Design, development, and validation of a strand-specific RT-qPCR assay for GI and GII human Noroviruses

Human noroviruses (HuNoV) are the major cause of viral gastroenteritis worldwide. Similar to other positive-sense single-stranded RNA viruses, norovirus RNA replication requires the formation of a negative strand RNA intermediate. Methods for detecting and quantifying the viral positive or negative sense RNA in infected cells and tissues can be used as important tools in dissecting virus replication. In this study, we have established a sensitive and strand-specific Taqman-based quantitative polymerase chain reaction (qPCR) assay for both genogroups GI and GII HuNoV. This assay shows good reproducibility, has a broad dynamic range and is able to detect a diverse range of isolates. We used tagged primers containing a non-viral sequence for the reverse transcription (RT) reaction and targeted this tag in the succeeding qPCR reaction to achieve strand specificity. The specificity of the assay was confirmed by the detection of specific viral RNA strands in the presence of high levels of the opposing strands, in both RT and qPCR reactions. Finally, we further validated the assay in norovirus replicon-bearing cell lines and norovirus-infected human small intestinal organoids, in the presence or absence of small-molecule inhibitors. Overall, we have established a strand-specific qPCR assay that can be used as a reliable method to understand the molecular details of the human norovirus life cycle.

Quantitative RT-PCR Detection of Human Noroviruses and Hepatitis A Virus in Surface Water and Fresh Produce

Vegetables can be considered as a vehicle for the transmission of human enteric viruses such as noroviruses (genogroups I and II) and hepatitis A virus (HAV) when irrigated with contaminated irrigation or when prepared by infected food handlers. In the current study, we investigated the presence of hepatitis A virus (HAV) and human noroviruses (genogroups I and II) in fresh produce and surface water used in cultivation of this produce using real-time PCR. Samples were collected from six different points in the Mansoura and Giza regions, Egypt. Our analysis showed that at least one virus was found in 41.6% (30/72) of surface water samples and 27% (13/48) of fresh produce samples. HAV (23/72) with a mean viral concentration = 4 × 106 genome copies/litter (GC/L) was the most frequently identified virus in surface irrigation water samples, followed by human norovirus genogroup II (HNoV GII) (15/72, with a mean concentration = 1.2 × 106 GC/L, and human noroviruses genogroup I (HNoV GI) (12/72, with a mean concentration = 1.4 × 104 GC/L). Additionally, HAV (10/48) with a mean concentration = 5.2 × 105 genome copies/gram (GC/g) was also the most frequently detected virus in the fresh produce samples, followed by HNoV GII (8/48, with a mean concentration = 1.7 × 104 GC/g), meanwhile HNoV GI (6/48) was less detected virus with a mean concentration = 3 × 103 GC/g. This work suggests a wide prevalence of human enteric viruses in surface irrigation waters and fresh produce, which is of concern when the fresh produce is eaten raw. Thus, Additional monitoring for viral pathogens in irrigation water and food is needed to increase produce safely.

Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.

Oyster heat shock protein 70 plays a role in binding of human noroviruses

Human noroviruses (HuNoVs) are important foodborne pathogens causing acute gastroenteritis. Oysters are an important vehicle for transmission of HuNoVs. Histo-blood group antigen (HBGA)-like substances are considered the primary ligands for bioaccumulation of HuNoVs in oyster tissues. In this study, proteinaceous ligands for specific binding of HuNoVs were mined from oyster tissues using a bacterial cell surface display system. The macromolecular target was captured and identified in proteomic analysis. The distribution of viral particles, oyster heat shock protein 70 (oHSP 70), and type A HBGA (positive control) in oyster tissue was investigated by multiplex immunofluorescence assays after artificial contamination with HuNoVs (GII.4). Our results demonstrated that oHSP 70 is a candidate vital ligand for specific binding of HuNoVs in oyster tissues. In addition, P proteins (GI.1 and GII.4) and viral particles (GI.1 and GII.4) were captured by recombinant oHSP 70 in an enzyme-linked immunosorbent assay with sample signal/negative signal of 7.8, 6.3, 17.0, and 8.8, respectively. The findings suggested that oHSP 70 plays an important role in the binding of these foodborne viruses. Importance Human noroviruses (HuNoVs) are the most important pathogen for non-bacterial epidemic gastroenteritis cases. Foodborne transmission plays an important role in HuNoVs infection. Oysters, filter-feeding epibenthic bivalves, can be contaminated by faecal discharge in harvest water. A new proteinaceous ligand for HuNoVs other than HBGA is identified in oyster tissues. The significance of our research is in identifying and verifying the ligands in oyster tissues for HuNoVs binding. Our data will allow a better understanding of HuNoVs attachment and transmission with oysters, leading to control of undesired foodborne disease.

Surveillance to Prevent the Spread of Norovirus Outbreak from Asymptomatic Food Handlers during the PyeongChang 2018 Olympics

The human noroviruses are major causes of nonbacterial gastroenteritis, and are transmitted by both food and water, as well as via a person-to-person route. Asymptomatic norovirus infection of food handlers may play a role in transmission. The outbreak of noroviruses infections has been recognized in PyeongChang Winter Olympics, starting with security staffs from February 3, 2018. The Ministry of Food and Drug Safety in Korea conducted a noroviruses surveillance from asymptomatic food handlers of food catering facilities related to the Olympics to prevent the spread of the noroviruses. A total of 707 rectal swab samples from food handlers were collected and examined for norovirus using real-time RT-PCR and conventional RT-PCR. Five of 707 detected noroviruses. Genotypes of the norovirus-positive samples were determined with sequencing analysis. Identified genotypes of norovirus in asymptomatic food handlers included GI.3, GII.4, and GII.17. The GII.17 was prevalent among the genotypes, accounting for 3 of 5 detections. Food handlers with noroviruses detected in rectal swab are excluded from cooking and all foods handled by infected food handlers were discarded. Surveillance of norovirus infection for food handlers contributed to preventing noroviruses spread.

Fucoidan But Not 2′-Fucosyllactose Inhibits Human Norovirus Replication in Zebrafish Larvae

Human noroviruses (hNoVs) cause heavy disease burden worldwide and there is no clinically approved vaccination or antiviral hitherto. In this study, with the use of a zebrafish larva in vivo platform, we investigated the anti-hNoV potentials of fucoidan (from brown algae Fucus vesiculosus) and 2′-Fucosyllactose (2′-FL). As a result, although both fucoidan and 2′-FL were able to block hNoV GII.4 virus-like particle (VLPs) from binding to type A saliva as expected, only fucoidan, but not 2′-FL, was able to inhibit the replication of hNoV GII.P16-GII.4 in zebrafish larvae, indicating the possible needs of higher molecular weights for fucosylated carbohydrates to exert anti-hNoV effect.

Norovirus Extraction from Frozen Raspberries Using Magnetic Silica Beads

Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.