key: cord-281679-xmbnpawj
authors: Meekins, David A.; Morozov, Igor; Trujillo, Jessie D.; Gaudreault, Natasha N.; Bold, Dashzeveg; Artiaga, Bianca L.; Indran, Sabarish V.; Kwon, Taeyong; Balaraman, Velmurugan; Madden, Daniel W.; Feldmann, Heinz; Henningson, Jamie; Ma, Wenjun; Balasuriya, Udeni B. R.; Richt, Juergen A.
title: Susceptibility of swine cells and domestic pigs to SARS-CoV-2
date: 2020-08-16
journal: bioRxiv
DOI: 10.1101/2020.08.15.252395
sha: 
doc_id: 281679
cord_uid: xmbnpawj

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.

The emergence of SARS-CoV-2, the causative agent of COVID-19, has resulted in a global pandemic with over 20 million cases and 740,000 deaths as of August 13, 2020 [1, 2] . SARS-CoV-2 causes a respiratory disease in humans with a broad clinical presentation, ranging from asymptomatic or mild illness to severe fatal disease with multi-organ failure [3] [4] [5] [6] . SARS-CoV-2 is rapidly transmissible via contact with infected respiratory droplets and can also be transmitted by asymptomatic carriers [6] [7] [8] .

To curb viral spread, countries have instituted varying levels of social distancing policies, which have significant negative economic and social impacts [9] . Mitigating the effects of this unprecedented pandemic will necessitate the development of effective vaccines and therapeutics, which will require well-characterized and standardized preclinical animal models. SARS-CoV-2 is a member of the Betacoronavirus genus that includes the pathogenic human viruses SARS-CoV-1 and MERS-CoV [2, [10] [11] [12] . While details of the origin of SARS-CoV-2 are unknown, evidence indicates it emerged from a zoonotic spillover event, with bats and perhaps pangolins as probable origin species [2, [13] [14] [15] .

The potential for a reverse zoonotic event, i.e. human-to-animal transmission, is possible and of significant concern to animal and public health [16] [17] [18] . Instances of natural human-to-animal transmission of SARS-CoV-2 have been reported with COVID-19 patients in domestic settings (dogs and cats), zoos (lions and tigers), and farms (mink) [18] [19] [20] . Therefore, investigations into the infectivity of SARS-CoV-2 in various animal species with human contact are essential to assess and control the risk of a spillover event and to establish the role these animals may play in the ecology of the virus.

Several studies have determined the susceptibility of different animal species to SARS-CoV-2 via experimental infection [20, 21] . Cats, hamsters, and ferrets are highly susceptible to SARS-CoV-2 infection, demonstrate varying clinical and pathological disease manifestations, readily transmit the virus to naïve animals, and mount a virusspecific immune response [22] [23] [24] [25] [26] [27] [28] . Dogs are mildly susceptible to experimental SARS-CoV-2 infection, with limited viral replication but with clear evidence of seroconversion in some animals [22] . Poultry species seem to be resistant to SARS-CoV-2 infection [22, 26] . These findings establish the respective utility of different animal species as pre-clinical models to study SARS-CoV-2.

Several lines of evidence suggest that pigs could be susceptible to SARS-CoV-2 infection. Pigs are susceptible to both experimental and natural infection with the related Betacoronavirus, SARS-CoV-1, and demonstrate seroconversion [29, 30] .

Structure-based analyses predict that the SARS-CoV-2 Spike (S) protein receptor binding domain (RBD) binds the pig angiotensin-converting enzyme 2 (ACE2) entry receptor with similar efficiency compared to human ACE2 [31] . Single-cell screening also indicated that pigs co-express ACE2 and the TMPRSS2 activating factor in a variety of different cell lines, and SARS-CoV-2 replicates in various pig cell lines [2, 26, 32, 33] . Despite these preliminary data indicating that pigs could be susceptible to SARS-CoV-2 infection, two recent studies revealed that intranasal inoculation of three and twelve pigs, respectively, with 10 5 pfu or TCID50 of SARS-CoV-2 did not lead to any detectable viral replication or seroconversion [22, 26] . However, the single route of intranasal inoculation used in these studies suggests that additional investigations are necessary before definitive conclusions can be made regarding susceptibility of pigs to SARS-CoV-2.

