Serological studies confirm the novel astrovirus HMOAstV-C as a highly prevalent human infectious agent

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.

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.

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.

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.

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.