## 4 Discussion
Recombinant RSV-G\({}_{\text{A}}\) or RSV-G\({}_{\text{B}}\) proteins blocked binding of human immune globulin in each assay, while recombinant RSV-N protein had no effect, providing additional evidence of assay specificity. Interestingly, rabbit antisera generated in response to RSV-G\({}_{\text{A}}\) antigens (i.e., anti-UV-A2, anti-G\({}_{\text{A}}\) protein, and anti-LHWT-G\({}_{\text{A}}\) peptide) reacted with both \(\text{Ruc-G}_{\text{A}}\) and \(\text{Ruc-G}_{\text{B}}\) antigens, although binding was higher against the homologous antigen. Cross-reactivity was likely due to binding within the central conserved region defined by RSV-G amino acids 164-176, which are 100% conserved across subtypes. In contrast, the antiserum to the LHWT-G\({}_{\text{B}}\) peptide only bound to homologous \(\text{Ruc-G}_{\text{B}}\) antigen, suggesting that RSV-G\({}_{\text{A}}\) elicits a more potent and broadly cross-reactive response while RSV-G\({}_{\text{B}}\) antigen may elicit a lower and more focused IgG antibody repertoire. Serum from adolescents 10-14 years of age also contained anti-RSV IgG antibodies that bound both \(\text{Ruc-G}_{\text{A}}\) and \(\text{Ruc-G}_{\text{B}}\) proteins. This was not surprising since it is likely that most children experience multiple, remote RSV exposures of both antigenic subtypes by this age. In contrast with the cross-reactivity seen using antisera from animals given multiple parenteral doses of RSV virus or protein, anti-RSV-G IgG responses observed in sera from mice experimentally infected intranasally with RSV-A2 or RSV-B1 strains once or twice were subtype specific. Similarly, we documented seroconversion among a small number of infants seronegative for RSV antibodies at 9 months of age who developed subtype specific anti-RSV-G\({}_{\text{A}}\) or -G\({}_{\text{B}}\) IgG responses by 15-18 months of age suggesting they had been infected with either a RSV subtype A or B strain in the interval between sampling. Other investigators have also noted subtype-specific neutralizing antibody and binding responses in serum following primary or early RSV infection in infants and toddlers [19]. The significance of subtype-specific responses against RSV-G\({}_{\text{A}}\) or RSV-G\({}_{\text{B}}\) proteins suggests that these differences early in life could potentially contribute to susceptibility of infants and young children to RSV lower respiratory tract infection (LRTI) if antibody responses against RSV-F are absent, low, or not sustained. Although many current RSV vaccines focus primarily on RSV-F protein, the importance of immune responses targeting the other major surface glycoprotein, RSV-G, should not be overlooked. There have been many discoveries about RSV-G in recent years, including its role in mediating attachment to CX3 CR1 whereby the virus initiates infection of bronchial epithelial cells, as well as the putative role of secreted RSV-G in modulating the host immune response [7]. Even though RSV-G shows more antigenic diversity than RSV-F, the central conserved region was shown to be an important target for MAb development and antibodies directed to this region were shown to provide effective prophylaxis against RSV infection in animals [10; 12; 31]. RSV-G antigenic sites outside this central domain can also elicit protective immunity in animals against RSV infection [15]. Since it is apparent that RSV-G can elicit both neutralizing and non-neutralizing antibody responses, a serological assay that determines anti-RSV-G antibody status may be a valuable tool in RSV sero-surveillance. There are several limitations to this study. We did not have sequential serum samples from infants or young children before and after documented RSV infection, nor did we have samples from children infected with other respiratory viruses to further define specificity of the assay. RSV-G genes and sequences from known laboratory adapted strains were used in the construction of the luciferase-tagged antigens used for the immunoprecipitation assay.