**2. Key notes from norovirus and hepatitis A virus**

#### **2.1. Norovirus genome and molecular diversity**

Although "winter-vomiting disease" was described in 1929 [6], the responsible viral agent was not discovered until 1972 by Kapikian [7] from fecal materials derived from an outbreak of gastroenteritis among elementary school children in 1968 in Norwalk, Ohio. The virus was named Norwalk virus and was designated as the prototype strain for the group of viruses now called Noroviruses; in the literature, they were previously referred to as small round-struc‐ tured viruses (SRSVs) by their surface morphology or Norwalk-like viruses. The Norwalk agent was the first enteric virus identified that specifically caused acute gastroenteritis in humans. Successful cloning and sequencing of the NoV genomes have led to progress in understanding viral genome organization and classification. Noroviruses are a member of the family *Caliciviridae* whose name is derived from the Greek word *Calyx* for cup [8]. The viral genome is composed of a single strand of polyadenylated positive-sense, non-enveloped RNA size of approximately 7.6 kb. The linear RNA is organized into three open reading frames (ORFs), designated ORF1, ORF2, and ORF3. ORF1 encodes a 194-kDa protein that is cleaved by the viral cysteine protease into six non-structural proteins including p48, NTPase, p22, Vpg, protease, and RNA-dependent RNA polymerase. ORF2 codes for the major capsid protein VP1, which folds in two major domains: a shell (S) and a protruding (P) domain. The P domain is comprised of P1 and P2 subdomains. P2 is a highly variable region that is thought to be involved in the binding of the histo-blood group antigens (HBGAs), which are regarded as receptors and host-susceptibility factors for human infection [8, 9].

According to the literature, NoVs have been genetically segregated into at least five gen‐ ogroups (GI, GII, GIII, GIV, and GV) based on the complete amino acid sequences of the major capsid protein of 164 NoV strains [10]. New genogroups consisting of canine NoVs have also been proposed [11-12]. GI, GII, and GIV are known to cause gastroenteritis in humans. GI and GII contain the majority of human strains, with GV viruses regarded as uncommon human pathogens. GIII viruses were identified in cow, while GV viruses infect mice. Due to their enormous genetic diversity, the viruses within genogroups can be further classified into genetic clusters or genotypes that are defined as containing 14—44% VP1 amino acid sequence difference, where strains have 0—14% difference [10]. Accordingly, there are currently nine recognized genogroup I clusters and 22 genogroup II clusters. Despite their great molecular diversity, Genogroup II, genotype 4 (GII.4) variants have been responsible for the majority of outbreaks and cases in recent years, particularly those associated with person-to-person transmission [13—14]. The genotype GII.4 was first identified to predominate in outbreaks of gastroenteritis in the mid-1990s in countries on five continents [15], and new emerging variants have continued to evolve since then and have become the etiological agents for each of the four global gastroenteritis epidemics [16]. Although the majority of reported NoV outbreaks and cases are derived from person-to-person transmission, it is estimated that approximate‐ ly14% of them are attributed to food, and 37% of the foodborne outbreaks are caused by mixtures of GII.4 and other genotypes, 10% by all genotype GII.4, and 27% by all other single genotypes [17].

#### **2.2. HAV genome and genotypes**

are a means of infection [3—5]. Of all the foodborne viruses, hNoVs and HAV are the most important foodborne viral pathogens with regard to the severity of the associated illnesses and frequent occurrence worldwide [2]. Both hNoVs and HAV display high environmental stability on contaminated objects, are abundantly excreted in human feces (e.g., exceeding 107 viral particles per gram of stool), and have a low infectious dose (1 to 100 infectious viral particles) [2], all of which contribute to the ease of transmission of the viruses within a community. It is commonly noted that one of the most efficient ways to prevent and control the foodborne viral infections is to implement a reliable surveillance system using rapid, sensitive, and precise diagnostics to identify the associated pathogens. Human NoVs do not replicate in cell culture. Wild-type HAV strains are not readily cultivated *in vitro* and the detection is impaired by their slow and inefficient growth in cell culture and lack of apparent cytopathic effect. Cell culture-based systems for determining virus infectivity are currently not available for hNoVs and wild-type HAV. Traditional diagnosis of these foodborne viral pathogens has been reliant on electron microscopy and immunological tests, but these methods lack sufficient sensitivity. While they may be useful for the detection of the viruses in clinical specimens that contain high amounts of viruses, for foods, which harbor potentially small quantities of viruses and may yet cause illness, it is not feasible to use these traditional laboratory methods to detect the viruses. This has led to the development of new, more sensitive and robust detection methods. In recent years, the majority of newly developed detection approaches are nucleic acid-oriented. Nucleic acid-based molecular methods have demonstrated a large improvement in speed, sensitivity, and accuracy of the detection of hNoV and HAV, bringing new insights into the etiology and diagnosis of foodborne viral disease. This chapter will touch upon a number of nucleic acid-based methods that have been devel‐

oped and applied to detect the two epidemiologically important foodborne viruses.

