Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples

Abstract

Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption.

Keywords: fish; hepatitis A; norovirus; pathogenic human viruses; tetraplex qPCR assay.

Figure 1

Population density on the Portuguese coast and localisation of the studied fish sampling regions (adapted from https://www.portugal.gov.pt/pt/gc21/governo/programa/programa-nacional-para-a-coesao-territorial-/ficheiros-coesaoterritorial/programa-nacional-para-a-coesao-territorial-o-interior-em-numeros-territorio-pdf.aspx (accessed on 13 February 2021)); * indicates aquaculture sampling sites.

Figure 2

Comparative standard curves of single and multiplex qPCR assays targeting plasmid and in vitro transcribed RNA for NoV GI, NoV GII, HAV, and mengovirus (vMC0). Virus genomic quantification in wastewater samples using qPCR based on the construction of standard curves using plasmid and in vitro transcribed RNA are represented as circles and stars, respectively. The linear regression line was obtained plotting the known quantities of serially diluted standard samples against the cycle quantification (Cq) of the samples. The slopes of the regression line were used to calculate the amplification efficiency (Ef) of the qPCR reactions according to the formula Ef = 10 (−1/slope).

Figure 3

Comparative qPCR quantifications for positive samples to NoV GI, NoV GII and HAV genomes using plasmid and in vitro transcribed RNA standard curves.

Figure 4

Phylogenetic analysis of the norovirus sequence detected in a pool of brains of seabass caught on the Portuguese coast by targeting part of the gene encoding the open reading frame 1 (ORF1)-ORF2 junction region. All viral sequences used are from human hosts and are identified by their accession number and genogroup; a fish is highlighting the viral sequence identified in this study. Scale bar indicates nucleotide substitutions per site and bootstrap values higher than 75% are displayed by *.