viruses Article Susceptibility of Domestic Goat (Capra aegagrus hircus) to Experimental Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) B.1.351/Beta Variant Leira Fernández-Bastit 1,2 , Núria Roca 1,2 , Miguel Romero-Durana 3 , Jordi Rodon 1,2 , Guillermo Cantero 1,2 , Óscar García 1,2 , Carlos López 1,2 , Mònica Pérez 1,2 , Rosa López 1,2 , Jorge Carrillo 4,5,6 , Nuria Izquierdo-Useros 4,5,6 , Julià Blanco 4,5,6,7 , Bonaventura Clotet 4,7,8 , Joan Pujols 1,2 , Júlia Vergara-Alert 1,2 , Joaquim Segalés 1,9, * and Cristina Lorca-Oró 1,2, * 1 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 2 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 3 Life Sciences Department, Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, 08034 Barcelona, Spain 4 IrsiCaixa AIDS Research Institute, 08916 Badalona, Spain 5 Germans Trias i Pujol Research Institute (IGTP), Can Ruti Campus, 08916 Badalona, Spain 6 Infectious Disease Networking Biomedical Research Center (CIBERINFEC), Carlos III Health Institute, 28029 Madrid, Spain 7 Infectious Diseases and Immunity, Faculty of Medicine, University of Vic-Central University of Catalonia (UVic-UCC), 08500 Barcelona, Spain 8 Lluita Contra la SIDA Foundation, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain Citation: Fernández-Bastit, L.; Roca, 9 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, N.; Romero-Durana, M.; Rodon, J.; 08193 Cerdanyola del Vallès, Spain Cantero, G.; García, Ó.; López, C.; * Correspondence: joaquim.segales@irta.cat (J.S.); cristina.lorca@irta.cat (C.L.-O.) Pérez, M.; López, R.; Carrillo, J.; et al. Susceptibility of Domestic Goat Abstract: A wide range of animal species are susceptible to the severe acute respiratory syndrome (Capra aegagrus hircus) to Experimental Infection with Severe coronavirus 2 (SARS-CoV-2) infection. Natural and/or experimental infections have been reported in Acute Respiratory Syndrome pet, zoo, farmed and wild animals. Interestingly, some SARS-CoV-2 variants, such as B.1.1.7/Alpha, Coronavirus 2 (SARS-CoV-2) B.1.351/Beta, and B.1.1.529/Omicron, were demonstrated to infect some animal species not suscepti- B.1.351/Beta Variant. Viruses 2022, 14, ble to classical viral variants. The present study aimed to elucidate if goats (Capra aegagrus hircus) are 2002. https://doi.org/10.3390/ susceptible to the B.1.351/Beta variant. First, an in silico approach was used to predict the affinity v14092002 between the receptor-binding domain of the spike protein of SARS-CoV-2 B.1.351/Beta variant and Academic Editor: F. Javier Salguero angiotensin-converting enzyme 2 from goats. Moreover, we performed an experimental inoculation with this variant in domestic goat and showed evidence of infection. SARS-CoV-2 was detected in Received: 5 August 2022 nasal swabs and tissues by RT-qPCR and/or immunohistochemistry, and seroneutralisation was Accepted: 6 September 2022 confirmed via ELISA and live virus neutralisation assays. However, the viral amount and tissue Published: 9 September 2022 distribution suggest a low susceptibility of goats to the B.1.351/Beta variant. Therefore, although Publisher’s Note: MDPI stays neutral monitoring livestock is advisable, it is unlikely that goats play a role as SARS-CoV-2 reservoir species, with regard to jurisdictional claims in and they are not useful surrogates to study SARS-CoV-2 infection in farmed animals. published maps and institutional affil- iations. Keywords: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); Beta variant; goat; ruminant; susceptibility; experimental infection Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and The causative agent of the coronavirus infectious disease 2019 (COVID-19) pandemic, conditions of the Creative Commons severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is mainly transmitted via Attribution (CC BY) license (https:// respiratory droplets or aerosols from human to human [1]. Additionally, SARS-CoV-2 creativecommons.org/licenses/by/ can infect several animal species that may act as intermediate hosts and play a role in the 4.0/). transmission and maintenance of the virus. Considering that the origin of this pandemic is Viruses 2022, 14, 2002. https://doi.org/10.3390/v14092002 https://www.mdpi.com/journal/viruses Viruses 2022, 14, 2002 2 of 13 apparently zoonotic [2–4], it is important to determine the risk of infection