Norovirus RNA in serum associated with increased fecal viral load in children: Detection, quantification and molecular analysis

Abstract

Worldwide, norovirus (NoV) is a major cause of acute gastroenteritis (AGE) responsible for pandemics every ~3 years, and over 200,000 deaths per year, with the majority in children from developing countries. We investigate the incidence of NoV in children hospitalized with AGE from Belém, Pará, Brazil, and also correlated viral RNA levels in their blood and stool with clinical severity. For this purpose, paired stool and serum samples were collected from 445 pediatric patients, ≤9 years between March 2012 and June 2015. Enzyme-linked immunosorbent assay (EIA) was used to detect NoV in stool and reverse transcription quantitative PCR (RT-qPCR) used to quantify NoV RNA levels in sera (RNAemia) and in the positive stool. Positives samples were characterized by the partial ORF1/2 region sequence of viral genome. NoV antigen was detected in 24.3% (108/445) of stool samples, with RNAemia also present in 20.4% (22/108). RNAemia and a high stool viral load (>107 genome copies/gram of faeces) were associated with longer hospitalizations. The prevalent genotypes were GII.4 Sydney_2012 (71.6%-58/81) and New Orleans_2009 (6.2%-5/81) variants. Eight other genotypes belonging to GII were detected and four of them were recombinant strains. All sera were characterized as GII.4 and shared 100% similarity with their stool. The results suggest that the dissemination of NoV to the blood stream is not uncommon and may be related to increased faecal viral loads and disease severity.

Fig 1. Distribution by age group of norovirus-positive patients in serum/stool samples collected from children hospitalized for acute gastroenteritis in the city of Belém, Pará, Brazil, from March/2012 to June/2015.

Fig 2. Monthly frequency of norovirus-positivity and temporal distribution of genotypes in stool samples from children hospitalized for acute gastroenteritis in the city of Belém, Brazil, from March/2012 to June/2015.

Fig 3. Viral load quantification by RT-qPCR in serum and stool samples from cases of norovirus infection in children hospitalized for acute gastroenteritis in the city of Belém, Pará, Brazil, from March/2012 to June/2015.

Dots in red represent positive serum samples, blue dots indicate positive stool samples without RNAemia, and green dots represent positive stool samples with presence of RNA in serum.

Fig 4. Phylogenetic cladogram generated by the maximum likelihood test using partial nucleotide sequences (360 bp) of the C region of the capsid of norovirus strains obtained from 47 stools of children hospitalized for acute gastroenteritis at two clinics in the city of Belém, Brazil, from March / 2012 to June / 2015.

Fig 5. Phylogenetic cladogram generated by the maximum likelihood test using partial nucleotide sequences (360 bp) from the C region of norovirus strains from 10 paired samples of sera and feces from children hospitalized for acute gastroenteritis at two clinics in the city of Belém, Brazil, from March/2012 to June/2015.

Fig 6

Simplot analysis of the ORF1-ORF2 overlap sequence (530 bp) of the strains VIR613F (MG023180)/ VIR699F (MG023186)/ VIR715F (MG023183) (a), VIR554F (MG023188) /VIR693F (MG023184) (b), VIR 138F (MG023190) (c), VIR 560 (MG023187) (d). The assay was performed using standards parameters of the program with a window size of 200 bp, a step size of 20 bp and with the Kimura (2-parameter) model. The accession numbers of the prototypes used in the analyses were the following: GII.P13/GII.13 (EU921354.2), GII.17/GII.17 (AY502009.1), GII.P22/GII.22 (AB233471), GII.P5/GII.5 (AF397156), GII.P7/GII.7 (JQ751043), GII.P6/GII.6 (AB039778), GI.Pb/GI.6 (AB081723), GI.6/GI.6 (AF093797), GI.Pb/GI.6 (AB354289). The y-axis indicates the nucleotide sequence similarity between the recombinant sequence and reference strains. The y-axis indicates nucleotide position.