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Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are

completely resistant to PRRSV infection

Article in Antiviral Research · January 2024

DOI: 10.1016/j.antiviral.2024.105793

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 Antiviral Research 221 (2024) 105793

Contents lists available at ScienceDirect

Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are

completely resistant to PRRSV infection
Brianna Salgado a, 1, Rafael Bautista Rivas a, 1, Derek Pinto a, Tad S. Sonstegard b,
Daniel F. Carlson b, Kyra Martins b, Jonathan R. Bostrom b, Yamlak Sinebo b, Raymond R.
R. Rowland a, Alberto Brandariz-Nuñez a, *
a
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Champaign, IL, USA
b
Acceligen, Eagan, Minnesota, USA

A R T I C L E I N F O A B S T R A C T

Keywords: CD163 expressed on cell surface of porcine alveolar macrophages (PAMs) serves as a cellular entry receptor for
CD163 porcine reproductive and respiratory syndrome virus (PRRSV). The extracellular portion of CD163 contains nine
PRRSV scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Genomic editing of
Gene-edited pigs
pigs to remove the entire CD163 or just the SRCR5 domain confers resistance to infection with both PRRSV-1 and
PSTII
Exon 13
PRRSV-2 viruses. By performing a mutational analysis of CD163, previous in vitro infection experiments showed
Antiviral breeding resistance to PRRSV infection following deletion of exon 13 which encodes the first 12 amino acids of the 16
amino acid PSTII domain. These findings predicted that removal of exon 13 can be used as a strategy to produce
gene-edited pigs fully resistant to PRRSV infection. In this study, to determine whether the deletion of exon 13 is
sufficient to confer resistance of pigs to PRRSV infection, we produced pigs possessing a defined CD163 exon 13
deletion (ΔExon13 pigs) and evaluated their susceptibility to viral infection. Wild type (WT) and CD163 modified
pigs, placed in the same room, were infected with PRRSV-2. The modified pigs remained PCR and serologically
negative for PRRSV throughout the study; whereas the WT pigs supported PRRSV infection and showed PRRSV
related pathology. Importantly, our data also suggested that removal of exon 13 did not affect the main phys­
iological function associated with CD163 in vivo. These results demonstrate that a modification of CD163 through
a precise deletion of exon 13 provides a strategy for protection against PRRSV infection.

1. Introduction PRRSV is an enveloped single-stranded positive-sense RNA virus

In 1989, Keffaber was the first to describe a mystery swine disease
associated with a diverse set of clinical signs (wellsKeffaber, 1989). Soon
afterwards, a similar syndrome appeared in Europe. The etiological
agent, porcine reproductive and respiratory syndrome virus (PRRSV),
was identified and sequenced in Europe in 1991 and called Lelystad
virus, followed in 1999 by the complete sequence of a North American
isolate, VR2332 (Allende et al., 1999; Benfield et al., 1992; Collins et al.,
1992; Nelsen et al., 1999; Wensvoort et al., 1991). Clinical signs after
infection include reproductive failure during late gestation such as
abortions, respiratory distress in young pigs, and poor growth perfor­
mance (Zimmerman et al., 2019).
(Cavanagh, 1997; Snijder and Meulenberg, 1998) belonging to the genus
Betaarterivirus, family Arteriviridae, order Nidovirales (Adams et al.,
2017). PRRSV has a polycistronic genome of approximately 15.4 kb in
length and harbors at least 10 open reading frames (ORFs), encoding 14
nonstructural proteins (nsp 1-12) and 8 structural proteins (Kappes and
Faaberg, 2015; Su et al., 2021). PRRSV is divided into two distinct
species, Betaarterivirus suid 1 (PRRSV-1) and Betaarterivirus suid 2
(PRRSV-2) (Brinton et al., 2021). Even though both species produce
similar clinical signs, PRRSV-1 and PRRSV-2 possess only about 70%
identity at the nucleotide level (Kuhn et al., 2016b). Despite both species
circulate in US swine herds, PRRSV-2 isolates remain the overwhelming
challenge in US swine herds (Kuhn et al., 2016a). PRRSV penetrates the

* Corresponding author. College of Veterinary Medicine, University of Illinois at Urbana-Champaign (UIUC). 2810 Veterinary Medicine Basic Sciences Building,
2001 Lincoln Avenue, Urbana, IL, 61802, USA.
E-mail address: brandari@illinois.edu (A. Brandariz-Nuñez).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.antiviral.2024.105793
Received 13 November 2023; Received in revised form 18 December 2023; Accepted 2 January 2024
Available online 4 January 2024
0166-3542/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
B. Salgado et al. Antiviral Research 221 (2024) 105793

host principally through the respiratory and reproductive mucosal sur­
faces and mainly infects tissue-specific macrophages in the nose, lungs,
tonsils, and pharyngeal lymphoid tissues (Duan et al., 1997). PRRSV is
now endemic in most swine-producing countries causing approximately
$500–600 million per year in losses to US producers alone (Holtkamp
et al., 2013; Neumann et al., 2005).
CD163 is a 130 kDa type I transmembrane protein that belongs to the
class B of the scavenger receptor cysteine-rich (SRCR) superfamily
(SRCR-SF) (Fabriek et al., 2005, 2007; Law et al., 1993; Ritter et al.,
1999; Su et al., 2021). CD163 is a scavenger receptor expressed exclu­
sively in cells of the monocyte-macrophage lineage (Law et al., 1993;
Ritter et al., 1999). CD163-positive macrophages, particularly porcine
alveolar macrophages (PAMs), are the main PRRSV target during acute
infection (Duan et al., 1997). The CD163 gene is composed of 17 exons,
which code for a peptide signal sequence followed by nine tandem re­
peats of the scavenger receptor cysteine-rich (SRCR) domain and two
linker peptide sequences referred to as proline serine threonine (PST)
domains, which are located after SRCR6, and SRCR9. The protein is
anchored to the cytoplasmic membrane by a transmembrane domain
followed by a short cytoplasmic tail (Kristiansen et al., 2001; Onofre
et al., 2009; Su et al., 2021; Subramanian et al., 2013). A schematic
representation of porcine CD163 protein is shown in Fig. 1A. The main
biological function of CD163 is the removal of cell-free hemoglobin (Hb)
and Hb/haptoglobin (Hp-Hb) complexes from the blood plasma, (Kris­
tiansen et al., 2001; Onofre et al., 2009; Subramanian et al., 2013).
CD163 binds and internalizes the Hp-Hb complex by binding Hp to the
SRCR3 domain (Kristiansen et al., 2001; Onofre et al., 2009; Sub­
ramanian et al., 2013). The Hb degradation products, biliverdin, bili­
rubin, and carbon monoxide are potent anti-inflammatory molecules
(Soares and Bach, 2009). Thus, one important anti-inflammatory role of
CD163 is the protection of oxidative toxicity that results from free he­
moglobin (Kristiansen et al., 2001; Soares and Bach, 2009). The

