Received: 15 July 2020 | Revised: 24 September 2020 | Accepted: 24 September 2020 DOI: 10.1111/tbed.13861 R A P I D C O M M U N I C AT I O N Pigs are not susceptible to SARS-CoV-2 infection but are a model for viral immunogenicity studies Júlia Vergara-Alert1,2 | Jordi Rodon1 | Jorge Carrillo3 | Nigeer Te1 | Nuria Izquierdo- Useros3,4 | María Luisa Rodríguez de la Concepción3 | Carlos Ávila-Nieto3 | Víctor Guallar5,6 | Alfonso Valencia5,6 | Guillermo Cantero1 | Julià Blanco3,4,7 | Bonaventura Clotet3,4,7 | Albert Bensaid1,2 | Joaquim Segalés2,8,9 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de Abstract la Universitat Autònoma de Barcelona, Conventional piglets were inoculated with severe acute respiratory syndrome coro- Barcelona, Spain 2 navirus 2 (SARS-CoV-2) through different routes, including intranasal, intratracheal, OIE Collaborating Centre for the Research and Control of Emerging and Re-emerging intramuscular and intravenous ones. Although piglets were not susceptible to SARS- Swine Diseases in Europe (IRTA-CReSA), CoV-2 and lacked lesions or viral RNA in tissues/swabs, seroconversion was observed Barcelona, Spain 3 IrsiCaixa AIDS Research Institute, in pigs inoculated parenterally (intramuscularly or intravenously). Badalona, Spain 4 Germans Trias i Pujol Research Institute KEYWORDS (IGTP), Badalona, Spain immunogenicity model, inoculation routes, lack of susceptibility, pig, SARS-CoV-2 5 Barcelona Supercomputing Center (BSC), Barcelona, Spain 6 Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain 7 University of Vic–Central University of Catalonia (UVic-UCC), Vic, Spain 8 UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, Barcelona, Spain 9 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, Barcelona, Spain Correspondence Júlia Vergara-Alert and Joaquim Segalés, Centre de Recerca en Sanitat Animal (CReSA), Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Edifici CReSA, Campus UAB, 08193 Bellaterra, Barcelona, Spain. Emails: julia.vergara@irta.cat (J. V.-A.); joaquim.segales@irta.cat (J. S.) Funding information Grifols Pharmaceutical Piglets inoculated by different routes are not susceptible to SARS-CoV-2, but those inoculated parenterally were immunized against the virus. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH Transbound Emerg Dis. 2021;68:1721–1725.wileyonlinelibrary.com/journal/tbed | 1721 1722 | VERGARA-ALERT et al. The coronavirus disease 2019 (COVID-19) is an infectious disease muscles; total volume 2 ml) or intravenous (IV, 2 ml), with a final dose that has caused a global pandemic with more than 36 million in- of 105.8 tissue culture infectious dose (TCID50) of the SARS-CoV-2 fected people from around 200 countries or territories, with more isolate (GISAID ID EPI_ISL_510689) per each animal. The IT and IV than 1 million deaths to date (World Health Organization (WHO), groups were anaesthetized with 10 mg/kg of ketamine and 0.8 mg/ 2020). The causative agent of COVID-19, the severe acute respira- kg xylazine prior inoculation. A passage-2 SARS-CoV-2 was propa- tory syndrome coronavirus 2 (SARS-CoV-2), is assumed to be origi- gated and titrated in Vero E6 cells (ATCC CRL-1586), following the nated in bats, since the bat-borne coronavirus RaTG13 is the closest same protocol as for other coronaviruses (Rodon et al., 2019). Two genetic relative to date (Andersen et al., 2020; Zhou et al., 2020). extra pigs were used as negative controls. Several species have been studied to determine their potential role All animals were seropositive against porcine respiratory coro- as intermediate hosts (Shi et al., 2020). Moreover, animal models navirus, as determined by a commercial ELISA (INgezim Corona to recapitulate a COVID-19-like disease are considered a major re- Diferencial 2.0 [TGEV/PRCV]). Taking into account that no anti- search line and required for the development of therapeutic drugs body cross-reactivity between alpha- and beta-coronaviruses has and prophylactic compounds. been described (Okba et al., 2020), the animals were kept into the Besides several modelling studies proposing potential ani- study. Initial reactivity against PRCV was expected since this virus mal species susceptible to SARS-CoV-2 (Damas et al., 2020; Qiu is ubiquitous in the European swine livestock (Saif et al., 2012; Vidal et al., 2020; Veljkovic et al., 2020), multiple experimental infec- et al., 2019). tions have already shown a broad range of susceptible animals. Animal experiments were approved by the Institutional Specifically, Egyptian fruit