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Protective Effect of Food Against Inactivation of Human Coronavirus OC43 by Gastrointestinal Fluids

Protective Effect of Food Against Inactivation of Human Coronavirus OC43 by Gastrointestinal Fluids The involvement of the gastrointestinal (GI) tract in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been reported in multiple studies. Since it has been demonstrated that human intestinal epithelial cells support productive viral replication and that a substantial portion of infected individuals shed the virus in feces, the possibility of fecal–oral and fecal-respiratory modes of transmission have been proposed for SARS-CoV-2. In order to establish viral replication in the intestine, enteric viruses need to retain their infectivity in often low pH gastric fluids, and in intestinal fluids, which contain digestive enzymes and bile salts. In this study, we examined whether human coronaviruses OC43 (HCoV-OC43) can remain infectious in simulated GI fluids that models human fasting-state and fed-state, in the presence or absence of food. We demonstrated that except for fasting-state gastric fluid (pH 1.6), the virus can remain infectious in all other gastrointestinal u fl ids for 1 h. Furthermore, we demonstrated that presence of food could signic fi antly improve viral survival in gastric u fl ids. Therefore, this study provides evidence that ingestion with food could protect the virus against inactivation by the GI fluids. Keywords Human coronavirus OC43 · Gastrointestinal fluids · Survival · Infectivity assay Introduction aerosols (Meyerowitz et al., 2021). Although gastrointestinal (GI) manifestations are reported in 11.4–61.1% of COVID- Coronaviruses are enveloped viruses with a genome com- 19 patients (Kariyawasam et al., 2021; Leal et al., 2021) posed of a non‐segmented positive sense single-stranded and multiple lines of evidence suggest that the infectious RNA with a size of approximately 30 kb (Fehr & Perlman, viral particles are excreted in the feces of 41% of infected 2015). Based on phylogenetic clustering, coronaviruses are individuals (Britton et al., 2021; Shi et al., 2020), there is divided into four genera: Alphacoronavirus, Betacoronavi- no conclusive evidence for fecal–oral or fecal-respiratory rus, Gammacoronavirus and Deltacoronavirus. Coronavi- transmission. ruses that infect humans (HCoVs), belong to the alpha and It has been reported that the viral load in feces could beta genera. Severe acute respiratory syndrome (SARS)- reach to 10  RNA copies/g, even higher than that in naso- CoV-2, which is responsible for the coronavirus disease pharyngeal swabs (Wolfel et al., 2020). The presence and 2019 (COVID-19) pandemic, is a Betacoronavirus that persistence of such large amounts of viral RNA in feces causes mild to severe respiratory infections, as well as gas- cannot only be explained by ingestion of viral particles rep- trointestinal symptoms (Cimolai, 2020; Kariyawasam et al., licated in the respiratory system, but suggests potential rep- 2021). Human-to-human transmission of SARS-CoV-2 is lication of SARS-CoV-2 in the intestinal tract (Guo et al., mainly attributed to contaminated respiratory droplets and 2021). Angiotensin-converting enzyme 2 (ACE2), which is the main receptor for SARS-CoV-2 entry, is abundantly expressed in the lung and upper respiratory epithelia, as * Neda Nasheri well as in the duodenum and small intestine, with lower neda.nasheri@hc-sc.gc.ca levels in the stomach and large intestine (Hikmet et  al., National Food Virology Reference Centre, Bureau 2020). Although the mere expression of viral receptors in of Microbial Hazards, Health Canada, Ottawa, ON, Canada the GI system does not mean these cells are permissive to Department of Biochemistry, Microbiology and Immunology, respiratory virus infection. The possibility of enteric propa- Faculty of Medicine, University of Ottawa, Ottawa, ON, gation is further supported by the fact that human intestinal Canada Vol:.