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CORD-19:a78663bdb83f4bdb19a1a4f304cb18e31a9e846a JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-110 Sentence denotes Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions HHS Public Access
T2 112-120 Sentence denotes Abstract
T3 121-279 Sentence denotes Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong.
T4 280-462 Sentence denotes Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water.
T5 463-619 Sentence denotes Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity.
T6 620-689 Sentence denotes In this study, we examined coronavirus stability on lettuce surfaces.
T7 690-978 Sentence denotes A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 10 4 to 1.7 × 10 6 throughout the experimental period of 30 days.
T8 979-1137 Sentence denotes Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce.
T9 1138-1365 Sentence denotes UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated.
T10 1366-1490 Sentence denotes Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans.
T11 1492-1569 Sentence denotes Coronavirus (CoV) is an enveloped, single-stranded, positive-sense RNA virus.
T12 1570-1701 Sentence denotes Although respiratory CoVs are largely secreted by the nasal route, the role of fecal/oral transmission has not been fully explored.
T13 1702-1865 Sentence denotes Earlier studies have demonstrated that CoV stability largely depends on environmental temperature (Ijaz et al., 1985; Siddell et al., 1983; Tennant et al., 1994) .
T14 1866-1976 Sentence denotes CoVs are inactivated at 56 °C within 10-15 min, at 37 °C after several days, and at 4 °C after several months.
T15 1977-2126 Sentence denotes However, at −60 °C, CoVs can survive for many years without loss of infectivity (Andries et al., 1978; McIntosh et al., 1974; Siddell et al., 1983) .
T16 2127-2445 Sentence denotes Survival studies of human coronavirus (HCoV)-229E and HCoV-OC43 demonstrated that both viruses survived in a saline solution up to 6 days at room temperature, but after drying on hospital surfaces, they survived as long as 3 h; although survival differences between the two viruses were observed (Sizun et al., 2000) .
T17 2446-2590 Sentence denotes These findings suggest that surfaces and suspensions can be sources of contamination for hospital-acquired CoV infections (Sizun et al., 2000) .
T18 2591-2810 Sentence denotes During the Severe Acute Respiratory Syndrome (SARS) epidemic in Hong Kong, it was thought that aerosolized droplets of feces were responsible for the transmission of a cluster of 420 SARS cases (McKinney et al., 2006) .
T19 2811-3001 Sentence denotes Consequently, the World Health Organization (WHO) urged that research be undertaken to better define the modes of transmission of SARS-CoV through sewage, feces, food and water (WHO, 2003) .
T20 3002-3192 Sentence denotes A more recent WHO report has listed hepatitis E virus (HEV), Highly Pathogenic Avian Influenza (HPAI) virus H5N1, SARS-CoV and Nipah virus as having the potential for foodborne transmission.
T21 3193-3334 Sentence denotes Initial foodborne transmission is a route to enter the human population, which can then shift and spread through human-to-human transmission.
T22 3335-3503 Sentence denotes Furthermore, virus long-term survival would not be a requirement when contamination occurs at the end of the food processing chain or in fresh produce (FAO/WHO, 2008) .
T23 3504-3624 Sentence denotes The risks of contamination of agricultural fields and field workers by animal enteric/respiratory CoV cannot be ignored.
T24 3625-3730 Sentence denotes Furthermore, in times of outbreaks food handlers may also be responsible for food contamination with CoV.
T25 3731-3872 Sentence denotes Because human enteric coronavirus causes a mild disease of lower incidence, foodborne transmission of CoV has not compelled an investigation.
T26 3873-4086 Sentence denotes A recent environmental survival study of two animal CoVs demonstrated that these viruses remained infectious in water and sewage at 27 °C for periods of weeks and at 4 °C for up to a year (Casanova et al., 2009) .
T27 4087-4300 Sentence denotes However, the role of ready-to-consume produce as a vehicle for potential zoonotic transmission or a source for genetic recombination during coinfections between animal and human CoVs has not yet been investigated.