in susceptible species, as well as the risk of reverse zoonosis [5,6]. Moreover, to better understand the epidemiology of this zoonotic pathogen under the “One Health” approach, it is crucial to identify the susceptibility of animal species and to investigate if they could act as potential reservoirs and/or intermediate hosts, especially those that are in close contact with human populations as domestic livestock. In an increasingly connected world, the emergence of multiple variants since the start of the COVID-19 pandemic has urged the implementation of rapid measures to control the spreading of the virus. Several variants of concern (VOCs) have been identified with different infectivity or transmissibility properties in humans and animal species. As of 31st August 2022, more than 599 million cases in humans and 700 outbreaks or infection events in animals, involving 23 species in 36 countries, have been reported [7,8]. Natural SARS-CoV-2 infections were reported in zoo animals (i.e., felids, but also gorillas, otters, and white-tailed deer, among others), farmed animals (mainly minks), and pets (i.e., cats, dogs, ferrets and hamsters) [9–12]. The susceptibility of non-human primates, hamsters, ferrets, cats, deer, rabbits, raccoon dogs, fruit bats, and skunks has also been demonstrated by experimental infections [13–19]. Importantly, wild-type mice are not susceptible to early pandemic variants of SARS-CoV-2 under experimental conditions, but susceptible to certain variants such as B.1.1.7/Alpha, BA.1.1/Omicron, and especially B.1.351/Beta [20–23]. This latter variant was first reported in South Africa in late 2020 and showed nine mutations in the spike (S) protein compared to the ancestral variant [24], allowing it to partially escape from previously acquired neutralising antibodies (nAbs) against SARS-CoV-2 and to increase its transmissibility. Indeed, the described novel viral variants acquired mutations along the genome, but most importantly in the receptor-binding domain (RBD) of the SARS- CoV-2 S protein which is responsible for the recognition of the angiotensin-converting enzyme 2 (ACE2) host receptor [25,26]. The affinity between the RBD and the ACE2 receptor as well as the distribution in different tissues of the ACE2 in animal species is a putative indicator of host susceptibility to infection [27]. These differences between variants raise concerns regarding how the risk of new mutations in SARS-CoV-2 could increase susceptibility in novel species, with the danger of becoming zoonotic reservoirs. The interactions between livestock animals and wildlife increase cross-transmission of infectious diseases which can reduce food safety and also result in economic impact [28]. Among ruminants, white-tailed deer has been found to be the most susceptible species to ancestral and Alpha SARS-CoV-2 variants, showing high seroprevalence in free-ranging animals in the USA [29]. Moreover, subclinical infection has been demonstrated following experimental challenge, as well as viral transmissibility between in-contact white-tailed deer and vertically from doe to foetus [15,30]. Regarding other ruminant species, sheep, cattle, and goat have shown low susceptibility to SARS-CoV-2 ancestral variant [31] or the combination of ancestral plus Alpha/B.1.1.7 variants [32]. The domestic goat (Capra aegagrus hircus) is a small ruminant species raised worldwide in a broad range of production systems for its meat, milk, hide, and hair [33]. Since goats are in constant contact with humans and other SARS-CoV-2 susceptible animal species, it is of high interest to investi- gate their susceptibility to this viral infection. As mentioned previously, previous studies reported limited susceptibility of livestock (cattle, sheep, and goats) to SARS-CoV-2 USA- WA1/2020 reference isolate [31,32] under experimental conditions; however, the emergence of new SARS-CoV-2 variants raises the possibility of host range expansion [20,34]. Since the potential reservoirs and intermediate hosts of SARS-CoV-2 are still being investigated and little information is available regarding the susceptibility of livestock to different variants, the aim of the present study was to evaluate the susceptibility of the domestic goat to the B.1.351/Beta variant of SARS-CoV-2, one of the most important VOCs that already showed expanded species tropism [21,23,34]. Viruses 2022, 14, 2002 3 of 13 2. Materials and Methods 2.1. In Silico Study We ran MODELLER v10.1 [35] to generate ten different homology models of goat ACE2 in complex with the spike RBD of the SARS-CoV-2 ancestral variant. The crystallographic structure of human ACE2 in complex with the spike RBD of the ancestral variant (PDB ID 6M0J) was used as a template. Then, using the MODELLER models as input, we used FoldX v5 [36] to predict the changes in binding affinity induced by the mutations of the variants. We considered the following mutations located at the RBD: K417N, E484K, N501Y for B.1.351/Beta, L452R, T478K for B.1.617.2/Delta, K417T, E484K, N501Y for P.1/Gamma, and G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H for B.1.1.529/Omicron (BA.1) variants. We ran FoldX modules repairPDB followed by BuildModel. We fixed all FoldX parameters to their default values except parameter vdwDesign, which was set to zero. As a positive control, we performed the same steps using the human ACE2 sequence to predict the changes in binding affinity caused by the mutations of the SARS-CoV-2 VOC in humans. 2.2. Cells and Virus Vero E6 cells (ATCC® repository, CRL-1586™) and SARS-CoV-2 B.1.351/Beta variant isolate (passage 3; hCoV-19/Spain/CT-IrsiCaixaR008CC8B3/2021; GISAID ID EPI_ISL_3164134) were prepared as previously reported [37] in Dulbecco’s modified Eagle Medium, DMEM (Lonza, Basel, Switzerland) supplemented with 5% foetal calf serum (FCS; EuroClone, Milan, Italy), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine (all from Gibco Life Technologies, Madrid, Spain). The infectious titre of the SARS-CoV-2 stock was calculated by determining the dilution that caused cytopathic effect (CPE) in 50% of the inoculated Vero E6 cells (50% tissue culture infectious dose endpoint, TCID50 ). 2.3. Experimental Study Design This experimental study was approved by the Institutional Animal Welfare Committee of the Institute of Agrifood Research and Technology (CEEA-IRTA) and by the Ethical Commission of Animal Experimentation of the Autonomous Government of Catalonia (CEA-OH/11586/3) and conducted by certified staff. Experiments involving SARS-CoV-2 were performed at the Biosafety Level-3 (BSL-3) facilities of the Biocontainment Unit of IRTA-CReSA (Barcelona, Spain). A total of 18 goats of 2–3 months of age were acquired from a Spanish commercial farm (La botiga d’Ullastrell S.L, Barcelona, Spain) and included in the study. Three animals were used as non-inoculated controls and were necropsied on the day of animal arrival. The remaining 15 animals were allocated inside an experimental box of the animal BSL-3 facilities, and after 7 days of acclimation, they were intranasally (IN) inoculated with 2 mL of 106 TCID50 /mL of SARS-CoV-2 B.1.351/Beta variant (1 mL in each nostril) using a diffusor device (LMA™ MAD® Nasal, Teleflex LLC). At 2, 4, 7, 10 and 18 days post-inoculation (dpi), three goats/day were euthanised and complete necropsies were performed (Figure 1). At necropsy, nasal and rectal swabs (NS/RS), blood, and the following tissues were collected: nasal turbinate (NT; caudal and cranial portions), olfactory bulb, parotid gland, trachea, pharynx, tonsil, lung (apical, medial, and caudal portions), lymph nodes (LN; cervical, mesenteric, and mediastinal ones), kidney, spleen, jejunum, colon, and heart. Clinical signs and rectal temperatures were recorded daily during the whole study. Viruses Viruses 2022, 2022, 14, 14, 2002 2002 4 4ofof13 13 Schematicrepresentation Figure1.1.Schematic Figure representationof ofthe thestudy studydesign. design.Three Threeout outof ofeighteen eighteengoats goatswere werenecropsied necropsied on the on the day day of of arrival arrival and and served served as as non-inoculated non-inoculated controls. controls. Nasal Nasal andand rectal rectal swabs, swabs, blood, blood, and and tissuesamples tissue sampleswere were collected. collected. After Afteracclimatisation, acclimatisation,the theremaining remainingfifteen fifteengoats goats were were intranasally intranasally inoculated inoculated with with the SARS-CoV-2 B.1.351/Beta variant SARS-CoV-2 B.1.351/Beta variant (2 (2 mL of11× mL of × 1066 TCID TCID5050/animal). /animal).AtAt2,2,4,4,7,10, 7,10, and and1818days dayspost-infection, post-infection,three threegoats goatswere werenecropsied necropsiedeach eachday dayand andnasal nasaland andrectal rectalswabs, swabs,blood, blood, and andtissues tissues were were collected. collected. Clinical Clinical signs signs and and rectal rectal temperature temperature were were recorded recorded daily. daily. 