(1234567890) 1 3 Food and Environmental Virology (2022) 14:212–216 213 cells are highly permissive to infection with SARS-CoV- composed of Eagle’s minimal essential medium, supple- 2 (Lamers et al., 2020; Zhou et al., 2020). The ability to mented with 0.22% (w/v) sodium bicarbonate, 500 µg/mL retain infectivity in the GI fluids is essential for a microbe penicillin/streptomycin), 1% Glutamax-1, 1% non-essential to establish infection in the human intestinal tract, and it has amino acids, 1% amphotericin B and 10% (v/v) fetal bovine been reported that SARS-CoV, SARS-CoV-2, middle-east serum (FBS) (ThermoFisher Scientific Inc, Canada). MEM respiratory syndrome (MERS), and HCoV-229E can tolerate maintenance media was used for dilution media of experi- fed-state gastric fluid and fasting-state intestinal fluid, but mental samples, with the same composition as growth media not fasting-state gastric fluids and fed-state intestinal fluids but with 2% FBS. (Chak-Yiu Lee et al., 2020; Zhou et al., 2017). Coronaviruses are enveloped, and as such, are expected Quantification of HCoV‑OC43 Using Median Tissue to be susceptible to gastric acid and bile, therefore theoreti- Culture Infectious Dose (TCID ) Assay cally, it is unlikely that these viruses retain their infectiv- ity and reach the lower GI tract to be excreted in the feces Quantification of infectious HCoV-OC43 was conducted as (Hirose et al., 2017). On the other hand, many of the mam- described previously (Dallner et al., 2021). Briefly, MRC-5 mal and avian‐associated coronaviruses are well known to cells were grown for 2 to 3 days and were seeded in 96 well cause gastroenteritis in their host species, including poul- plates at a cell concentration of 5 × 10 cells/100 µL well, try, swine, bovine, equine, canine, and feline hosts (Cimolai up to 24 h in advance of the assay, in order to reach 90% et al., 2020; Pusterla et al., 2018; Wang, et al., 2019). Fur- confluence. Maintenance media was used to dilute gastric thermore, evidence of the presence of SARS-CoV has been fluid treated, intestinal fluid treated, or maintenance media found in the intestinal mucosal epithelium and lymphoid control samples. Cell monolayers were washed once with tissue of human fatal cases (Gu et al., 2005; Shi et al., 2005). 1 × PBS (pH 7.35) and 100 µL of each dilution was used for Notably, SARS-CoV-2 antigen has been detected in duode- quantification of tissue culture infectious dose, measured num of infected golden hamster model (Sia et al., 2020). in quadruplicate for each sample. HCoV-OC43 stock was These observations suggest that at least some coronaviruses used as the positive control, diluted in maintenance media. are resistant to gastrointestinal fluids and enzymes. Uninoculated maintenance media was the negative control. Given the limited access to biosafety level 3 (BSL-3) con- MRC-5 plates were then incubated at 33 °C for 5 days. Sam- tainment facilities required to safely handle SARS-CoV-2, ples were removed from the 96 well plate, discarded, and scientists have turned to surrogate viruses that enable stud- stained with 0.1% crystal violet. Cells were observed under ies of viral replication as well as survival and inactivation the microscope and the cytopathic effect for each sample at biosafety level 2 (BSL-2). This approach has expanded was recorded. T CID /mL was calculated using the Reed- the current knowledge regarding the molecular and applied Muench method (Reed & Muench, 1938). TCID values aspects of highly pathogenic coronaviruses. were converted to PFU by multiplying by 0.7, which is a The aim of this study is to examine the potential protec- constant value obtained based upon Poisson distribution tive effect of food against HCoV inactivation by gastrointes- (ATCC, 2021). The percentage viral recovery of HCoV- tinal fluids, using HCoV-OC43 as a surrogate for more path- OC43 from 2 g cucumbers was determined by this equation: ogenic coronaviruses. In a previous study, we demonstrated Recovered infectious virus(PFU) that HCoV-229E and HCoV-OC43 survive significantly Viral recovery(%) = × 100 longer on cucumbers than apples and tomatoes (96 h vs 16 h) Inoculated infectious virus (PFU) when stored at 22 °C (Blondin-Brosseau et al., 2021). As an extension of the previous study, we investigated the potential Sample Preparation and HCoV‑OC43 Treatment for HCoV to remain infectious following ingestion, in the presence or absence of cucumber slices. with Simulated Gastrointestinal Fluids English cucumbers (PLU code 4593, Ontario, Canada) were purchased from a local grocery store in Ottawa, Ontario, Materials and Methods Canada. Simulated gastric and intestinal fluids were pre- pared according to manufacturer’s instructions (Biorelevant, Cell line and Virus London, UK). Simulated gastric fluids were supplemented with 0.1 mg/mL pepsin (Millipore Sigma, Canada). The sur- Human lung fibroblast cells, MRC-5 (ATCC# CCL-171), and human coronavirus OC43 (HCoV-OC43; ATCC# face of intact