Ultraviolet Light Inactivation of Murine Norovirus and Human Norovirus GII: PCR May Overestimate the Persistence of Noroviruses Even When Combined with Pre-PCR Treatments

Transmission of gastroenteritis-causing noroviruses may be significant via contaminated surfaces. Measures for control, e.g. disinfection with ultraviolet irradiation (UV), are therefore necessary for interrupting this transmission. Human norovirus (HuNoV) GII.4 and Murine norovirus (MuNoV) were used to study the efficacy of UV for virus inactivation on dry glass surfaces. MuNoV inactivation was measured using viability assay and the reduction in viral RNA levels for both viruses using reverse transcription quantitative PCR (RT-QPCR). For each UV dose, two parallel sample groups were detected using RT-QPCR: one group was enzymatically pre-PCR treated with Pronase and RNAse enzymes, while the other was not treated enzymatically. In the viability assay, loss of infectivity and a 4-log reduction of MuNoV were observed when the viruses on glass slides were treated with a UV dose of 60 mJ/cm2 or higher. In the RT-QPCR assay, a steady 2-log decline of MuNoV and HuNoV RNA levels was observed when UV doses were raised from 0 to 150 mJ/cm2. A distinct difference in RNA levels of pretreated and non-pretreated samples was observed with UV doses of 450–1.8 × 103 mJ/cm2: the RNA levels of untreated samples remained over 1.0 × 10³ PCR units (pcr-u), while the RNA levels of enzyme-treated samples declined below 100 pcr-u. However, the data show a prominent difference between the persistence of MuNoV observed with the infectivity assay and that of viral RNA detected using RT-QPCR. Methods based on genome detection may overestimate norovirus persistence even when samples are pretreated before genome detection

Molecular Detection and Genotyping of Noroviruses

Noroviruses (NoVs) are a major cause of gastroenteritis worldwide in humans and animals and are known as very infectious viral agents. They are spread through feces and vomit via several transmission routes involving person-to-person contact, food, and water. Investigation of these transmission routes requires sensitive methods for detection of NoVs. As NoVs cannot be cultivated to date, detection of these viruses relies on the use of molecular methods such as (real-time) reverse transcriptase polymerase chain reaction (RT-PCR). Regardless of the matrix, detection of NoVs generally requires three subsequent steps: a virus extraction step, RNA purification, and molecular detection of the purified RNA, occasionally followed by molecular genotyping. The current review mainly focused on the molecular detection and genotyping of NoVs. The most conserved region in the genome of human infective NoVs is the ORF1/ORF2 junction and has been used as a preferred target region for molecular detection of NoVs by methods such as (real-time) RT-PCR, NASBA, and LAMP. In case of animal NoVs, broad range molecular assays have most frequently been applied for molecular detection. Regarding genotyping of NoVs, five regions situated in the polymerase and capsid genes have been used for conventional RT-PCR amplification and sequencing. As the expected levels of NoVs on food and in water are very low and inhibition of molecular methods can occur in these matrices, quality control including adequate positive and negative controls is an essential part of NoV detection. Although the development of molecular methods for NoV detection has certainly aided in the understanding of NoV transmission, it has also led to new problems such as the question whether low levels of human NoV detected on fresh produce and shellfish could pose a threat to public health. © 2012 Springer Science+Business Media New York