Fig. 1. Generation of pigs lacking CD163 exon 13. (A) Diagram of porcine CD163 protein and gene. CD163 protein SRCR (ovals) and PST (rectangles) domains along
with transmembrane domain (TM) and cytoplasmic tail are shown on top. The corresponding gene exons are displayed in the bottom figure. (B) Targeted deletion of
CD163 exon 13 in somatic cells. Two Cas9-gRNA (red arrows) were designed to removed exon 13 entirely, along with 66bp of intron 12 and 58bp of intron 13.
Porcine fetal fibroblasts (PFS) were co-transfected with both gRNAs, a Cas9 coding plasmid, and a HDR (Homology-directed repair) template to direct precise
deletion of 160bp (Δ160) from the CD163 gene. Relative efficiency of exon 13 deletion upon transfection was assessed by PCR. The PCR analysis showed candidate
clones homozygous containing the Δ160 allele (2, 3, 5, 6, 8, 9, 10, 11). Banding consistent with heterozygous or wild type (WT) clones were also detected (1, 4, 7).
(C) Genotypes were confirmed by PCR amplification across intron 12 to intron 13 and sequencing. The unmodified genome PCR product is predicted to generate a
493 bp band, whilst exon 13 deletion is predicted to result in a 333 bp PCR product. (D) cDNA sequence comparison of CD163 WT with CD163 lacking exon 13
(ΔExon13) following sequencing of cDNA obtained from RNA extracted from PAMs is shown. The DNA sequence corresponding to exon 13 is indicated in red. (E)
Predicted peptide sequence comparison of WT CD163 and CD163 variant with exon 13 deleted. The bold underlined 16 amino acid peptide sequence encoded by
exon 13 and exon 14 is the PSTII domain (Stoian et al., 2022a; Van Gorp et al., 2010a). The amino acids coded for by exon 13 are indicated in red.