cucumbers were disinfected with 70% ethanol, dried, and chopped into 2 g amounts. Each 2 g sample of VR-1558) were obtained from the American Type Culture Collection (ATCC) (Cedarlane, Canada). Cells were grown cucumber was inoculated, in triplicate, with 100 µL of 10 TCID /mL HCoV-OC43, applied as drops to provide an and maintained at 37  °C and 5% C O in culture media 2 50 1 3 214 Food and Environmental Virology (2022) 14:212–216 even cover of the cucumber surface, then left at room tem- No Cucumber perature for 10 min to dry. Each sample was then mixed with 5 mL of fed-state gastric fluid, (FEDGAS pH 6, pH 4.5, or 0 min pH 3), fasting-state gastric fluid (FaSSGF, pH 1.6), fed-state 10 min intestinal fluid (FeSSIF pH 5), or fasting-state intestinal fluid 60 min (FaSSIF pH 6.5). Instead of gastric or intestinal fluid, main- tenance media was added to inoculated cucumber samples as a control and to calculate viral recovery. Samples were then incubated at 37 °C for 0, 10 or 60 min with rocking at 320 rpm. At the end of incubation, the gastric or intestinal fluid was neutralized to pH 7 using 2.5 M sodium hydroxide, and then processed immediately for viral quantification. With Cucumber HCoV‑OC43 Treatment with Simulated Gastrointestinal Fluids Without Food 0 min 10 min Simulated gastric and intestinal fluids were prepared as 6 60 min described above. In triplicate, 100uL of 10 TCID /mL HCoV-OC43 was added directly to each type of gastric or intestinal fluid and incubated at 37 °C for 0, 10 or 60 min with rocking at 320 rpm. The gastric or intestinal fluid was neutralized to pH 7 using 2.5 M sodium hydroxide and sam- ples were processed immediately. Statistical Analysis Fig. 1 In vitro stability of HCoV-OC43 in simulated human gastro- Statistical analysis was performed using GraphPad Prism intestinal fluids a in the absence of food b on two grams of cucum- v9.0 (GraphPad Software, San Diego, CA) (2-way ANOVA ber slices. HCoV-OC43 inoculum (10 TCID ) was added directly multiple comparisons) was used to determine significant to each type of gastric or intestinal fluid and incubated at 37  °C for 0, 10 or 60 min. FaSSIF: fasting-state simulated intestinal fluid. FeS- differences between treatments. Student t-test was used SIF: fed-state simulated intestinal fluid. FEDGAS: fed-state gastric to determine the statistical significance of the difference fluid. FaSSGF: fasting-state gastric fluid. Data are presented as log between treatments in the presence or absence of cucumber. reduction in viral infectivity compared to the media treatment. Data obtained from three independent experiments. Error bars are standard deviations Results and Discussion In order to determine whether HCoV-OC43 can survive in (FaSSGF, pH 1.6). The presence of food did not lead 4 5 to recovery of any infectious virus after treatment with gastrointestinal fluid, 7 × 10 PFU/mL (1 × 10 TCID /mL) of the virus, in the presence or absence of food (cucumber (FaSSGF, pH 1.6) for 10 and 60 min (Fig. 1b, and Sup- plementary Table 1). We compared this to fed-state gastric slices), were exposed to fasting (pH 1.6) and fed-state gas- tric fluids (pH 3, 4.5, and 6), supplemented with pepsin, as fluids (FEDGAS) at different stages of stomach empty - ing (early: pH 6, mid: pH 4.5, and late: pH 3), as well as well as fasting- and fed-state intestinal fluids, pH 6.5 and 5, respectively, for 0 to 60 min. The extraction efficiency fed-state intestinal fluid (FeSSIF, pH 5) and fasting-state intestinal fluid (FaSSIF, pH 6.5). The results showed that of HCoV-OC43 from cucumber slices in the presence of maintenance media was 11.1%. The detection limit (LOD) HCoV-OC43 remain infectious for 60  min in all other types of fed-state gastric fluids and both intestinal fluids. for HCoV-OC43 on cucumbers was previously determined by our group to be 32 PFU (Dallner et al., 2021). However, the infectivity was reduced in fluids modeling the mid and late stages of stomach emptying (FEDGAS pH Figure  1a demonstrates the log reduction compared with the media control following treatments in the absence 4.5 and pH 3, respectively) compared to FEDGAS pH 6.5, possibly due to the higher acidic conditions. Importantly, of food and Fig. 1b demonstrates the log reduction in viral infectivity in the presence of food. As shown in Fig. 1a, the presence of food significantly improved the viral sur - vival in FEDGAS pH 6.5, 4.5 and pH 3 (Supplementary and b, HCoV-OC43 infectivity was rapidly lost after 10  min of treatment