Three-Week Old Pigs Are Not Susceptible to Productive Infection with SARS-COV-2

Abstract

As the COVID-19 pandemic moves into its third year, there remains a need for additional animal models better recapitulating severe COVID to study SARS-CoV-2 pathogenesis and develop countermeasures, especially treatment options. Pigs are known intermediate hosts for many viruses with zoonotic potential and are susceptible to infection with alpha, beta and delta genera of coronaviruses. Herein, we infected young (3 weeks of age) pigs with SARS-CoV-2 using a combination of respiratory and parenteral inoculation routes. Pigs did not develop clinical disease, nor macroscopic or microscopic pathologic lesions upon SARS-CoV-2 infection. Despite occasional low levels of SARS-CoV-2 genomic RNA in the respiratory tract, subgenomic RNA and infectious virus were never found, and SARS-CoV-2-specific adaptive immune responses were not detectable over the 13-day study period. We concluded that pigs are not susceptible to productive SARS-CoV-2 infection and do not serve as a SARS-CoV-2 reservoir for zoonotic transmission.

Keywords: SARS-CoV-2; disease; infection; pathology; replication; transmission; young pigs.

Figure 1

Clinical parameters of SARS-CoV-2 infection in 3-week-old pigs. Young pigs (3 weeks of age) were infected with SARS-CoV-2 by a combination of respiratory (intranasal, oropharyngeal and intratracheal) and parenteral (intravenous) routes (total dose 2 × 10⁶ TCID₅₀)⁶ TCID₅₀. Pigs were monitored daily for clinical signs until euthanasia on 3 DPI (#340–343) and 13 DPI (#344–347). The graphs display clinical scores (A), body temperature (B), body weight (C) and radiographic scores (D) for individual animals (the indicated color code is identical for animals in all figure parts).

Figure 2

Pathology parameters of SARS-CoV-2 infected 3-week-old pigs. SARS-CoV-2 infected young pigs were euthanized on 3 DPI and 13 DPI, necropsied for tissue harvest and tissues prepared for histopathology. Negative control tissue samples were provided from previous age-matched studies. H&E did not find any SARS-CoV-2 related pathology, and IHC targeting SARS-CoV-2 nucleocapsid (N) protein remained negative. None of the pigs developed significant lung pathology, as shown here for one representative animal of the 3 DPI and 13 DPI. Except for a singular focus within a section of lung from pig #341 (3-DPI group), none of the lung samples showed SARS-2 immunoreactivity. The magnifications are indicated (40× and 400×).

Figure 3

Immune responses to SARS-CoV-2 infection in 3-week-old pigs. Blood samples were collected on 0 DPI and 13 DPI to determine adaptive immune responses. (A,B) Humoral immune responses. Serum was analyzed for the presence of IgM (A) and IgG (B) antibodies directed against the nucleocapsid (N) protein, as well as the S1 subunit and the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Dashed line indicates mean background absorbance of wells receiving no serum. For statistical comparisons, we used a two-way ANOVA with Sidak’s multiple comparisons test (ns: p > 0.05, ** p < 0.01, *** p < 0.001). (C,D) Cellular immune responses. PBMCs were isolated and SARS-CoV-2 specific T cells were quantified by counting IFN-γ+ spot forming unit (SFU) following stimulation with a 15-mer SARS-CoV-2 S peptide pool (2 µg/mL) (C) or N peptide pool (2 µg/mL) (D). Individual SFU counts per 10⁶ stimulated PBMCs after deduction of background counts are shown. Mean SFUs ± SEM are plotted (n = 4; ns: not significant, Wilcoxon non-parametric test).