Serological Studies Confirm the Novel Astrovirus HMOAstV-C as a Highly Prevalent Human Infectious Agent Peter D. Burbelo1*, Kathryn H. Ching1, Frank Esper2, Michael J. Iadarola1, Eric Delwart3, W. Ian Lipkin4, Amit Kapoor4 1 Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Department of Pediatrics, University Hospitals Case Medical Center, Cleveland, Ohio, United States of America, 3 Blood Systems Research Institute and Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, United States of America, 4 Center for Infection and Immunity, Columbia University, New York, New York, United States of America Abstract Molecular identification of a microbe is the first step in determining its prevalence of infection and pathogenic potential. Detection of specific adaptive immune responses can provide insights into whether a microbe is a human infectious agent and its epidemiology. Here we characterized human anti-IgG antibody responses by luciferase immunoprecipitation systems (LIPS) against two protein fragments derived from the capsid protein of the novel HMOAstV-C astrovirus. While antibodies to the N-terminal fragment were not informative, the C-terminal capsid fragment of HMOAstV-C showed a high frequency of immunoreactivity with serum from healthy blood donors. In contrast, a similar C-terminal capsid fragment from the related HMOAstV-A astrovirus failed to show immunoreactivity. Detailed analysis of adult serum from the United Sates using a standardized threshold demonstrated HMOAstV-C seropositivity in approximately 65% of the samples. Evaluation of serum samples from different pediatric age groups revealed that the prevalence of antibodies in 6–12 month, 1–2 year, 2–5 year and 5–10 year olds was 20%, 23%, 32% and 36%, respectively, indicating rising seroprevalence with age. Additionally, 50% (11/22) of the 0–6 month old children showed anti-HMOAstV-C antibody responses, likely reflecting maternal antibodies. Together these results document human humoral responses to HMOAstV-C and validate LIPS as a facile and effective approach for identifying humoral responses to novel infectious agents. Citation: Burbelo PD, Ching KH, Esper F, Iadarola MJ, Delwart E, et al. (2011) Serological Studies Confirm the Novel Astrovirus HMOAstV-C as a Highly Prevalent Human Infectious Agent. PLoS ONE 6(8): e22576. doi:10.1371/journal.pone.0022576 Editor: Man-Seong Park, College of Medicine, Hallym University, Korea Received April 5, 2011; Accepted June 24, 2011; Published August 4, 2011 Copyright: 2011 Burbelo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Division of Intramural Research, National Institute of Dental and Craniofacial Research. This study is supported in part by the National Institute of Allergy and Infectious Diseases K23 AI065829 (F.E.), AI090196 (A.K.) and AI57158 (Northeast Biodefense Center - A.K., W.I.L), and the Department of Defense (A.K., W.I.L.) and the Blood Systems Research Institute and NIH R01220 HL083254 (E.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: burbelop@nidcr.nih.gov Introduction The family Astroviridae consists of small (28–30 nm in diameter), non-lipid enveloped, single-stranded positive-sense RNA viruses with genomes ranging in size from 6.4 to 7.3 kb. The genome includes three open reading frames (ORFs) designated ORF1a, ORF1b and ORF2. ORF1a encodes the non-structural poly- protein 1a, while the longer ORF1b encodes polyprotein 1ab including the RNA dependent RNA polymerase (RdRp) expressed through a ribosomal frameshift at the ORF1a/1b junction. ORF2 encodes the viral capsid structural polyprotein [1,2]. To date the Astroviridae family consists of two genera, Astrovirus and Mamas- trovirus, which infect avian and mammalian hosts, respectively. These astroviruses, transmitted through the fecal-oral route can cause gastroenteritis in numerous avian and mammalian species, including humans [3,4]. All eight known human astrovirus serotypes belonging to the first identified human Astrovirus species (HAstV) have been associated with gastroenteritis [5,6,7,8,9]. Clinical symptoms of HAstV infection in humans usually lasts between two and four days [10] and consists of watery diarrhea and less commonly, vomiting, headache, fever, abdominal pains and anorexia. HAstV can also cause significant disease in the elderly and immunocompromised patients [11]. Several metagenomics studies have recently used random amplification and mass sequencing of nucleic acid extracted from human stool to systematically catalogue viruses, phage and bacteria present in patients with diarrhea [12,13,14,15]. For example, a novel astrovirus species, AstV-MLB1, was identified in stool from children and adults, including some with diarrhea [13,16,17,18]. In addition, a new group of astroviruses, provi- sionally named HMOAstV/AstV-VA, were discovered by con- sensus PCR using stool samples from different continents [13,18]. Phylogenic analysis of the HMOAstV viruses revealed that they consisted of three subgroups, HMOAstV-A, HMOAstV-B, and HMOAstV-C. HMOAstV-C RNA was also identified in individ- uals from a gastroenteritis outbreak in a daycare center in Virginia and tentatively named AstV-VA1 [19]. Despite the identification of novel astroviruses in human stool, it is unclear if these new viruses are pathogenic or whether they are passengers associated with ingested food. PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e22576