2
B. Salgado et al.
N-terminus of CD163 was identified as critical for the infection of PAMs
with African swine fever virus (ASFV) (Sánchez-Torres et al., 2003).
However, CD163 knockout pigs were susceptible to infection with ASFV
(Popescu et al., 2017). Therefore, in vitro results do not always translate
to the context of in vivo infection biology, highlighting the importance of
in vivo models to study the PRRSV biology and pathogenesis.
Numerous studies demonstrated that CD163 serves as the primary
cellular entry receptor for PRRSV. For example, overexpression of
CD163 renders non-susceptible cell lines permissive to PRRSV infection
(Calvert et al., 2007; Lee et al., 2010; Patton et al., 2009; Su et al., 2021;
Wang et al., 2013). The role of CD163 as the essential receptor medi­
ating PRRSV infection was confirmed by genetic modification experi­
ments demonstrating that pigs possessing a complete deletion of the
CD163 gene were resistant to both PRRSV-1 and PRRSV-2 isolates (Wells
et al., 2017; Yang et al., 2018; Whitworth et al., 2016; Prather et al.,
2017; Xu et al., 2020). The CD163 knockout pigs possessed elevated
levels of haptoglobin (hp) in blood, confirming the loss of the main
CD163 biological function (Wells et al., 2017; Whitworth et al., 2016; Xu
et al., 2020). Previous mutational analysis of CD163 to study the
contribution of the different CD163 domains to PRRSV infection
revealed that SRCR5, along with PSTII, and SRCR7-9 domains are
important for both PRRSV-1 and PRRSV-2 infection in vitro (Stoian et al.,
2022a, 2022b; Van Gorp et al., 2010b). The role of SRCR5 domain in
viral infection was confirmed by genetic modified experiments demon­
strating that pigs possessing a clean deletion of exon 7, which codes for
the SRCR5 domain, in the CD163 gene were resistant to both PRRSV-1
and PRRSV-2 infections (Burkard et al., 2017, 2018; Wang et al.,
2015). In addition, gene-modified pigs possessing a partial deletion of
the SRCR5 domain also were fully resistant to PRRSV-2 infection (Guo
et al., 2019), further confirming the importance of SRCR5 domain in
viral infection. Pigs possessing a replacement of SRCR 5 with the ho­
molog, human CD163-like (hCD163L1) SRCR8 were resistant to
PRRSV-1 but supported PRRSV-2 infection to near normal levels (Chen
et al., 2019; Wells et al., 2017), making this gene-editing strategy inef­
ficient in fighting both PRRSV-1 and PRRSV-2 infections. Recently, by
performing a deep mutational analysis within CD163, we identified
specific regions in CD163 that are required for both PRSV-1 and
PRRSV-2 infections in vitro (Stoian et al., 2022a). Concretely, we found a
specific pentapeptide sequence present in both CD163 SRCR5 and
SRCR7 domains that was essential for PRRSV infection. Resistance to
PRRSV infection was also observed after removal of four amino acid of
interdomain region that connects SRCR4 and SRDCR5 domains. Addi­
tionally, deletion of the exon 13 that encodes the first 12 amino acids of
the 16 amino acids PSTII domain resulted in the complete loss of
infection (Stoian et al., 2022a). The results from in vitro studies predict
that the deletion of exon 13 could be used as a strategy to generate
gene-edited pigs resistant to PRRSV infection.
The aim of the current study was to determine whether the deletion
of CD163 exon 13 would be sufficient to confer resistance of pigs to both
type 1 and type 2 viruses. For this purpose, we generated pigs with a
defined CD163 exon 13 deletion (ΔExon13 pigs) and evaluate the sus­
ceptibility of gene-modified pigs and alveolar macrophages isolated
from these pigs to PRRSV infection. Our data demonstrated that modi­
fying CD163 gene by deleting exon 13 remarkably inhibited PRRSV-2
replication in vivo. Furthermore, the PAMs isolated from ΔExon13 pigs
were resistant to both PRRSV-1 and PRRSV-2 infections. Our results also
suggested that the deletion of exon 13 had no adverse effects on the main
biological function associated with CD163 in vivo. These findings suggest
that modifying CD163 gene by a complete deletion of exon 13 may
provide a potential strategy for the control and elimination of PRRSV.
2. Materials and methods
2.1. Generation of gene-edited pigs
Pigs possessing a clean deletion of CD163 exon 13 were prepared
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Antiviral Research 221 (2024) 105793
using the CRISPR/Cas9 technology as previously described (Wells et al.,
2017; Whitworth et al., 2014; Workman et al., 2021), and the exon 13
deletion is diagrammed in Fig. 1. For the CD163 gene, the sgRNA were
designed to target exon 13 entirely, along with 66bp of intron 12 and
58bp of intron 13. Porcine fetal fibroblasts (PFS) were co-transfected
with both gRNAs, a Cas9 coding plasmid, and a HDR (Homology-dir­
ected repair) template to direct precise deletion of exon 13 from the
CD163 gene. After identifying the positive clones, somatic cell nuclear
transfer (SCNT) was conducted as previously described (Wells et al.,
2017; Whitworth et al., 2016).
2.2. Genotyping
Genomic DNA was extracted from ear biopsy or tail clippings taken
from piglets as previously described (Wells et al., 2017; Whitworth et al.,
2014). The region spanning exon 13 was amplified by PCR using the
following primers: the sense primer, 5′-GCCTTCACCGAGGAAAGGAA-3′,
and the antisense primer, 5′-AATGCGATGAGACAGGCCAA-3′, producing
a 493 bp product from the intact allele and a 333 bp product if complete
removal of exon 13 had occurred. PCR products were analyzed by sep­
aration on a 1% agarose gel and subsequent Sanger sequencing.
2.3. RNA phenotyping
RNA was isolated from WT and ΔExon13 PAMs using the GeneJET
RNA Purification Kit (Thermofisher, cat# K0702) according to the
manufacturer’s instructions. The Superscript™ IV One-Step RT-PCR
System (Thermo Fisher, cat# 12,594,025) and the following primers:
the sense primer, 5′- ATGGTGCTACTTGAAGACTCTGG-3′, and the
antisense primer, 5′- TCATTGTACTTCAGAGTGGTCTCC-3′ were used
according to the manufacturer’s specifications to amplify the CD163
gene. PCR products were purified and the absence of exon 13 in PAMs
from gene-edited pigs was confirmed by DNA sequencing.
2.4. Ethics statement
All experiments and protocols involving the use of animals and vi­
ruses were approved by the University of Illinois Institutional Animal
Care and Use Committee (IACUC) and were performed in accordance
with the Federation of Animal Science Societies Guide for the Care and
Use of Agricultural Animals in Research and Teaching and the USDA
Animal Welfare Act and Animal Welfare Regulations.
2.5. Infection of pigs
Four days prior to transfer of the animals to a specific-pathogen-free
unit in the animal facility, blood samples were taken from all animals by
jugular venipuncture. Sera were separated and screened by using the
Idexx PRRSV X3 ELISA to confirm that none of the animals had previ­
ously been exposed to PRRSV. Animals were acclimated in the animal
facility for one week prior to challenge. All WT pigs used in the infection
experiment were born from natural breeding, and they were matched by
age and breed with the genetic-edited pigs. The eight modified-pigs and
two WT pigs used for PRRSV NVSL viral challenge were both about 49
days old. Each pig was infected with the PRRSV-2 isolate NVSL (Ladinig
et al., 2015). Infectivity of NVSL stocks was evaluated using a TCID50
assay on PAMs immediately after production and prior to challenge.
Viral inoculation was administered intranasally (IN) (2 ml: 106
TCID50/mL) and intramuscularly (2 ml: 106 TCID50/mL). After viral
challenging, the WT and gene-edited pigs were maintained in the same
room. The ratio of control to edited pigs was approximately 1–4.
Experimental staff were blind as to the nature of the gene-edit and the
identification of gene-edited pigs, thus avoiding the potential for
observer bias. During the 14 days of PRRSV challenge, clinical obser­
vations data including rectal temperature, demeanor, nasal discharge,
coughing, and respiration were recorded regularly. In addition, piglet
B. Salgado et al.
survival rate was recorded, blood was collected, and the piglets were
weighed on day 0 (prior to challenge) and on day 14 (prior to eutha­
nasia). Serum samples were collected on day 0 prior to challenge and on
days 3, 7, 10, and 14 prior to euthanasia. No animal reached the criteria
for premature termination during the challenge. All surviving pigs were
slaughtered at 14 days post-infection (dpi) and lung tissue was examined
for disease symptoms.
2.6. Measurement of PRRSV viremia
RNA was isolated from serum using Ambion’s MagMax 96 RNA Viral
Isolation Kit (Applied Biosystems) according to the manufacturer’s in­
structions. PRRSV RNA was quantified using EZ-PRRSV MPX 4.0 real-
time reverse transcription (RT)-PCR target-specific reagents (Tetra­
core) according to the manufacturer’s instructions as previously
described (Kittawornrat et al., 2014). The assay covers two target re­
gions of the PRRSV-1 and 2 genes with several primer and probes. PCR
was performed on a 7500 real-time PCR system (Applied Biosystems) in
a 96-well format using the recommended cycling parameters. The PCR
assay results were reported as log10 PRRSV RNA copy number per 50 μl
reaction volume, which approximates the number of copies per milliliter
of serum.
2.7. Detection of viral RNA in pig tissues
Total RNAs from different tissues were extracted with TRIzol reagent
(Invitrogen). PRRSV RNA was quantified using EZ-PRRSV MPX 4.0 real-
time reverse transcription (RT)-PCR target-specific reagents (Tetracore)
as indicated above.
2.8. Measurement of PRRSV antibody
The blood of WT pigs and ΔExon13 edited-pigs were collected at
different times after viral challenge and the sera were separated. The
sera from all samples were subjected to PRRSV antibody detection by
using the commercially available enzyme-linked immunosorbent assay
(ELISA), IDEXX PRRS X3 Ab (IDEXX, ME). The antibody level was
determined to be negative or positive according to the S/P value. If the
S/P ratio is < 0.4, the sample is considered negative for PRRSV anti­
bodies. If the S:P ratio is ≥ 0.4, the sample is considered positive.
2.9. Necropsy and histopathology
At 14 dpi, animals were euthanized. During necropsy, the lungs were
removed and analyzed. Accurate photographs were taken from the
dorsal and ventral sides for scoring of macroscopic lung lesions. Lung
pathology was analyzed in a blind fashion. For histopathology, lung
tissues of pigs exposed to PRRSV challenge were collected. The tissues
were fixed in 10% neutral buffered formalin. Formalin-fixed sections
were embedded in paraffin and routinely processed for histological ex­
amination with hematoxylin and eosin (H and E) staining.
2.10. Immunofluorescence and confocal microscopy
For indirect immunofluorescence microscopy, PAMs from WT and
gene-modified pigs grown on coverslips were fixed with 4% para­
formaldehyde. Fixed cells were blocked in PBS containing 2% BSA. After
incubation with anti-CD163 mouse antibody (Clone: 2A10) overnight at
4 ◦ C, the cells were incubated for 1 h with Alexa 594 goat anti-mouse IgG
secondary antibody and DAPI (49,69-diamidino-2-phenylindole). Im­
ages were obtained with a Nikon A1R laser scanning confocal micro­
scope. The images were processed with NIS-elements software (Nikon).
2.11. Measurement of CD163 surface expression on PAMs
Analysis of cell surface expression of CD163 by fluorescence-
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Antiviral Research 221 (2024) 105793
activated cell sorter (FACS) was performed as described previously
(Wells et al., 2017). Approximately 1 × 106 PAMs were incubated for 15