T28 4301-4687 Sentence denotes The potential zoonotic transmission of CoVs was first suggested by studies in which human enteric coronavirus (HECoV)-4408 isolated from a diarrheic child was found to be indistinguishable genetically, antigenically and cytopathogenically from bovine CoV (BCoV) (Zhang et al., 1994) and retained infectivity and pathogenicity when inoculated into gnotobiotic calves (Han et al., 2006) .
T29 4688-4832 Sentence denotes In addition, BCoV is evolutionarily closely related to HCoV-OC43 (Vijgen et al., 2005) and to a lesser extent to SARS-CoV (Zhang et al., 2005) .
T30 4833-4977 Sentence denotes Therefore, in the present study, we used BCoV strain 88 as a model to examine the stability and the potential for foodborne transmission of CoV.
T31 4978-5091 Sentence denotes A total of 10 Romaine lettuce heads were purchased in separate occasions from local markets and used immediately.
T32 5092-5148 Sentence denotes Undamaged leaves were chosen for use in the experiments.
T33 5149-5264 Sentence denotes Prior to use, the leaves were washed twice in deionized water; the water was drained and the leaves were air-dried.
T34 5265-5370 Sentence denotes Leaves were surface-sterilized with UV light for 20 min on each side and then cut into 4 × 4 cm sections.
T35 5371-5624 Sentence denotes The cell adapted bovine coronavirus strain 88 (BCoV-88) was propagated in human rectal tumor (HRT)-18 cells, diluted to a final concentration of 1.2 × 10 5 plaque forming units (PFU) in either minimum essential medium (MEM) or a bovine fecal suspension.
T36 5625-5784 Sentence denotes Two bovine fecal suspensions (0.1% and 10%) were used to dilute the virus, representing light and heavy fecal contaminations (1 ppm and 100 ppm), respectively.
T37 5785-5932 Sentence denotes Because enteric viruses are shed in titers up to 10 11 particles per gram of feces, the virus was diluted to simulate 1 ppm of viral contamination.
T38 5933-6115 Sentence denotes Each piece of prepared lettuce was spiked with 100 µl of the virus preparation, allowed to dry for 2 h in a biosafety cabinet and then stored at 4 °C in a refrigerator until elution.
T39 6116-6293 Sentence denotes Bovine feces were obtained from a healthy cow, resuspended at 0.1% and 10% in minimum essential medium (MEM), centrifuged at 3000×g and filtered through a 0.2 µM syringe filter.
T40 6294-6394 Sentence denotes The suspensions were confirmed to be BCoV negative by qRT-PCR before being used, as described later.
T41 6395-6506 Sentence denotes Experiments were duplicated using feces from a healthy young calf, also confirmed negative for BCoV by qRT-PCR.
T42 6507-6588 Sentence denotes To establish an optimal elution method, a pilot procedure was conducted on day 0.
T43 6589-6804 Sentence denotes Virus from triplicate lettuce pieces was eluted with MEM + 2% fetal bovine serum (FBS, Gibco), Tris-glycine + 1% FBS or phosphate-buffered saline (PBS)-Triton X-100 + 0.5% FBS, immediately following the drying step.
T44 6805-7025 Sentence denotes The eluents were then precipitated with 10% polyethylene glycol (PEG) 6000 (Calbiochem, EMD Biosciences, La Jolla, CA) and 2.5% NaCl at 4 °C with agitation for 2 h followed by centrifugation at 3500×g for 30 min at 4 °C.
T45 7026-7142 Sentence denotes The pellet was reconstituted with MEM + 2% FBS and subsequently analyzed by qRT-PCR for detecting viral genomic RNA.
T46 7143-7238 Sentence denotes Similar results were obtained with both MEM + 2% FBS and Tris-glycine + 1% FBS elution buffers.
T47 7239-7379 Sentence denotes However, significantly lower viral RNA copy numbers were detected when the elution buffer containing Triton X-100 was used (data not shown).
T48 7380-7555 Sentence denotes Because MEM + 2% FBS would interfere less with an infectivity assay than the buffer containing Tris-glycine, MEM + 2% FBS was selected as the eluent in subsequent experiments.
T49 7556-7637 Sentence denotes Virus was eluted in triplicate samples on days 0, 2, 5, 7, 12, 14, 20, 26 and 30.
