key: cord-0255647-lhtepnrf
authors: Featherstone, Austin B.; Dass, Sapna Chitlapilly
title: A biosafety level 2 surrogate for studying SARS-CoV-2 survival in food processing environmental biofilms
date: 2021-11-01
journal: bioRxiv
DOI: 10.1101/2021.10.29.466519
sha: 6d7f3f546afe72e4923d98f17a32804810a96063
doc_id: 255647
cord_uid: lhtepnrf

Meat processing plants have been at the center of the SARS-CoV-2 pandemic. There are several factors that contribute to the persistence of SARS-CoV-2 in meat processing plants and one of the factors is the formation of a multi-species biofilm with virus. Biofilm can act as a reservoir in protecting, harboring, and dispersing SARS-CoV-2 from biofilm to the meat processing facility environment. We used Murine Hepatitis Virus (MHV) as a surrogate for SARS-CoV-2 virus and meat processing facility drain samples to develop mixed-species biofilms on commonly found materials in processing facilities (Stainless-Steel (SS), PVC and tiles). The results showed that MHV was able to integrate into the environmental biofilm and survived for a period of 5 days at 7°C. There was no significate difference between the viral-environmental biofilm biovolumes developed on different materials SS, PVC, and tiles. There was a 2-fold increase in the virus-environmental biofilm biovolume when compared to environmental biofilm by itself. These results indicate a complex virus-environmental biofilm interaction which is providing enhanced protection for the survival of viral particles with the environmental biofilm community.

Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-48

CoV-2) belongs to the genus β coronaviruses [1] . Studies suggests that MHV is a good persistent 49 surrogate model for SARS-CoV-2 [1, 2] . In a recent study on wastewater, there were no 50 statistically significant difference between RNA decay of SARS-CoV-2 and MHV [1] . 51

Biofilms in food processing facilities are a major threat to food safety, one of the main carriers 52 of food borne pathogens [3, 4] . Biofilms are multicellular assemblage of prokaryotic and 53 eukaryotic cells that are enclosed in a polysaccharide material and attached to a surface [5] . 54

Bacterial and fungal biofilms have so far been the focus of research on biofilm in food processing 55 facilities, research on the presence of viral particles in the mixed-species biofilm community is 56 spars. There are several factors that could potentially contribute to consider biofilms as an ideal 57 site to harbor SARS-CoV-2 in meat processing facilities. The temperature of the meat processing 58 facilities is maintained at 4-7 ºC. The SARS-CoV-2 virions are stable at colder temperatures and 59 stable for several days on stainless steel (SS), copper, plastic, PVC and cardboard [6] , which are 60 commonly used materials in meat processing facilities. Thus, making these facilities high risk of 61 harboring and transmitting the SARS-CoV-2 [7] . Although bacteria do not support virus infection, 62 they can promote viral fitness [8] . Specifically, some viruses use components of the bacterial 63 envelope to enhance their stability [8] [9] [10] . Moreover, bacterial communities and biofilms can 64 impact the infection of mammals by viruses [8, 9, 11] . Furthermore, from a biophysics perspective, 65 the virus stability could also be enhanced by the thin liquid film produced by bacterial biofilm [6, 66 12] . 67

There is a critical gap of knowledge in understanding the stability and infectious state of the virus 68 in multi-species biofilms, particularly in meat processing plants. In this study, MHV (SARS-CoV-69

The National Cattlemen's Beef Association [13] (predicts that the cattle industry will be potentially 115 experience a $13.6 billion loss due to the novel coronavirus SARS-CoV-2, impacting ranchers 116 across the country [13] . This loss stems from the rampant spreading of SARS-CoV-2 virus among 117 meat processing plant workers and resulting in the closure of many meats processing and packing 118 plants. The bottleneck in the supply chain between the livestock producers, feedlot operators and 119 the processors are creating an impending breakdown in the nation's meat supply. Additionally, the 120 factors that contributes to the persistent high level of COVID-19 infection within the meat 121 processing plant could be the conducive environment for the infectious virus particle to be stable 122 for a prolonged period. The temperature of the meat processing chain are maintained at 4-7°C. The 123 SARS-CoV-2 virions are stable at colder temperatures; hence these facilities are at high risk of 124 harboring and transmitting the SARS-CoV-2. These virus particle can be stable for several days 125 on stainless steel, copper, plastic, PVC and cardboard [6] , which are commonly used materials 126 throughout the farm-to-plate chain [7], thus making the meat processing facility a hotspot for 127

We utilized MHV as a surrogate for SARS-CoV-2 as they are useful for assessing methods 129 where the viral particles enhanced the biovolumes of the biofilm [2, [19] [20] [21] . 152

