Our objective was to determine the seroprevalence of anti- HMOAstV-C antibodies in children and adults in the US. Traditionally, ELISA assays are used for serological testing and require purified virus or recombinant viral proteins. Unfortunate- ly, ELISA tests often show cross-reactivity, have inherent high background signals and require extensive optimization and standardization. Luciferase Immunoprecipitation System (LIPS) is a new technology that employs luciferase-tagged antigens in a liquid phase immunoprecipitation assay [20]. LIPS offers several advantages over ELISA including low backgrounds, highly quantitative data and the ability to generate diagnostically useful serodeterminations without pre-determined cut-off values [20,21]. To determine if the novel HMOAstV-C astrovirus is a prevalent human virus, LIPS was used to screen for antibodies against conserved regions of the HMOAstV-C viral capsid. Materials and Methods Serum samples Serum samples from adult, healthy blood donors (n = 106) were collected without any clinical information under IRB approved protocols at the National Institutes of Health, Bethesda, Maryland. Children serum samples were from Rainbow Babies and Children’s Hospital, Cleveland, Ohio and obtained under IRB approval from University Hospitals – Case Medical Center. A total of 103 serum samples from children from different ages were analyzed as follows: 0-6 months (n = 22), 6–12 months (n = 15), 1-2 years (n = 22), 2-5 years (n = 22) and 5-10 years old (n = 22). Samples were collected from September 2009 through March 2010. Other than the age of the individual from whom the serum was obtained, no other clinical information was available. Based on the similarity of HMOAstV-C astrovirus with animal astroviruses [13], serum samples from different domesticated animals, including horses, pigs and rabbits, were tested. Pig and horse serum samples were the kind gift of Yanjin Zhang and Utpal Pal (VA-MD Regional College of Veterinary Medicine, Univ. of Maryland). Rabbit serum samples were obtained from NIH laboratories and commercial vendors. All serum samples were stored at 280uC, thawed, and then left at 4uC prior to processing for LIPS analysis. Generation of Ruc-Astrovirus antigen fusion constructs Templates for capsid coding sequences of HMOAstV-C and HMOAstV-A were generated by RT-PCR amplification using human stool as described [13]. Due to the possibility of antibody cross-reactivity to different regions of the HMOAstV-C capsid, two different fragments encompassing the N-terminal (amino acids 1-393) and C-terminal fragment (amino acids 402-704) were generated by PCR. The primer adapter sequences used to clone each protein fragment are as follows: N-terminal capsid fragment of HMOAstV-C, 59-GAGGGATC CATGGCTGGTAAA- CAGCCC-39 and 59-GAGCTCGAGTCAAGGGCCTGTGT- TAGGTGC-39 and C-terminal capsid fragment of HMOAstV- C, 59- GAGGGATCCAACACCACTACAGGGTCA-39 and 59- GAGCTCGAGTCAATCCAGTGGGGTCAATCT-39. A third C-terminal capsid fragment from the HMOAstV-A astrovirus (amino acids 396-700), spanning a similar region to HMOAstV-C was constructed with the primers: 59-GAGGGATCCAGCA- CAGCCTCAGCGG TT -39 and 59-GAGCTCGAGT- CAATCCTTAGGCTTCTTTCT-39. The three capsid frag- ments were subcloned downstream of Renilla luciferase (Ruc) using the pREN2 vector [22]. DNA sequencing was used to confirm the integrity of the three DNA constructs. The sequence for the C-terminal capsid fragment of HMOAstV-C has been HMOAstV-C Is a Highly Prevalent Human Infection deposited in GenBank with accession (JF313458). Plasmid DNA was then prepared from these two different pREN2 expression vectors using a Qiagen Midi preparation kit. Following transfec- tion of mammalian expression vectors, crude protein extracts were obtained as described for use as antigen [23]. LIPS assays Briefly, human and animal sera were processed in a 96-well format at room temperature as previously described [23]. Serum samples were first diluted 1:10 in assay buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1% Triton X-100) using a 96-well polypropylene microtiter plate. Antibody titers were measured by adding 40 ml of buffer A, 10 ml of diluted sera (1 ml equivalent), and 16107 light units (LU) of each of the Ruc- HMOAstV antigen fragments containing crude Cos1 cell extract to wells of a polypropylene plate and incubated for 60 minutes at room temperature on a rotary shaker. Next, 5 ml of a 30% suspension of Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL) in PBS were added to the bottom of each well of a 96-well filter HTS plate (Millipore, Bedford, MA). To this filter plate, the 100 ml antigen-antibody reaction mixture was trans- ferred and incubated for 60 minutes at room temperature on a rotary shaker. The washing steps of the retained protein A/G beads were performed on a Biomek Workstation or Tecan plate washer with a vacuum manifold. After the final wash, LU were measured in a Berthold LB 960 Centro microplate luminometer (Berthold Technologies, Bad Wilbad, Germany) using coelenter- azine substrate mix (Promega, Madison, WI). All LU data were obtained from the average of at least two separate experiments. Sequence analyses Using the C-terminal capsid fragment of HMOAstV-C as the query sequence, a BLAST search was performed against the non- redundant NCBI protein databases. From this analysis, the highest homology was with HMOAstV-B and HMOAstV-A astroviruses. Viral capsid sequences were aligned using the global alignment program COBALT (www.ncbi.nlm.nih.gov/guide/sequence- analysis/) with default parameters. Data analysis GraphPad Prism software (San Diego, CA) was used for statistical analysis. For the calculation of sensitivity and specificity, a cut-off limit was used, which was derived from the combined value of the mean value plus 3 standard deviations (SD) of the replica samples containing only buffer, Ruc-extract and protein A/G beads. Human blood donor samples highly positive for anti- HMOAstV-C antibodies were used as internal positive controls to standardize the LIPS parameters for testing of serum samples. Results Identification of human antibody responses to the capsid of HMOAstV-C While most bona fide antigenic targets used in LIPS assays show high sensitivity and specificity [20], the exact antigens useful for diagnosis of HMOAstV-C are not known. As a screening approach and to potentially eliminate cross-reactivity spanning the full-length capsid regions of these viruses, we chose to first test two different protein fragments encompassing conserved N- and C-terminal capsid fragments of HMOAstV-C. From LIPS screening of 45 adult blood donor samples, the HMOAstV-C N- terminal capsid fragment showed higher background binding to the mock protein A/G beads than to clinical samples and was judged not to be immunoreactive (Fig. 1). However, the C- PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e22576