with the fasting-state gastric fluid Table 1). However, the presence of food did not lead to a 1 3 FEDGAS pH 6 FEDGAS pH 4.5 FEDGAS pH 3 FaSSGF (pH 1.6) FaSSIF (pH 6.5) FeSSIF (pH 5) FEDGAS pH 6 FEDGAS pH 4.5 FEDGAS pH 3 FaSSGF (pH 1.6) FaSSIF (pH 6.5) FeSSIF (pH 5) Log reduction compared to media Log reduction compared to media Food and Environmental Virology (2022) 14:212–216 215 significant difference in viral survival in the presence of will aid in the implementation of effective disease control fed-state or fasting-state intestinal fluids (Supplementary measures. Table 1). In general, HCoV-OC43 retained the most infec- Altogether, this study demonstrates that coronaviruses tivity at the fasting-state intestinal fluids, followed by the could be remain infectious in human GI fluid if ingested, early stage fed-state gastric fluid (pH 6). and therefore, it is possible that infection could begin in Treatment with FaSSGF (pH 1.6) rapidly inactivated the the intestine. Nevertheless, it seems that the proportion of virus, at T the infectious viral titer was reduced by more the virus that remains infectious in stool is not remarkable than 2 log and longer treatment times of 10 and 60 min com- (Cuicchi et al., 2021). pletely inactivated the virus (< LOD, i.e. 3.8 log reduction Supplementary Information The online version contains supplemen- in infectivity). This finding is in accordance with previous tary material available at https://doi. or g/10. 1007/ s12560- 022- 09520-5 . studies that have reported complete inactivation of HCoVs in fasting-state gastric fluids (Chak-Yiu Lee et al., 2020; Zhou Acknowledgements This study is financially supported by the Bureau et  al., 2017). Consistent with previous reports, treatment of Microbial Hazards, Health Canada. The authors would like to thank Dr. Alex Gill and Dr. John Austin for thorough review of the with each of the GI fluids at 10 and 60 min significantly manuscript. reduced viral infectivity compared to the treatment with maintenance media. Furthermore, there was a significant Funding A-base Health Canada. decrease in viral infectivity following treatment with FeS- SIF, compared to FaSSIF at T . Potentially, this was due to Data Availability The information is provided in the article. Further the deleterious effect of bile on the viral membrane. How - details can be provided upon request. ever, in contrast to previous studies (Chak-Yiu Lee et al., 2020; Zhou et al., 2017), treatment with FeSSIF in the pres- Declarations ence or absence of cucumber, did not lead to complete inac- tivation of HCoV-OC43 (Fig. 1a, and b). Nonetheless, in Conflict of interest None to be declared. the absence of food, there is approximately 3 logs reduction Open Access This article is licensed under a Creative Commons Attri- in viral infectivity at 10 and 60 min treatment with FeSSIF bution 4.0 International License, which permits use, sharing, adapta- (Supplementary Table 1). tion, distribution and reproduction in any medium or format, as long Our results are in accordance with the observations as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes made in a study from COVID-19 patients undergoing gas- were made. The images or other third party material in this article are trointestinal endoscopy, in which SARS-CoV-2 RNA was included in the article's Creative Commons licence, unless indicated detected and quantified in the gastric fluid and intestinal flu - otherwise in a credit line to the material. If material is not included in ids obtained (Miyake et al., 2021). This indirectly indicates the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will that the virus was able to endure gastrointestinal fluids. need to obtain permission directly from the copyright holder. To view a While fecal-respiratory and fecal–oral modes of transmis- copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . sion have been demonstrated for some respiratory viruses including Hantavirus and Nipah virus (O'Brien et al., 2020; Witkowski et al., 2017), these modes of transmission remain References controversial for SARS-CoV-2 (Britton et al., 2021). To date, only a few reports have indicated the possibility of ATCC. Is it possible to determine from the TCID[50] how many plaque fecal-respiratory transmission of SARS-CoV-2 (Meyerow- forming units to expect?. 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Protective Effect of Food Against Inactivation of Human Coronavirus OC43 by Gastrointestinal Fluids