T50 7638-7813 Sentence denotes Twenty milliliters of elution buffer was added to each lettuce piece in a 50 ml conical tube which was agitated for 15 min on an orbital shaker at 100 rpm at room temperature.
T51 7814-7888 Sentence denotes Virus was then precipitated with 10% PEG 6000 and 2.5% NaCl, as described.
T52 7889-7988 Sentence denotes The pellet was reconstituted with the elution buffer (MEM + 2% FBS) to 250 µl and stored at −70 °C.
T53 7989-8090 Sentence denotes Experiments were repeated on three different occasions, with 9 samples per time point for each group.
T54 8091-8320 Sentence denotes To estimate the amount of virus particles lost during drying and elution, each virus dilution was kept at 4 °C, sampled in triplicate at each time point, precipitated and reconstituted in the same way as the lettuce spiked virus.
T55 8321-8513 Sentence denotes Viral RNA was extracted from 90 µl of resuspended pellet using the MagMax™ viral RNA isolation kit and the MagMAX™ Express Magnetic Particle Processor (Applied Biosystems/ Ambion, Austin, TX).
T56 8514-8610 Sentence denotes Extracted RNA samples were either analyzed immediately by qRT-PCR or stored at −80 °C until use.
T57 8611-8862 Sentence denotes For qRT-PCR, we used primers and a Locked Nucleic Acid (LNA) Cy5 labeled fluorescent probe (Integrated DNA Technology, Coralville, IA) for the open reading frame (ORF) 1b region of CoV genomic RNA (Escutenaire et al., 2007; Muradrasoli et al., 2009) .
T58 8863-8984 Sentence denotes A synthetic oligonucleotide complementary to the probe was used to generate a standard curve (Escutenaire et al., 2007) .
T59 8985-9126 Sentence denotes Primers and probes for human 18S RNA (Cat# 4308329, Applied Biosystems, Foster City, CA) were used as internal controls (Poon et al., 2004) .
T60 9127-9170 Sentence denotes BCoV-88 RNA was used as a positive control.
T61 9171-9297 Sentence denotes RNA from mock infected cell supernatant was used as a negative control and RNAse-free water was used as a nontemplate control.
T62 9298-9457 Sentence denotes For all assays, 8 µl of RNA was transferred to a Qiagen Rotor-Gene strip tube containing 12 µl of the Rotor Gene™ Multiplex qRT-PCR mix (Qiagen, Valencia, CA).
T63 9458-9706 Sentence denotes Cycling conditions were 50 °C for 30 min, 95 °C for 10 min, 5 touchdown amplification steps of 94 °C for 30 s and 56 °C for 30 s, decreasing by 2 °C every second cycle down to 48 °C for 30 s, and then 50 cycles of 94 °C for 30 s and 46 °C for 60 s.
T64 9707-9782 Sentence denotes Amplification was detected using a Rotor Gene Q 6 plex machine from Qiagen.
T65 9783-9895 Sentence denotes A plaque assay (Hasoksuk et al., 2008) was used to quantify infectious virus recovered from the lettuce surface.
T66 9896-10057 Sentence denotes Briefly, 6-well plates containing 3-to 5-day-old monolayers of HRT-18 cells were rinsed and incubated with FBS-free MEM for 3 h at 37 °C in a 5% CO 2 atmosphere.
T67 10058-10207 Sentence denotes The culture medium was then aspirated and serial dilutions of the recovered virus preparations from the lettuce leaves were added to duplicate wells.
T68 10208-10356 Sentence denotes Following virus adsorption for 1.5 h at 37 °C in a 5% CO 2 atmosphere, the inoculum was removed and the cell monolayer was rinsed with FBS-free MEM.
T69 10357-10602 Sentence denotes The plates were then overlaid with MEM containing 0.8% Noble agar, 0.1% neutral red, 0.15 µg/ml of trypsin (Cat#1426 -Sigma, St. Louis, MO), 1% diethylaminoethyl dextran, 1% antibiotic-antimycotic (Gibco, Carlsbad, CA) and 3% sodium bicarbonate.
T70 10603-10802 Sentence denotes Trypsin was added to the overlay media to mimic host cell proteases by activating viral spike protein, increasing coronavirus infectivity (Bertram et al., 2011; Gaertner et al., 1991; Holmes, 2001) .
