HMOAstV-C Is a Highly Prevalent Human Infection Figure 2. Comparison of the C-terminal capsid fragment of HMOAstV-C with related viruses. From BLASTP analysis, the highest homology of the HMOAstV-C capsid fragment was with HMOAstV-B (68% identity) and HMOAstV-A (30% identity) astroviruses. Identical amino acid residues with HMOAstV-C are shaded. doi:10.1371/journal.pone.0022576.g002 demonstrate markedly different serologic responses to similarly homologous antigens in subjects infected with related pathogens [25,26]. In these studies, variations in both linear and conforma- tional epitopes are more clearly differentiated using the LIPS liquid assay than using ELISAs [20]. Further studies examining the humoral responses against the HMOAstV-B capsid, as well as additional proteins from the HMOAstV viruses should resolve this issue. Third, the anti-HMOAstV-C capsid antibody titers detected by LIPS assay in human samples was substantially higher than the background binding associated with mock protein A/G beads alone. This magnitude is comparable to those seen by LIPS against other infection agents such as Borrelia burgdorferi [27] and Kaposi Sarcoma associated virus [28]. Our strategy of using buffer blanks instead of seronegative uninfected samples has been used in other seroepidemiologic investigations and provide comparable diagnostic thresholds as seronegative samples [24,29]. Fourth, the relatively low immunoreactivity against HMOAstV-C in young children (age 6-12 months) and a corresponding increase in seroprevalence with childhood age is consistent with human infection. Lastly, additional studies evaluating rabbit, pig and horse samples showed that anti-HMOAstV-C antibodies were rare or absent in animals. The lack of immunoreactivity against HMOAstV-C in animals, despite the greater homology of HMOAstV-C to animal Astroviruses [13], supports the notion that humans are a host for these viruses. The approach of analyzing humoral responses to a panel of different animal serums side-by-side human samples by LIPS is useful for understanding the host range for this and other potential pathogens. Figure 3. LIPS detection of antibodies to the C-terminal capsid fragment of HMOAstV-C in human and animal serum samples. Immunoreactivity to the HMOAstV-C was determined in 106 healthy adult US blood donors, 6 rabbits, 16 horses, 16 pigs and 14 buffer only controls. Raw LU values are shown without subtracting background binding to protein A/G beads. The short solid line represents the mean titer for each group. The dashed line represents the diagnostic cut-off, derived from the mean plus 3 SD of 19 replica buffer blank samples. doi:10.1371/journal.pone.0022576.g003 Figure 4. Prevalence of HMOAstV-C antibodies with childhood age. A total of 103 child serum samples were analyzed by LIPS including from the following age brackets: 0–6 months (n = 22), 6–12 month (n = 15), 1–2 year (n = 22), 2–5 year (n = 22), and 5–10 year olds (n = 22). Raw LU values are shown without subtracting background binding to protein A/G beads. The dashed line represents the diagnostic cut-off value derived from the mean plus 3SD of replica buffer blank samples. The fraction and percent seropositive for HMOAstV-C are shown above each group. doi:10.1371/journal.pone.0022576.g004 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e22576