Feline Calicivirus, Murine Norovirus, Porcine Sapovirus, and Tulane Virus Survival on Postharvest Lettuce

Abstract

bHuman norovirus (HuNoV) is the leading cause of foodborne illnesses, with an increasing number of outbreaks associated with leafy greens. Because HuNoV cannot be routinely cultured, culturable feline calicivirus (FCV), murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV) have been used as surrogates. These viruses are generated in different cell lines as infected cell lysates, which may differentially affect their stability. Our objective was to uniformly compare the survival of these viruses on postharvest lettuce while evaluating the effects of cell lysates on their survival. Viruses were semipurified from cell lysates by ultrafiltration or ultracentrifugation followed by resuspension in sterile water. Virus survival was examined before and after semipurification: in suspension at room temperature (RT) until day 28 and on lettuce leaves stored at RT for 3 days or at 4°C for 7 and 14 days. In suspension, both methods significantly enhanced the survival of all viruses. On lettuce, the survival of MNV in cell lysates was similar to that in water, under all storage conditions. In contrast, the survival of FCV, SaV, and TV was differentially enhanced, under different storage conditions, by removing cell lysates. Following semipurification, viruses showed similar persistence to each other on lettuce stored under all conditions, with the exception of ultracentrifugation-purified FCV, which showed a higher inactivation rate than MNV at 4°C for 14 days. In conclusion, the presence of cell lysates in viral suspensions underestimated the survivability of these surrogate viruses, while viral semipurification revealed similar survivabilities on postharvest lettuce leaves. Human noroviruses (HuNoVs) are the leading cause of acute viral gastroenteritis in the United States (1). These viruses are 28 to 35 nm in diameter, nonenveloped, single-stranded RNA viruses that are members of the Caliciviridae family. An increasing number of HuNoV outbreaks have been associated with leafy greens (2). For example, food was the primary vehicle of transmission in 1,008 HuNoV outbreaks reported between 2009 and 2012 in the United States, and leafy greens were the primary food associated with these outbreaks (1). Therefore, there is a need to understand the occurrence and survival/persistence of HuNoV on leafy greens. However, assessing the survival of HuNoVs on leafy greens is hampered by the fact that these viruses are still refractory to routine growth in cell culture; hence, their infectivity cannot be readily quantified (3, 4). Therefore, surrogate culturable viruses are often used as proxies to investigate HuNoV transmission routes, disinfection, and survival in the environment.Among members of the Caliciviridae family, feline calicivirus (FCV) has been the most widely used HuNoV surrogate since the 1990s (5). In 2004, murine norovirus (MNV), a virus genetically more closely related to HuNoV, was propagated in cell culture (6) and, since then, it has been widely used as a HuNoV surrogate (7). In 2004, bile acids in i...

Abiotic Stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and Its Surrogates on Preharvest Leafy Greens

“…The survival of these viruses may differ between preharvest and postharvest environments, and survival on the leaf surfaces may differ from that within the leaf tissues. Multiple studies that have investigated the postharvest survival of HuNoV on fresh produce have shown that this virus or its calicivirus surrogates (MNV, SaV, and TV) can survive during the chilling storage period and well beyond the produce shelf life (17,30,(39)(40)(41)(42). For example, HuNoV inoculated onto cut lettuce pieces persisted at 4°C for the entire 14-day study period with only a 1-to 1.5-log-unit reduction in RNA titers (39).…”

“…Following abiotic stress treatment, viral contamination of plant leaves was performed inside a biohazard hood. The suspension of feces containing HuNoV and the cell lysates of MNV, SaV, and TV were buffer exchanged to sterile water (to mimic contamination through irrigation water) using Amicon 100K Ultra-15 centrifugal devices (Millipore, Billerica, MA) that were centrifuged at 4,000 ϫ g for 0.5 h at 4°C as described previously (30). The titer of HuNoV RNA used in subsequent experiments was 6.3 Ϯ 0.1 log 10 genomic equivalent (GE)/ml.…”

Tissue Distribution and Visualization of Internalized Human Norovirus in Leafy Greens

“…Cell culture propagation of SaV and TV was performed as described previously (27) using the following cell lines: an LLC-porcine kidney cell line (LLC-PK1, ATCC CL-101) and an LLC-monkey kidney cell line (LLC-MK2, ATCC CCL-7), respectively. The HuNoV used in this study was HS194 strain (GenBank accession no.…”

“…One research team has used HuNoV in their studies and found significant internalization of the virus in lettuce and green onion under hydroponic growth conditions (24,25), while another team failed to detect any internalization of HuNoV in lettuce under hydroponic or soil growth conditions (26). This discrepancy, in addition to the fact that different surrogate viruses have been found to exhibit various degrees of similarity to HuNoV on leafy greens (21,27), necessitates a better assessment of the internalization dynamics of HuNoV in leafy greens, especially lettuce. Therefore, the objectives of this study were to (i) investigate whether lettuce and spinach can internalize HuNoV into the edible leaves, (ii) simultaneously assess the behavior of surrogate viruses TV and SaV in comparison with that of HuNoV, and (iii) determine whether HuNoV particles can be visualized inside lettuce and spinach tissues.…”

Eligibility of Feline Calicivirus for a Surrogate of Human Norovirus in Comparison with Murine Norovirus, Poliovirus and Coxsackievirus

“…The use of FCV as a surrogate of HuNoV should be avoided. MNV, taxonomically closely related to HuNoV, is known to be sensitive to ethanol Akasaka et al, 2016;Cromeans et al, 2014;Zonta et al, 2016 . Other surrogates including Tulane virus, porcine enteric calicivirus, porcine sapovirus, echovirus 12 and coliphage MS2 are also used Cromeans et al, 2014;Esseili et al, 2015;Kniel, 2014 . As a robust enterovirus, the use of CoV as a surrogate virus could be considered.…”