min at room temperature in 1 ml of PBS with 10% normal mouse serum
to block Fc receptors. The cells were pelleted by centrifugation and
resuspended in 5 μl of fluorescein isothiocyanate (FITC)-conjugated
mouse anti-porcine CD163 MAb (clone 2A10/11; Bio-Rad). After a 30
min incubation, the cells were washed twice with PBS containing 1%
bovine serum albumin (BSA) (Sigma) and immediately analyzed on a BD
LSR Fortessa flow cytometer (BD Biosciences). Unstained and an isotype
control consisting of PAMs cells from WT and gene-edited pigs stained
with an FITC-conjugated mouse IgG1 isotype control (BIO-RAD) were
included in each experiment. All the data were processed with FCS Ex­
press 7 software (De Novo Software). All experiments were done in
triplicate and repeated at least three times. A minimum of 50,000 cells
were analyzed for each sample.
2.12. Measurement of haptoglobin (hp)
The amount of Hp in serum was measured using a commercial
porcine-specific Hp enzyme-linked immunosorbent assay (ELISA) kit
(Genway Biotech Inc.) as previously described (Wells et al., 2017). As­
says were performed in triplicate for each sample.
2.13. Infection of alveolar macrophages
The preparation and infection of macrophages were performed as
previously described (Wells et al., 2017). Lungs were removed from
euthanized pigs and lavaged by pouring 100 ml of cold PBS into the
trachea. The tracheas were clamped, and the lungs were gently
massaged. The alveolar contents were poured into 50-ml centrifuge
tubes and stored on ice. The PAMs were sedimented by centrifugation at
1200×g for 10 min at 4 ◦ C. The pellets were resuspended and washed
once in cold sterile PBS. The cell pellets were resuspended in freezing
medium (45% RPMI 1640, 45% FBS, 10% DMSO) and stored in liquid
nitrogen until use. The frozen cells were thawed on ice, counted, and
adjusted to 5 × 105 cells/ml in medium (RPMI 1640 supplemented with
10% FBS, PenStrep, and Gentamycin). Approximately 105 PAMs per
well were added to 48-well plates and incubated overnight at 37 ◦ C in
5% CO2. The cells were gently washed to remove nonadherent cells.
Serial 1:10 dilutions of virus in medium were added to triplicate wells.
At 24 h post-infection (hpi), the cells were washed with PBS and fixed for
15 min with 4 % paraformaldehyde. Fixed cells were permeabilized with
0.5 % Triton X-100 in PBS and then blocked in PBS containing 2 % BSA.
Cells were then stained with PRRSV N-protein-specific mAb (SDOW-17;
Rural Technologies Inc.) diluted 1:60 in PBS containing 2% BSA. After 1
h incubation at room temperature, the cells were stained with Alexa
Fluor 488-labelled anti-mouse IgG (Thermo Fisher Scientific) diluted
1:1000 in PBS containing 2% BSA. The plates were incubated for 30 min
in the dark at 37 ◦ C, washed with PBS, and viewed under a fluorescence
microscope (Nikon ECLIPSE TE2000-S).
2.14. Viruses
The PRRSV-2 isolate, a P129 strain expressing a green fluorescent
protein (GFP) (Pei et al., 2009), the PRRSV-1 isolate, Lelystad (Meu­
lenberg et al., 1993), the type 2 prototype PRRSV, ATCC VR-2332
(Benfield et al., 1992), and the PRRSV-2 isolate NVSL (Ladinig et al.,
2015) were propagated and titrated on MARC-145 cells, as previously
described (Cafruny et al., 2008). P129 is a virulent strain of PRRSV
isolated in 1995 from an outbreak of highly virulent PRRSV in Southern
Indiana, USA (Lee et al., 2005). For virus titration, the samples were
serially diluted 1:10. Dilutions were performed in quadruplicate and the
titration endpoint was determined as the last well showing CPE (cyto­
pathogenic effect). The log 10 50% tissue culture infectious dose
(TCID50)/ml was calculated as previously described (Stoian et al.,
2022b).
B. Salgado et al.
2.15. Antibodies
The mouse anti-porcine CD163 monoclonal antibody (Clone: 2A10/
11) and the anti-nucleocapsid monoclonal antibody (SDOW17) were
purchased from Bio-Rad and Rural Technologies Inc., respectively. The
Alexa 594- and Alexa 488-conjugated antibodies against mouse and
rabbit IgG, respectively, were from Thermo Fischer. The anti-CD163
antibody (abcam #ab87099) was purchased from Abcam. The anti-
Actin pan mAb MA5-11869 was purchased from Thermo Fisher
Scientific.
2.16. Western blotting
Immunoblotting was performed as previously described (Rowland
and Brandariz-Nuñez, 2021). Briefly, cellular proteins were extracted
with a whole-cell extract buffer (50 mM Tris [pH 7.5], 150 mM NaCl,
0.5% Triton X-100, 10% glycerol, 1 mM EDTA, protease inhibitor
cocktail [Sigma]), resolved on 4–12% Bis-Tris NuPAGE gels (Invi­
trogen), and transferred to nitrocellulose membranes using a trans-blot
turbo transfer system (Bio-Rad). Detection of proteins bands was per­
formed by using anti-CD163 antibody diluted 1:1000 or anti-Actin mAb
diluted 1:1000. Secondary antibodies against rabbit and mouse conju­
gated to HRP were obtained from Cell-signaling. Protein bands were
detected by using SuperSignal West femto maximum sensitivity sub­
strate (Thermo Fisher) and imaged on a FluorChem R system
(ProteinSimple).
2.17. Statistical analysis
All data are presented as mean ± standard deviation. The mean and
standard deviation values were calculated using GraphPad Prism 9.
Statistical analysis was performed using two-tailed unpaired Student’s t-
test. A P value of <0.05 was considered to be statistically significant.
Number of repeats are specified in the figure legends.
3. Results
3.1. ΔExon13 modified-pigs are resistant to PRRSV-2 infection
Previous studies demonstrated that an intact PSTII domain is
required for both PRRSV-1 and PRRSV-2 infections (Stoian et al., 2022a;
Van Gorp et al., 2010b). Furthermore, the deletion of PSTII, which is
located just proximal to the plasma membrane did not alter CD163
expression (Stoian et al., 2022a). As shown in Fig. 1E, the 16 amino acid
PSTII peptide sequence of porcine CD163 is encoded by two different
exons. The first 12 amino acids are contributed entirely by exon 13,
while the remaining four amino acids are encoded by exon 14. We
demonstrated that deleting the first 10 amino acids coded by exon 13 is
sufficient to prevent PRRSV infection in vitro experiments (Stoian et al.,
2022a). Based on these results, we hypothesized that removing the
entire exon 13 will produce the same impact on viral infection. There­
fore, the next logical step is to construct gene-edited pigs lacking exon
13 and evaluate whether the modified pigs are resistant to PRRSV-2
infection. For this purpose, founder-generation (F0) animals carrying a
deletion of exon 13 in the CD163 gene were generated by using the
CRISPR/Cas9 technology as previously described (Wells et al., 2017;
Whitworth et al., 2014; Workman et al., 2021). To generate pigs lacking
CD163 exon 13, a sgRNA delivery plasmid was designed to remove the
entire exon 13 in porcine fetal fibroblasts (PEFs). PEFS containing the
desired exon 13 deletion in the CD163 gene were used as nuclear
transfer donors (Fig. 1A and B). Genotyping revealed that all the
gene-modified animals possessed a precise deletion of exon 13 (Fig. 1C).
Sequencing of cDNA generated by reverse transcription PCR (RT-PCR)
from RNA extracted from the wild-type (WT) PAMs and ΔExon13 PAMs
verified that all editing events had resulted in complete deletion of exon
13 (Fig. 1D).
5
Antiviral Research 221 (2024) 105793
Next, we evaluated whether the ΔExon13 pigs were resistant to
PRRSV-2 infection. At seven weeks of age, the WT pigs and CD163
modified-pigs were inoculated with a virulent PRRSV-2 isolate, NVSL,
which was isolated in 1997 from a herd undergoing an abortion storm
(Halbur and Bush, 1997). WT and ΔExon13 pigs inoculated with NVSL
strain were placed in the same pen, which allowed the continuous
exposure of CD163 gene-edited pigs to virus shed from the WT pen mates
during the 14-day infection period. As shown in Fig. 2A, the ΔExon13
pigs were negative for the presence of viral nucleic acid at all time
points. As expected, the WT pigs were productively infected, with mean
viremia levels approaching 106 templates/reaction at 7 days
post-infection (dpi). At 14 dpi, PRRSV antibodies were detected in WT
pigs but remained undetectable in all ΔExon13 pigs (Fig. 2B), con­
firming that the gene-modified pigs were resistant to PRRSV-2 infection.
In addition to PAMs, PRRSV can infect lymphoid tissues, such as the
thymus, tonsils, spleen, and lymph nodes (Duan et al., 1997; Lamon­
tagne et al., 2003; Wang et al., 2011). Therefore, to further confirm that
the CD163 gene-modified pigs are not susceptible to PRRSV-2 infection,
we analyzed the presence of PRRSV in lung and lymphoid tissues (tonsils
and lymph nodes) from the two groups of animals after viral challenge
by real-time PCR. Results showed that PRRSV was detected in all tissues
examined in the WT group, while PRRSV was undetectable in ΔExon13
pigs (Table 1).
After the viral challenging we observed that WT pigs exhibited res­
piratory distress, lethargic and decreased demeanor at 5 and 10 dpi,
which are typical clinical symptoms of PRRSV, whereas the ΔExon13
group displayed no abnormalities except for a brief diarrhea in two pigs
at 3 dpi and 8 dpi, respectively. The rectal temperatures were signifi­
cantly elevated on day 7 of the challenge in the WT pigs, whereas no
clinical fever (over 40 ◦ C) was observed in the ΔExon13 pigs throughout
the 14 day post viral challenge observation period (Supplemental
Fig. 1A). In addition, the body weight of the gene-edited pigs increased,
after the 14 day post viral challenge (Supplemental Fig. 1B). Gross pa­
thology showed that the lungs from the WT pigs were swollen, with
severe bleeding, and obvious lesions and signs of interstitial pneumonia,
while the lungs from the Exon13 pigs appeared normal (Fig. 2C). At the
microscopic level, the lungs from the WT pigs showed signs of severe
broncho interstitial pneumonia, with the absence of distinct alveoli, and
accompanied by the infiltration of mononuclear cells, all characteristic
features associated with PRRSV (Halbur et al., 1996) (Fig. 2D). In
contrast, these pathological signs were not observed in the lung tissue of
the genetic edited pigs (Fig. 2D). The absence of viral nucleic acid
combined with the absence of PRRSV-associated pathology and a PRRSV
seronegative status confirmed that all ΔExon13 pigs were completely
free of PRRSV, even though all ΔExon13 pigs were virus inoculated and
continually exposed to PRRSV during the 14-day study period. Alto­
gether, these results provide compelling evidence that the ΔExon13 pigs
are resistant to PRRSV-2 infection.
3.2. CD163 is expressed on the cell surface of PAMs from ΔExon13 pigs
The role of CD163 as the primary cellular receptor was confirmed by
genetic modification experiments demonstrating that CD163 knockout
pigs were resistant to PRRSV infection (Wells et al., 2017; Yang et al.,
2018; Whitworth et al., 2016; Prather et al., 2017). When analyzing the
resistance of ΔExon13 pigs to PRRSV infection, one possibility for not
detecting viral infection was that the CD163 mutant lacking the amino
acids encoded by exon 13 was not expressed on the cell surface of PAMs
derived from gene-edited pigs, preventing access of the virus to the re­
ceptor. To confirm that the CD163 variant is properly expressed on the
cell membrane, we first analyzed the surface expression of CD163 by
performing immunofluorescence experiments on not permeabilized
PAMs isolated from WT and ΔExon13 pigs fixed in paraformaldehyde.
Cell surface labeling revealed that CD163 was abundantly present on the
surface of PAMs from gene-edited pigs (Fig. 3A). To further confirm that
the CD163 variant is properly expressed on the cell surface, we
B. Salgado et al. Antiviral Research 221 (2024) 105793