T71 10803-10885 Sentence denotes After solidification of the agar, the plates were inverted and incubated at 37 °C.
T72 10886-10921 Sentence denotes Plaques were counted from days 4-6.
T73 10922-11027 Sentence denotes The final titers were calculated by averaging counted plaques on duplicate wells of the highest dilution.
T74 11028-11282 Sentence denotes Because of the long period of incubation required for plaque formation in the plaque assay, cell toxicity was observed when the recovered virus was originally diluted in fecal suspensions, despite rinsing the monolayer with FBS-free MEM after adsorption.
T75 11283-11359 Sentence denotes A cell culture immunofluorescence assay was used to circumvent this problem.
T76 11360-11519 Sentence denotes Briefly, 3-4-dayold 96-well plates containing mono-layers of HRT-18 cells were rinsed with FBS-free MEM and incubated for 3 h at 37 °C in a 5% CO 2 atmosphere.
T77 11520-11640 Sentence denotes Serial dilutions of recovered virus were added to duplicate wells, followed by the addition of 0.04 µg trypsin per well.
T78 11641-11748 Sentence denotes Plates were then centrifuged for 1 h at 500×g at room temperature and incubated at 37 °C, 5% CO 2 for 20 h.
T79 11749-11991 Sentence denotes Plates were then fixed with 80% acetone in water, stained with guinea pig anti-BCoV antiserum (BEI Resources cat# NR-455) and FITC-conjugated goat anti-guinea pig IgG (KPL, Inc, Gaithersburg, MD), and observed using an inverted UV microscope.
T80 11992-12158 Sentence denotes The final titers were calculated by averaging counted fluorescing cells in duplicate wells of the highest dilution of the virus where fluorescing cells were observed.
T81 12159-12488 Sentence denotes To determine if residual virus would remain on the lettuce surface after the elution procedure, BCoV-88 was concentrated by ultracentrifugation onto a 20% sucrose cushion, labeled with SYBR-Gold (Invitrogen, Carlsbad, CA) and purified using a 20-60% continuous sucrose gradient at 105,000×g for 18 h using a Beckman SW41TI rotor.
T82 12489-12609 Sentence denotes Labeled virus was diluted in MEM+ 2% FBS, 0.1% fecal suspension or 10% fecal suspension and was spiked onto the lettuce.
T83 12610-12683 Sentence denotes After drying, the virus was eluted from the lettuce surface as described.
T84 12684-12830 Sentence denotes Lettuce pieces were then observed using a UV microscope and/or fluorescence confocal microscope for detection of residual virus on lettuce leaves.
T85 12831-13061 Sentence denotes Results from (log-transformed) qRT-PCR and infectivity assays were compared among and within the groups using two way ANOVA testing for group, time, and the interaction of group and time, using the SigmaStat® program (Systat Inc).
T86 13062-13112 Sentence denotes Statistical significance was assessed at p < 0.05.
T87 13113-13359 Sentence denotes To determine the stability of the viral genomic RNA on the lettuce surface under household refrigeration conditions, we used qRT-PCR to detect and quantify viral RNA at various time points after storage of virus-spiked lettuce at 4 °C (Fig. 1A) .
T88 13360-13590 Sentence denotes Whereas viral RNA was detected from the lettuce surface at all time points (up to 30 days) throughout the experiment, RNA levels varied significantly among the different experimental groups (6.6 × 10 4 to 1.7 × 10 6 copy numbers).
T89 13591-13761 Sentence denotes In general, viral RNA levels were significantly higher (p < 0.05) when the spiked virus was diluted with MEM + 2% FBS than when diluted with fecal suspensions (Fig. 1A) .
T90 13762-13981 Sentence denotes Analysis of variance within groups showed a significant decrease (p < 0.05) in the amount of RNA detected from lettuce after day 5 for MEM + 2% FBS and 0.1% fecal suspensions, and after day 12 for 10% fecal suspensions.
T91 13982-14202 Sentence denotes When the virus was stored in suspensions at 4 °C, there was no significant decrease in the amount of viral RNA detected throughout the 30 day experimental period between MEM + 2% FBS and 0.1% fecal suspension (Fig. 1B) .