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1867-0334
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10.1007/s12560-022-09520-5
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Abstract

The involvement of the gastrointestinal (GI) tract in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been reported in multiple studies. Since it has been demonstrated that human intestinal epithelial cells support productive viral replication and that a substantial portion of infected individuals shed the virus in feces, the possibility of fecal–oral and fecal-respiratory modes of transmission have been proposed for SARS-CoV-2. In order to establish viral replication in the intestine, enteric viruses need to retain their infectivity in often low pH gastric fluids, and in intestinal fluids, which contain digestive enzymes and bile salts. In this study, we examined whether human coronaviruses OC43 (HCoV-OC43) can remain infectious in simulated GI fluids that models human fasting-state and fed-state, in the presence or absence of food. We demonstrated that except for fasting-state gastric fluid (pH 1.6), the virus can remain infectious in all other gastrointestinal u fl ids for 1 h. Furthermore, we demonstrated that presence of food could signic fi antly improve viral survival in gastric u fl ids. Therefore, this study provides evidence that ingestion with food could protect the virus against inactivation by the GI fluids. Keywords Human coronavirus OC43 · Gastrointestinal fluids · Survival · Infectivity assay Introduction aerosols (Meyerowitz et al., 2021). Although gastrointestinal (GI) manifestations are reported in 11.4–61.1% of COVID- Coronaviruses are enveloped viruses with a genome com- 19 patients (Kariyawasam et al., 2021; Leal et al., 2021) posed of a non‐segmented positive sense single-stranded and multiple lines of evidence suggest that the infectious RNA with a size of approximately 30 kb (Fehr & Perlman, viral particles are excreted in the feces of 41% of infected 2015). Based on phylogenetic clustering, coronaviruses are individuals (Britton et al., 2021; Shi et al., 2020), there is divided into four genera: Alphacoronavirus, Betacoronavi- no conclusive evidence for fecal–oral or fecal-respiratory rus, Gammacoronavirus and Deltacoronavirus. Coronavi- transmission. ruses that infect humans (HCoVs), belong to the alpha and It has been reported that the viral load in feces could beta genera. Severe acute respiratory syndrome (SARS)- reach to 10  RNA copies/g, even higher than that in naso- CoV-2, which is responsible for the coronavirus disease pharyngeal swabs (Wolfel et al., 2020). The presence and 2019 (COVID-19) pandemic, is a Betacoronavirus that persistence of such large amounts of viral RNA in feces causes mild to severe respiratory infections, as well as gas- cannot only be explained by ingestion of viral particles rep- trointestinal symptoms (Cimolai, 2020; Kariyawasam et al., licated in the respiratory system, but suggests potential rep- 2021). Human-to-human transmission of SARS-CoV-2 is lication of SARS-CoV-2 in the intestinal tract (Guo et al., mainly attributed to contaminated respiratory droplets and 2021). Angiotensin-converting enzyme 2 (ACE2), which is the main receptor for SARS-CoV-2 entry, is abundantly expressed in the lung and upper respiratory epithelia, as * Neda Nasheri well as in the duodenum and small intestine, with lower neda.nasheri@hc-sc.gc.ca levels in the stomach and large intestine (Hikmet et  al., National Food Virology Reference Centre, Bureau 2020). Although the mere expression of viral receptors in of Microbial Hazards, Health Canada, Ottawa, ON, Canada the GI system does not mean these cells are permissive to Department of Biochemistry, Microbiology and Immunology, respiratory virus infection. The possibility of enteric propa- Faculty of Medicine, University of Ottawa, Ottawa, ON, gation is further supported by the fact that human intestinal Canada Vol:.