ABSTRACT
Viruses can infect animals, plants, and bacteria. Viruses are composed of either single-stranded or double-stranded nucleic acid, which can be either DNA or RNA. Viral infections in humans usually result in an immune response and disease. In this chapter, we utilize human sapovirus, a cause of gastroenteritis, as a model to illustrate the various ways to prepare viral samples for molecular detection and identication. In recent years, a number of important ndings concerning human sapovirus were discovered, which were likely, in part, due to improved detection techniques and increased surveillance. Human sapovirus strains were detected in water samples, which included untreated wastewater specimens, treated wastewater samples, and river samples;1,2 human sapovirus strains were detected in clams destined for human consumption in Japan;3 porcine sapovirus was detected in oysters destined for human consumption in the USA;4 and the number of sapovirus-associated outbreaks of gastroenteritis,
especially involving adults, appears to be steadily increasing, suggesting that sapovirus virulence and/or prevalence may be increasing.5-8
The virus family Caliciviridae contains four genera Sapo virus, Norovirus, Lagovirus, and Vesivirus, which include Sapporo virus, Norwalk virus, Rabbit hemorrhagic disease virus, and Feline calicivirus, respectively. Human sapovirus is an etiological agent of gastroenteritis. The prototype strain of human sapovirus, the Sapporo virus, was originally discovered from an outbreak in an orphanage in Sapporo, Japan, in 1977.9 In that study, Chiba et al. identi-ed viruses with the typical animal calicivirus morphology, the Star-of-David structure, by electron microscopy (EM). Besides having this classical structure, sapovirus particles are typically 41-46 nm in diameter and have a cup-shaped depression and/or ten spikes on the outline. The sapovirus
genome is a single-stranded, positive sense RNA molecule of approximately 7.5 kb that is polyadenylated at the 3′ end. Sapovirus can be divided into ve genogroups (GI-GV) Figure 8.1), among which GI, GII, GIV, and GV are known to infect humans, whereas sapovirus GIII infects porcine species (Figure 8.1). The sapovirus GI, GIV, and GV genomes are each predicted to contain three main open reading frames (ORFs), whereas sapovirus GII and GIII have two ORFs. Sapovirus ORF1 encodes for nonstructural proteins, including the VPg, protease, and RNA dependent RNA polymerase (RdRp), and a major capsid protein (VP1). Sapovirus ORF2 and ORF3 encoded proteins of yet unknown functions. Recently, naturally occurring intragenogroup recombinant sapovirus strains were identied (i.e., strains Mc10 and C12).10 When polymerase-based grouping was performed, Mc10 and C12 strains clustered together, but when capsid-based grouping
was performed, these two strains belonged in two distinct genotypes. In a more recent study, intergenogroup recombinant sapovirus strains were identied (i.e., strains SW278 and Ehime1107).11 Phylogenetic analysis of the nonstructural region (i.e., genome start to capsid start) grouped SW278 and Ehime1107 strains into GII, while the structural region (i.e., capsid start to genome end) grouped these strains into GIV. By comparing the sequence similarity across the length of the genomes using SimPlot software,12 a potential recombination site was discovered, at a point where the similarity analysis showed a sudden drop in nucleotide identity after the RdRp region, indicating that a recombination event occurred at the RdRp-capsid junction. Since the discovery of these intragenogroup and intergenogroup recombinant sapovirus strains classication numbering schemes have conicted between the different research groups. Ideally, both polymerase and
capsid genes should be analyzed. However, this is not always practical, due to amplication difculties, time and cost of reagents. Therefore, many phylogenetic studies have used the 5′ terminus of the capsid gene.