2.4. Virus 2.4. VirusDetection Detectionand andIsolation Isolation Tissues, NS, Tissues, NS,and andRSRSwere werecollected collectedduring duringnecropsy necropsyand andwere weretransferred transferred into into cry- cryo- otubes containing 500 µL DMEM supplemented with 100 U/mL tubes containing 500 μL DMEM supplemented with 100 U/mL penicillin, 100 μg/mL penicillin, 100 µg/mL ◦ C until use. Blood was streptomycin, and streptomycin, and 22 mM mM glutamine, glutamine, vortexed vortexed and and stored stored atat − 75°C −75 until use. Blood was (1800×g,g,10 ◦ ◦ C until centrifuged (1800× centrifuged 10min minatat44 °C)C) and and sera sera were were collected collected andand kept at − kept at 20 °C −20 until further use. Detection of SARS-CoV-2 RNA was performed via genomic further use. Detection of SARS-CoV-2 RNA was performed via genomic and subgenomic and subgenomic reverse transcription-quantitative reverse transcription-quantitativePCR PCR(RT-qPCR) (RT-qPCR)in inall all collected collectedsamples, samples,as as previously previously described [38,39]. Briefly, viral RNA was extracted using the Indimag Pathogen Kit(Indical described [38,39]. Briefly, viral RNA was extracted using the Indimag Pathogen Kit (Indi- Biosciences, cal Leipzig, Biosciences, Germany) Leipzig, Germany)on aon Biosprint 96 workstation a Biosprint 96 workstation (Qiagen, Hilden, (Qiagen, Germany) Hilden, Ger- in accordance many) with thewith in accordance manufacturers’ instructions. the manufacturers’ Then, RT-qPCR instructions. was carried Then, RT-qPCR wasout using carried AgPath-IDTM out One-Step One-Step using AgPath-IDTM RT-PCR Reagents RT-PCR (Applied Reagents Biosystems, Life Technologies), (Applied Biosystems, Life Technol-and amplification was performed using a 7500 Fast Real-Time PCR System ogies), and amplification was performed using a 7500 Fast Real-Time PCR System (Ap-(Applied Biosystems, Life Technologies, plied Waltham, Biosystems, Life MA, USA). Technologies, Samples Waltham, MA,with a Ct USA). value 30) at 7 dpi (Figure the nine remaining goats were positive with low viral loads (Ct > 30) at 7 dpi (Figure 3a). 3a). Moreover, low levels of viral RNA (Ct > 23) were observed in caudal and cranial NT Moreover, low levels of viral RNA (Ct > 23) were observed in caudal and cranial NT (Figure (Figure3b),3b),lymphoid lymphoidtissues tissues (tonsil (tonsil and and cervical cervical and/or and/ormediastinal mediastinalLN) LN)(Figure (Figure3c,d), 3c,d), and and respiratory tract (trachea and lung) (Figure 3e,f). SARS-CoV-2 RNA fromtonsil respiratory tract (trachea and lung) (Figure 3e,f). SARS-CoV-2 RNA from tonsiland and LN LN samples samples werewere detected detected until until 18 18 dpi dpi inin goats goats (Figure (Figure 3c,d). 3c,d). Regarding Regarding subgenomic subgenomic RNA, RNA,tonsil tonsilsamples sampleswerewerepositive positiveat at2, 2,4, 4,and and77dpi dpi(Ct (Ct>>23) 23)as aswell wellas ascranial cranialand andcaudal caudal NT at 2 dpi (Ct > 25). Blood and all other samples (RS, olfactory bulb, parotid NT at 2 dpi (Ct > 25). Blood and all other samples (RS, olfactory bulb, parotid gland, gland, tra- chea, spleen, trachea, kidney, spleen, jejunum, kidney, and and jejunum, heart) tested heart) negative tested (Ct ≥(Ct negative 40)≥via 40)genomic and sub- via genomic and genomic RT-qPCR. subgenomic RT-qPCR. Viruses 2022, 14, 2002 7 of 13 Viruses 2022, 14, 2002 7 of 13 Figure3. Figure Detection of 3. Detection of SARS-CoV-2 SARS-CoV-2genomic genomicRNA RNA(gRNA) (gRNA) byby RT-qPCR. RT-qPCR.SARS-CoV-2 SARS-CoV-2 loads in (a)innasal loads (a) swabs; (b) cranial and caudal nasal turbinate; (c) tonsil; (d) mediastinal, cervical, and mesenteric nasal swabs; (b) cranial and caudal nasal turbinate; (c) tonsil; (d) mediastinal, cervical, and mesen- lymph teric nodes; lymph (e) trachea; nodes; and (f) (e) trachea; andlung. Horizontal (f) lung. bars bars Horizontal indicate median indicate viral viral median loads.loads. Dotted lines Dotted lines reflect the limit of detection (Ct reflect the limit of detection (Ct = 40).= 40). Onlysamples Only sampleswith with a Ct a Ct < 30