(1234567890) 1 3 Food and Environmental Virology (2022) 14:212–216 213 cells are highly permissive to infection with SARS-CoV- composed of Eagle’s minimal essential medium, supple- 2 (Lamers et al., 2020; Zhou et al., 2020). The ability to mented with 0.22% (w/v) sodium bicarbonate, 500 µg/mL retain infectivity in the GI fluids is essential for a microbe penicillin/streptomycin), 1% Glutamax-1, 1% non-essential to establish infection in the human intestinal tract, and it has amino acids, 1% amphotericin B and 10% (v/v) fetal bovine been reported that SARS-CoV, SARS-CoV-2, middle-east serum (FBS) (ThermoFisher Scientific Inc, Canada). MEM respiratory syndrome (MERS), and HCoV-229E can tolerate maintenance media was used for dilution media of experi- fed-state gastric fluid and fasting-state intestinal fluid, but mental samples, with the same composition as growth media not fasting-state gastric fluids and fed-state intestinal fluids but with 2% FBS. (Chak-Yiu Lee et al., 2020; Zhou et al., 2017). Coronaviruses are enveloped, and as such, are expected Quantification of HCoV‑OC43 Using Median Tissue to be susceptible to gastric acid and bile, therefore theoreti- Culture Infectious Dose (TCID ) Assay cally, it is unlikely that these viruses retain their infectiv- ity and reach the lower GI tract to be excreted in the feces Quantification of infectious HCoV-OC43 was conducted as (Hirose et al., 2017). On the other hand, many of the mam- described previously (Dallner et al., 2021). Briefly, MRC-5 mal and avian‐associated coronaviruses are well known to cells were grown for 2 to 3 days and were seeded in 96 well cause gastroenteritis in their host species, including poul- plates at a cell concentration of 5 × 10 cells/100 µL well, try, swine, bovine, equine, canine, and feline hosts (Cimolai up to 24 h in advance of the assay, in order to reach 90% et al., 2020; Pusterla et al., 2018; Wang, et al., 2019). Fur- confluence. Maintenance media was used to dilute gastric thermore, evidence of the presence of SARS-CoV has been fluid treated, intestinal fluid treated, or maintenance media found in the intestinal mucosal epithelium and lymphoid control samples. Cell monolayers were washed once with tissue of human fatal cases (Gu et al., 2005; Shi et al., 2005). 1 × PBS (pH 7.35) and 100 µL of each dilution was used for Notably, SARS-CoV-2 antigen has been detected in duode- quantification of tissue culture infectious dose, measured num of infected golden hamster model (Sia et al., 2020). in quadruplicate for each sample. HCoV-OC43 stock was These observations suggest that at least some coronaviruses used as the positive control, diluted in maintenance media. are resistant to gastrointestinal fluids and enzymes. Uninoculated maintenance media was the negative control. Given the limited access to biosafety level 3 (BSL-3) con- MRC-5 plates were then incubated at 33 °C for 5 days. Sam- tainment facilities required to safely handle SARS-CoV-2, ples were removed from the 96 well plate, discarded, and scientists have turned to surrogate viruses that enable stud- stained with 0.1% crystal violet. Cells were observed under ies of viral replication as well as survival and inactivation the microscope and the cytopathic effect for each sample at biosafety level 2 (BSL-2). This approach has expanded was recorded. T CID /mL was calculated using the Reed- the current knowledge regarding the molecular and applied Muench method (Reed & Muench, 1938). TCID values aspects of highly pathogenic coronaviruses. were converted to PFU by multiplying by 0.7, which is a The aim of this study is to examine the potential protec- constant value obtained based upon Poisson distribution tive effect of food against HCoV inactivation by gastrointes- (ATCC, 2021). The percentage viral recovery of HCoV- tinal fluids, using HCoV-OC43 as a surrogate for more path- OC43 from 2 g cucumbers was determined by this equation: ogenic coronaviruses. In a previous study, we demonstrated Recovered infectious virus(PFU) that HCoV-229E and HCoV-OC43 survive significantly Viral recovery(%) = × 100 longer on cucumbers than apples and tomatoes (96 h vs 16 h) Inoculated infectious virus (PFU) when stored at 22 °C (Blondin-Brosseau et al., 2021). As an extension of the previous study, we investigated the potential Sample Preparation and HCoV‑OC43 Treatment for HCoV to remain infectious following ingestion, in the presence or absence of cucumber slices. with Simulated Gastrointestinal Fluids English cucumbers (PLU code 4593, Ontario, Canada) were purchased from a local grocery store in Ottawa, Ontario, Materials and Methods Canada. Simulated gastric and intestinal fluids were pre- pared according to manufacturer’s instructions (Biorelevant, Cell line and Virus London, UK). Simulated gastric fluids were supplemented with 0.1 mg/mL pepsin (Millipore Sigma, Canada). The sur- Human lung fibroblast cells, MRC-5 (ATCC# CCL-171), and human coronavirus OC43 (HCoV-OC43; ATCC# face of intact cucumbers were disinfected with 70% ethanol, dried, and chopped