T92 14203-14453 Sentence denotes However, the amount of viral RNA detected from the 10% fecal suspension was generally lower at all time points, and significantly lower (p < 0.05) at days 0, 2, 14, 20, 26 and 30, when compared to the 0.1%s fecal suspension or MEM + 2% FBS treatment.
T93 14454-14712 Sentence denotes To better estimate long-term stability of coronavirus RNA, the recovery of viral RNA was expressed as a percentage of the log10 copy number of viral RNA revered at various time points relative to the viral RNA recovered at day 0 which was normalized to 100%.
T94 14713-14852 Sentence denotes As shown in Fig. 1C and D, there was less than 20% reduction in viral RNA recovery from spiked lettuce or stored in suspension for 30 days.
T95 14853-14986 Sentence denotes These results demonstrate that CoV RNA was stable on the lettuce surface and in the suspensions tested throughout the 30 days period.
T96 14987-15203 Sentence denotes To assess the stability of infectious virus on the lettuce surface, we used a plaque assay to detect and quantify infectious virus recovered from lettuce after spiking and storage at 4 °C for various periods of time.
T97 15204-15312 Sentence denotes The infectivity of recovered virus varied significantly depending upon the type of diluent used ( Fig. 2A) .
T98 15313-15476 Sentence denotes Overall, the infectivity (PFU) was significantly higher at each time point for virus diluted in MEM + 2% FBS than for those in either of the two fecal suspensions.
T99 15477-15781 Sentence denotes Specifically, virus infectivity from MEM + 2% FBS decreased gradually from approximately 10,000 PFU on day 0-10 PFU on day 20 and was below the detectable level by day 25, whereas the CoV infectivity from the 0.1% fecal suspension decreased similarly until day 12, but was no longer detectable by day 14.
T100 15782-15912 Sentence denotes In contrast, no infectious virus was recovered from lettuce spiked with virus diluted in a 10% fecal suspension from day 2 onward.
T101 15913-16161 Sentence denotes When the virus was stored in MEM + 2% FBS or in 0.1% fecal suspension, the reduction of infectivity over 30 days was much slower than that on the lettuce surface, but it was similar on the lettuce surface and in the 10% fecal suspension (Fig. 2B) .
T102 16162-16285 Sentence denotes Virus infectivity did not change significantly in MEM + 2% FBS diluent from day 0 to day 25, decreasing slightly by day 30.
T103 16286-16547 Sentence denotes When the number of plaque forming units was normalized to 100% recovery at day 0 ( Fig. 2C and D) , a 90% reduction (1 log) every 5-6 days was observed in infectious virus recovered from lettuce surfaces spiked with CoV in MEM + 2% FBS or 0.1% fecal suspension.
T104 16548-16685 Sentence denotes A 90% reduction in recovery was observed every 14 days from virus stored in 0.1% fecal suspension, and 30 days if stored in MEM + 2% FBS.
T105 16686-16885 Sentence denotes These results demonstrate that infectious virions are more stable in suspension than when dried on the lettuce surface and that viral stability is influenced by the type of diluent used in the study.
T106 16886-17087 Sentence denotes These results also suggest that the 10% fecal suspension might contain factors that can inactivate virus during storage, thus reducing infectivity, or inhibiting plaque formation during a plaque assay.
T107 17088-17261 Sentence denotes To distinguish these two possibilities, we used an immunofluorescence assay to determine the fluorescence foci following infection of cell cultures with the recovered virus.
T108 17262-17384 Sentence denotes We found that overall there were more focus forming units (FFU) than PFU detected by the plaque assay (Fig. 3 vs Fig. 2) .
T109 17385-17740 Sentence denotes However, significant amounts of infectious virus (~3 log 10 FFU) were detected by the cell culture fluorescence assay at day 14 in samples recovered from both lettuce spiked with virus diluted in 10% fecal suspension (Fig. 3) and the corresponding diluent, whereas the plaque assay resulted in no detectable levels of virus infectivity at day 2 (Fig. 2) .
