Western blot (WB) against the recombinant antigens (E0, E2 and NS3). ND: non-determined. Serum samples identified as probable outliers by the Box and Whisker statistic test are indicated in italics. a ELISA result expressed as the difference between the OD 405nm value obtained with the recombinant antigen (E0, E2, NS3) and the one obtained with the negative control (Sf9 antigen).

106

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

Table 5 Evaluation of the performance of the recombinant assays (ELISAE2 , ELISAE0 and ELISANS3 ) to detect seroconversion in sequentially collected serum samples from one British crossbred calve experimentally infected with 2.5 × 107 DICT50 of BVDV NADL strain Serum

VNT

ELISAE2 a , cut-off 0.104

ELISAE0 a , cut-off 0.113

ELISANS3 a , cut-off: 0.083

Balc48, 0 dpib Balc48, 8 dpi Balc48, 15 dpi Balc48, 25 dpi Balc48, 47 dpi Balc48, 68 dpi

− − − + + +

− (0) − (0) − (0.004) + (0.267) + (0.507) + (0.269)

− (0) − (0) − (0) + (0.429) + (0.519) + (0.316)

− (0) − (0.011) − (0.028) + (0.2) + (0.421) + (0.325)

Results (+ or −) are indicated for the different assays: viral neutralization test (VNT) and recombinant ELISAs. a ELISA result expressed as the difference between the OD 405nm value obtained with the recombinant antigen (E0, E2, NS3) and the one obtained with the negative control (Sf9 antigen). b Days post infection.

by the ELISAE2 and positive by the ELISAE0 . The absence of reactivity of these samples against the NS3 antigen was also confirmed by Western blot. Since the finding of negative samples recognizing NS3 and/or E0 antigens was not expected, and taking into account that BVDV type 2 also circulates in Argentina, although in a very low proportion, about 10% (Jones et al., 2001), samples negative by virus neutralization and by ELISAE2 , but positive by ELISAE0 or by ELISANS3 were also evaluated by virus neutralization with BVDV type 2 (data not shown). The obtained results indicated that the antibodies present in seven out of eight samples (49.11, 49.13, 49.15, 58.1VP, 58.2VP, 64.1VP and 48.13OD) had neutralizing activity against BVDV type 2, suggesting that the E0 and NS3 recombinant antigens had the ability of detecting specific antibodies not only to BVDV type 1 but also to BVDV type 2. Finally, in order to evaluate the performance of the assays developed for detecting seroconversion, the recombinant ELISAs were applied to the testing of serum samples collected sequentially from one British crossbred calve infected experimentally with 2.5 × 107 DICT50 of BVDV NADL strain. Table 5 shows that the seroconversion could be detected on day 25 postinfection (pi) by the three recombinant ELISAs, in agreement with the viral neutralization test. Although further confirmation with more samples collected sequentially should be carried out, this result suggests that the recombinant assays developed in this work can be employed to test seroconversion against BVDV (type 1) in a comparable way to the reference test using the same type of BVDV. 4. Discussion BVDV specific antibodies can be classified into two functional groups. Antibodies to the viral glycoproteins (E0 and mainly E2) neutralize the virus. These antibodies cross react with different strains of BVDV, but when sera raised against one virus strain are tested against other isolates, different antibody titres may be obtained (Brock, 1995). Conversely, the highly immunogenic non-structural viral protein NS2/NS3, which is essential for the replication of the virus, is conserved antigenically between all pestiviruses (Collett, 1992). Antibodies to the NS2/NS3 protein do not neutralize BVDV, but since they can be