Fig. 2. ΔExon13 edited-pigs are resistant to PRRSV-2 infection. (A) Viremia during challenge with the PRRSV-2 isolate NVSL. Serum samples were collected at days
0, 3, 7, 10, and 14 dpi (days post-infection), and viral RNA was isolated and quantified by RT-PCR (real-time PCR). Results are shown as log10 copies per reaction. WT
(N = 2); ΔExon13 (N = 8). Data are expressed as means ± SD. (B) Antibody response to PRRSV-2 during the challenge. Serum samples were analyzed for the presence
of PRRSV antibodies using the Idexx PRRSV X3 ELISA at 14 dpi. A value of <0.40 is negative and a value of ≧0.4 is positive. Each bar represents data for a single
animal. KO, ΔExon13 pigs; WT, wild type pigs (C) Representative lungs from pigs at necropsy at 14 dpi. (D) H and E stained thin sections of lung showing
representative pathology at 14 dpi. Bar = 100 μm.

expression of CD163.
Table 1
Another possibility for the resistance of ΔExon13 pigs to PRRSV is
PRRSV RT-qPCR results in lung and lymphoid tissues.
that the deletion of the exon 13 peptide sequence created an overall
Pig ID Genotype Lung Trachbronch. Tonsil Subman. conformational change in CD163, preventing virus recognition of other
Lymph node Lymph node
domains, such as SRCR5. Previously, to evaluate the impact of removal
2 Wild Type 22.9 28.4 20.2 24.6 of SRCR5 domain on the overall structure of CD163, the structure of
8 Wild Type 21.3 32.9 27.3 33.5
CD163 lacking SRCR5 domain was predicted by using RaptorX (Burkard
1 ΔExon13 >37 >37 >37 >37
3 ΔExon13 >37 >37 >37 >37 et al., 2018; Källberg et al., 2012). They found that all the SRCR domains
4 ΔExon13 >37 >37 >37 >37 in both full-length and ΔSRCR5 CD163 were predicted to adopt a similar
5 ΔExon13 >37 >37 >37 >37 globular structure and retain the pearl-on-a-string configuration of the
6 ΔExon13 >37 >37 >37 >37 native CD163 protein (Burkard et al., 2018). To analyze the effect of the
7
deletion of the peptide sequence of PSTII coded by exon 13 on the
ΔExon13 >37 >37 >37 >37
9 ΔExon13 >37 >37 >37 >37
10 ΔExon13 >37 >37 >37 >37 structure of CD163, we decided to predict the overall 3D structure of
CD163 lacking exon 13 peptide sequence by using AlphaFold, since this
server generates more accurate 3D models than the RaptorX (Jumper
quantified its plasma membrane levels in live cells by et al., 2021; Pereira et al., 2021; Xu, 2019). Importantly, our predicted
fluorescence-activated cell sorter (FACS). As shown in Fig. 3B, the structure for WT CD163 resembled the crystal structure for porcine
CD163 protein from ΔExon13 PAMs were efficiently expressed on the CD163 SRCR5-9 recently deposited in the RSCB Protein Data Bank (PDB
cell surface. These results confirmed that the negative effect on infection accession number: 8H7J). Next, we compared the predicted structure of
in gene-modified pigs was not due to the absence of CD163 on the sur­ WT CD163 generated by using AlphaFold with its mutant version
face of the plasma membrane. In addition, this suggests that the (Jumper et al., 2021). The results indicated that both WT and mutant
ΔExon13 version of CD163 is likely to be properly folded since the CD163 were predicted to adopt an identical globular structure, since the
anti-CD163 clone 2A10/11 antibody used in these experiments only CD163 variant closely mimics the structure of WT CD163 (Fig. 4).
recognizes the protein in a non-reduced, native conformation (Bullido Therefore, our structural predictions suggest that removal of the exon 13
et al., 1997; Burkard et al., 2017; Stoian et al., 2022a). Additionally, portion of PSTII does not induce a misfolding of the CD163 protein.
these results confirm our previous findings that showed that the CD163
variant lacking the peptide sequence coded by the exon 13 was properly
exposed on cell surface of transfected cells (Stoian et al., 2022a). Next, 3.3. Circulating haptoglobin levels in WT and ΔExon13 pigs
we analyzed the expression levels of CD163 by immunoblot analysis,
and the results showed that there was no significant difference in the As a scavenging receptor, CD163 is responsible for removing Hb-Hp
expression of CD163 between ΔExon13 and WT PAMs (Supplemental complexes from the blood (Fabriek et al., 2005; Kristiansen et al., 2001;
Fig. 3), indicating that removal of exon 13 does not affect to the proper Madsen et al., 2004; Onofre et al., 2009; Subramanian et al., 2013).