T110 17741-17936 Sentence denotes When the number of fluorescent foci was normalized to 100% at day 0 (Fig. 3C) , a 90% decrease in recovery was observed every 7 days for virus recovered from spiked lettuce of all three diluents.
T111 17937-18119 Sentence denotes Similar observation was made from virus stored in 0.1 and 10% fecal suspensions (Fig. 3D) , whereas reduction of 90% was only observed after 25 days for virus stored in MEM + 2% FBS.
T112 18120-18147 Sentence denotes Combined with data in Figs.
T113 18148-18376 Sentence denotes 2 and 3 , these results indicate that viral infectivity was less stable in fecal suspensions than in MEM + 2% FBS and that an immunofluorescence assay may be more sensitive than the plaque assay for quantifying infectious virus.
T114 18377-18530 Sentence denotes An immunofluorescence assay may be particularly useful with samples containing higher concentrations of feces that could interfere with plaque formation.
T115 18531-18833 Sentence denotes To determine the efficiency of elution and to test the hypothesis that the degree of fecal contamination could affect the effectiveness of virus elution, we used SYBR-Gold to label virus particles, then diluted the labeled virus with one of three diluents and spiked the virus onto the lettuce surface.
T116 18834-19017 Sentence denotes Following the same procedure for processing, storage and virus elution as described earlier, residual virus particles on the lettuce surface were subjected to microscopic examination.
T117 19018-19189 Sentence denotes Results confirmed that residual coronavirus was present on the lettuce surface after elution (Fig. 4) with increased virus accumulation on the lettuce veins ( Fig. 4E-G ).
T118 19190-19367 Sentence denotes It appears that slightly more residual virus remained on the lettuce surface when the spiked virus was diluted in fecal suspensions (Comparing panels C and D with B in Fig. 4 ).
T119 19368-19530 Sentence denotes In this study, we tested the hypothesis that contaminated vegetables may serve as a vehicle for coronavirus transmission to humans through consumption of produce.
T120 19531-19734 Sentence denotes Specifically we used BCoV as a surrogate of the Genus Betacoronavirus, which includes SARS-CoV, to evaluate the stability of coronaviruses on the lettuce surface under household refrigeration conditions.
T121 19735-19814 Sentence denotes Our data showed that BCoV on lettuce retained infectivity for at least 14 days.
T122 19815-19953 Sentence denotes We also observed that higher numbers of infectious particles were detected using fluorescent focus forming assay compared to plaque assay.
T123 19954-20276 Sentence denotes Plaque assay relies on the induction of cell death to form plaques and is sensitive to changes in the assay condition, whereas fluorescent focus assay relies on the detection of virus antigen within infected cells, which may increase the detection and visualization of the infectious virus particles (Flint et al., 2009) .
T124 20277-20561 Sentence denotes Interestingly, as assessed by a plaque assay, significantly lower amounts of infectious virus were recovered from lettuce surfaces spiked with virus samples that were diluted in a 10% fecal suspension as compared to those diluted in MEM + 2% FBS and a 0.1% fecal suspension (Fig. 2) .
T125 20562-20670 Sentence denotes The reason for such a rapid reduction in infectivity in virus diluted in 10% fecal suspensions is not known.
T126 20671-20789 Sentence denotes It is possible that the high fecal concentration contains inhibitory factors, such as intestinal proteases or lipases.
T127 20790-21119 Sentence denotes The abundant proteolytic or lipolytic enzymes present in the intestine, including those from bacterial flora, are required for digestion of food and may destroy the virion surface spike glycoprotein, which is sensitive to protease cleavage and the virus envelope, which is sensitive to lipases (Chen, 1985; Krempl et al., 2000) .
T128 21120-21219 Sentence denotes Thus, the proteolytic and lipolytic effects of fecal suspensions might render BCoV less infectious.
T129 21220-21428 Sentence denotes A similar finding has been reported for canine CoV: at 4 °C, fecal dilutions of 1:1 resulted in a rapid loss of viral infectivity, but a fecal dilution of 1:1000 preserved infectivity (Tennant et al., 1994) .
T130 21429-21579 Sentence denotes However, viral genomic RNA levels were more stable and steady on the lettuce surface for the duration of the experiment, compared to infectious virus.