detected readily by other serological tests, this antigen is important in BVDV serology (Sandvik, 1999). In this study, the possibility of employing the most immunogenic proteins of BVDV (E0, E2 and NS3) as antigens in three different ELISAs for the detection of anti-BVDV antibodies in cattle sera was evaluated. The antigens were expressed in a baculovirus system and the appropriate conditions for extraction were determined. When all the parameters of each ELISA were established, the assays developed were applied to the testing of 200 bovine serum samples, previously characterized by the viral neutralization test. The three assays showed high levels of sensitivity and specificity when compared with the reference test. Examining the results obtained by the three recombinant assays, two different situations were observed. Firstly, those samples that although positive by the reference test were negative by ELISA. At this point it was possible to appreciate the complementary value of the three assays considering that the simultaneous performance of the three assays could identify almost all the positive samples. Secondly, there were samples that although negative by viral neutralization were positive by ELISA. Here again, two kind of samples were distinguished: the samples that, though positive, showed values next to the cutoff point and, therefore, the diagnosis by this method may be doubtful. In these cases, the use of another test such as Western blot, would be advisable. On the other hand, the samples whose absorbance values were clearly positive. This was observed in the ELISAs based on E0 and NS3 and demonstrated their ability to detect anti-BVDV antibodies that were not detected by the ELISA based on the E2 glycoprotein or by the reference test with BVDV type 1. The ELISAE0 and ELISANS3 were of particular interest since they demonstrated the ability of both antigens (E0 and NS3) to detect the presence of specific antibodies that were not detectable by the viral neutralization test with BVDV type 1. A possible explanation for this observation is that the viral neutralization test only detects antibodies with neutralizing activity, but these antibodies do not represent the total population of antiBVDV antibodies. Thus, the inclusion of E0 and NS3 would extend the range of antibodies that can be detected in comparison with E2 alone. Considering that the E2 glycoprotein carries the neutralizing epitopes, and that E0 and NS3, although

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

they are immunogenic, do not elicit the production of neutralizing antibodies, it is possible that these antigens detect different antibodies, with respect to those detected by the viral neutralization test. Therefore, when comparing the performance of the BVDV ELISAs with the reference test against BVDV type 1, it could be expected that the major level of coincidence would be obtained with the ELISA based on E2 glycoprotein. Moreover, the three negative samples that were classified as positive by the ELISAE0 (Table 4), were negative when they were evaluated by ELISAE2 , confirming the observation mentioned previously. The evaluation of the positive samples with ELISAE0 demonstrated something similar, as 14 out of 60 positive sera were negative by this test. The use of a diagnostic assay based on E0 has therefore the advantage of allowing the detection of antibodies that cannot be detected by the reference test, but nevertheless, it would be disadvantageous if the virus neutralization was intended to be replaced by this assay, as a considerable number of positive samples are classified as negative by the ELISAE0 . Something similar was observed among the results achieved by the ELISA based on NS3, which detected specific antibodies in three samples classified previously as negative (Table 4). These samples were also negative by the other two ELISA tests, revealing the ability of the NS3 antigen to detect anti-BVDV antibodies in samples classified as negative by the reference test and by the ELISAs based on structural proteins. Another possibility is that the antibodies detected by the E0 or NS3 antigens had been induced by BVDV type 2 and, therefore, they were not detected by the viral neutralization test with BVDV type 1. This hypothesis was confirmed by the detection of anti-BVDV type 2 antibodies in samples, which reacted with the E0 or NS3 antigens, but had not reacted against the E2 antigen or BVDV type 1 by the viral neutralization assay. This result is in agreement with the fact that E0 and NS3 proteins are more conserved among BVDV strains (Ridpath and Bolin, 1995; Ridpath, 2003) and suggests that their inclusion in the design of a diagnostic test allows to extend the spectrum of antibodies that can be detected. Probably, a combination of both situations could occur simultaneously. Previous reports demonstrated the ability of BVDV antigens obtained in insect cells to be used for serological diagnosis. Vanderheijden et al. (1993) compared bacteria- and baculovirusexpressed NS3 protein from the BVDV Osloss strain for the detection of BVDV infection from cattle and concluded that baculovirus-expressed BVDV antigens were superior. On the other hand, Reddy et al. (1997) developed an ELISA based on the precursor region of E0 and E1 (NADL strain) obtained in the baculovirus system and described the advantages of this expression system in comparison with recombinants expressed in bacteria and conventional whole-cell ELISA. The present report improves the results obtained in this field so far, because it describes the expression and simultaneous use of the three main antigenic proteins (structural and non-structural) of BVDV, demonstrating the contribution of each one to clearly determine the pattern of anti-BVDV antibodies in 200 bovine serum samples evaluated in this study. Several investigators have suggested possible applications of the NS3 protein for diagnosis. If effective recombinant subunit