In the present study, we determined the susceptibility of swine cell lines and domestic pigs to SARS-CoV-2 infection. Two different porcine cell lines were found to be permissive to SARS-CoV-2 infection showing cytopathic effects (CPE). Domestic pigs were challenged via simultaneous oral/intranasal/intratracheal inoculation with a 10 6 TCID50 dose of SARS-CoV-2. SARS-CoV-2 did not replicate in pigs and none of them seroconverted. Furthermore, the virus was not transmitted from SARS-CoV-2 inoculated animals to sentinels. The present findings, combined with the other studies [22, 26] , confirm that pigs seem resistant to SARS-CoV-2 infection despite clear susceptibility of porcine cell lines. Pigs are therefore unlikely to play an important role in the COVID-19 pandemic as a virus reservoir or as a pre-clinical animal model to study SARS-CoV-2 pathogenesis or develop novel countermeasures.

SARS-CoV-2 USA-WA1/2020 isolate (GenBank accession # MN985325) [34] was Eighteen pigs (mix of males and females, five weeks of age) were used in the study. Pigs were acquired from a source guaranteed free of swine influenza virus (SIV), porcine circovirus-2 (PCV-2), and porcine reproductive and respiratory syndrome virus (PRRSV) infection. The study outline is illustrated in Figure 1 . Upon arrival, pigs were acclimated for 3 days prior to SARS-CoV-2 inoculation. Nine pigs were designated as uninfected negative controls and housed in separate BSL-2 facilities. Three of these uninfected negative control pigs were humanely euthanized at 3 days post challenge (DPC) to provide negative control clinical and tissue samples. The nine principal infected pigs were housed in the same room in two separate groups (4 or 5 pigs each; Gross pathological examinations on major organs were performed and respiratory tissue samples were collected and either stored in 10% neutral-buffered formalin or stored as fresh samples at -80˚C. Blood and swab samples were all filtered using a 0.2µm filter prior to storage at -80˚C.

RNA was isolated from blood, swabs, and tissue samples, using a magnetic bead-based protocol in a BSL-3+ laboratory at the BRI at KSU. Lung tissue homogenates (200 mg per 1mL DMEM; 20% w/v) were prepared by thawing tissue, mincing it into 1mm sections, followed by lysis in a 2 mL sure-lock tube containing 5 mm stainless steel homogenization beads using the TissueLyser LT (Qiagen, Germantown, MD, USA) for 30 seconds at 30 hz followed by 1 min of 30 hz while keeping the sample cold. Following clarification via a 3-minute centrifugation (3,000xg; room temperature), supernatants were mixed with an equal volume of RLT lysis buffer.

Blood and clinical swabs were directly mixed with an equal volume of RLT lysis buffer.

200 µL of each sample lysate was used to extract RNA using a magnetic bead-based 

During post mortem examinations, the upper and lower respiratory tract, central nervous system, lymphatic and cardiovascular systems, gastrointestinal and urogenital systems, and integument were evaluated. Lungs were removed in toto and the percentage of the lung surface that was affected by macroscopic lesions was estimated by single veterinarian experienced in evaluating gross porcine lung pathology as previously described [36, 37] . Lungs were evaluated for gross pathology such as edema, congestion, discoloration, atelectasis, and consolidation. Tissue samples of interest were collected and either fixed in 10% neutral-buffered formalin for histopathological examination or frozen at -80˚C for RT-qPCR testing. Tissues were fixed in formalin for 7 days, then transferred to 70% ethanol (ThermoFisher Scientific, Waltham, MA, USA) prior to trimming and paraffin embedding following standard automated protocols used in the histology section of the Kansas State Veterinary Diagnostic Laboratory.

Following embedding, tissue sections were cut and stained with hematoxylin and eosin and evaluated by a board-certified veterinary pathologist who was blinded to the treatment groups.

