Mechanisms of Antiviral Action of Plant Antimicrobials against Murine Norovirus (2024)

Viruses and cells.

MNV (strain S7-PP3), obtained from Yukinobu Tohya (Department of Veterinary Medicine, Nihon University, Kanagawa, Japan), was propagated on RAW 264.7 (ATCC TIB-71) cell line monolayers with Dulbecco's modified Eagle medium (DMEM; Mediatech Inc., Manassas, VA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT), 10 mM HEPES buffer (Mediatech Inc.), 0.113% sodium bicarbonate (Fisher Scientific, Fair Lawn, NJ), and 1.0% antibiotic-antimycotic (Mediatech Inc.). The cells were incubated at 37°C with 5% CO₂ as described previously (16). MNV was concentrated and purified using polyethylene glycol precipitation and Vertrel XF extraction to promote monodispersion of the virus and the removal of lipids (73, 74). The virus stocks were stored at −80°C until used in experiments.

Viral titrations were performed using the Reed-Muench method (75) to determine the viral dilution in which 50% of the wells containing cells were visibly affected (the 50% tissue culture infective dose [TCID₅₀]). Serial 10-fold dilutions of the virus sample were assayed in 96-well tissue culture plates (Nunclon, Roskilde, Denmark) containing RAW 264.7 cell monolayers and 50 μl of DMEM containing 10% FBS (DMEM-FBS), with incubation at 37°C with 5% CO₂ as described above. A volume of 50 μl of each dilution was used to inoculate eight replicate wells to ensure adequate precision of the assay. Each well was checked every day for 5 days for cytopathic effects (CPE). The highest dilution in which >50% of the wells exhibited CPE was used to determine the virus TCID₅₀/ml. The use of such a method for MNV has been widely reported in the literature (25, 71, 72, 76, 77, 78, 79).

Poliovirus type 1 (PV1; strain LSc-2ab) was obtained from the Department of Virology and Epidemiology at the Baylor College of Medicine (Houston, TX) and was included as a process control in several experiments. PV1 was propagated on monolayers of BGM cells (Buffalo green monkey kidney cells) obtained from D. Dahling (Environmental Protection Agency, Cincinnati, OH) with minimal essential medium (MEM; Irvine Scientific, Santa Ana, CA) containing 5% calf serum (HyClone Laboratories) with an incubation temperature of 37°C and an atmosphere of 5% CO₂. PV1 was purified in the same manner as described previously for MNV, and the titer was determined using 10-fold serial dilutions in plaque-forming assays on BGM cell monolayers as described by Bidawid et al. (80).

Antimicrobial preparation.

Lemongrass and allspice oils were obtained from Lhasa Karnak Herbal Co. (Berkeley, CA). No information was available regarding the concentration of citral in this specific lemongrass oil product; however, citral may account for up to 85% of the composition of lemongrass oil (81). Purified citral (mixture of cis and trans, >96%) was purchased from Sigma-Aldrich (St. Louis, MO). The antimicrobials were diluted to the specific concentrations used in the experiments with sterile phosphate-buffered saline (PBS; pH 7.4; Sigma-Aldrich). Since alcohol could potentially enhance the antimicrobial effect of a solution, it was not used in these experiments to suspend these viscous antimicrobials. Though the oils did not dissolve completely in the PBS, the results from these experiments were found to be consistent and repeatable.

Antimicrobial efficacy experiments.

Allspice oil, lemongrass oil, and citral were evaluated in separate experiments at concentrations of 2.0% and 4.0% (vol/vol). The experiments were performed at room temperature (∼24°C) in triplicate in PBS (1-ml volume in 5-ml polystyrene tubes) (Becton, Dickinson and Company, Franklin Lakes, NJ). MNV was added separately to each of the tubes (to a final concentration of ∼10⁶ to 10⁷ TCID₅₀/ml), and the tubes were placed on an orbital shaker (model G33; New Brunswick Scientific, Edison, NJ) with agitation at 300 rpm. Control tubes (no antimicrobials added) containing MNV in PBS were also included in each experiment. The control tubes were sampled immediately (0 h) by removing 100 μl from each and placing this volume in 900 μl of DMEM-FBS. At time intervals of 0.5, 6, and 24 h of exposure, 100-μl samples were removed from each tube and diluted in 900 μl of DMEM-FBS to neutralize the antimicrobials. All samples were placed at −80°C until subsequent assays were performed using the TCID₅₀ cell culture method (as described previously) to determine the infectious virus titer.

Antimicrobial neutralization and cytotoxicity experiments.

Neutralization tests were performed with both the 2.0% and 4.0% concentrations of lemongrass oil, citral, and allspice oil in which a PBS solution containing the desired concentration of the antimicrobial was placed into either DMEM-FBS or PBS (1 ml into 9 ml). The solution was mixed thoroughly, and then approximately 1 × 10⁷ TCID₅₀ of MNV was added. The solution was mixed again and then allowed to sit for 5 min at room temperature. Tenfold serial dilutions of the solutions were assayed on RAW 264.7 cells as described previously. If the antimicrobials were completely neutralized in the DMEM-FBS or PBS solution, it was expected that there would be no reduction in MNV numbers in comparison to the controls with either PBS or DMEM-FBS alone.

In a separate experiment, 10-fold serial dilutions of the 2.0% and 4.0% concentrations of the lemongrass oil, citral, and allspice oil were added to RAW 264.7 cells which were then examined daily for 6 days for any signs of cell toxicity.

Mechanism-of-action experiments.

To determine the mechanism(s) of antiviral action, another set of tests was performed. Each antimicrobial was added to three replicate test tubes containing approximately 1.0 × 10⁶ TCID₅₀/ml of MNV in 2 ml of PBS and a 4.0% (vol/vol) concentration of the plant antimicrobial (i.e., allspice oil, lemongrass oil, or citral). These were tested as described previously. After each exposure period (0.5, 6, or 24 h), volumes of 50 μl were removed from each tube (including the control tubes) and placed separately into four microcentrifuge tubes (replicates), three containing 450 μl of DMEM-FBS and one that contained 450 μl of PBS (Fig. 1). Two of the tubes containing DMEM-FBS (Fig. 1, tubes A and B) were used immediately in an RNase I protection experiment to assess the virus capsid integrity. The other two tubes were stored at −80°C until used in a cell binding experiment (Fig. 1, tube C with DMEM-FBS) to assess the ability of the treated MNV to bind to host RAW 264.7 cells and for transmission electron microscopy (TEM) imaging (Fig. 1, tube D with PBS) to directly observe any physical changes to the MNV particles following antimicrobial treatments. These experiments are described in detail below.

Open in a separate window

FIG 1

Sampling strategy for mechanism of action experiment. At each time exposure, four 50-μl volumes were removed from each of the replicate test tubes (1, 2, and 3) and placed into separate microcentrifuge tubes containing 450 μl of either Dulbecco's modified Eagle medium with 10% fetal bovine serum (tubes A, B, and C) or phosphate-buffered saline (tube D). These four tubes were used in subsequent experiments or assays (described in box) to determine the antiviral mechanism(s) of action.