107

vaccines against BVDV became available, simultaneous vaccination and anti-NS3 serology for monitoring of natural infection with BVDV should be possible (Sandvik, 1999). On the other hand, in the case of animals vaccinated with a conventional inactivated vaccine, such as those allowed commonly in several countries, the detection of anti-NS3 antibodies would distinguish between vaccinated and natural infected cattle, because of the non-structural nature of the NS3 polypeptide (Bolin and Ridpath, 1990). In this study, it was not possible to evaluate this aspect of the ELISANS3 mainly due to the lack of information about the clinical history of the animals from which the samples were obtained. Nevertheless, both mentioned points represent potential applications of the ELISANS3 , which converts it in a very valuable diagnostic tool. In conclusion, the results obtained suggest that simultaneous application of the three assays developed allows the detection of anti-BVDV antibodies in cattle sera by a simple, rapid and secure methodology, which does not require the manipulation of infectious material. Although the recombinant antigens employed were obtained from a BVDV type1 strain, the inclusion of E0 and NS3 proteins allowed the detection of anti-BVDV type 2 antibodies. Therefore, although this multiplex ELISA must be evaluated with a higher number of samples with known history of BVDV prevalence, it represents a promising diagnostic tool with potential value for serological surveillance of BVDV. Acknowledgments The authors gratefully acknowledge Dr. Mar´ıa Isabel Craig (Virology Institute, INTA) for her assistance with viral neutralization assays against BVDV type 2, and Dr. Laura Marangunich because of the statistic assistance. This research was supported by grants from ANPCyT (PICT N◦ 08-00067-01290). References Baker, J.C., 1995. The clinical manifestations of bovine viral diarrhea infection. Vet. Clin. North Am. 11, 425–445. Bolin, S.R., 1993. Immunogens of bovine viral diarrhea virus. Vet. Microbiol. 37, 263–271. Bolin, S.R., Ridpath, J.F., 1990. Range of viral neutralizing activity and molecular specificity of antibodies induced in cattle by inactivated bovine viral diarrhea virus vaccines. Am. J. Vet. Res. 51, 703–707. Brock, K.V., 1995. Diagnosis of bovine viral diarrhea virus infections. Vet. Clin. North Am. Food Anim. Pract. 11, 549–561. Collett, M.S., 1992. Molecular genetics of pestiviruses. Comp. Immunol. Microbiol. Infect. Dis. 15, 145–154. Collett, M.S., Wiskerchen, M., Welniak, E., Belzer, S.K., 1991. Bovine viral diarrhea virus genomic organization. Arch. Virol. Suppl. 3, 19–27. Collett, M.S., Larson, R., Belzer, S., Retzel, E., 1988a. Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology 165, 200–208. Collett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K., Purchio, A.F., 1988b. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165, 191–199. Donis, R.O., Corapi, W., Dubovi, E.J., 1988. Neutralizing monoclonal antibodies to bovine viral diarrhoea virus bind to the 56K to 58K glycoprotein. J. Gen. Virol. 69, 77–86. Edwards, S., 1990. The diagnosis of bovine virus diarrhoea-mucosal disease in cattle. Rev. Sci. Tech. (Off. Int. Epizoot.) 9, 115–130.

