Overview of Caliciviridae and Norwalk Virus: Structure, Replication, Transmission, Clinical Features, and Laboratory Diagnosis

Introduction to Caliciviridae Family:

Caliciviridae is a family of viruses having positive-sense, single stranded RNA which is non-segmented. The family Caliciviridae consists of several genera, Lagovirus, Nebovirus, Norovirus, Sapovirus and Vesivirus. Members of Caliciviridae are significant causes of viral gastroenteritis in both humans and a variety of animals. The first human calicivirus is called Norovirus (Norwalk virus). Around 90% of epidemic, non-bacterial outbreaks of gastroenteritis in people worldwide are believed to be caused by norovirus.

Structure of Caliciviruses:

Caliciviruses are tiny, positive-sense RNA-containing viruses with an icosahedral capsid that is 35 to 40 nm in diameter and a genome that is around 7.5 kb long. The virion surface has cup-shaped indentations; the 7.5 kb single-stranded RNA genome is polyadenylated at its 3′ terminus. Viral protein genome-linked, or VPg, is a protein that functions as a primer during RNA synthesis and is covalently bonded to the 5′ end of positive strand viral RNA.

Viral Replication Cycle:

• The virus enters the host cell through endocytosis, which is mediated by attachment to host receptors.
• The viral genomic RNA is uncoated and released into the cytoplasm.
• The replication proteins are produced by removing VPg from the viral RNA and translating it into a processed ORF1 polyprotein.
• Viral factories are where replication takes place.
• The genomic ssRNA (+) is converted into a dsRNA genome.
• Viral mRNAs and new ssRNA(+) genomes are produced by transcription and replication of the dsRNA.
• The capsid protein and VP2 are produced via subgenomic RNA translation.
• New virus particles are assembled and released through cell lysis.
• There are outbreaks all year long, with cooler months seeing a seasonal increase.

Noroviruses are highly contagious due to their extremely low infectious dose, high shedding levels, and extended environmental persistence. Although norovirus-related sickness is typically thought to be minor and self-limiting, older and immunocompromised people are increasingly experiencing more severe results. The Norwalk virus is a tiny, spherical, non-enveloped virus with a feathery shape and an amorphous surface structure. Its diameter is between 27 and 40 nm.
Single-stranded, positive sense RNA, approximately 7.5 or 7.6 kb in size, is present in the virion. The virion RNA functions as both the genome and the viral messenger RNA and is contagious.

Genome Organization (ORF1, ORF2, ORF3):

• There is a poly(A) tract at the 3′ terminus and a VPg protein at the 5′ terminus. Eight viral proteins are encoded by three open reading frames (ORFs), known as ORF-1, ORF-2, and ORF-3.
• There are three open reading frames (ORF) in the genome. The polyprotein that ORF1 encodes is cleaved to create RNA-dependent RNA polymerase, protease, and nonstructural proteins.
  The VP1-major capsid protein is encoded by ORF2. The VP2-minor capsid protein is encoded by ORF3. The norovirus genome is contagious. The structural elements of the virion, viral protein 1 (VP1) and VP2, are encoded by ORF-2 and ORF-3, respectively. The polyprotein that ORF-1 encodes is broken down by proteases into six nonstructural proteins, such as RNA-dependent RNA polymerase and norovirus protease. The virus, which was isolated from excrement, is a member of the single-stranded, RNA-containing calicivirus group and has a single structural protein with a molecular weight of roughly 60,000.

Mode of Transmission:

Foodborne or person-to-person transmission by fomites or aerosolization of contaminated bodily fluids (vomitus, fecal material) accounts for the majority of epidemics.

Clinical Manifestations of Norovirus Infection:

Many human noroviruses attach to HBGAs on the surface of epithelial cells, but noroviruses cannot bind to the cells of people who lack these antigens (i.e., non-secretors); norovirus infection is linked to malfunctioning of the epithelial barrier. The primary cause of epidemic viral gastroenteritis in adults is the Norwalk virus. The characteristics of epidemic nonbacterial gastroenteritis include: (1) the lack of bacterial pathogens; (2) gastroenteritis with quick onset and recovery and comparatively mild systemic symptoms; and (3) an epidemiologic pattern of a highly contagious disease that spreads quickly without any age or geographic preference. Depending on the major clinical feature, reports of various epidemics have used a variety of descriptive words (e.g., epidemic viral gastroenteritis, viral diarrhea, winter vomiting illness). Norwalk viral gastroenteritis has an incubation period of 24–48 hours. The symptoms include diarrhea, nausea, vomiting, low grade fever, cramping in the abdomen, headache, and malaise. The clinical course is short, lasting 12 to 60 hours. Although hospitalization is rarely necessary, the condition can be incapacitating during the symptomatic phase. Vomiting is more common with Norwalk infections. The most frequent issue in both young and old people is dehydration.

Laboratory Diagnosis of Norovirus:

• Antiviral antibodies can be found using radioimmunoassays and ELISA (enzyme-linked immunosorbent assay) tests.
• The most used method for identifying human caliciviruses in clinical specimens (vomitus, feces) and environmental samples (contaminated food, water) is reverse transcriptase-polymerase chain reaction.
• The selection of polymerase chain reaction primer pairs is crucial due to the genetic variability among circulating strains.
• Virus particles in stool samples are often found using electron microscopy.
• However, Norwalk virus particles are typically found in low concentrations and are challenging to identify; immunoelectron microscopy should be used to do so.
• Antibody responses can be detected by ELISA immunoassays based on recombinant virus-like particles; a recent infection is indicated by a fourfold or higher increase in IgG antibody titer in acute and convalescent-phase sera. Nevertheless, not all Norwalk virus antigenic types can be detected by the antigens, and the required reagents are not easily accessible.
•  Due to the wide variety of antigen types, antigen-detection tests typically comprise a mixture of norovirus-specific antibodies and exhibit some fluctuation in sensitivity.
• For point-of-care diagnosis, rapid antigen-detection tests have been commercialized.
• Enzyme immunoassays are also accessible and exhibit comparable sensitivity and specificity to immunochromatography testing.
• The main method for diagnosing norovirus infection is direct virus detection, namely viral-RNA detection techniques.
• Among the most often utilized detection techniques are end-point RT-PCR and real-time RT-PCR assays that focus on the genomic region connecting the primary capsid protein-coding area and RNA polymerase.
• Norovirus is a target in multiplexed molecular detection tests; sequencing this brief amplified region yields genotype information that is helpful in molecular epidemiology investigations.