Tissue Distribution and Visualization of Internalized Human Norovirus in Leafy Greens

Abstract

Lettuce has been implicated in human norovirus (HuNoV) outbreaks. The virus is stable on the leaf surface for at least 2 weeks; however, the dynamics of virus internalization have not been fully investigated. The purpose of this study was to assess the internalization and distribution of HuNoV and two surrogate viruses, porcine sapovirus (SaV) and Tulane virus (TV), in lettuce and spinach. Viral inoculations through the roots of seedlings and the petiole of leaves from mature plants were performed, and the viruses were tracked on days 1 and 6 post-root inoculation and at 16 h and 72 h post-petiole inoculation. Confocal microscopy was used to visualize root-internalized HuNoV. In both lettuce and spinach, (i) HuNoV was internalized into the roots and leaves at similar RNA titers, whereas surrogate viruses were more restricted to the roots, (ii) all three viruses were stable inside the roots and leaves for at least 6 days, and (iii) HuNoV disseminated similarly inside the central veins and leaf lamina, whereas surrogate viruses were more restricted to the central veins. Infectious TV, but not SaV, was detectable in all tissues, suggesting that TV has greater stability than SaV. HuNoV was visualized inside the roots' vascular bundle and the leaf mesophyll of both plants. In conclusion, using surrogate viruses may underestimate the level of HuNoV internalization into edible leaves. The internalization of HuNoV through roots and cut leaves and the dissemination into various spinach and lettuce tissues raise concerns of internal contamination through irrigation and/or wash water. IMPORTANCE Human noroviruses are the leading cause of foodborne outbreaks, with lettuce being implicated in the majority of outbreaks. The virus causes acute gastroenteritis in all age groups, with more severe symptoms in children, the elderly, and immunocompromised patients, contributing to over 200,000 deaths worldwide annually. The majority of deaths due to HuNoV occur in the developing world, where limited sanitation exists along with poor wastewater treatment facilities, resulting in the contamination of water resources that are often used for irrigation. Our study confirms the ability of lettuce and spinach to internalize HuNoV from contaminated water through the roots into the edible leaves. Since these leafy greens are consumed with minimal processing that targets only surface pathogens, the internalized HuNoV presents an added risk to consumers. Thus, preventive measures should be in place to limit the contamination of irrigation water. In addition, better processing technologies are needed to inactivate internalized viral pathogens.

Human Intestinal Enteroids to Evaluate Human Norovirus GII.4 Inactivation by Aged-Green Tea

“…Fecal filtrates from GII.4 Sydney[P16] and GII.4 Sydney[P4 New Orleans] human norovirus-positive fecal samples were prepared by passing clarified stool extracts through four serial filters (5, 1, 0.45, and 0.22 μm) as described previously ( Costantini et al, 2018 ) and stored at −70°C until the time of testing. Tulane virus was grown in LLC-MK2 cell line (ATCC CCL-7) cultured in Opti-MEM (Gibco Life Technologies, Grand Island, NY) supplemented with 2% FBS and 1% antibiotic-antimycotic cocktail (STR/Pen; Life Technologies Corporation, Carlsbad, CA), as described previously ( Cromeans et al, 2014 ; Esseili et al, 2018 ).…”

Human Norovirus Surrogate Is Highly Stable in Berry Smoothies and under In Vitro Simulated Digestion

“…The TV stock was generated in the LLC-MK2 cell line (ATCC CCL-7, Gaithersburg, MD, USA) as described in our previous publications [8,9]. The stock virus was ultrafiltered using Amicon 100 KDa Ultra-15 centrifugal devices (Millipore, MA, USA) to semi-purify the virus from cell culture lysate and further concentrate the virus.…”

“…The stock virus was ultrafiltered using Amicon 100 KDa Ultra-15 centrifugal devices (Millipore, MA, USA) to semi-purify the virus from cell culture lysate and further concentrate the virus. The TCID 50 assay was performed as described in our previous publications [8,9]. The stock TV titer was ~6.5 log TCID 50 /mL.…”

“…Historically, the lack of efficient approaches to culturing HuNoV in cell lines limited the ability to devise control strategies that prevent norovirus foodborne outbreaks. Surrogate culturable viruses, such as murine norovirus (MNV) and Tulane virus (TV), are often used to gain insights into HuNoV inactivation under various interventions, with TV being preferred to study HuNoV persistence in food and in the environment, because it binds to the histo-blood group antigens similar to HuNoV [7][8][9].…”

Human Norovirus Histo-Blood Group Antigen (HBGA) Binding Sites Mediate the Virus Specific Interactions with Lettuce Carbohydrates

“…Our group has shown that many factors enhance HuNoV persistence on the surface of lettuce leaves, including the presence of phytopathogens and physical damage [14,15,16]. The virus was also shown to internalize through the roots of lettuce seedlings and was detected by confocal microscopy inside the leaf mesophyll [17]. In addition, our group has shown that HuNoV GII.4 virus-like particles (VLPs) can bind specifically to lettuce cell wall carbohydrates extracted from leaves [16].…”