To detect SARS-CoV-2 antibodies in sera, indirect ELISAs were performed observed. Neutralizing sera from SARS-CoV-2-infected cats from a separate study [38] was used as a positive control.

To determine the consensus sequence of the USA-WA/1/2020 virus and to analyze if there were any nucleic acid substitutions in the SARS-CoV-2 virus after passage in porcine cell lines, RNA was extracted from cell culture supernatant as described above. The RNA was then subjected to RT-PCR amplification using a tiledprimer approach to amplify the entire SARS-CoV-2 genome as described previously [39] . Briefly, the PCR amplicons were pooled and subjected to library preparation for Next Generation Sequencing using the Nextera XT library prep kit (Illumina, San Diego, CA, USA). The library was normalized and sequenced using a MiSeq nano v2 2x250 sequencing kit. The sequence was then analyzed by mapping reads to the parent sequence (Genbank accession # MN985325) [34] to generate a consensus sequence.

The SARS-CoV-2 USA-WA1/2020 isolate, which was isolated from a human patient in Washington State, USA, was used as the parent stock for the study [34] . The 

To determine the effect of SARS-CoV-2 infection in domestic pigs, nine sixweek-old SARS-CoV-2 seronegative piglets were inoculated with a total of 1 x 10 6 TCID50 of the USA-WA1/2020 isolate, which was passaged once in swine ST cells ( Figure 1 ). The challenge material (total 4 mL) was administered orally (1 mL), intranasally (1 mL; 0.5 mL each nostril) and intratracheally (2 mL) after sedation of the animals. At 1-day post challenge (DPC), six uninoculated sentinel contact pigs were comingled with the principal inoculated animals (3 animals per pen). Daily rectal temperatures were recorded for each pig and clinical signs were monitored daily, including observations for signs of lethargy, hyporexia, respiratory distress (coughing, labored breathing, nasal discharge), and digestive issues (diarrhea or vomiting). No significant temperature elevation or change in rectal temperature was observed in the principal inoculated nor sentinel contact pigs throughout the study (Figure 3) .

Moreover, no obvious clinical signs were observed for any of the principal inoculated nor sentinel pigs throughout the 21-day observation period.

To detect viral replication in the principal and sentinel pigs, clinical samples were subjected to RT-qPCR to detect the SARS-CoV-2 N gene ( Table 2) Table 2 ). The only the exception was a low suspect positive result in a nasal swab at 1 DPC in a principal inoculated pig #161, for which one of two qPCR replicates yielded a low fluorescent amplification curve with a Ct of 37 (Table 2) . Moreover, viral RNA was not detected in any lung sample collected at post-mortem examination on 4, 8 and 21 DPC (Table 2 ). In addition, gross and histopathological analysis of trachea and lung from the principal challenged pigs did not reveal the presence of any obvious pathological lesions ( Table   3 , Figure 4 ). These results indicate that SARS-CoV-2 failed to replicate in the respiratory and digestive tract as well as the blood in orally/intranasally/intratracheally inoculated pigs throughout an observation period of 21 days. This is confirmed by the fact that the principal infected pigs failed to transmit SARS-CoV-2 to co-mingled sentinel animals.

To determine whether the orally/intranasally/intratracheally inoculated pigs 

SARS-CoV-2 is a zoonotic agent, and a detailed understanding of the susceptibility of various animal species to SARS-CoV-2 is central to controlling its spread [16, 17] . In addition, the development of animal models that emulate COVID-19 in humans is essential for pre-clinical testing of novel vaccines and therapeutics [20] . In this study, we inoculated nine pigs with a high dose of SARS-CoV-2 that was passaged once in porcine cells. Simultaneous oral/intranasal/intratracheal inoculation did not result in any detectable viral RNA in the blood, the oral/nasal/rectal cavities, or the lungs. Also, none of the co-mingled, sentinel contact pigs shed viral RNA. Moreover, a virus-specific immune response characteristic of infection was not observed within the 21-day study period in the principal infected or sentinel pigs. The transient nature of the IgM and IgG response observed in pig #848 could indicate cross-reactivity of antibodies directed against a porcine coronavirus such as porcine epidemic diarrhea virus [40] . Such antibodies could be maternally derived and therefore transient as the lack of SARS-CoV-2 specific reactivity by the end of the study might suggest. In contrast to previous SARS-CoV-2 swine studies [22, 26] , the present study used a more stringent inoculation procedure (intratracheal and oral, in addition to intranasal) and 1 log higher titer of virus inoculum (10 6 vs 10 5 ). In addition, the inoculum in the present study was passaged once in porcine ST cells. These results, combined with previous intranasal pig inoculation studies [22, 26] , indicate that pigs seem to be resistant to SARS-CoV-2 infection, are unlikely to be a SARS-CoV-2 carrier animal species, and are also not suitable as an animal model for research.