(i) RNase I protection experiment.

To one of these tubes (Fig. 1, tube B), 1.0 μl of RNase I (100 U; Ambion Inc., Austin, TX) was added. This tube and another (Fig. 1, tube A without RNase I) were then placed in a 37°C incubator for 30 min. RNase I should degrade any viral RNA that is exposed to the environment (e.g., if the RNA is no longer protected by an intact capsid). The tube without RNase I was included as a control. Following the 30-min incubation, the samples were immediately placed at −80°C to halt the enzymatic activity and held at this temperature for storage.

Immediately prior to the nucleic acid extraction step, the samples were thawed and 200 μl of each was added (separately) to 600 μl of the ZR viral RNA buffer of the ZR viral RNA kits (Zymo Research, Irvine, CA). This freeze-thaw step should denature the RNase I enzyme. The ZR viral RNA buffer should also denature the enzyme, and β-mercaptoethanol (BME) was added to each sample to inhibit RNases (as per the manufacturer's instructions). A volume of 2 μl of purified PV1 (∼2.0 × 10⁶ genome copies total) was added to each of the samples as a process control (explained in detail below). The viral RNA (from both MNV and PV1) was purified from each sample using the ZR viral RNA kits according to the manufacturer's protocol, with the exception that the final volume was adjusted to 20 μl.

The reverse transcription (RT) step was performed using high-capacity cDNA reverse transcription kits (Applied Biosystems, Foster City, CA) in the following manner. Three microliters of extracted virus RNA was added to 3 μl of RT mixture containing 0.6 μl of 10× reverse transcription buffer, 0.24 μl of 25× deoxynucleoside triphosphate (dNTPs), 0.6 μl of 10× random hexamers, 15 U of MultiScribe reverse transcriptase, and 6 U of RNase inhibitor (Applied Biosystems). This RT reaction mixture was incubated at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min to inactivate the enzyme.

A TaqMan-based quantitative PCR (qPCR) assay which targets a 129-nucleotide sequence in the open reading frame 1 (ORF1)-ORF2 junction region of MNV was performed in a 25-μl reaction volume containing 2.5 μl of cDNA from the RT reaction, 12.5 μl of LightCycler 480 Probes Master (Roche Diagnostics, Mannheim, Germany), 400 nM (each) concentrations of the primers MNV-S (5′-CCGCAGGAACGCTCAGCAG-3′) and MNV-AS (5′-GGYTGAATGGGGACGGCCTG-3′), and 300 nM TaqMan MGB probe MNV-TP (5′-FAM-ATGAGTGATGGCGCA-MGB-NFQ-3′) as described previously (76). The qPCR amplification was performed using a LightCycler 480 Real-Time PCR Instrument II (Roche Diagnostics) with the following conditions: denaturation at 95°C for 15 min to activate the DNA polymerase, followed by 50 cycles of amplification with denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. Tenfold serial dilutions (from 10² to 10⁸ copies per PCR tube) of the standard plasmid DNA containing an insert of approximately 500 nucleotides including the ORF1-ORF2 junction region of the MNV-S7 PP3 strain were used to generate a standard curve for the quantification of MNV cDNA copy numbers (76). The average copy number of two PCR tubes was used in these calculations. The amplification data were analyzed using the LightCycler 480 software (version 1.5; Roche Diagnostics).

A subsequent separate qPCR assay was performed for the PV1 process control (an enterovirus) using the cDNA from the RT as the template. The qPCR was performed as described previously for MNV, but with primers and probes specific to the enteroviruses (forward primer EV1F [400 nM], 5′-CCCTGAATGCGGCTAAT-3′; reverse primer EV1R [400 nM], 5′-TGTCACCATAAGCAGCCA-3′; and EV probe [120 nM], 5′-FAM [6-carboxyfluorescein]-ACGGACACCCAAAGTAGTCGGTTC-BHQ [Black Hole Quencher]-1-3′) and with the following conditions: denaturation at 95°C for 10 min, followed by 50 cycles with denaturation at 94°C for 15 s and annealing and extension at 60°C for 1 min. This assay results in a 143-bp product (82). Since 2 μl of the PV1 process control was added prior to nucleic acid extraction to each of the samples, the PV1 concentration (copy number) that was determined by qPCR in the control samples (which did not contain any extraction-, RT-, and/or qPCR-inhibiting substances) was used to determine if there was any inhibition in the samples with antimicrobials (leading to reduced MNV amplification and an underestimate of the viral copy number).

(ii) Cell binding experiment.

A cell binding assay was performed to assess the ability of the MNV treated with the plant antimicrobials to bind to host cells. RAW 264.7 cell monolayers were prepared in 24-well plates, and the growth medium was carefully aspirated from each well. The cells were rinsed with Tris-buffered saline (TBS; 2.53 g/liter of Trizma base, 6.54 g/liter of NaCl, 0.3 g/liter of KCl, 0.046 g/liter of Na₂HPO₄ [anhydrous], 4 liters of ultrapure H₂O), and then the TBS was removed. Following this, 100 μl from the third replicate tube from each sample (Fig. 1, tube C) was added to two wells and the plate was incubated at 4°C for 1 h to prevent the virus from entering the cells (62), with gentle agitation every 15 min to ensure that the cells remained covered to prevent them from drying. In addition to the MNV treated with allspice oil, lemongrass oil, or citral, numerous controls were also included in duplicate wells (Table 1).

After the 1-h incubation period, the cells were washed three times with TBS (with careful aspiration as before) to remove any unbound virus particles or virus RNA, and then an additional 198 μl of PBS was added to each well. This was followed first by the addition of 600 μl of the extraction buffer of the ZR viral RNA kit (Zymo Research) and finally by addition of 2 μl of the PV1 process control (∼2.0 × 10⁸ total genome copies). This entire 800-μl volume was then extracted using the ZR viral RNA kits as described previously. The extracts were used as the template in the MNV and PV1 RT-qPCR assays as described previously. The 1-h incubation period at 4°C does not allow for viral replication or the development of any CPE in the cells. This therefore was used to determine the number of viruses that were able to bind to the cells (while discounting those that did not bind), but it did not measure successful infection of the cells or assess virus viability.

(iii) TEM imaging.

To directly observe any structural changes to the MNV particles following treatment with the plant antimicrobials, TEM imaging was performed. A drop (5 to 10 μl) of each sample from the 24-h exposure from the mechanism of action experiments (Fig. 1, tube D with PBS) was applied to separate glow discharge carbon-coated EM grids. The grids were then stained with 2% aqueous uranyl acetate for 3 min, dried, and examined using an FEI CM12S TEM (FEI Electronics Instruments, Co., Hillsboro, OR) operated at 80 kV. The images were captured using an AMT 420 camera (Advanced Microscopy Techniques, Woburn, MA). The 24-h exposure was chosen since it was likely to produce the greatest antiviral effects which could then be observed under TEM to better understand the mechanisms of action.

Statistical analyses.

