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The coronavirus disease 2019 (COVID-19) is an infectious disease that
has caused a global pandemic with more than 7.5 million infected people
from around 200 countries or territories, with more than 425,000 deaths
to date1. The causative agent of COVID-19, the severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is assumed to be
originated in bats, since the bat-borne coronavirus RaTG13 is the
closest genetic relative to date2. Several species
have been studied to determine their potential role as intermediate
hosts. Moreover, animal models to recapitulate a COVID-19-like disease
have focused another major research line and are required for the
development of therapeutic drugs and prophylactic compounds.
Besides several modelling studies proposing potential animal species
susceptible to SARS-CoV-23–5, multiple experimental
infections have already shown a broad range of susceptible animals.
Specifically, Egyptian fruit bat, ferret, golden Syrian hamster, cat,
mice expressing humanized angiotensin-converting enzyme 2 (ACE2), BALB/c
mice (using a mutated SARS-CoV-2 by several cell culture passages) and
some non-human primate species are permissive to viral infection,
developing from subclinical to mild-to-moderate respiratory
disease6–11. From an experimental point of view, dog
susceptibility to SARS-CoV-2 is limited, since inoculated animals can
partly seroconvert8. In contrast, the intranasal
inoculation of chicken, duck and pig resulted in no evidence of
infection8.
Pig is commonly used in research because of the similarities existing
with humans in terms of anatomy, genetics, physiology and, also,
immunology. Indeed, experiments in pigs are likely to be more predictive
of therapeutic and preventive treatments in humans than experiments in
rodents12. However, since pigs are not susceptible to
SARS-CoV-2 infection when inoculated intranasally8,
the possibility to develop a swine infection model with this virus using
other potential inoculation routes deserves investigation. The main
rational to test pigs is that the ACE2 receptor of this species is
functional either by transfecting swine ACE2 in HeLa cells (which do not
express constitutively the human ACE2)2 or that
pseudoparticles with the S protein of SARS-CoV-2 are able to infect
swine kidney cells13. In consequence, to set up a
putative COVID-19 pig model, we investigated the effect of different
natural and non-natural routes of SARS-CoV-2 inoculation in domestic
pigs (Sus scrofa domesticus ).
For the purpose, four groups of five 5-to-6-week-old conventional
piglets (Landrace x Large White) were selected and inoculated by means
of different routes: intranasal (IN, 1.5 mL/nostril; total volume of 3
mL), endotracheal (IT, 3 mL), intramuscular (IM, 1 mL in each side of
the neck muscles; total volume 2 mL) or intravenous (IV, 2 mL), with a
final dose of 105.8 tissue culture infectious dose
(TCID50) of the SARS-CoV-2 isolate (GISAID ID
EPI_ISL_418268) per each animal. SARS-CoV-2 was produced and titrated
in Vero E6 cells (ATCC CRL-1586). Two extra pigs were used as negative
controls. Animal experiments were approved by the Institutional Animal
Welfare Committee of the Institut de Recerca i Tecnologia
Agroalimentàries (CEEA-IRTA) and by the Ethical Commission of Animal
Experimentation of the Autonomous Government of Catalonia and conducted
by certified staff. Experiments with SARS-CoV-2 were performed at the
Biosafety Level-3 (BSL-3) facilities of the Biocontainment Unit of
IRTA-CReSA (Barcelona, Spain).
On 2- and 22-days post-inoculation (dpi), two and three animals/group
(IT, IM and IV), respectively, were euthanized. Since IN inoculation was
already demonstrated as non-effective to cause SARS-CoV-2
infection8, pigs inoculated by this route were
euthanized on days 1 and 2 pi to assess evidence of a possible transient
early infection in tissues. Negative control animals were euthanized
prior to the start of the experiment. Complete necropsies were performed
in all animals. Several tissues (frontal, medial and caudal turbinates;
proximal, medial and distal trachea; large and small bronchus, left
cranial, mediodorsal and caudal lung areas; kidney; liver; heart; and
spleen) were taken, fixed by immersion in 10% neutral-buffered
formalin, embedded in paraffin and sectioned at 3 µm to prepare slides.
