Diagnosis of Noncultivatable Gastroenteritis Viruses, the Human Caliciviruses

Abstract

Gastroenteritis is one of the most common illnesses of humans, and many different viruses have been causally associated with this disease. Of those enteric viruses that have been established as etiologic agents of gastroenteritis, only the human caliciviruses cannot be cultivated in vitro. The cloning of Norwalk virus and subsequently of other human caliciviruses has led to the development of several new diagnostic assays. Antigen detection enzyme immunoassays (EIAs) using polyclonal hyperimmune animal sera and antibody detection EIAs using recombinant virus-like particles have supplanted the use of human-derived reagents, but the use of these assays has been restricted to research laboratories. Reverse transcription-PCR assays for the detection of human caliciviruses are more widely available, and these assays have been used to identify virus in clinical specimens as well as in food, water, and other environmental samples. The application of these newer assays has significantly increased the recognition of the importance of human caliciviruses as causes of sporadic and outbreak-associated gastroenteritis

Porcine enteric caliciviruses: Genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology

“…Direct EM is not a very sensitive technique with a detection limit of 10 6 particles per ml of stool for enteric viruses [29]. Compared to SaVs, NoVs do not have the distinctive calicivirus morphology (cupshaped surface depressions) and they are harder to differentiate from other small round viruses including enteroviruses, astroviruses and picornaviruses in stool specimens [6,66].…”

“…The primers mainly determine the specificity of the RT-PCR reactions. Although nested RT-PCR can increase sensitivity up to 10-1,000 times, it is less used because of the high risk of cross-contamination [29]. Real-time RT-PCR is more sensitive and it has become an alternative method for detection of low amounts of virus, especially from environmental samples [69,70].…”

“…At present, the most accepted RNA extraction method for fecal samples is a mixture of guanidinium thiocyanate (GTC), phenol and chloroform extraction and precipitation of RNA by alcohol [15,29,50]. The principal reagent is commercially available from several companies, such as TRIzol from Invitrogen (Carlsbad, CA).…”

“…The majority of specific primers have been designed based on the most conserved RdRp region of the genome [29,68]. However, sequence analysis of the polymerase region of a wide range of virus strains indicated that this region is also variable with the nucleotide identity as low as 53% between strains of different genogroups and 60-64% within genogroup [59].…”

“…Internal control (IC) RNA-The RT-PCR inhibitors are common and sample-dependent in food, shellfish and fecal samples [6,29] causing false-negative results. These inhibitors may include body fluids, food and bacterial cell constituents [79].…”