6
B. Salgado et al. Antiviral Research 221 (2024) 105793

Fig. 3. Cell surface expression of CD163 on PAMs from WT and ΔExon13 pigs. (A) Representative confocal fluorescence images showing the surface expression of
CD163 in non-permeabilized PAMs from WT pigs and ΔExon13 pigs. Cells were stained with mouse anti-CD163 monoclonal antibody (Clone: 2A10), followed by
Alexa 594-goat anti-mouse IgG (red). Nuclei were stained with DAPI (blue). Non-permeabilized PAMs stained with an isotype control are also shown. Similar results
were obtained in three separate experiments and representative data are shown. (B) The histograms plots show cell surface expression of CD163 measured by FACS.
Live PAMs from WT and ΔExon13-modified pigs were stained with fluorescein isothiocyanate (FITC)-conjugated mouse anti-porcine CD163 MAb (Clone: 2A10). The
anti-CD163 antibody staining results are shown as percentage of CD163-positive cells (blue). Histograms of the isotype controls are shown on the bottom (pink).
FITC-conjugated mouse IgG1 was used as an isotype control. Similar results were obtained in three independent experiments and representative data are shown.

Therefore, the level of Hp in serum provides a convenient method for
evaluating the CD163 physiological function in ΔExon13 pigs. Hp levels
in sera from WT and CD163 gene-edited pigs were measured at seven
weeks of age and prior to infection with PRRSV-2. The results showed
that five of the eight gene-edited pigs had similar Hp levels compared to
WT pigs (Fig. 5), suggesting that the overall functional state of PAMs
isolated from ΔExon13 pigs remained intact. Furthermore, our struc­
tural models agrees with the results that show that CD163 activity is not
compromised in most of the gene-modified pigs, since removal of the
first portion of PSTII did not affect the folding of CD163 SRCR3 domain
which is involved in the binding and internalization of the Hp-Hb
complexes (Kristiansen et al., 2001; Onofre et al., 2009) (Fig. 4).
3.4. PAMs from ΔExon13 pigs are resistant to both PRRSV-1 and PRRSV-
2 infections in vitro
Since PAMs are the PRRSV main target cells in vivo (Duan et al.,
1997), we next determine whether the PRRSV resistance of ΔExon13
pigs arose from antiviral properties of PAMs. For this purpose, we iso­
lated PAMs from CD163 gene-edited pigs and analyzed whether they
were resistant to both PRRSV-1 and PRRSV-2 infections.
To assess the infectability of the ΔExon13 PAMs with PRRSV-2,
PAMs were infected with three strains, P129, a virulent isolate from
an outbreak in Indiana (Lee et al., 2005); the prototypic PRRSV isolate,
VR-2332 (Benfield et al., 1992), and NVSL (Ladinig et al., 2015). For
PRRSV-1, we tested the prototype isolate, Lelystad (Meulenberg et al.,
1993).
PAMs from WT pigs and gene-modified pigs were infected at an MOI
= 1, and at 24 h post infection (hpi) the infected cells were detected by

