Figure 1. LIPS detection of antibodies to a C-terminal capsid fragment of HMOAstV-C. Antibodies to the N- and C-terminal capsid protein of HMOAstV-C and the C-terminal capsid protein of HMOAstV- CA capsid protein fragments were analyzed in 45 adult serum samples. Each symbol represents individual serum samples tested with each protein fragments and LU values were adjusted by subtracting background binding to protein A/G beads. The short solid line represents the mean antibody titer for each group. doi:10.1371/journal.pone.0022576.g001 terminal capsid fragment of HMOAstV-C protein demonstrated a robust response up to 125,900 LU above background (Fig. 1). The HMOAstV-C immunoreactivity in these human seropositive samples was highly reproducible and could be completely eliminated by pre-adsorption with protein A/G beads (data not shown). A third protein fragment derived from a similar C- terminal capsid region of the related HMOAstV-A virus failed to show immunoreactivity with any of the human samples tested (Fig.1). These results suggest that detectable human antibody responses are specific for HMOAstV-C and not for HMOAstV-A astrovirus. Sequence analysis of the C-terminal fragment of HMOAstV-C revealed 68% and 30% amino acid identity with corresponding capsid region from the novel Astrovirus, HMOAstV-B and HMOAstV-A astroviruses, respectively (Fig. 2). Importantly, no significant homology of the HMOAstV-C protein fragment was detected with the capsids derived from the eight known serotypes of HAstV strains (data not shown). High prevalence of anti-HMOAstV-C antibodies in adult humans To determine if HMOAstV-C infection is unique to humans, additional LIPS analysis was performed to compare immunore- activity in animal and human serum samples. For these studies, immunoreactivity against the C-terminal capsid fragment of HMOAstV-C was examined in 106 healthy adult US blood HMOAstV-C Is a Highly Prevalent Human Infection donors side-by-side serum samples from different domesticated animals. Buffer blanks in the LIPS format were used as negative controls to evaluate seropositivity [24] and a cut-off value was calculated from the mean plus 3 SD of 19 replica samples containing only buffer, Ruc-extract and protein A/G beads. Using a threshold of .9,525 LU, 65% (69 of 106) of healthy adult US blood donors were seropositive (Fig. 3). In contrast, no immunoreactivity was detected in any of the rabbit (n = 6), or pig (n = 16) serum samples (Fig. 3). Of 16 horse serum samples, one sample (6.25%) was immunoreactive to HMOAstV-C antigen (Fig. 3). As a control, several antigens were tested and revealed highly robust antibody titers in these same animal serum samples against known animal pathogens (data not shown). Collectively, these results suggest that antibodies against HMOAstV-C are relatively common in human adults, but are rare or non-existent in pigs, rabbits and horses. Increased exposure to HMOAstV-C with age We next explored differences in HMOAstV-C seroprevalence amongst different aged children in the United States. LIPS analysis revealed that the prevalence of infection in 6–12 month, 1–2 year, 2–5 year and 5–10 year olds was 20%, 23%, 32% and 36%, respectively. These results suggest a trend of increased antibody prevalence with increasing age, although no statistical difference was observed between age groups (Fig. 4). Additionally, 50% (11/22) of the 0-6 month old children showed anti- HMOAstV-C antibody responses, which likely reflects the presence of maternal antibodies. No statistical difference in antibody titers was observed between these different groups of children. All of the different children age groups showed a significantly lower prevalence of HMOAstV-C antibodies than adults (Fischer Exact T test; p,0.005). These results are consistent with increased infection by HMOAstV-C over time. Discussion Although several novel viruses have been identified in human stool [12,13,14,15], few studies have examined humoral responses to them. We have established a robust, sensitive serology platform that is ideally suited for pathogen discovery applications. LIPS holds several advantages over ELISA for studying humoral responses against potentially new infectious agents stemming from the low background binding, high sensitivity innate to this assay and the ability to rapidly test different antigens/antigen fragments using a standard format with little or no assay optimization [20]. Here we used LIPS assays to demonstrate humoral responses to the HMOAstV-C Astrovirus capsid protein and provide experi- mental data supporting human infection. Several different lines of evidence indicate that the HMOAstV- C capsid fragment is a target of humoral responses. First, only the C-terminal capsid region of HMOAstV-C was immunoreactive with human serum samples. Although RNA from HMOAstV-A was previously identified in human stool samples, we were unable to detect human immunoreactivity to this analogous C-terminal capsid region of HMOAstV-A. The exact reason for the lack of immunoreactivity is not clear, but it is possible that this capsid region of HMOAstV-A is less antigenic or its conformational folding is not recapitulated in the current Renilla luciferase fusion protein using the LIPS system. Second, the HMOAstV-C astrovirus capsid region used in LIPS was relatively unique and had no significant homology with the eight known serotypes of HAstV strains. Based on its homology, it is possible that some of the observed HMOAstV-C capsid immunoreactivity is against the related HMOAstV-B capsid. However, our previous LIPS studies PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e22576