Although "winter-vomiting disease" was described in 1929 [6], the responsible viral agent was not discovered until 1972 by Kapikian [7] from fecal materials derived from an outbreak of gastroenteritis among elementary school children in 1968 in Norwalk, Ohio. The virus was named Norwalk virus and was designated as the prototype strain for the group of viruses now called Noroviruses; in the literature, they were previously referred to as small round-struc‐ tured viruses (SRSVs) by their surface morphology or Norwalk-like viruses. The Norwalk agent was the first enteric virus identified that specifically caused acute gastroenteritis in humans. Successful cloning and sequencing of the NoV genomes have led to progress in understanding viral genome organization and classification. Noroviruses are a member of the family *Caliciviridae* whose name is derived from the Greek word *Calyx* for cup [8]. The viral genome is composed of a single strand of polyadenylated positive-sense, non-enveloped RNA size of approximately 7.6 kb. The linear RNA is organized into three open reading frames (ORFs), designated ORF1, ORF2, and ORF3. ORF1 encodes a 194-kDa protein that is cleaved by the viral cysteine protease into six non-structural proteins including p48, NTPase, p22, Vpg,

**2. Key notes from norovirus and hepatitis A virus**

**2.1. Norovirus genome and molecular diversity**

38 Nucleic Acids - From Basic Aspects to Laboratory Tools

HAV, first identified in 1973 by electron microscopy, is the most common cause of infectious hepatitis with annually causing about 1.4 million clinical cases and 200 million asymptomatic carriers worldwide [18]. HAV is one of the most frequent causes of foodborne viral infection. In the United States, it is estimated that approximately 270,000 people become annually infected with hepatitis A, and most of the infection cases are not reported to health authorities [19]. Epidemics associated with contaminated food or water can occur involving hundreds of thousands of people, such as the epidemic in Shanghai, China in 1988 affected almost 300,000 people due to the consumption of HAV-contaminated clams [20]. Like other enteric viruses, HAV is resilient to environmental stressors. The virus is able to retain infectivity in acidic environments below pH3, and after refrigeration and freezing. HAV is a non-enveloped positive single-stranded RNA virus with a genome of approximately 7.5 kb in length. The virus is classified within the genus *Hepatovirus* of the family of the *Picornaviridae* [21]. The viral genome consists of (i) a 5´-untranslated region (5´-UTR) of about 735 nucleotides; (ii) a single open reading frame (ORF) that is organized into three functional regions termed P1, P2, and P3; (iii) 3´-untranslated region (3´-UTR) with a polyadenylated A tail [22]. The P region encodes the viral capsid polypeptides VP1—VP4, and the P2 and P3 regions encode the non-structural protein. The 5´-UTR is the most conserved region of the genome and therefore is favored for primer design in polymerase chain reaction (PCR) to detect most genotypes. HAV displays a high level of antigenic conservation throughout the viral genome. An immunological study identified the existence of a single human serotype of HAV [23], but ample genetic diversity still exists to classify HAV into six genotypes based upon differences of a 186-bp nucleotide sequence in the VP1—P2A junction region [24—26]. Genotype I, II, and III are associated with human infection, while genotype IV, V, and VI are found in simians [27]. A genotype VII, designated SLF88, was proposed in an earlier study [24], but further analysis of the complete genome and capsid region of additional strains indicated that the genotype VII should be reclassified as genotype IIB [25, 26]. Genotype I and III can be each further divided into subgenotypes IA, IB, IIIA, and IIIB. Genotype I that comprises 80% human HAV strains studied is remarkably prevalent around the globe; subgenotype IA is more common than IB [24]. Since genotype I predominates worldwide, genotyping alone is rarely used to determine the source of a chain of HAV transmissions or outbreaks. Genotype III includes most of the remaining human HAV strains. Genotype II contains two subgenotypes: IIA and IIB.
