Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Ross River Virus in Vertebrate Sera

Ross River virus (RRV) is a mosquito-borne alphavirus that is endemic in Australia and Papua New Guinea. The primary hosts of the virus include humans, kangaroos, wallabies, and horses (2, 11, 12, 15). RRV is the major etiological agent of epidemic polyarthritis in humans, which is characterized by severe arthritis with possible development of a rash and mild fever or chills and for which there is no specific treatment. Effective surveillance of virus activity in its natural transmission cycle, with appropriate warnings to the public, is the best measure available for minimizing this disease. Traditionally, hemagglutination inhibition (HI), neutralization, and immunofluorescence assays (2, 7, 13) have been the means for detecting RRV antibodies in both human and animal sera. Enzyme-linked immunosorbent assay (ELISA) has also been used to specifically detect RRV immunoglobulin M in human sera (6, 17). Previously, epitope-blocking assays were developed for sensitive and specific detection of seroconversions to the medically important flaviviruses Murray Valley encephalitis virus (8, 9) and West Nile virus (3, 4, 10) in avian and mammalian sera. In this study, an epitope-blocking ELISA was developed for the rapid detection of RRV antibodies in both animal and human sera to improve the efficiency of seroepidemiological studies.

This study used seven isolates of RRV, obtained over 30 years from different regions in Australia, as well as the closely related alphaviruses Chikungunya virus, Getah virus, Barmah Forest virus (BFV), Semliki Forest virus, and Sindbis virus (Table 1). RRV ELISA antigen was produced by propagation of the prototype RRV strain T48 on Vero cells in serum-free medium. Virus supernatant was clarified at 4,000 × g for 15 min at 4°C and stored in 1-ml aliquots at −80°C. Polyclonal antisera were produced in New Zealand half-lop rabbits by intravenous inoculation with 50 μg purified virus/200 μl phosphate-buffered saline and bled at day 14 postinoculation (Table 1). Hyperimmune antisera were not used due to the enhanced cross-reactions observed after multiple immunizations. Nonreactive control sera were collected from nonimmune animals. Clinical samples of human sera (PathCentre, Western Australia [WA] State Health Department, QE11 Medical Centre, Nedlands, Australia) and samples from kangaroos and horses were collected as part of an ongoing seroepidemiological study of RRV in parts of WA (13). These samples were previously tested for RRV antibodies by standard assays (1, 6, 7). Titers are presented as the reciprocal of the highest dilution of antibody to completely neutralize or inhibit RRV. In developing the epitope-blocking ELISA, the protocol described by Hall et al. (9) was adapted. U-bottomed 96-well polyvinyl chloride plates were coated with an optimal concentration of RRV ELISA antigen at 50 μl/well under appropriate biological containment and incubated overnight at 4°C in coating buffer (0.1 M carbonate/bicarbonate, pH 9.6). Antigen-coated plates were washed twice with wash buffer, and nonspecific sites were blocked with 100 μl blocking buffer (0.05 M Tris, 1 mM EDTA, 0.15 M NaCl, 0.05% [vol/vol] Tween 20, 0.2% [wt/vol] casein, pH 8.0) for 1 hour at room temperature (RT). Reference or test sera were added (50 μl/well) in duplicate at dilutions of 1/10 and 1/100 in blocking buffer and incubated for 2 hours at RT. Nonimmune chicken and rabbit sera were used as nonreactive controls. Without removal of serum, 50 μl of monoclonal antibody (MAb) (hybridoma culture supernatant diluted in blocking buffer) was added to each well, and after gentle agitation the plates were incubated at RT for 1 hour. Plates were washed four times and bound MAb detected by incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Bio-Rad) diluted in blocking buffer for 1 hour at RT. Plates were washed six times and enzyme activity visualized by the addition of 100 μl substrate solution [1 mM 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid] (ABTS) and 3 mM H₂O₂ in a citrate/phosphate buffer, pH 4.2). Quantitative results were determined by measuring the optical density (OD) at 405 nm, and percent inhibitions were calculated as 100 − [OD (test)/OD (negative control) × 100]. A threshold of 20% inhibition by the test serum was considered “positive” for RRV antibodies (9).

Three MAbs (3B2, G8, and B10), produced to the E2 protein of reference RRV strains as previously described (5, 16), were assessed in the assay. The most sensitive and specific reactions were obtained using MAb 3B2, which was specifically inhibited from binding to RRV antigen in the presence of antisera to all seven reference RRV strains (Table 1). Thus, unless there is a major antigenic shift in circulating RRV strains, detection of RRV antibodies directed to this immunodominant epitope should be effective. Furthermore, using this MAb, there was no cross-reactivity (<20% MAb inhibition) with rabbit antisera to the closely related alphaviruses (Table 1). The results with field samples of kangaroo and horse sera showed ELISA to be as sensitive (100%) and specific (95%) as conventional neutralization for detecting RRV antibodies in these known vertebrate hosts (Table 2).

When used to test a panel of clinical RRV-positive human samples, the blocking ELISA exhibited 97% sensitivity and 98% specificity for the detection of antibody (Tables 3 and 4). This assay's ability to differentiate between seroconversion to BFV and RRV is also important, because both viruses are causative agents of epidemic polyarthritis and they circulate in the same geographical region (13). The acute-phase samples that were negative by ELISA (5007112N and 5028481W) (Table 3) may represent individuals who fail to mount a detectable immune response to the 3B2 epitope early in infection, as the convalescent-phase paired sample to 507112N (5013418) (Table 4) was subsequently shown to be positive by ELISA. The definitive criterion for confirming a recent viral infection is a fourfold or greater increase in the neutralization or HI titer of a paired serum sample (14). We propose that a twofold or greater increase in the percent inhibition of MAb by paired serum samples in the blocking ELISA be used as the criterion for serodiagnosis of recent RRV infection (Table 4). Past seroconversions to RRV could be identified by a greater than 20% inhibition by a single serum sample (Table 3) (9). In conclusion, the RRV epitope-blocking ELISA described in this paper provides a feasible alternative for the rapid diagnosis of clinical human RRV infection and for application in preclinical serosurveillance of susceptible vertebrate hosts (12, 15).

• American Society for Microbiology