7
B. Salgado et al.
Fig. 4. Structure prediction for CD163 mutant lacking the peptide sequence coded for by exon 13. Protein structure prediction was performed using Alphafold
(Jumper et al., 2021) and viewed using UCSF Chimaera (Pettersen et al., 2004). The red portion shows the peptide sequence encoded by exon 13. The numbers refer
to the locations of the SRCR domains. The protein domains critical for infection are colored as follows: SRCR5 (blue), SRCR7 (cyan), SRCR8 (orange) and SRCR9
(magenta). PM: plasma membrane.
staining them with an anti-PRRSV N protein (green) antibody as previ­
ously described (Stoian et al., 2022a, 2022b; Wells et al., 2017). As
expected, the WT PAMs were infected by all the viruses. In contrast, the
ΔExon13 PAMs obtained from two different pigs were negative for
infection by all the PRRSV strains (Fig. 6). To further confirm that PAMs
isolated from ΔExon13 pigs does not support viral infection, we per­
formed a TCID50 assay on supernatants collected at 48 hpi to analyze
whether infectious virions were produced. As expected, viruses pro­
duced from PAMs of WT origin were infectious. By contrast, ΔExon13
PAMs did not support virus production (Supplemental Fig. 2). Taken
together, these results demonstrated that PAMs from CD163 gene-edited
pigs were completely resistant to both PRRSV-1 and PRRSV-2 infections.
4. Discussion
Previous studies show that pigs possessing a complete deletion of
CD163 or of the targeted deletion of SRCR5 domain of the viral receptor
are resistant to infection with PRRSV-1 and PRRSV-2 (Burkard et al.,
2017, 2018; Whitworth et al., 2016; Xu et al., 2020; Yang et al., 2018).
Pigs possessing a partial deletion of SRCR5 also show resistance (Guo
et al., 2019). However, resistance derived from the partial knockout of
SRCR5 is likely the result of the removal of important disulfide bonds,
which are required to maintain SRCR conformational integrity (Stoian
et al., 2022b). In previous work, we showed that substitution of SRCR5
domain with human CD163L1 SRCR8 conferred resistance of pigs to
infection with PRRSV-1 but not PRRSV-2 (Chen et al., 2019; Wells et al.,
2017), demonstrating that both PRRSV-1 and PRRSV-2 require SRCR5
for infection, but the two genotypes recognize SRCR5 differently. All
these findings support the idea that CD163 is an essential receptor and
its SRCR5 domain is critical for PRRSV infection. Therefore, the next
logical step in the construction of PRRSV-resistant pigs is to find the
minimum peptide sequence that grants resistance to both PRRSV-1 and
8
Antiviral Research 221 (2024) 105793
PRRSV-2 infections while maintaining CD163 structural and functional
integrity. In this study, we successfully generated pigs in which the first
12 amino acids of PSTII domain of CD163 encoded by exon 13 were
removed. The results of this report show that gene-edited pigs harboring
a clean deletion of CD163 exon 13 were healthy under our experimental
conditions and resistant to PRRSV infection while maintaining the key
biological functions of CD163. However, it is important to point out that
the presence of possible off-target mutations in the genome of the
gene-modified pigs that could have been incorporated in the
CRISPR-Cas9 assay remains to be determined, which would be a critical
step to produce a commercial-scale founder population of pigs resistant
to PRRSV.
In previous in vitro experiments, we and others demonstrated that
removal of the PSTII domain in CD163 has a negative effect on infection
with PRRSV-1 and PRRSV-2 isolates (Stoian et al., 2022a; Van Gorp
et al., 2010b). Specifically, we showed that removal of the first 10 amino
acids of PSTII domain encoded by exon 13 affects the ability of CD163 to
support viral infection (Stoian et al., 2022a). These results predicted that
the deletion of exon 13 can be used as a strategy for conferring resistance
to PRRSV infection in vivo. To verify that these findings translated to an
in vivo model, we demonstrated in this study that ΔExon13 pigs were
completely resistant to infection with a highly virulent PRRSV-2 subtype
2 strain. The genetic modified pigs showed no clinical or pathological
signs of infection, and no viral replication was observed. These results
confirm that our previous in vitro results directly translate to the in vivo
scenario. The removal of exon 13 left the remaining four amino acids,
Gly-Arg-Ser-Ser (GRSS), as a short spacer domain separating SRCR9
from the cell surface. In human and non-human primates, the four amino
acids coded for by exon 14, Arg-Ser-Ser-Arg (RSSR), form an ADAM17
cleavage site (Etzerodt et al., 2014). During inflammation, RSSR cleav­
age releases soluble CD163, which can be used as a marker for inflam­
mation in the blood (Etzerodt et al., 2014). Pasternak et al. (2019)
B. Salgado et al.
Fig. 5. Serum haptoglobin levels. Serum Hp concentrations of WT and
ΔExon13-edited pigs. WT group: N = 2; ΔExon13 group: N = 8. Each dot
represents data for a single pig. Results are means ± standard deviation (SD)
values. Statistical analysis of the data was performed by using Student’s t-test.
ns, not significant. Results are shown for seven week-old pigs prior to infection.
documented the presence of soluble porcine CD163 in blood (Pasternak
et al., 2019). However, porcine CD163 possesses only the first three
amino acids, RSS, of the ADAM17 cleavage site (see Fig. 1). Whether or
not the RSS peptide sequence in porcine CD163 functions as an ADAM17
cleavage domain remains unclear. In support of the RSS cleavage, Zhu
et al. (2020) showed a direct relationship between production of
ADAM17 and accumulation of soluble CD163 in the media of cultured
porcine macrophages (Zhu et al., 2020). The release of soluble CD163 in
the ΔExon13 pigs remains to be determined.
Fig. 6. Infection of PAMs from WT and ΔExon13 pigs with different PRRSV-1 and PRRSV-2 isolates. PAM cells were infected with the viruses at a MOI = 1 at 24 h
after placement in culture. At 24 h post-infection (hpi), the cells were fixed and stained with PRRSV N protein antibody, followed by Alexa 488-goat anti-mouse IgG
(green). Nuclei were counterstained with DAPI (blue). Similar results were obtained in three separate experiments and representative data are shown.
9
Antiviral Research 221 (2024) 105793
The complete resistance of ΔExon13 pigs to PRRSV infection created
several possibilities that could explain the absence of infection. First, the
combined intranasal-intramuscular inoculation of pigs was not sufficient
to establish a productive infection in the ΔExon13 pigs. To overcome
this possibility, the ΔExon13 pigs were co-mingled with the WT pigs,
which allowed the constant exposure of the ΔExon13 pigs to virus shed
by the acutely infected WT piglets. After infection, the two WT pigs
attained a relatively high level of viremia, between 105 and 106 copies of
genomic RNA copies per PCR reaction (see Fig. 2). During PRRSV
infection, PRRSV is readily shed through respiratory droplets, feces,
urine and other body fluids indicating continuous exposure during the
period of systemic infection (Chae, 2021; Zimmerman et al., 2019). A
second possibility for resistance is that the removal of the first 12 amino
acids of PST-II blocked surface expression of CD163 on macrophages.
However, our results indicated that CD163 is properly expressed on the
cell surface of PAMs isolated from the ΔExon13 pigs, which is in
agreement with our previous work that showed that the same mutant
version of CD163 is located on the surface when is exogenously
expressed in HEK293T cells (Stoian et al., 2022a). Therefore, the resis­
tance to PRRSV observed in gene-edited pigs was not due to the absence
of CD163 variant on the cell surface. Remarkably, the CD163 mutant
was detected on the surface of ΔExon13 PAMs by using a
native-confirmation antibody, which suggests that the protein variant
was properly folded. In agreement with this notion, our previous
biochemical binding experiments showed that removing the PSTII re­
gion coded by exon 13 did not affect the ability of CD163 to interact with
the different viral envelope glycoproteins, suggesting that the CD163
variant is in a native conformational state (Stoian et al., 2022a). Our
structural models to analyze the effect of the deletion of the PSTII
portion encoded by exon 13 indicated that this modification would yield
a protein that basically mimics the overall structure of full-length
CD163. The major difference between WT and ΔExon13 structures
was found in a change in orientation of SRCR9 (see Fig. 4). These data
further support our conclusions that suggested the proper folding and
expression of the CD163 present in PAMs isolated from ΔExon13 pigs.
CD163 is directly implicated in the clearance of excess of cell-free Hb
and Hp-Hb complexes from the blood (Van Gorp et al., 2010a). Thus, the
measurement of the blood levels of Hp constitutes a suitable method to
evaluate the biological function of CD163. In a previous study, we
showed that nursery-age pigs lacking CD163 possessed significantly
elevated levels of haptoglobin (Wells et al., 2017). The results showed
that mean levels in the CD163 KO pigs were approximately elevated
10-fold. Results in Fig. 5 show a variation in serum Hp levels in the
B. Salgado et al.
ΔExon13 pigs, ranging between 0.02 mg/ml and 0.6 mg/ml. Five pigs
showed levels similar to WT pigs. For the three pigs showing elevated
HP, other factors, besides decreased CD163 could influence circulating
Hp levels. The fact that the CD163 activity is not compromised after
deletion of a portion of PSTII domain agrees with previous work that
showed that SRCR3 domain of CD163 is implicated in the binding and
internalization of the Hp-Hb complexes (Kristiansen et al., 2001; Onofre
et al., 2009). Altogether these data support the notion that the CD163