Biofilms are formed in many areas of food processing environments, including floors, drains, 153 difficult to clean surfaces such as the back of the conveyor belts and pipes [22, 23] . These surfaces 154 become hot spots that attract biofilm development due to poor accessibility and difficulty for 155 regular maintenance of hygiene and sanitation maintenance [24] . Furthermore, nearly all biofilms 156 in food processing environments consist of multiple species of microorganisms, and the complex 157 interactions within the community significantly influence the architecture, activity, and sanitizer 158 tolerance of the biofilm [14, 15, 23, 25] . If viral particles present in a meat processing plant are 159 protected within the the biofilms, regular sanitizer treatment will not eliminate the biofilm or 160

virus [4, 14, 26, 27] . 161

In addition, to providing sheltered and protected living for viral particles from sanitizers, microbial 162 communities in the biofilm could also enhance the dispersal of viruses through several active 163 processes: Viruses can bind to bacteria [9] , and they can be transported over large distances by 164 bacterial motility. The bacteria can also colonize new areas by continuously producing new 165 extracellular matrix (ECM), actively swarming outwards [14, 15] , thus creating new locations 166

where the virus can harbor throughout the meat-processing facility. Even without binding to the 167 viruses, bacteria will generate strong flows [16] that mix the surrounding fluid [17] . Therefore, 168 viral particles could be spread rapidly from biofilms in the meat facility. Microbial biofilms should 169 thus be considered as potential reservoirs of pathogenic viruses. Indeed, they are probably 170 responsible for numerous persistent viral infections [20] . Thus, biofilms should thus be considered 171 as potential reservoirs of pathogenic viruses. SARS-CoV-2 is well documented to be airborne and 172 spread through aerosols [6, 28] . Hence, once the virus is airborne, the colder temperature and the 173 airflow from HVAC systems in the meat processing facility will enable the spread of the SARS-174

CoV-2 throughout the plant, in agreement with recent fluid mechanics simulations [29] . 175

The above findings led us to conclude that multi-species biofilms may provide a safe reservoir for 176 SARS-CoV-2 to persist as an infectious agent due to the advantages conferred by the biofilm 177 structure and facilitate the dispersal of the viral particle through the collective microbial motility. 178

This could be one of the reasons for the higher rates of COVID-19 cases in meat processing 179 facilities. Due to the nature of the work, it will be difficult with workplace physical distancing, 180 personal hygiene, crowded living and transportation conditions among the meat processing 181 frontline workers [30] . 182

In summary, our results suggest that environmental multi-species biofilm from meat processing 185 plant can harbor viral particles (MHV which is used as a surrogate for SARS-CoV-2) and survive 186 for prolonged periods of time protecting them from sanitizers. Thus, facilitating a safe reservoir 187 for virus to persist and periodically disperse the viral particle to the meat processing facility, thus 188 making these facilities the hotspot for higher COVID 19 infections. Further studies will be required 189 to decipher molecular mechanism of how these interaction takes place, and this knowledge will for infectivity were produced as previously described [31] . 207

virus stock onto cultured L2 cells and performed a solid double overlay plaque assay as previously 209 described [32] . 210

MHV stocks were cultured to a viral titer of 10 4 VPUs/mL prior to the start of the experiment [32] . At the end of the incubation period, each chip was harvested for the biofilm biomass (Fig 4) by 229 lifting the chip with sterile forceps, scraping biofilm from both sides with a sterile cell scraper and 230 rinsing the chip with 1000 µL of LB-NS. 231

The homogenate was mixed several times by pipetting. The drain biofilm biomass was determined 232 by 10 fold dilution of the homogenate in LB-NS and plated on TSA plates for colony enumeration 233 after overnight incubation at 37C. The remaining homogenate was used for qPCR and plaque assay 234 analysis. 235

Viral RNA from each samples were extracted and purified to perform RT-qPCR to determine the 237 relative copy numbers for MHV. Viral RNA was extracted using NEB's Monarch Total RNA 238

Miniprep Kit and following the Tough-to-Lyse procedure following manufacturers protocol. 239

Purified RNA samples were quantified by using a Thermo Fisher Scientific ND-1000 240 spectrophotometer. RNA samples were stored at -20°C. Real Time thermal cycler. Thermal cycling conditions were set to 55°C for reverse transcription 251 for 10 minutes, denaturation and Taq polymerase activation at 95°C for 1 minute, and 40 cycles at 252 95°C for 15 seconds followed by 60°C for 30 seconds for data collection. RT-qPCR reactions were 253 performed in quadruplicate for each sample and the sample quantification cycle (Cq) was used for 254 data analysis. Data for each sample was compared using positive and negative controls performed 255 in duplicate. 256

Plaque assay from biofilm homogenate were performed to determine the MHV infectivity rate in 258 biofilm. 300 µL of each biofilm homogenate samples were filtered through a .2 µm filter before 259 being serially diluted in DMEM with 2% FBS and 1% Streptomycin/Penicillin mix. Previously 260 published protocol was followed for the double layer overlay plaque assay [32] . was analyzed by unpaired t-test. n.: not significant; *: P < 0.05; ***: P < 0.001; ****: P < 0.0001. 275 

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