into 2 g amounts. Each 2 g sample of VR-1558) were obtained from the American Type Culture Collection (ATCC) (Cedarlane, Canada). Cells were grown cucumber was inoculated, in triplicate, with 100 µL of 10 TCID /mL HCoV-OC43, applied as drops to provide an and maintained at 37  °C and 5% C O in culture media 2 50 1 3 214 Food and Environmental Virology (2022) 14:212–216 even cover of the cucumber surface, then left at room tem- No Cucumber perature for 10 min to dry. Each sample was then mixed with 5 mL of fed-state gastric fluid, (FEDGAS pH 6, pH 4.5, or 0 min pH 3), fasting-state gastric fluid (FaSSGF, pH 1.6), fed-state 10 min intestinal fluid (FeSSIF pH 5), or fasting-state intestinal fluid 60 min (FaSSIF pH 6.5). Instead of gastric or intestinal fluid, main- tenance media was added to inoculated cucumber samples as a control and to calculate viral recovery. Samples were then incubated at 37 °C for 0, 10 or 60 min with rocking at 320 rpm. At the end of incubation, the gastric or intestinal fluid was neutralized to pH 7 using 2.5 M sodium hydroxide, and then processed immediately for viral quantification. With Cucumber HCoV‑OC43 Treatment with Simulated Gastrointestinal Fluids Without Food 0 min 10 min Simulated gastric and intestinal fluids were prepared as 6 60 min described above. In triplicate, 100uL of 10 TCID /mL HCoV-OC43 was added directly to each type of gastric or intestinal fluid and incubated at 37 °C for 0, 10 or 60 min with rocking at 320 rpm. The gastric or intestinal fluid was neutralized to pH 7 using 2.5 M sodium hydroxide and sam- ples were processed immediately. Statistical Analysis Fig. 1 In vitro stability of HCoV-OC43 in simulated human gastro- Statistical analysis was performed using GraphPad Prism intestinal fluids a in the absence of food b on two grams of cucum- v9.0 (GraphPad Software, San Diego, CA) (2-way ANOVA ber slices. HCoV-OC43 inoculum (10 TCID ) was added directly multiple comparisons) was used to determine significant to each type of gastric or intestinal fluid and incubated at 37  °C for 0, 10 or 60 min. FaSSIF: fasting-state simulated intestinal fluid. FeS- differences between treatments. Student t-test was used SIF: fed-state simulated intestinal fluid. FEDGAS: fed-state gastric to determine the statistical significance of the difference fluid. FaSSGF: fasting-state gastric fluid. Data are presented as log between treatments in the presence or absence of cucumber. reduction in viral infectivity compared to the media treatment. Data obtained from three independent experiments. Error bars are standard deviations Results and Discussion In order to determine whether HCoV-OC43 can survive in (FaSSGF, pH 1.6). The presence of food did not lead 4 5 to recovery of any infectious virus after treatment with gastrointestinal fluid, 7 × 10 PFU/mL (1 × 10 TCID /mL) of the virus, in the presence or absence of food (cucumber (FaSSGF, pH 1.6) for 10 and 60 min (Fig. 1b, and Sup- plementary Table 1). We compared this to fed-state gastric slices), were exposed to fasting (pH 1.6) and fed-state gas- tric fluids (pH 3, 4.5, and 6), supplemented with pepsin, as fluids (FEDGAS) at different stages of stomach empty - ing (early: pH 6, mid: pH 4.5, and late: pH 3), as well as well as fasting- and fed-state intestinal fluids, pH 6.5 and 5, respectively, for 0 to 60 min. The extraction efficiency fed-state intestinal fluid (FeSSIF, pH 5) and fasting-state intestinal fluid (FaSSIF, pH 6.5). The results showed that of HCoV-OC43 from cucumber slices in the presence of maintenance media was 11.1%. The detection limit (LOD) HCoV-OC43 remain infectious for 60  min in all other types of fed-state gastric fluids and both intestinal fluids. for HCoV-OC43 on cucumbers was previously determined by our group to be 32 PFU (Dallner et al., 2021). However, the infectivity was reduced in fluids modeling the mid and late stages of stomach emptying (FEDGAS pH Figure  1a demonstrates the log reduction compared with the media control following treatments in the absence 4.5 and pH 3, respectively) compared to FEDGAS pH 6.5, possibly due to the higher acidic conditions. Importantly, of food and Fig. 1b demonstrates the log reduction in viral infectivity in the presence of food. As shown in Fig. 1a, the presence of food significantly improved the viral sur - vival in FEDGAS pH 6.5, 4.5 and pH 3 (Supplementary and b, HCoV-OC43 infectivity was rapidly lost after 10  min of treatment with the fasting-state gastric fluid Table 1). However, the