T131 21580-21871 Sentence denotes This suggests that the rapid decrease in CoV infectivity in the 10% fecal suspension was not due mainly to degradation of the viral genome, but more likely due to the loss of a functional spike glycoprotein on the virion surface that confers infectivity or to the loss of the virus envelope.
T132 21872-22052 Sentence denotes However, neither the structure of the spike glycoprotein, nor the integrity of the virions or the viral genomic RNA recovered from the lettuce surface was determined in this study.
T133 22053-22250 Sentence denotes Microscopic observation of labeled virus on the lettuce surface indicates that some residual virus particles remained attached after elution, suggesting that virus elution was incomplete (Fig. 4) .
T134 22251-22499 Sentence denotes A quantitative assay to enumerate virus particles on the lettuce surface after elution may be required to establish if the differences in infectivity among the three diluents are the result of time-related inactivation or time-increased attachment.
T135 22500-22838 Sentence denotes However, qRT-PCR results for detecting viral genomic RNA after virus elution from the lettuce surface support the former possibility because the difference in viral genome copies among the three diluents was lower than that for CoV titers in viral infectivity assay and remained relatively constant throughout the 30 days (comparing Figs.
T136 22839-22849 Sentence denotes 1 and 2) .
T137 22850-23050 Sentence denotes It is not clear, however, whether the lower levels of viral genomic RNA, especially in the 10% fecal suspensions after elution ( Fig. 1 ) resulted from RNA degradation or incomplete elution (Fig. 4) .
T138 23051-23174 Sentence denotes For non-enveloped viruses, the presence of biosolids increased viral attachment to the lettuce surface (Wei et al., 2009 ).
T139 23175-23333 Sentence denotes Adsorption of non-enveloped virus particles may be linked to the virus adsorbent isoelectric point, hydrophobicity and ionic strength (Charles et al., 2008) .
T140 23334-23481 Sentence denotes Similar studies are needed for enveloped viruses and also to establish the role of biosolids on coronavirus environmental maintenance and survival.
T141 23482-23655 Sentence denotes Our results demonstrate that coronavirus was stable during the shelf-life of romaine lettuce, and that a wash procedure (elution) did not completely remove residual viruses.
T142 23656-23800 Sentence denotes Thus, it is possible that contaminated ready-to-consume produce may be a potential vehicle for zoonotic transmission of coronaviruses to humans.
T143 23801-23959 Sentence denotes However, the epidemiologic significance of the amount of recovered infectious particles cannot be evaluated, since the coronavirus infectious dose is unknown.
T144 23960-24147 Sentence denotes Developing countries have an increasing role in food production for the world market thus increasing the necessity of monitoring food production beyond our borders (Newell et al., 2010) .
T145 24148-24334 Sentence denotes Although the probability of foodborne transmission of certain newly emerging viruses may be low, this transmission route may cause higher rates of disease and morbidity (FAO/WHO, 2008) .
T146 24335-24502 Sentence denotes Awareness regarding the possible roles of water, fresh produce and fecal contamination in coronavirus transmission is required at times of human coronavirus outbreaks.
T147 24503-24687 Sentence denotes Several coronaviruses can replicate in the epithelial cells of the respiratory tract as well as the enteric tract, and they can be transmitted by the respiratory route (Holmes, 2001) .
T148 24688-24845 Sentence denotes Bovine coronavirus has been described as a pneumoenteric virus that infects the upper and lower respiratory tract and intestine of cattle and wild ruminants.
T149 24846-24998 Sentence denotes It is shed in feces and nasal secretions, causing respiratory and enteric syndromes such as shipping fever and winter dysentery in cattle (Saif, 2010) .
T150 24999-25189 Sentence denotes During the SARS outbreak, diarrhea was a common symptom of SARS, occurring in approximately 38% of the patients, demonstrating that SARS also had an intestinal tropism (Leung et al., 2003) .
T151 25190-25344 Sentence denotes However, foodborne transmission of SARS Coronavirus was not documented during the SARS outbreak and oral-fecal transmission of SARS is yet to be explored.