For the cell culture infectivity (antimicrobial efficacy) assays, the data were reported as the logarithmic reduction in infectivity using the formula −log₁₀ (N_t/N₀), where N₀ was the concentration of MNV particles measured via cell culture infectivity at time zero and N_t was the infectious particle concentration at time t. A two-tailed Student t test was used to compare the reductions in cell culture infectivity observed with the controls or with the antimicrobial treatments. The reductions observed for the antimicrobial treatments at each time exposure were compared to the reduction in the controls, if any. Differences were considered statistically significant if the t test resulted in a P value of ≤0.05. Differences between the reductions in cell culture infectivity observed between the two concentrations of each antimicrobial were also evaluated for statistical significance.

In order to allow for statistical comparisons between the reductions in cell culture infectivity observed with different plant antimicrobials in separate experiments, the average reduction in each experiment for the controls (after 24 h) was subtracted from the reductions reported for each sample exposed to an antimicrobial (for all time exposure intervals) in order to normalize the reductions. These normalized data were used only for the Student t tests; the values reported in the tables are thus the actual, nonnormalized results.

For the RNase I protection assays, the data were reported as the log₁₀ reduction [−log₁₀ (N_t/N₀)] in the amplifiable virus copy number as determined by RT-qPCR in comparison to the untreated controls. A two-tailed Student t test was used to compare any differences between the controls and the antimicrobial treatments. The reduction (if any) in the viral copy number in each sample was compared to the corresponding control samples (i.e., with or without RNase I). Also, comparisons were performed between the samples that had been treated with antimicrobials with or without subsequent RNase I digestion. Differences were considered statistically significant if the results of the Student t test indicated that the P value was ≤0.05.

Antimicrobial neutralization and cytotoxicity experiments.

No reductions in MNV were observed in the neutralization tests with any of the plant antimicrobials in comparison to the DMEM-FBS or PBS controls (with no antimicrobials). Therefore, this method was confirmed to completely neutralize the antimicrobials at these concentrations. This dilution method was used for all subsequent assays.

Cell toxicity was observed in the RAW 264.7 cells with allspice oil, lemongrass oil, and citral in the 10⁻¹ dilution wells. These 10⁻¹ wells were therefore not included in the determination of the viral TCID₅₀/ml in the subsequent cell culture assays. The 10⁻² wells were the lowest dilution which could be accurately read. This effectively increased the limit of detection of these assays 10-fold (to 6.3 × 10² TCID₅₀/ml).

Antimicrobial efficacy experiments.

The antiviral efficacy of each plant antimicrobial was determined by comparison to the reductions (if any) in the cell culture infectivity of MNV observed in the controls (with no antimicrobials) at the same time interval. The results for lemongrass oil and its active component citral are shown in Tables 2 and ​and3,3, respectively. Concentrations of 2.0% and 4.0% were used for both antimicrobials. Both concentrations of lemongrass oil and citral produced significant reductions in MNV cell culture infectivity within 6 h of exposure in comparison to those of the controls (P ≤ 0.05). The reductions observed with 4.0% lemongrass oil were significantly greater than those with the 2.0% concentration following 6 and 24 h of exposure (P = 0.005 and P = 0.01, respectively). No significant differences (P > 0.05) were found between the two citral concentrations for any of the exposure times. Both the lemongrass oil and the citral required 24 h to reach at least a 2.0-log₁₀ reduction (range of 2.19 log₁₀ to 3.00 log₁₀). There were no statistically significant differences (P > 0.05) between the reductions observed between lemongrass oil and citral for either concentration or for any of the exposure times.

The antiviral efficacies of 2.0% and 4.0% allspice oil are shown in Table 4. The 4.0% concentration produced a significant reduction (P = 0.014 in comparison to the control) within 30 min of exposure, whereas the reduction observed with the 2.0% concentration was not significant within 30 min or 6 h of exposure (P = 1.0 and P = 0.12, respectively) but became significant after 24 h of exposure (P = 0.001). The reductions observed for the two concentrations were significantly different from each other after 30 min (P = 0.01) and 6 h (P = 0.0005) of exposure, but the reductions after 24 h of exposure (2.97 log₁₀ and 3.41 log₁₀ for the 2.0% and 4.0% allspice oil concentrations, respectively) were not (P = 0.21). Similar to the case with lemongrass oil and citral, reductions greater than 2.0 log₁₀ were not observed until 24 h of exposure, though the 4.0% allspice oil yielded a reduction near to this within 6 h of exposure (1.83 log₁₀).

The reductions observed with 2.0% allspice oil and 2.0% lemongrass oil differed significantly (P = 0.03) after 24 h of exposure (2.97 log₁₀ versus 2.19 log₁₀, respectively). The reductions observed with 4.0% allspice oil were greater than those with 4.0% lemongrass oil for all time exposures; the reductions were significantly greater after 30 min and 6 h (P = 0.005 and P = 0.001, respectively) but not after 24 h (P = 0.09). Therefore, allspice oil may have greater and faster-acting antiviral efficacy than lemongrass oil. No significant differences were found between citral and allspice oil at either concentration or any of the exposure times (P > 0.05), suggesting that these two antimicrobials have comparable antiviral efficacies against MNV.

RNase I protection experiment.

An RNase I protection experiment was performed in order to assess if the MNV capsid was degraded by allspice oil, lemongrass oil, or citral. Two of the four replicate tubes from each sample (Fig. 1, tubes A and B) were used. One of these tubes (tube B) was treated with RNase I to digest any exposed viral RNA; both tubes (with and without RNase I digestion) were then subjected to RT-qPCR to determine the degradation of the viral capsid as well as the viral RNA by comparing the amounts of amplifiable viral RNA in the digested and undigested samples to the corresponding controls (no antimicrobial treatment; with and without RNase I digestion).

The RT-qPCR results (log₁₀ reduction in viral copy number) for the RNA protection experiment with all three antimicrobials (at a 4.0% concentration) are shown in Fig. 2. No reductions were observed in the controls (with no exposure to antimicrobials) after 24 h, regardless of whether they had been digested with RNase I (data not shown). For both lemongrass oil and citral, small reductions in virus RNA copy numbers (≤0.44-log₁₀ reductions with and without RNase I digestion) were observed (Fig. 2A and ​andB).B). A few of these reductions were significant (indicated by single asterisks) in comparison to the corresponding (i.e., with or without RNase I digestion) no-antimicrobial control: the 30-min and 24-h exposures to lemongrass oil with RNase (P = 0.005 and P = 0.02, respectively) and the 30-min exposure to citral without RNase I digestion (P = 0.01). None of the reductions differed significantly between the samples treated with lemongrass oil or citral with and without RNase I digestion, with the exception of lemongrass oil after 30 min of exposure (P = 0.05; indicated by double asterisks).

Open in a separate window

FIG 2

Results of the RNase I protection assay for MNV after exposure to a concentration of 4.0% of lemongrass oil (A), citral (B), or allspice oil (C). The log₁₀ genome copy numbers of MNV RNA recovered were determined by RT-qPCR after exposure to each antimicrobial (for 30 min, 6 h, or 24 h) followed by RNase I digestion. The P values for the Student t tests comparing the log₁₀ virus copy numbers recovered are also presented. Values with statistical significance (P ≤ 0.05) for differences between the antimicrobial-treated samples and their corresponding untreated control (with or without RNase I digestion) are indicated with an asterisk; values with statistical significance (P ≤ 0.05) for differences between the antimicrobial-treated samples either with or without RNase I digestion are indicated with double asterisks.