bat, ferret, golden Syrian hamster, Animal Welfare Committee of the Institut de Recerca i Tecnologia cat, mice expressing humanized angiotensin-converting enzyme Agroalimentàries (CEEA-IRTA) and by the Ethical Commission 2 (ACE2), BALB/c mice (using a mutated SARS-CoV-2 by sev- of Animal Experimentation of the Autonomous Government of eral cell culture passages) and some non-human primate species Catalonia and conducted by certified staff. Experiments with are permissive to viral infection, developing from subclinical to SARS-CoV-2 were performed at the Biosafety Level-3 (BSL-3) mild-to-moderate respiratory disease (Bao et al., 2020; Halfmann facilities of the Biocontainment Unit of IRTA-CReSA (Barcelona, et al., 2020; Kim et al., 2020; Rockx et al., 2020; Shi et al., 2020; Spain). Yu et al., 2020). From an experimental point of view, dog sus- On 2 and 22 days post-inoculation (dpi), two and three ani- ceptibility to SARS-CoV-2 is limited, since inoculated animals can mals/group (IT, IM and IV), respectively, were euthanized. Since partly seroconvert (Shi et al., 2020). In contrast, the intranasal IN inoculation was already demonstrated as non-effective to inoculation of chicken, duck and pig resulted in no evidence of cause SARS-CoV-2 infection (Shi et al., 2020), pigs inoculated infection (Schlottau et al., 2020; Shi et al., 2020). by this route were euthanized on days 1 and 2 pi to assess evi- Pig is commonly used in research because of the similarities ex- dence of a possible transient early infection in tissues. Negative isting with humans in terms of anatomy, genetics, physiology and, control animals were euthanized prior to the start of the ex- also, immunology. Indeed, experiments in pigs are likely to be more periment. Samples were collected and processed as previously predictive of therapeutic and preventive treatments in humans than described (Vergara-Alert et al., 2017). Briefly, complete necrop- experiments in rodents (Meurens et al., 2012). However, since pigs sies were performed in all animals. Several tissues (frontal, me- are not susceptible to SARS-CoV-2 infection when inoculated intra- dial and caudal turbinates; proximal, medial and distal trachea; nasally (Schlottau et al., 2020; Shi et al., 2020), the possibility to de- large and small bronchus, left cranial, mediodorsal and caudal velop a swine infection model with this virus using other potential lung areas; kidney; liver; heart; and spleen) were taken, fixed inoculation routes deserves investigation. The main rational to test by immersion in 10% neutral-buffered formalin, embedded in pigs is that the ACE2 receptor of this species is functional either by paraffin and sectioned at 3 µm to prepare slides. Histology transfecting swine ACE2 in HeLa cells (which do not express con- slides were stained with haematoxylin and eosin (HE) to assess stitutively the human ACE2) (Zhou et al., 2020) or that pseudopar- potential microscopic lesions. Besides, the same tissues plus ticles with the S protein of SARS-CoV-2 are able to infect swine ileum, cervical lymph node (LN), mediastinal LN, mesenteric LN, kidney cells (Letko et al., 2020). Furthermore, the ACE2 protein is olfactory bulb, tonsil, thymus, parotid salivary gland, adrenal, expressed in all major tissues of pigs as assessed by immunohisto- pancreas, brainstem, eyelids and bone marrow were also taken chemistry (Xiao et al., 2020). In consequence, to set up a putative in Dulbecco's modified Eagle medium (DMEM) in tubes with COVID-19 pig model, we investigated the effect of different natural beads to perform SARS-CoV-2 upE gene detection by RT-qPCR and non-natural routes of SARS-CoV-2 inoculation in domestic pigs (Corman et al., 2020). Nasal and rectal swabs were also taken (Sus scrofa domesticus). (daily during the first week and at 14 and 22 dpi) to analyse For the purpose, four groups of five 5- to 6-week-old conven- them for the presence of viral RNA by means of the above-men- tional piglets (Landrace × Large White) were selected and inoculated tioned RT-qPCR. Serum samples collected on days 0, 14 and 22 by means of different routes: intranasal (IN, 1.5 ml/nostril; total vol- pi were tested for the presence of antibodies against SARS- ume of 3 ml), intratracheal (IT, 3 ml) as previously described (Garcia- CoV-2 spike S1 + S2 and nucleocapsid (N) proteins by in-house Morante et al., 2016), intramuscular (IM, 1 ml in each side of the neck ELISAs (Institut de Recerca de la sida (Irsicaixa), 2020). Also, a VERGARA-ALERT et al. | 1723 F I G U R E 1 (a) Antibody detection