The results of the present and previous SARS-CoV-2 inoculation studies in pigs are intriguing in light of the findings that the porcine ACE2 receptor seems highly compatible with the SARS-CoV-2 RBD, suggesting that pigs could be susceptible to SARS-CoV-2 infection [2, 31] . Pigs are susceptible to both experimental and natural infection with SARS-CoV-1 [29, 30] . However, the experimental SARS-CoV-1 infection was via simultaneous intranasal/oral/intraocular/intravenous inoculation [29] , thus the actual route(s) of SARS-CoV infection cannot be determined. Recently, several porcine cell lines have been shown to be permissive to SARS-CoV-2 infection [26, 33] ; in addition, single-cell screening studies showed that porcine ACE2/TMPRSS2 expression are compatible with infection [32] . In contrast to previous reports that some porcine cell lines are susceptible to SARS-CoV-2 infection, but show no CPE [26, 33] , we found that both ST and PK-15 cell lines are susceptible to infection and observed CPE after two or four passages, respectively. The absence of SARS-CoV-2 replication and transmission in the present and two previous pigs studies [22, 26] seems to lessen the need to monitor pig populations for SARS-CoV-2 during the ongoing pandemic. However, the evidence described above suggests pig susceptibility should not be disregarded, because all pig studies to date have used rather young pigs and commercially available pig breeds/genetics. We also have to be aware that unforeseen genetic changes in the SARS-CoV-2 genome may result in a better compatibility of the virus for pigs in the future.

Pigs are considered to be an excellent model for studying human infectious diseases based on their relatedness to humans in terms of anatomy and immune responses and they have been found to be much more predictive for the efficacy of therapeutics when compared to rodent models [41] . However, the results presented here indicate that pigs are not a suitable preclinical model for SARS-CoV-2 pathogenesis studies and the development and efficacy testing of therapeutics and/or vaccines. A recently available article indicates that while pigs are not susceptible to SARS-CoV-2 infection, neutralizing antibody responses were detected in pigs infected via intramuscular or intravenous inoculation routes [42] ; this indicates that pigs could be used for immunogenicity studies related to SARS-CoV-2. However, the use of pigs to monitor SARS-CoV-2 immune responses must be careful to screen for cross-reactive maternal antibodies derived from other coronaviruses [43] . Alternate pre-clinical animal models, namely non-human primates, Syrian hamsters, transgenic or transduced mice expressing human ACE2, ferrets, or even cats need to be considered to gain additional insights into SARS-CoV-2 pathogenesis and virulence. Comprehensive characterization of SARS-CoV-2 pathogenesis in preclinical animal models and the establishment of standardized infection and testing protocols will be crucial for the development of much-need countermeasures to combat COVID-19. 

1 Swabs/blood were tested from samples on 0, 3, 5, 7, 10, and 14 DPC. Lung tissue was collected 4, 8, and 21 DPC.

2 Swabs/blood were tested from samples on 0, 3, 5, and 10 DPC. Lung tissue was collected on 21 DPC 3 Swabs/blood were tested on 0 DPC. Lung tissue was collected on 3 DPC for these uninfected controls.

*One pig (#161) had a ct signal of 37.72 (3.82x10 4 copy number/mL) for 1/2 of RT-qPCR wells on 1 DPC. Magnification is 10x for main images and 20x for inserts. 

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We gratefully thank the staff of KSU Biosecurity Research Institute, the 

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.