All of the reductions observed for the samples treated with allspice oil were highly significant (P ≤ 0.0001) in comparison to the no-antimicrobial controls, with more than 3- and 2-log₁₀ reductions with and without RNase I digestion, respectively (single asterisk in Fig. 2C). Following treatment with allspice oil, the log₁₀ reductions were greater in the samples that were subsequently digested with RNase I; these differences were statistically significant (P ≤ 0.05; double asterisks in Fig. 2C) for all exposure times (i.e., 30 min, 6 h, and 24 h).

No inhibition was observed in the RNA extraction, the RT, or the qPCR steps with the PV1 process control for any of the samples from the RNase I protection experiment (data not shown).

Cell binding experiment.

The results for the cell binding experiment to determine if the antimicrobials inhibited the ability of MNV to bind to host cells are shown in Table 5. Under the experimental conditions, the positive control (MNV with no antimicrobials) was able to bind to the RAW 264.7 cells; nevertheless, although each control well was originally inoculated with approximately 5.6 log₁₀ MNV genome copies, only 2.7 log₁₀ remained bound to the cells at the end of the assay. The multiple wash steps may have removed much of the MNV (or possibly cells with bound MNV) from the wells.

MNV did not bind nonspecifically to the cell culture plates either in the control (negative control 2 in Table 1; not detected by RT-qPCR; data not shown) or the 4.0% allspice oil-treated samples (no-cell control 3 in Table 1); however, MNV treated with 4.0% lemongrass oil (no-cell control 1 in Table 1) or 4.0% citral (no-cell control 2 in Table 1) was able to bind to the cell culture plates (2.6 log₁₀ and 2.5 log₁₀ virus genome copies, respectively). The MNV RNA did not bind to the RAW 264.7 cells or the cell culture plate (naked RNA controls 1 and 2 in Table 1; not detected by RT-qPCR; data not shown). Finally, the extracted RNA from the RAW 264.7 cells did not result in nonspecific amplification in the RT-qPCR assays (negative control 1 in Table 1; data not shown).

The numbers of virus particles that were able to bind to the RAW 264.7 cells following treatment with 4.0% lemongrass oil and citral were initially (after 30 min of exposure) lower than the number of untreated viruses binding to cells in the control samples. Interestingly, the number of bound virus particles increased with longer exposures to both antimicrobials, particularly for the lemongrass oil treatment. This is directly opposite the cell culture infectivity (as shown in Table 2 and ​and3),3), which decreased over time.

In contrast, the number of 4.0% allspice oil-treated virus particles that were able to bind to the cells was initially comparable to that of the untreated control, 2.8 log₁₀ genome copies per well after 30 min and 6 h of exposure, but fell to 2.2 log₁₀ genome copies after 24 h of exposure.

No inhibition was observed with the extraction, RT, or qPCR steps for the PV1 process control for any of the samples from the cell binding experiment (data not shown). The values for the virus genome copy numbers determined by the RT-qPCR were considered accurate. Nevertheless, the number of bound viruses was likely overestimated to some degree due to the nonspecific direct binding of the lemongrass- and citral-treated virus particles to the plates (see no-cell control data in Table 5).

TEM imaging.

TEM imaging was used to directly observe MNV particles following treatment with the plant antimicrobials to determine if there were any structural changes to the virus particles. The TEM images for the untreated MNV, the MNV after exposure to 4.0% lemongrass oil and 4.0% citral for 24 h, and the MNV after exposure to 4.0% allspice oil for 30 min are shown in Fig. 3. The MNV following a 24-h exposure to allspice oil was included in the original TEM imaging, yet very few particles (one or two per entire grid) could be found. In addition, there was also a larger amount of debris present, making it more difficult to identify MNV on the grids (images not shown). Therefore, the 30-min exposure, with presumably less damage to the virus particles from the effects of the antimicrobial, was examined.

Open in a separate window

FIG 3

Transmission electron microscope images of MNV. (A) Untreated (no antimicrobials) MNV control (examples of MNV particles are indicated by arrows); (B) MNV following exposure to 4.0% allspice oil for 30 min (examples of MNV particles are indicated by arrows); (C) MNV following exposure to 4.0% lemongrass oil for 24 h; (D) MNV following exposure to 4.0% citral for 24 h.

Untreated MNV particles range from approximately 20 nm to 35 nm in diameter and have an icosahedral symmetry (appear spherical in most images) (Fig. 3A). The MNV particles exposed to allspice oil appeared to be slightly larger (∼25 to 75 nm) but still morphologically similar to the untreated MNV control and seemingly intact (Fig. 3B). Following exposure to allspice oil for 24 h, the few virus particles that were observed were similar in size and appearance (∼60 to 70 nm) (images not shown).

The MNV treated with lemongrass oil and its active component, citral, were greatly expanded in size. The virus particles following treatment with lemongrass oil ranged in size from approximately 100 to 500 nm, with an average size of ∼300 nm (Fig. 3C). The MNV particles treated with citral were even larger, with an average size of ∼600 nm (range of ∼350 to 750 nm) (Fig. 3D). The MNV particles treated with these two antimicrobials appeared to be intact. In addition, there was an appreciable amount of increased texture to the surfaces of the treated virus particles in the TEM images. This appeared to be a buildup of small round components on the surface of the virus capsid rather than clumping of virus particles together, since the size of the textured particles was too small and the particles were too numerous to be MNV. No such large particles were observed in control grids which included only the antimicrobial in PBS and no virus particles.

This work was partially funded by the Organic Agriculture Research and Extension Initiative (grant 2010-51300-21760) from the U.S. Department of Agriculture.

We thank Tony Day of the Arizona Research Labs AHSC EM Core Facility at The University of Arizona for his invaluable assistance with the TEM imaging and Sadhana Ravishankar from the School of Animal and Comparative Biomedical Sciences at The University of Arizona for kindly providing the plant antimicrobials used in this study.