variant lacking the peptide sequence encoded by exon 13 is structurally
and functionally intact.
Our infection experiments performed in PAMs isolated from the
genetically edited pigs and their WT comtemporaries showed that
removal of the first 12 amino acids of PSTII domain generated PAMs that
were completely resistant to both PRRSV-1 and PRRSV-2 infections. This
shows that a targeted removal of exon 13 is sufficient to achieve com­
plete resistance to PRRSV infection. These findings mimic the infection
results obtained with CD163 knockout pigs that were fully resistant to
infection with a type 1 and type 2 PRRSV isolates (Whitworth et al.,
2016; Xu et al., 2020; Yang et al., 2018). However, compared to CD163
knockout animals, we have demonstrated that a precise deletion of exon
13 of CD163 can successfully generated PRRSV genotype 1 and genotype
2 resistant pigs that express a CD163 variant on the surface of PAMs,
presumably in a native confirmation. In addition, we further showed
that ΔExon13 animals retain the ability to perform key biological
functions associated with CD163 expression, such as Hb-Hp uptake.
Therefore, we were able to use targeted genome editing tools to produce
swine resistant to viral infection, whilst retaining biological function of
the targeted gene.
Our in vivo experiments confirmed previous mutational analysis of
CD163 that showed that PSTII domain is critical for PRRSV infection
(Stoian et al., 2022a; Van Gorp et al., 2010b). However, the role of PSTII
domain in PRRSV infection remains unclear. One possibility is that PSTII
forms a physical interaction with the PRRSV virion, and the resistance to
infection is due to the loss of virus-receptor binding. However, our
previous binding experiments showed that CD163 lacking the PSTII
portion encoded by exon 13 has the ability to associate with individual
viral envelope glycoproteins (Stoian et al., 2022a). Furthermore,
removal of the peptide sequence encoded by exon 13 did not affect
PRRSV attachment and internalization, indicating that PRRSV can be
endocytosed properly in cells expressing the CD163 variant (Stoian
et al., 2022a). These previous observations suggested that negative ef­
fect on infection is not result of a loss of interaction between the domain
on the receptor and the virion. Another explanation is that deletion of
PSTII may disrupt the overall conformation of CD163, making viral re­
ceptor binding domains inaccessible to the virus. However, our in vivo
results suggest that the CD163 variant is in a native conformational
state, since the gene-edited pigs retain the biological activity of CD163
and its cell surface expression in macrophages. A third possibility is that
PSTII may function as a spacer domain, placing SRCR9 away from the
cell surface. The predicted structures in Fig. 4 revealed a change in the
orientation of SRCR9 relative to the cell surface caused by the deletion of
the exon 13 decapeptide. Previous studies showed that PRRSV colo­
calized with CD163 in the early endosomes after viral attachment and
internalization, suggesting that the viral uncoating occurs in these
intracellular organelles (Van Gorp et al., 2009; Yu et al., 2020). One
possible explanation for the resistance of the ΔExon13 pigs to PRRSV
infection, is that the change in the orientation of SRCR9 induced by
removing a significant portion of PSTII might increase the affinity of the
viral receptor for PRRSV, trapping the virions in the early endosomes.
This would block virions uncoating and inhibit the genome release into
the cytoplasm. Subsequently virions trapped in the early endosomes
might be transported to the later step of endocytic pathway, and finally
be degraded in the lysosomes. Further experiments are needed to clarify
the exact function of PSTII on PRRSV infection.
Previous studies support a “Single Domain” model for the interaction
between PRRSV and CD163, in which the SRCR5 is the principal contact
10
Antiviral Research 221 (2024) 105793
between CD163 and PRRSV envelope proteins (Su et al., 2021). This
model is further supported by previous neutralization experiments that
showed that an anti-CD163 SRCR5-specific antibody can prevent PRRSV
infection (Van Gorp et al., 2010b). However, our recent binding ex­
periments showed that deletion of SRCR5 or PSTII domain, both critical
for viral infection, did not disrupt the ability of CD163 to interact with
the viral envelope glycoproteins (Stoian et al., 2022a). These observa­
tions suggest that the PRRSV glycoproteins may interact with multiple
peptide sequences on the CD163 molecule, including the SRCR5
domain, PSTII, SRCR7-9 domains and other specific sequences impor­
tant for PRRSV infection (Stoian et al., 2022a). In contrast to the “Single
Domain” model, our previous results suggested an alternative, “Multi-­
Domain” model for describing the interaction between PRRSV and
CD163. This hypothesis is supported by the results presented here that
show PRRSV resistance in ΔExon13-edited pigs. In this model, different
regions of CD163 receptor can bind similar viral epitopes, suggesting
that CD163 has multiple binding sites that are critical for recognition by
the viral envelope glycoproteins. In this model, the removal of any
domain implicated in the interaction with PRRSV is sufficient to inhibit
infection. Interestingly, our structural predictions for CD163 showed
that the different CD163 domains essential for PRRSV infection are
located in close proximity within the 3-D model structure (See Fig. 4),
which agrees with the recent crystal structure for porcine CD163
SRCR5-9 recently deposited in the RSCB Protein Data Bank (PDB
accession number: 8H7J). This suggest that the interaction of PRRSV
with CD163 occurs in a specific region of the protein in which all the
domains important for viral infection are present, presumably forming
multiple interactions with the viral envelope glycoproteins (Fig. 4). In
agreement with the idea that a receptor can contain multiple interaction
sites is the existence of two binding sites for the receptor on the polio­
virus type 1 capsid (McDermott et al., 2000). In addition, the finding of
two types of receptor-interaction sites for rhinovirus type 3 has been
reported (Casasnovas et al., 1998; Casasnovas and Springer, 1995). Our
previous binding experiments do not rule out the possibility that CD163
is indirectly interacting with the viral envelope glycoproteins and that
other cell proteins are implicated in the CD163-viral glycoprotein as­
sociation (Stoian et al., 2022a). Thus, we cannot strictly rule out the
conclusion that the CD163 deletions do not interfere with the in­
teractions between CD163 and the viral envelope glycoproteins.
Therefore, more evidence is required to evaluate whether it exists a
direct interaction between the receptor domains/regions and the viral
glycoproteins. Another possibility is that the CD163 regions/domains,
that are required for infection have other cellular binding partners that
are involved in PRRSV infection. For example calpain 1, which forms an
association with CD163 was reported as a novel cellular protein impli­
cated in PRRSV infection (Yu et al., 2020). Hence, it will be important to
evaluate whether other cellular factors are also involved in PRRSV
infection though their interaction with different CD163 regions essential
for viral infection.
In conclusion, this study demonstrates that it is possible to generate a
CD163 variant that is resistant to viral infection, but structurally and
functionally intact. Incorporation of ΔExon13 animals in pig breeding
could significantly reduce the economic losses associated with PRRSV
infection, improve animal welfare, and develop preventative cures for
one of the most serious diseases in the global swine industry. For pork
industry applications, the CD163 modification presented here will need
to be comprehensively characterized for subtle phenotypes related to
feed efficiency, growth performance and susceptibility of CD163-modi­
fied pigs to infection by other pathogens.
CRediT authorship contribution statement
Brianna Salgado: Conceptualization, Formal analysis, Investiga­
tion, Methodology, Data curation. Rafael Bautista Rivas: Conceptual­
ization, Data curation, Formal analysis, Methodology, Writing – original
draft. Derek Pinto: Data curation, Formal analysis, Investigation,
B. Salgado et al.
Methodology. Tad S. Sonstegard: Conceptualization, Data curation,
Formal analysis, Investigation, Methodology. Daniel F. Carlson:
Conceptualization, Data curation, Formal analysis, Investigation,
Methodology. Kyra Martins: Conceptualization, Data curation, Formal
analysis, Investigation, Methodology. Jonathan R. Bostrom: Concep­
tualization, Data curation, Formal analysis, Investigation, Methodology.
Yamlak Sinebo: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology. Raymond R.R. Rowland: Conceptualiza­
tion, Data curation, Formal analysis, Funding acquisition, Investigation,
Methodology, Project administration, Resources, Supervision, Valida­
tion, Writing – review & editing. Alberto Brandariz-Nuñez: Concep­
tualization, Data curation, Formal analysis, Funding acquisition,
Investigation, Methodology, Project administration, Resources, Super­
vision, Validation, Visualization, Writing – original draft, Writing – re­
view & editing.
Declaration of competing interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
The work is in support of an ongoing U.S. patent application.
Data availability
No data was used for the research described in the article.
Acknowledgments
This study was supported by USDA NIFA grant (no. 2023-67015-
40731). We are grateful for the assistance of Lorraine Keller in the flow
cytometry analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.antiviral.2024.105793.
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