108

S. Chimeno Zoth, O. Taboga / Journal of Virological Methods 138 (2006) 99–108

Harlow, E., Lane, D., 1988. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory. Houe, H., 1999. Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Vet. Microbiol. 64, 89–107. Hulst, M.M., Himes, G., Newbigin, E., Moormann, R.J.M., 1994. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology 200, 558–565. Jacobson, R.H., 1998. Validation of serological assays for diagnosis of infectious diseases. Rev. Sci. Tech. (Off. Int. Epizoot.) 17 (2), 507–526. Jones, L.R., Zandomeni, R., Weber, E.L., 2001. Genetic typing of bovine viral diarrhea virus isolates from Argentina. Vet. Microbiol. 81, 367–375. Justewicz, D.M., Magar, R., Marsolais, G., Lecomte, J., 1987. Bovine viral diarrhea virus-infected MDBK monolayer as antigen in enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies in bovine sera. Vet. Immunol. Immunopathol. 14, 377–384. Lindberg, A.L.E., 2003. Bovine viral diarrhoea virus infections and its control. A review. Vet. Quart. 25 (1), 1–16. Maisonnave, J., Rossi, C.R., 1982. A microtiter test for detecting and titrating non-cytopathogenic bovine viral diarrhea virus. Arch. Virol. 72, 279–287. Maniatis, T., Fristch, E.F., Sambrock, J. (Eds.), 1987. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Mu˜noz, D.P., Lager, I.A., Mersich, S., Zabal, O., Ulloa, E., Schudel, A.A., Weber, E.L., 1996. Fetal infections with bovine viral diarrhea virus in Argentina. Brit. Vet. J. 152, 175–182. Ode´on, A.C., Risatti, G., Kaiser, G.G., Leunda, M.R., Odriozola, E., Campero, C.M., Donis, R.O., 2003. Bovine viral diarrhea virus genomic associations in mucosal disease, enteritis and generalized dermatitis outbreaks in Argentina. Vet. Microbiol. 96, 133–144. O’Reilly, D., Miller, L.K., Luckow, V.A., 1994. Baculovirus Expression Vectors. A Laboratory Manual. W.H. Freeman and Company, New York. Pacheco, J., Lager, I., 2000. Comparaci´on de las t´ecnicas de seroneutralizaci´on, inmunofluorescencia y ELISA para la detecci´on de anticuerpos contra el Virus de la Diarrea Viral Bovina. Rev. Med. Vet. 81, 13–15. Petric, M., Yolken, R.H., Dubovi, E.J., Wiskerchen, M.A., Collett, M.S., 1992. Baculovirus expression of pestivirus non-structural proteins. J. Gen. Virol. 73, 1867–1871.

Pinto, G.B., Hawkes, P., Zabal, O., Ulloa, E., Lager, I.A., Weber, E.L., Schudel, A.A., 1993. Viral antibodies in bovine fetuses in Argentina. Res. Vet. Sci. 55, 385–388. Reddy, J.R., Kwang, J., Okwumabua, O., Kapil, S., Loughin, T.M., Lechtenberg, K.F., Chengappa, M.M., Minocha, H.C., 1997. Application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle. Vet. Microbiol. 57 (2–3), 119–133. Reed, L.J., Muench, H.A., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Ridpath, J.F., 2003. BVDV genotypes and biotypes: practical implications for diagnosis and control. Biologicals 31, 127–131. Ridpath, J.F., Bolin, S.R., 1995. The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype: detection of a large genomic insertion in a non-cytopathic BVDV. Virology 212, 39–46. Rweyemamu, M.M., Fernandez, A.A., Espinosa, A.M., Schudel, A.A., Lager, I.A., Mueller, S.B.K., 1990. Incidence, epidemiology and control of bovine viral diarrhea virus in South America. Rev. Sci. Tech. (Off. Int. Epizoot.) 9, 207–214. Sandvik, T., 1999. Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle. Vet. Microbiol. 64 (2–3), 123–134. Silva-Krott, I.U., Kennedy, M.A., Potgieter, L.N.D., 1994. Cloning, sequencing, and in vitro expression of glycoprotein gp48 of a non-cytopathogenic strain of bovine viral diarrhea virus. Vet. Microbiol. 39, 1–14. Sutula, C., Gillett, J., Morrissey, S., Ramsdell, D., 1986. Interpreting ELISA data and establishing the positive–negative threshold. Plant Dis. 8 (70), 722– 726. Vanderheijden, N., Moerlooze, L.De., Vandenbergh, D., Chappuis, G., Renard, A., Lecomte, C., 1993. Expression of the bovine viral diarrhoea virus Osloss p80: its use as ELISA antigen for cattle serum antibody detection. J. Gen. Virol. 74, 1427–1431. Weiland, E., Stark, R., Haas, B., R¨umenapf, T., Meyers, G., Thiel, H.J., 1990. Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J. Virol. 64, 3563–3569. Zoth, S.C., Taboga, O., Konig, G., Pereda, A., Palma, E.L., Piccone, M.E., 2001. Expresi´on y caracterizaci´on de dos prote´ınas estructurales del virus de la diarrea viral bovina (VDVB). Rev. Argent. Microbiol. 33, 15–21.