Published ahead of print 6 June 2014

2. Atmar RL, Estes MK.2006. The epidemiologic and clinical importance of norovirus infection. Gastroenterol. Clin. North Am.35:275–290. 10.1016/j.gtc.2006.03.001 [PubMed] [CrossRef] [Google Scholar]

3. Widdowson MA, Sulka A, Bulens SN, Beard RS, Chaves SS, Hammond R, Salehi ED, Swanson E, Totaro J, Woron R, Mead PS, Bresee JS, Monroe SS, Glass RI.2005. Norovirus and foodborne disease, United States, 1991–2000. Emerg. Infect. Dis.11:95–102. 10.3201/eid1101.040426 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

4. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM.2011. Foodborne illness acquired in the United States—-major pathogens. Emerg. Infect. Dis.17:7–15. 10.3201/eid1701.P11101, 10.3201/eid1701.09-1101p1 [PMC free article] [PubMed] [CrossRef] [CrossRef] [Google Scholar]

5. Donaldson EF, Lindesmith LC, Lobue AD, Baric RS.2008. Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations. Immunol. Rev.225:190–211. 10.1111/j.1600-065X.2008.00680.x [PubMed] [CrossRef] [Google Scholar]

6. Carling PC, Bruno-Murtha LA, Griffiths JK.2009. Cruise ship environmental hygiene and the risk of norovirus infection outbreaks: an objective assessment of 56 vessels over 3 years. Clin. Infect. Dis.49:1312–1317. 10.1086/606058 [PubMed] [CrossRef] [Google Scholar]

7. Evans HS, Madden P, Douglas C, Adak GK, O'Brien SJ, Djuretic T, Wall PG, Stanwell-Smith R.1998. General outbreaks of infectious intestinal disease in England and Wales: 1995 and 1996. Commun. Dis. Public Health1:165–171 [PubMed] [Google Scholar]

8. Barker J, Stevens D, Bloomfield SF.2001. Spread and prevention of some common viral infections in community facilities and domestic homes. J. Appl. Microbiol.91:7–21. 10.1046/j.1365-2672.2001.01364.x [PMC free article] [PubMed] [CrossRef] [Google Scholar]

9. Watanabe T, Miyata H, Sato H.1989. Inactivation of laboratory animal RNA-virus by physicochemical treatment. Jikken Dobutsu38:305–311 [PubMed] [Google Scholar]

10. Said MA, Perl TM, Sears CL.2008. Gastrointestinal flu: norovirus in healthcare and long-term care facilities. Clin. Infect. Dis.47:1202–1208. 10.1086/592299 [PubMed] [CrossRef] [Google Scholar]

12. Barker J, Vipond IB, Bloomfield SF.2004. Effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces. J. Hosp. Infect.58:42–49. 10.1016/j.jhin.2004.04.021 [PubMed] [CrossRef] [Google Scholar]

13. Doultree JC, Druce JD, Birch CJ, Bowden DS, Marshall JA.1999. Inactivation of feline calicivirus, a Norwalk virus surrogate. J. Hosp. Infect.41:51–57. 10.1016/S0195-6701(99)90037-3 [PubMed] [CrossRef] [Google Scholar]

14. Smíd B, Valicek L, Rodák L, Stěpánek J, Jurák E.1991. Rabbit haemorrhagic disease: an investigation of some properties of the virus and evaluation of an inactivated vaccine. Vet. Microbiol.26:77–85. 10.1016/0378-1135(91)90043-F [PubMed] [CrossRef] [Google Scholar]

15. Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MP, Estes MK.2004. Laboratory efforts to cultivate noroviruses. J. Gen. Virol.85:79–87. 10.1099/vir.0.19478-0 [PubMed] [CrossRef] [Google Scholar]

16. Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, Belliot G, Krug A, Mackenzie JM, Green KY, Virgin HW., IV2004. Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol.2:e432. 10.1371/journal.pbio.0020432 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

17. Malik YS, Maherchandani S, Allwood PB, Goyal SM.2005. Evaluation of animal origin cell cultures for in vitro cultivation of noroviruses. J. Appl. Res.5:312–317 [Google Scholar]

18. Wobus CE, Thackray LB, Virgin HW., IV2006. Murine norovirus: a model system to study norovirus biology and pathogenesis. J. Virol.80:5104–5112. 10.1128/JVI.02346-05 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

19. Bright KR, Sicairos-Ruelas EE, Gundy PM, Gerba CP.2009. Assessment of the antiviral properties of zeolites containing metal ions. Food Environ. Virol.1:37–41. 10.1007/s12560-008-9006-1 [CrossRef] [Google Scholar]

20. Slomka MJ, Appleton H.1998. Feline calicivirus as a model system for heat inactivation studies of small round structured viruses in shellfish. Epidemiol. Infect.121:401–407. 10.1017/S0950268898001290 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

21. Clay S, Maherchandani S, Malik YS, Goyal SM.2006. Survival on uncommon fomites of feline calicivirus, a surrogate of noroviruses. Am. J. Infect. Control34:41–43. 10.1016/j.ajic.2005.05.013 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

22. Farkas T, Sestak K, Wei C, Jiang X.2008. Characterization of a rhesus monkey calicivirus representing a new genus of Caliciviridae. J. Virol.82:5408–5416. 10.1128/JVI.00070-08 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

23. Wei C, Farkas T, Sestak K, Jiang X.2008. Recovery of infectious virus by transfection of in vitro-generated RNA from Tulane calicivirus cDNA. J. Virol.82:11429–11436. 10.1128/JVI.00696-08 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

24. Esseili MA, Wang Q, Zhang Z, Saif LJ.2012. Internalization of sapovirus, a surrogate for norovirus, in romaine lettuce and the effect of lettuce latex on virus infectivity. Appl. Environ. Microbiol.78:6271–6279. 10.1128/AEM.01295-12 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

25. Wang Q, Zhang Z, Saif LJ.2012. Stability of and attachment to lettuce by a culturable porcine sapovirus surrogate for human caliciviruses. Appl. Environ. Microbiol.78:3932–3940. 10.1128/AEM.06600-11 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

26. Karst SM, Wobus CE, Lay M, Davidson J, Virgin HW., IV2003. STAT1-dependent innate immunity to a Norwalk-like virus. Science299:1575–1578. 10.1126/science.1077905 [PubMed] [CrossRef] [Google Scholar]

27. Sosnovtsev SV, Belliot G, Chang KO, Prikhodko VG, Thackray LB, Wobus CE, Karst SM, Virgin HW, Green KY.2006. Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells. J. Virol.80:7816–7831. 10.1128/JVI.00532-06 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

28. Cannon JL, Papafragkou E, Park GW, Osborne J, Jaykus LA, Vinjé J.2006. Surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus. J. Food Prot.69:2761–2765 [PubMed] [Google Scholar]

29. Bae J, Schwab KJ.2008. Evaluation of murine norovirus, feline calicivirus, poliovirus, and MS2 as surrogates for human norovirus in a model of viral persistence in surface water and groundwater. Appl. Environ. Microbiol.74:477–484. 10.1128/AEM.02095-06 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

30. Reichling J, Schnitzler P, Suschke U, Saller R.2009. Essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties—an overview. Forsch. Komplementmed.16:79–90. 10.1159/000207196 [PubMed] [CrossRef] [Google Scholar]

31. Dillon VM.1999. Natural anti-microbial systems. (a) Preservative effects during storage, p 1570–1576InRobinson RK, Batt CA, Patel P. (ed), Encyclopedia of food microbiology. Academic Press, Boston, MA [Google Scholar]

32. Ress NB, Hailey JR, Maronpot RR, Bucher JR, Travlos GS, Haseman JK, Orzech DP, Johnson JD, Hejtmancik MR.2003. Toxicology and carcinogenesis studies of microencapsulated citral in rats and mice. Toxicol. Sci.71:198–206. 10.1093/toxsci/71.2.198 [PubMed] [CrossRef] [Google Scholar]

