Internalization of Sapovirus, a Surrogate for Norovirus, in Romaine Lettuce and the Effect of Lettuce Latex on Virus Infectivity

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Noroviruses are the leading cause of food-borne outbreaks, including those that involve lettuce. The culturable porcine sapovirus (SaV) was used as a norovirus surrogate to study the persistence and the potential transfer of the virus from roots to leaves and from outer to inner leaves of lettuce plants. Treatment of lettuce with SaV was done through the roots of young plants, the soil, or the outer leaves of mature plants. Sampling of roots, xylem sap, and inner and outer leaves followed by RNA extraction and SaV-specific real-time reverse transcription (RT)-PCR was performed at 2 h and on postinoculation days (PID) 2, 5, 7, 14, and/or 28. When SaV was inoculated through the roots, viral RNA persisted on the roots and in the leaves until PID 28. When the virus was inoculated through the soil, viral RNA was detected on the roots and in the xylem sap until PID 14; viral RNA was detected in the leaves only until PID 2. No infectious virus was detected inside the leaves for either treatment. When SaV was inoculated through the outer leaves, viral RNA persisted on the leaves until PID 14; however, the virus did not transfer to inner leaves. Infectious viral particles on leaves were detected only at 2 h postinoculation. The milky sap (latex) of leaves, but not the roots' xylem sap, significantly decreased virus infectivity when tested<jats:italic>in vitro</jats:italic>. Collectively, our results showed the transfer of SaV from roots to leaves through the xylem system and the capacity of the sap of lettuce leaves to decrease virus infectivity in leaves.</jats:p>

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

“…Our results expand on those of the latter study and show that HuNoV RNA can persist on mature lettuce and spinach plant leaves for at least 14 days. In contrast, MNV, SaV, and TV showed increased inactivation over time (according to their infectivity and RNA titers), consistent with the findings of previous studies utilizing MNV and TV on preharvest spinach (46) and SaV on lettuce (28). These findings are also consistent with those from a recent systematic review showing that, on the basis of RNA data, HuNoV is more stable than its surrogates (47).…”

“…A number of studies reported that HuNoV and its surrogates can internalize inside lettuce and spinach plants under normal conditions (28,43,44) and in lettuce under certain biotic and extreme abiotic stress a Numbers in bold indicate a significant effect (P Ͻ 0.05) for that factor. Statistical analyses were performed by two-way ANOVA.…”

“…Seeds of romaine lettuce cultivar Tall Guzmaine Elite (Siegers Seed Co., Holland, MI) and baby's leaf hybrid spinach (Burpee, Warminster, PA) were grown in 200-cell trays containing Fafard superfine germinating mix (Conrad Fafard, Aguwam, MA) under greenhouse conditions with temperatures of ϳ23°C during the daytime and 15°C during the nighttime and with a 12-h photoperiod (28). At 2 weeks of age, the seedlings were transferred to 15-cm-diameter pots containing sterile soil (Wooster sandy loam) and were fertilized biweekly using Osmocote slow-release fertilizer.…”

“…Each leaf was weighed, cut into small pieces, and individually suspended in elution buffer. The viruses were eluted from the leaf samples using MEM supplemented with a 1% antibiotic-antimycotic cocktail (Invitrogen, Carlsbad, CA, USA) and 2% heat-inactivated (60°C for 1 h) fetal bovine serum (Thermo Scientific HyClone) as described previously (28). Briefly, the samples were shaken vigorously (vortexing for 1 min, followed by shaking at 250 rpm for 10 min at 4°C), and the resulting solutions were transferred to sterile 50-ml Falcon tubes and centrifuged at 2,095 ϫ g for 10 min to remove bacterial cells and plant debris.…”

Comprehensive Comparison of Cultivable Norovirus Surrogates in Response to Different Inactivation and Disinfection Treatments

“…Many studies comparing FCV and MNV with or without other surrogates, such as MS2 coliphage and hepatitis A virus, as well as human norovirus (using reverse transcription-quantitative PCR [RT-qPCR]), have been published (4,(18)(19)(20)(21)(22)(23)(24). However, because MNV is highly sensitive to alcohols (18), the focus has expanded to cultivable calici-viruses such as TuV (14,(25)(26)(27)(28) and to porcine enteric calicivirus (PEC), a genogroup III (GIII) sapovirus (29,30).…”