presence of food did not lead to a 1 3 FEDGAS pH 6 FEDGAS pH 4.5 FEDGAS pH 3 FaSSGF (pH 1.6) FaSSIF (pH 6.5) FeSSIF (pH 5) FEDGAS pH 6 FEDGAS pH 4.5 FEDGAS pH 3 FaSSGF (pH 1.6) FaSSIF (pH 6.5) FeSSIF (pH 5) Log reduction compared to media Log reduction compared to media Food and Environmental Virology (2022) 14:212–216 215 significant difference in viral survival in the presence of will aid in the implementation of effective disease control fed-state or fasting-state intestinal fluids (Supplementary measures. Table 1). In general, HCoV-OC43 retained the most infec- Altogether, this study demonstrates that coronaviruses tivity at the fasting-state intestinal fluids, followed by the could be remain infectious in human GI fluid if ingested, early stage fed-state gastric fluid (pH 6). and therefore, it is possible that infection could begin in Treatment with FaSSGF (pH 1.6) rapidly inactivated the the intestine. Nevertheless, it seems that the proportion of virus, at T the infectious viral titer was reduced by more the virus that remains infectious in stool is not remarkable than 2 log and longer treatment times of 10 and 60 min com- (Cuicchi et al., 2021). pletely inactivated the virus (< LOD, i.e. 3.8 log reduction Supplementary Information The online version contains supplemen- in infectivity). This finding is in accordance with previous tary material available at https://doi. or g/10. 1007/ s12560- 022- 09520-5 . studies that have reported complete inactivation of HCoVs in fasting-state gastric fluids (Chak-Yiu Lee et al., 2020; Zhou Acknowledgements This study is financially supported by the Bureau et  al., 2017). Consistent with previous reports, treatment of Microbial Hazards, Health Canada. The authors would like to thank Dr. Alex Gill and Dr. John Austin for thorough review of the with each of the GI fluids at 10 and 60 min significantly manuscript. reduced viral infectivity compared to the treatment with maintenance media. Furthermore, there was a significant Funding A-base Health Canada. decrease in viral infectivity following treatment with FeS- SIF, compared to FaSSIF at T . Potentially, this was due to Data Availability The information is provided in the article. Further the deleterious effect of bile on the viral membrane. How - details can be provided upon request. ever, in contrast to previous studies (Chak-Yiu Lee et al., 2020; Zhou et al., 2017), treatment with FeSSIF in the pres- Declarations ence or absence of cucumber, did not lead to complete inac- tivation of HCoV-OC43 (Fig. 1a, and b). Nonetheless, in Conflict of interest None to be declared. the absence of food, there is approximately 3 logs reduction Open Access This article is licensed under a Creative Commons Attri- in viral infectivity at 10 and 60 min treatment with FeSSIF bution 4.0 International License, which permits use, sharing, adapta- (Supplementary Table 1). tion, distribution and reproduction in any medium or format, as long Our results are in accordance with the observations as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes made in a study from COVID-19 patients undergoing gas- were made. The images or other third party material in this article are trointestinal endoscopy, in which SARS-CoV-2 RNA was included in the article's Creative Commons licence, unless indicated detected and quantified in the gastric fluid and intestinal flu - otherwise in a credit line to the material. If material is not included in ids obtained (Miyake et al., 2021). This indirectly indicates the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will that the virus was able to endure gastrointestinal fluids. need to obtain permission directly from the copyright holder. To view a While fecal-respiratory and fecal–oral modes of transmis- copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . sion have been demonstrated for some respiratory viruses including Hantavirus and Nipah virus (O'Brien et al., 2020; Witkowski et al., 2017), these modes of transmission remain References controversial for SARS-CoV-2 (Britton et al., 2021). To date, only a few reports have indicated the possibility of ATCC. Is it possible to determine from the TCID[50] how many plaque fecal-respiratory transmission of SARS-CoV-2 (Meyerow- forming units to expect?. 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Journal

Food and Environmental VirologySpringer Journals

Published: Jun 1, 2022

Keywords: Human coronavirus OC43; Gastrointestinal fluids; Survival; Infectivity assay

References