33. Adams TB, Cohen SM, Doull J, Feron VJ, Goodman JI, Marnett LJ, Munro IC, Portoghese PS, Smith RL, Waddel WJ, Wagner BM.2004. The FEMA GRAS assessment of cinnamyl derivatives used as flavor ingredients. Food Chem. Toxicol.42:157–185. 10.1016/j.fct.2003.08.021 [PubMed] [CrossRef] [Google Scholar]

34. Knowles JR, Roller S, Murray DB, Naidu AS.2005. Antimicrobial action of carvacrol at different stages of dual-species biofilm development by Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Appl. Environ. Microbiol.71:797–803. 10.1128/AEM.71.2.797-803.2005 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

35. Didry N, Dubreuil L, Pinkas M.1994. Activity of thymol, carvacrol, cinnamaldehyde and eugenol on oral bacteria. Pharm. Acta Helv.69:25–28. 10.1016/0031-6865(94)90027-2 [PubMed] [CrossRef] [Google Scholar]

36. Friedman M, Henika PR, Mandrell RE.2002. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J. Food Prot.65:1545–1560 [PubMed] [Google Scholar]

37. Peñalver P, Huerta B, Borge C, Astorga R, Romero R, Perea A.2005. Antimicrobial activity of five essential oils against origin strains of the Enterobacteriaceae family. APMIS113:1–6. 10.1111/j.1600-0463.2005.apm1130101.x [PubMed] [CrossRef] [Google Scholar]

38. Callaway TR, Carroll JA, Arthington JD, Pratt C, Edrington TS, Anderson RC, Galyean ML, Ricke SC, Crandall P, Nisbet DJ.2008. Citrus products decrease growth of E. coli O157:H7 and Salmonella Typhimurium in pure culture and in fermentation with mixed ruminal microorganisms in vitro. Foodborne Pathog. Dis.5:621–627. 10.1089/fpd.2008.0088 [PubMed] [CrossRef] [Google Scholar]

39. Koch C, Reichling J, Schneele J, Schnitzler P.2008. Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine15:71–78. 10.1016/j.phymed.2007.09.003 [PubMed] [CrossRef] [Google Scholar]

40. Ravishankar S, Zhu L, Olsen CW, McHugh TH, Friedman M.2009. Edible apple film wraps containing plant antimicrobials inactivate foodborne pathogens on meat and poultry products. J. Food Sci.74:M440–M445. 10.1111/j.1750-3841.2009.01320.x [PubMed] [CrossRef] [Google Scholar]

41. Ravishankar S, Zhu L, Reyna-Granados J, Law B, Joens L, Friedman M.2010. Carvacrol and cinnamaldehyde inactivate antibiotic-resistant Salmonella enterica in buffer and on celery and oysters. J. Food Prot.73:234–240 [PubMed] [Google Scholar]

42. Hammer KA, Carson CF, Riley TV.1999. Antimicrobial activity of essential oils and other plant extracts. J. Appl. Microbiol.86:985–990. 10.1046/j.1365-2672.1999.00780.x [PubMed] [CrossRef] [Google Scholar]

43. Hammer KA, Carson CF, Riley TV.2002. In vitro activity of Melaleuca alternifolia (tea tree) oil against dermatophytes and other filamentous fungi. J. Antimicrob. Chemother.50:195–199. 10.1093/jac/dkf112 [PubMed] [CrossRef] [Google Scholar]

44. Carson CF, Hammer KA, Riley TV.2006. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin. Microbiol. Rev.19:50–62. 10.1128/CMR.19.1.50-62.2006 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

45. Pinto E, Pina-Vaz C, Salgueiro L, Gonçalves MJ, Costa-de-Oliveira S, Cavaleiro C, Palmeira A, Rodrigues A, Martinez-de-Oliveira J.2006. Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species. J. Med. Microbiol.55:1367–1373. 10.1099/jmm.0.46443-0 [PubMed] [CrossRef] [Google Scholar]

46. Hayashi K, Hayashi T, Ujita K, Takaishi Y.1996. Characterization of antiviral activity of a sesquiterpene, triptofordin C-2. J. Antimicrob. Chemother.37:759–768. 10.1093/jac/37.4.759 [PubMed] [CrossRef] [Google Scholar]

47. Minami M, Kita M, Nakaya T, Yamamoto T, Kuriyama H, Imanishi J.2003. The inhibitory effect of essential oils on herpes simplex virus type-1 replication in vitro. Microbiol. Immunol.47:681–684. 10.1111/j.1348-0421.2003.tb03431.x [PubMed] [CrossRef] [Google Scholar]

48. Schnitzler P, Koch C, Reichling J.2007. Susceptibility of drug resistant clinical herpes simplex virus type 1 strains to essential oils of ginger, thyme, hyssop, and sandalwood. Antimicrob. Agents Chemother.51:1859–1862. 10.1128/AAC.00426-06 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

49. Garozzo A, Timpanaro R, Bisignano B, Furneri PM, Bisignano G, Castro A.2009. In vitro antiviral activity of Melaleuca alternifolia essential oil. Lett. Appl. Microbiol.49:806–808. 10.1111/j.1472-765X.2009.02740.x [PubMed] [CrossRef] [Google Scholar]

50. Yamada K, Ogawa H, Hara A, Yoshida Y, Yonezawa Y, Karibe K, Nghia VB, Yoshimura H, Yamamoto Y, Yamada M, Nakamura K, Imai K.2009. Mechanism of the antiviral effect of hydroxytyrosol on influenza virus appears to involve morphological change of the virus. Antiviral Res.83:35–44. 10.1016/j.antiviral.2009.03.002 [PubMed] [CrossRef] [Google Scholar]

51. Astani A, Reichling J, Schnitzler P.2010. Comparative study on the antiviral activity of selected monoterpenes derived from essential oils. Phytother. Res.24:673–679. 10.1002/ptr.2955 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

52. Astani A, Reichling J, Schnitzler P.2011. Screening for antiviral activities of isolated compounds from essential oils. Evid. Based Complement. Alternat. Med.2011:253643. 10.1093/ecam/nep187 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

53. Lai WL, Chuang HS, Lee MH, Wei CL, Lin CF, Tsai YC.2012. Inhibition of herpes simplex virus type 1 by thymol-related monoterpenoids. Planta Med.78:1636–1638. 10.1055/s-0032-1315208 [PubMed] [CrossRef] [Google Scholar]

54. Gómez LA, Stashenko E, Ocazionez RE.2013. Comparative study on in vitro activities of citral, limonene and essential oils from Lippia citriodora and L. alba on yellow fever virus. Nat. Prod. Commun.8:249–252 [PubMed] [Google Scholar]

55. Reichling J, Koch C, Stahl-Biskup E, Sojka C, Schnitzler P.2005. Virucidal activity of a β-triketone-rich essential oil of Leptospermum scoparium (manuka oil) against HSV-1 and HSV-2 in cell culture. Planta Med.71:1123–1127. 10.1055/s-2005-873175 [PubMed] [CrossRef] [Google Scholar]