Histology slides were stained with hematoxylin and eosin (HE) to assess
potential microscopic lesions. Besides, the same tissues plus ileum,
cervical lymph node (LN), mediastinal LN, mesenteric LN, olfactory bulb,
tonsil, thymus, parotid salivary gland, adrenal, pancreas, brainstem,
eyelids, and bone marrow were also taken in Dulbecco’s modified Eagle
medium (DMEM) in tubes with beads to perform SARS-CoV-2 UpE gene
detection by RT-qPCR14. Nasal and rectal swabs were
also taken (daily during the first week and at 14 and 22 dpi) to analyze
them for the presence of viral RNA by means of the abovementioned
RT-qPCR. Serum samples collected on days 0, 14 and 22 pi were tested for
the presence of antibodies against SARS-CoV-2 spike S1+S2 and
nucleocapsid (N) proteins by in-house ELISAs (Rodríguez de la Concepción
et al., 2020). Also, a virus neutralization assay was performed
following a previous protocol with a minor
modification15, the serial dilutions of sera and
SARS-CoV-2 were incubated for 1 h at 37°C prior the plate assay
performance.
All animals were daily monitored but none of them showed clinical signs
after SARS-CoV-2 inoculation. Also, no gross or microscopic lesions
attributable to SARS-CoV-2 infection were found in any of the studied
animals from all inoculation groups as well as control ones.
None of the pigs had nasal or rectal shedding of viral RNA. Proximal
trachea from one IN-inoculated animal was positive at 1 dpi for viral
RNA. The remaining tissues from this animal and the rest of pigs
resulted negative for RT-qPCR.
By 14 and 22 dpi, low levels of antibodies directed against the Spike
protein could be detected in all animals from IM and IV groups (Figure
1A). Furthermore, these pigs also showed neutralizing antibody titers at
22 dpi (ranging from 40-160 reciprocal dilutions) (Figure 1B). Also, low
antibody levels targeting the N protein were found in one out of three
IM and all IV inoculated animals by the end of the experiment (data not
shown). Importantly, one single animal from the IT group did not show
antibodies against the S but had antibodies against the nucleocapsid
protein as well as neutralizing titers (1:20-1:40) at day 0 pi, which
might suggest a potential cross-reaction with another coronavirus
infecting swine. Of note, these antibodies waned by the time the
experiment finished, suggesting they were of maternal origin or their
expression was down regulated during experimental procedure. In
addition, this animal did not show seroneutralizing antibodies at the 22
dpi.
Present data indicate that SARS-CoV-2 was not able to infect pigs by any
of the tested routes, namely IN, IT, IM and IV. Therefore, our efforts
confirm earlier experiments indicating lack of susceptibility of
infection by the pig8 Schlottau et al., 2020; Juergen
Richt, Kansas State University, USA, personal communication), although
it can be used for assessing the immunogenicity of the upcoming vaccine
candidates.
Importantly, the current study goes beyond other studies with SARS-CoV-2
and pigs since we tested a broader number of inoculation routes.
However, none of them resulted in a productive infection in piglets. A
significant outcome of this study was the evidence of seroconversion
against the Spike glycoprotein at days 14 and 22 pi and presence of
neutralizing antibodies at day 22 pi in pigs inoculated by parenteral
routes (IM and IV). Considering the short duration of the experiment (22
days), such seroconversion emphasizes the potential interest of the pig
to be used in immunogenicity studies for SARS-CoV-2. In fact, the
interest of swine as a suitable animal model for immunology, as well as
physiology, pharmacology and surgery, applicable to human medicine is
widely acknowledged16.
In conclusion, the present study confirms that piglets are not a
suitable animal model for COVID-19, but its potential usefulness as a
model for immunogenicity in preclinical vaccine development studies
deserves further investigation.