of pigs experimentally inoculated with SARS- CoV-2. Detection of antibodies against the spike protein (days 0, 14 and 22 pi) by ELISA in sera from animals inoculated intratracheally (No. 8–10, IT group), intramuscularly (No. 13–15, IM group) and intravenously (No. 18–20, IV group). (b) Antibody detection of pigs experimentally inoculated with SARS-CoV-2. Detection of neutralizing antibodies (days 0 and 22 pi) in sera from animals inoculated intratracheally (No. 8–10, IT group), intramuscularly (No. 13–15, IM group) and intravenously (No. 18–20, IV group). The graph shows the reciprocal serum dilution showing neutralization activity versus dpi. Dotted line indicates limit of detection of the assay (1/20 serum dilution). A value of 5 was assigned to undetectable neutralization activity. Unpaired Student's t tests were performed to assess whether neutralizing antibodies significantly increased at 22 dpi. ns, not significant; *p-value < .05 virus neutralization assay was performed following a previous another coronavirus infecting swine. Of note, these antibodies protocol with a minor modification (Rodon et al., 2020), the se- against the N protein decreased by the time the experiment fin- rial dilutions of sera and SARS-CoV-2 were incubated for 1 hr at ished, suggesting they were of maternal origin. In addition, this 37°C prior the plate assay performance. animal did not show seroneutralizing antibodies at the 22 dpi All animals were daily monitored but none of them showed clini- (Figure 1b). cal signs after SARS-CoV-2 inoculation. Also, no gross or microscopic Present data indicate that SARS-CoV-2 was not able to infect lesions attributable to SARS-CoV-2 infection were found in any of pigs by any of the tested routes, namely IN, IT, IM and IV. Therefore, the studied animals from all inoculation groups as well as control our efforts confirm earlier experiments indicating lack of suscepti- ones (data not shown). bility of infection by the pig (Schlottau et al., 2020; Shi et al., 2020), None of the pigs had nasal or rectal shedding of viral RNA. although it can be used for assessing the immunogenicity of the up- Proximal trachea from one IN-inoculated animal was positive at 1 dpi coming vaccine candidates. for viral RNA (Cq = 24.36). The remaining tissues from this animal Importantly, the current study goes beyond other studies and the rest of pigs resulted negative for RT-qPCR (qPCR detection with SARS-CoV-2 and pigs since we tested a broader number limit of 38.6 cycles). of inoculation routes. However, none of them resulted in a By 14 and 22 dpi, low levels of antibodies directed against productive infection in piglets. A significant outcome of this the Spike protein could be detected in all animals from IM and study was the evidence of seroconversion against the Spike IV groups (Figure 1a). Furthermore, these pigs also showed neu- glycoprotein at days 14 and 22 pi and presence of neutralizing tralizing antibody titres at 22 dpi (ranging from 74 to 317 SNT50 antibodies at day 22 pi in pigs inoculated by parenteral routes reciprocal dilution titre) (Figure 1b). Also, low antibody levels tar- (IM and IV). Considering the short duration of the experiment geting the N protein were found in one out of three IM and all IV (22 days), such seroconversion emphasizes the potential inter- inoculated animals by the end of the experiment (data not shown). est of the pig to be used in immunogenicity studies for SARS- Importantly, one single animal from the IT group did not show CoV-2. In fact, the interest of swine as a suitable animal model antibodies against the S but had antibodies against the N protein for immunology, as well as physiology, pharmacology and sur- as well as neutralizing titres (SNT50 reciprocal dilution titre of 29) gery, applicable to human medicine is widely acknowledged at day 0 pi, which might suggest a potential cross-reaction with (Rothkötter, 2009). 1724 | VERGARA-ALERT et al. In conclusion, the present study confirms that piglets are not a irsic​aixa.es/sites/​defau​lt/files/​detec​tion_of_sars-cov-2_antib​odies_ by_elisa_-_proto​col_by_irsic​aixa_prote​c ted.pdf suitable animal model for COVID-19, but its potential usefulness as a Kim, Y.-I., Kim, S.-G., Kim, S.-M., Kim, E.-H., Park, S.-J., Yu, K.-M., Chang, model for immunogenicity in preclinical vaccine development stud- J.-H., Kim, E. J., Lee, S., Casel, M. A. B., Um, J., Song, M.-S., Jeong, ies deserves further investigation. H. W., Lai, V. D., Kim, Y., Chin, B. 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