T152 25345-25465 Sentence denotes Viral RNA recovered from spiked lettuce or from virus stored in suspension detected by real-time RT-PCR for coronavirus.
T153 25466-25468 Sentence denotes A.
T154 25469-25563 Sentence denotes Log 10 of viral RNA copy number recovered from the lettuce surface over time after elution; B.
T155 25564-25651 Sentence denotes Log 10 of viral RNA copy number recovered from virus stored in suspension over time; C.
T156 25652-25774 Sentence denotes Log 10 of viral RNA copy number recovered from the lettuce surface over time after elution normalized to 100% at day 0; D.
T157 25775-25887 Sentence denotes Log 10 of viral RNA copy number recovered from virus stored in suspension over time normalized to 100% at day 0.
T158 25888-26028 Sentence denotes Different letters denote statistically significant differences at p < 0.05 (Two way ANOVA using all pairwise multiple comparison procedures:
T159 26029-26080 Sentence denotes Holm-Sidak method) among groups at same time point.
T160 26081-26101 Sentence denotes Statistical symbols:
T161 26102-26150 Sentence denotes A is statistically different from B at p < 0.05.
T162 26151-26252 Sentence denotes Legend: -◆-CoV in MEM + 290025; FBS; -○-CoV in 0.1% fecal suspension; -□-CoV in 10% fecal suspension.
T163 26253-26392 Sentence denotes Infectious coronavirus recovered from spiked lettuce or stored in suspensions and detected by cell culture immunofluorescence (CCIF) assay.
T164 26393-26395 Sentence denotes A.
T165 26396-26513 Sentence denotes Focus forming unit (FFU) (log 10 ) of infectious virus recovered from the lettuce surface over time after elution; B.
T166 26514-26605 Sentence denotes FFU (log 10 ) of infectious virus, recovered from virus stored in suspensions over time; C.
T167 26606-26730 Sentence denotes FFU (log 10 ) of infectious virus recovered from the lettuce surface over time after elution normalized to 100% at day 0; D.
T168 26731-26847 Sentence denotes FFU (log 10 ) of infectious virus, recovered from virus stored in suspensions over time normalized to 100% at day 0.
T169 26848-26988 Sentence denotes Different letters denote statistically significant differences at p < 0.05 (Two way ANOVA using all pairwise multiple comparison procedures:
T170 26989-27040 Sentence denotes Holm-Sidak method) among groups at same time point.
T171 27041-27061 Sentence denotes Statistical symbols:
T172 27062-27110 Sentence denotes A is statistically different from B at p < 0.05.
T173 27111-27207 Sentence denotes Legend: -◆-CoV in MEM + 2% FBS; -○-CoV in 0.1% fecal suspension; -□-CoV in 10% fecal suspension.
T174 27208-27302 Sentence denotes Residual SYBR-Gold labeled coronavirus on the lettuce surface after elution with MEM + 2% FBS.
T175 27303-27427 Sentence denotes A-D images were acquired using a Nikon Eclipse TS100 UV microscope using the Image NIS element program with a 40× objective.
T176 27428-27519 Sentence denotes E-H images are confocal micrographs using an Olympus Fluoview™ FV 1000 confocal microscope.
T177 27520-27632 Sentence denotes The laser setting was the excitation filter DM 488 and the Emission filters BA (505-525 nm) and BA (560-620 nm).
T178 27633-27706 Sentence denotes The objective setting was 10× in the image; 100 µm marker bars are shown.
T179 27707-27712 Sentence denotes A, E.
T180 27713-27806 Sentence denotes Lettuce leaf spiked with CoV SYBR-Gold labeled, diluted in MEM + 2% FBS before elution; B, F.
T181 27807-27898 Sentence denotes Lettuce leaf spiked with CoV SYBR-Gold labeled diluted, in MEM + 2% FBS after elution C, G.
T182 27899-28003 Sentence denotes Lettuce leaf spiked with CoV SYBR-Gold labeled, diluted in 0.1% fecal suspension after elution and D, H.
T183 28004-28097 Sentence denotes Lettuce leaf spiked with CoV SYBR-gold labeled diluted in 10% fecal suspension after elution.
T184 28098-28138 Sentence denotes Arrows indicate labeled virus particles.