Molecular Epidemiology of Norovirus (NoV) Infection in Mie Prefecture: The Kinetics of Norovirus Antigenemia in Pediatric Patients

Abstract

Few studies have shown the presence of norovirus (NoV) RNA in blood circulation but there is no data on norovirus antigenemia. We examined both antigenemia and RNAemia from the sera of children with NoV infections and studied whether norovirus antigenemia is correlated with the levels of norovirus-specific antibodies and clinical severity of gastroenteritis. Both stool and serum samples were collected from 63 children admitted to Mie National Hospital with acute NoV gastroenteritis. Norovirus antigen and RNA were detected in sera by ELISA and real-time RT-PCR, respectively. NoV antigenemia was found in 54.8% (34/62) and RNAemia in 14.3% (9/63) of sera samples. Antigenemia was more common in the younger age group (0-2 years) than in the older age groups, and most patients were male. There was no correlation between stool viral load and norovirus antigen (NoV-Ag) levels (r_s = -0.063; Cl -0.3150 to 0.1967; p = 0.6251). Higher levels of acute norovirus-specific IgG serum antibodies resulted in a lower antigenemia OD value (n = 61; r = -0.4258; CI -0.62 to -0.19; p = 0.0006). Norovirus antigenemia occurred more commonly in children under 2 years of age with NoV-associated acute gastroenteritis. The occurrence of antigenemia was not correlated with stool viral load or disease severity.

Keywords: ELISA; RNAemia; antigenemia; norovirus; real-time RT-PCR.

Figure 1

Viral load estimation from RT-qPCR and its correlation with NoV-Ag levels in serum and stool samples. (A) The red square dots represent positive serum samples, the dotted blue triangles represent positive stool samples without RNA present in their serum, and the circular green dots represent positive stool samples with RNA present in the serum. (B) Correlation between positive stool and antigenemia based on serum OD at 450 nm. ** p < 0.01, *** p < 0.001.

Figure 2

NoV IgG titration curves in serum samples from acute infections. (A) Sera from children with GII.4 Sydney/2012 (black lines, n = 21) and GI.2 genotype (green line, n = 2). (B) Other GII genotypes (red lines, n = 14) were titrated with two-fold serial dilutions, and IgG antibodies were analyzed against GII.4 Ni 1315 and GII.17 Kawasaki-308 genotypes. For GII.17 Kawasaki-308, (C) black lines, n = 17; green lines, n = 2; and (D) red lines, n = 12.

Figure 3

NoV pre-existing antibody titer responses for different age groups. * p < 0.05, ns: not significant.

Figure 4

Correlation of antigenemia to serum acute antibody titers.

Figure 5

Phylogenetic analyses generated by the maximum likelihood test using the partial nucleotide sequences (338, 302, and 342 bp) from the C region of the capsid of norovirus. The strains reported in this study are indicated with a blue circle (stool) and a red triangle (sera).