56. Loizzo MR, Saab AM, Tundis R, Statti GA, Menichini F, Lampronti I, Gambari R, Cinatl J, Doerr HW.2008. Phytochemical analysis and in vitro antiviral activities of the essential oils of seven Lebanon species. Chem. Biodivers.5:461–470. 10.1002/cbdv.200890045 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

57. Meneses R, Ocazionez RE, Martínez JR, Stashenko EE.2009. Inhibitory effect of essential oils obtained from plants grown in Colombia on yellow fever virus replication in vitro. Ann. Clin. Microbiol. Antimicrob.8:8. 10.1186/1476-0711-8-8 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

58. García CC, Acosta EG, Carro AC, Fernández Belmonte MC, Bomben R, Duschatzky CB, Perotti M, Schuff C, Damonte EB.2010. Virucidal activity and chemical composition of essential oils from aromatic plants of central west Argentina. Nat. Prod. Commun.5:1307–1310 [PubMed] [Google Scholar]

59. Jackwood MW, Rosenbloom R, Petteruti M, Hilt DA, McCall AW, Williams SM.2010. Avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo. Virus Res.149:86–94. 10.1016/j.virusres.2010.01.006 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

60. Ocazionez RE, Meneses R, Torres FA, Stashenko E.2010. Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro. Mem. Inst. Oswaldo Cruz105:304–309. 10.1590/S0074-02762010000300010 [PubMed] [CrossRef] [Google Scholar]

61. Wu S, Patel KB, Booth LJ, Metcalf JP, Lin HK, Wu W.2010. Protective essential oil attenuates influenza virus infection: an in vitro study in MDCK cells. BMC Complement. Altern. Med.10:69. 10.1186/1472-6882-10-69 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

62. Garozzo A, Timpanaro R, Stivala A, Bisignano G, Castro A.2011. Activity of Melaleuca alternifolia (tea tree) oil on influenza virus A/PR/8: study of the mechanism of action. Antiviral Res.89:83–88. 10.1016/j.antiviral.2010.11.010 [PubMed] [CrossRef] [Google Scholar]

63. Siddiqui YM, Ettayebi M, Haddad AM, Al-Ahdal MN.1996. Effect of essential oils on the enveloped viruses: antiviral activity of oregano and clove oils on herpes simplex virus type 1 and Newcastle disease virus. Med. Sci. Res.24:185–186 [Google Scholar]

64. Tait S, Salvati AL, Desideri N, Fiore L.2006. Antiviral activity of substituted hom*oisoflavonoids on enteroviruses. Antiviral Res.72:252–255. 10.1016/j.antiviral.2006.07.003 [PubMed] [CrossRef] [Google Scholar]

65. Cermelli C, Fabio A, Fabio G, Quaglio P.2008. Effect of eucalyptus essential oil on respiratory bacteria and viruses. Curr. Microbiol.56:89–92. 10.1007/s00284-007-9045-0 [PubMed] [CrossRef] [Google Scholar]

66. Su X, D'Souza DH.2011. Grape seed extract for control of human enteric viruses. Appl. Environ. Microbiol.77:3982–3987. 10.1128/AEM.00193-11 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

67. Li D, Baert L, Zhang D, Xia M, Zhong W, Van Coillie E, Jiang X, Uyttendaele M.2012. The effect of grape seed extract on human norovirus GII.4 and murine norovirus-1 in viral suspensions, on stainless steel discs, and in lettuce wash water. Appl. Environ. Microbiol.78:7572–7578. 10.1128/AEM.01987-12 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

68. Su X, Howell AB, D'Souza DH.2010. The effect of cranberry juice and cranberry proanthocyanidins on the infectivity of human enteric viral surrogates. Food Microbiol.27:535–540. 10.1016/j.fm.2010.01.001 [PubMed] [CrossRef] [Google Scholar]

69. Su X, Howell AB, D'Souza DH.2010. Antiviral effects of cranberry juice and cranberry proanthocyanidins on foodborne viral surrogates—a time dependence study in vitro. Food Microbiol.27:985–991. 10.1016/j.fm.2010.05.027 [PubMed] [CrossRef] [Google Scholar]

70. Su X, Sangster MY, D'Souza DH.2011. Time-dependent effects of pomegranate juice and pomegranate polyphenols on foodborne virus reduction. Foodborne Pathog. Dis.8:1177–1183. 10.1089/fpd.2011.0873 [PubMed] [CrossRef] [Google Scholar]

71. Elizaquível P, Azizkhani M, Aznar R, Sánchez G.2013. The effect of essential oils on norovirus surrogates. Food Control32:275–278. 10.1016/j.foodcont.2012.11.031 [CrossRef] [Google Scholar]

72. Gilling DH, Kitajima M, Torrey JR, Bright KR.2014. Antiviral efficacy and mechanisms of action of oregano essential oil and its primary component carvacrol against murine norovirus. J. Appl. Microbiol.116:1149–1163. 10.1111/jam.12453 [PubMed] [CrossRef] [Google Scholar]

73. Thurston-Enriquez JA, Hass CN, Jacangelo J, Gerba CP.2003. Chlorine inactivation of adenovirus type 40 and feline calicivirus. Appl. Environ. Microbiol.69:3979–3985. 10.1128/AEM.69.7.3979-3985.2003 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

74. Black S, Thurston JA, Gerba CP.2009. Determination of Ct values for chlorine resistant enteroviruses. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng.44:336–339. 10.1080/10934520802659653 [PubMed] [CrossRef] [Google Scholar]

75. Payment P, Trudel M.1993. Isolation and identification of viruses, p 32–33InPayment P, Trudel M. (ed), Methods and techniques in virology. Marcel Dekker, Inc, New York, NY [Google Scholar]

76. Kitajima M, Oka T, Takagi H, Tohya Y, Katayama H, Takeda N, Katayama K.2010. Development and application of a broadly reactive real-time reverse transcription-PCR assay for detection of murine noroviruses. J. Virol. Methods169:269–273. 10.1016/j.jviromet.2010.07.018 [PubMed] [CrossRef] [Google Scholar]

77. Sánchez G, Aznar R, Martínez A, Rodrigo D.2011. Inactivation of human and murine norovirus by high-pressure processing. Foodborne Pathog. Dis.8:249–253. 10.1089/fpd.2010.0667 [PubMed] [CrossRef] [Google Scholar]

78. Kim SW, Baek SB, Ha JH, Lee MH, Choi C, Ha SD.2012. Chlorine treatment to inactivate norovirus on food contact surfaces. J. Food Prot.75:184–188. 10.4315/0362-028X.JFP-11-243 [PubMed] [CrossRef] [Google Scholar]

79. Toffan A, Brutti A, De Pasquale A, Cappellozza E, Pascoli F, Cigarini M, Di Rocco M, Terregino C, Arcangeli G.2014. The effectiveness of domestic cook on inactivation of murine norovirus in experimentally infected Manila clams (Ruditapes philippinarum). J. Appl. Microbiol.116:191–198. 10.1111/jam.12346 [PubMed] [CrossRef] [Google Scholar]

80. Bidawid S, Malik N, Adegbunrin O, Sattar SS, Farber JM.2003. A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus. J. Virol. Methods107:163–167. 10.1016/S0166-0934(02)00214-8 [PubMed] [CrossRef] [Google Scholar]

81. de Bona da Silva C, Guterres SS, Weisheimer V, Schapoval EES.2008. Antifungal activity of the lemongrass oil and citral against Candida spp. Braz. J. Infect. Dis.12:63–66. 10.1590/S1413-86702008000100014 [PubMed] [CrossRef] [Google Scholar]

82. Gregory JB, Litaker RW, Noble RT.2006. Rapid one-step quantitative reverse transcriptase PCR assay with competitive internal positive control for detection of enteroviruses in environmental samples. Appl. Environ. Microbiol.72:3960–3967. 10.1128/AEM.02291-05 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

83. Friedman M, Henika PR, Levin CE, Mandrell RE.2004. Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice. J. Agric. Food Chem.52:6042–6048. 10.1021/jf0495340 [PubMed] [CrossRef] [Google Scholar]

84. Maizura M, Fazilah A, Norziah MH, Karim AA.2007. Antibacterial activity and mechanical properties of partially hydrolyzed sago starch-alginate edible film containing lemongrass oil. J. Food Sci.72:C324–C330. 10.1111/j.1750-3841.2007.00427.x [PubMed] [CrossRef] [Google Scholar]

85. Aiemsaard J, Aiumlamai S, Aromdee C, Taweechaisupapong S, Khunkitti W.2011. The effect of lemongrass oil and its major components on clinical isolate mastitis pathogens and their mechanisms of action on Staphylococcus aureus DMST 4745. Res. Vet. Sci.91:e31–e37. 10.1016/j.rvsc.2011.01.012 [PubMed] [CrossRef] [Google Scholar]

86. Padmakumari Amma KP, Rani MP, Sasidharan I, Sreekumar MM.2013. Comparative chemical composition and in vitro antioxidant activities of essential oil isolated from the leaves of Cinnamomum tamala and Pimenta dioica. Nat. Prod. Res.27:290–294. 10.1080/14786419.2012.668691 [PubMed] [CrossRef] [Google Scholar]

87. Du WX, Olsen CW, Avena-Bustillos RJ, McHugh TH, Levin CE, Mandrell R, Friedman M.2009. Antibacterial effects of allspice, garlic, and oregano essential oils in tomato films determined by overlay and vapor-phase methods. J. Food Sci.74:M390–M397. 10.1111/j.1750-3841.2009.01289.x [PubMed] [CrossRef] [Google Scholar]

88. Katsukawa M, Nakata R, Takizawa Y, Hori K, Takahashi S, Inoue H.2010. Citral, a component of lemongrass oil, activates PPARα and γ and suppresses COX-2 expression. Biochim. Biophys. Acta1801:1214–1220. 10.1016/j.bbalip.2010.07.004 [PubMed] [CrossRef] [Google Scholar]

89. Armaka M, Papanikolaou E, Sivropoulou A, Arsenakis M.1999. Antiviral properties of isoborneol, a potent inhibitor of herpes simplex virus type 1. Antiviral Res.43:79–92. 10.1016/S0166-3542(99)00036-4 [PubMed] [CrossRef] [Google Scholar]

90. Wen CC, Kuo YH, Jan JT, Liang PH, Wang SY, Liu HG, Lee CK, Chang ST, Kuo CJ, Lee SS, Hou CC, Hsiao PW, Chien SC, Shyur LF, Yang NS.2007. Specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus. J. Med. Chem.50:4087–4095. 10.1021/jm070295s [PubMed] [CrossRef] [Google Scholar]

91. Mantani N, Andoh T, Kawamata H, Terasawa K, Ochiai H.1999. Inhibitory effect of Ephedrae herba, an oriental traditional medicine, on the growth of influenza A/PR/8 virus in MDCK cells. Antiviral Res.44:193–200. 10.1016/S0166-3542(99)00067-4 [PubMed] [CrossRef] [Google Scholar]

92. Imanishi N, Tuji Y, Katada Y, Maruhashi M, Konosu S, Mantani N, Terasawa K, Ochiai H.2002. Additional inhibitory effect of tea extract on the growth of influenza A and B viruses in MDCK cells. Microbiol. Immunol.46:491–494. 10.1111/j.1348-0421.2002.tb02724.x [PubMed] [CrossRef] [Google Scholar]

93. Sanchez Palomino S, Abad MJ, Bedoya LM, Garcia J, Gonzales E, Chiriboga X, Bermejo P, Alcami J.2002. Screening of South American plants against human immunodeficiency vırus: preliminary fractionation of aqueous extract from Baccharis trinervis. Biol. Pharm. Bull.25:1147–1150. 10.1248/bpb.25.1147 [PubMed] [CrossRef] [Google Scholar]

94. Pilau MR, Alves SH, Weiblen R, Arenhart S, Cueto AP, Lovato LT.2011. Antiviral activity of the Lippia graveolens (Mexican oregano) essential oil and its main compound carvacrol against human and animal viruses. Braz. J. Microbiol.42:1616–1624. 10.1590/S1517-83822011000400049 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

95. Cliver DO.2009. Capsid and infectivity in virus detection. Food Environ. Virol.1:123–128. 10.1007/s12560-009-9020-y [PMC free article] [PubMed] [CrossRef] [Google Scholar]

96. Semple SJ, Pyke SM, Reynolds GD, Flower RLP.2001. In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus. Antiviral Res.49:169–178. 10.1016/S0166-3542(01)00125-5 [PubMed] [CrossRef] [Google Scholar]

97. Búfalo MC, Figueiredo AS, de Sousa JP, Candeias JM, Bastos JK, Sforcin JM.2009. Anti-poliovirus activity of Baccharis dracunculifolia and propolis by cell viability determination and real-time PCR. J. Appl. Microbiol.107:1669–1680. 10.1111/j.1365-2672.2009.04354.x [PMC free article] [PubMed] [CrossRef] [Google Scholar]

98. Lipson SM, Sethi L, Cohen P, Gordon RE, Tan IP, Burdowski A, Stotzky G.2007. Antiviral effects on bacteriophages and rotavirus by cranberry juice. Phytomedicine14:23–30. 10.1016/j.phymed.2006.11.009 [PMC free article] [PubMed] [CrossRef] [Google Scholar]

99. Nuanualsuwan S, Cliver DO.2002. Pretreatment to avoid positive RT-PCR results with inactivated viruses. J. Virol. Methods104:217–225. 10.1016/S0166-0934(02)00089-7 [PubMed] [CrossRef] [Google Scholar]

100. Nuanualsuwan S, Cliver DO.2003. Capsid functions of inactivated human picornaviruses and feline calicivirus. Appl. Environ. Microbiol.69:350–357. 10.1128/AEM.69.1.350-357.2003 [PMC free article] [PubMed] [CrossRef] [Google Scholar]