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\begin{document}

\title{Characterization and cross-protection of experimental infections with
SeCoV and two PEDV variants.}



\author[1]{Héctor Puente}%
\author[2]{Ivan Díaz}%
\author[1]{Hector Arguello }%
\author[1]{Óscar Mencía-Ares}%
\author[1]{Manuel Gómez-García}%
\author[1]{Lucía Pérez-Perez}%
\author[1]{Clara Vega}%
\author[3]{Marti Cortey}%
\author[3]{Marga Martín}%
\author[1]{Pedro Rubio}%
\author[1]{Ana Carvajal}%
\affil[1]{Universidad de Leon Departamento de Sanidad Animal}%
\affil[2]{IRTA Centre de Recerca en Sanitat Animal (CReSA IRTA-UAB) Universitat Autònoma de Barcelona Bellaterra Spain}%
\affil[3]{Universitat Autonoma de Barcelona Facultat de Veterinaria}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Global emergence and re-emergence of \emph{Porcine epidemic diarrhea
virus} (PEDV), an Alphacoronavirus which causes a highly contagious
enteric disease, have led to several studies addressing its variability.
The aim of this study was to characterize the infection of weaned pigs
with \emph{Swine enteric coronavirus} (SeCoV) -a chimeric virus most
likely originated from a recombination event between PEDV and
\emph{Transmissible gastroenteritis virus,} or its mutant \emph{Porcine
respiratory coronavirus-}, and two PEDV G1b variants, including a
recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to
determine the degree of cross-protection achieved against the
rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were
randomly allocated into four groups of 12 animals; piglets in groups B,
C and D were orally inoculated with a PEDV variant (B and D) or SeCoV
(C), while piglets in group A were mock inoculated and maintained as
controls. At day 20 post-infection all groups were exposed to
rPEDV-SeCoV; thus, group D was subjected to a homologous re-challenge,
groups B and C to a heterologous re-challenge (PEDV/rPEDV-SeCoV and
SeCoV/rPEDV-SeCoV, respectively) and group A was primary challenged
(-/rPEDV-SeCoV). Clinical signs, viral shedding, microscopic lesions and
specific humoral and cellular immune responses (IgG, IgA, neutralizing
antibodies and IgA and IFN-\selectlanguage{greek}γ-\selectlanguage{english}secreting cells) were monitored. After
primo-infection all three viral strains induced an undistinguishable
mild-to-moderate clinical disease with diarrhea as the main sign and
villus shortening lesions in the small intestine. In homologous
re-challenged pigs, no clinical signs or lesions were observed, and
viral shedding was only detected in a single animal. This fact may be
explained by the significant high level of rPEDV-SeCoV-specific
neutralizing antibodies found in these pigs before the challenge. In
contrast, prior exposition to a different PEDV G1b variant or SeCoV only
provided partial cross-protection, allowing rPEDV-SeCoV replication and
shedding in feces.%
\end{abstract}%



\sloppy


\emph{Characterization and cross-protection of experimental infections
with SeCoV and two PEDV variants.}

\textbf{Authors}

H\selectlanguage{ngerman}éctor Puente\textsuperscript{1}, Ivan Díaz\textsuperscript{2}, Héctor
Arguello\textsuperscript{1}, Óscar Mencía-Ares\textsuperscript{1},
Manuel Gómez-García\textsuperscript{1}, Lucía
Pérez-Perez\textsuperscript{1}, Clara Vega\textsuperscript{1}, Martí
Cortey\textsuperscript{3}, Margarita Martín\textsuperscript{3}, Pedro
Rubio\textsuperscript{1}, Ana Carvajal\textsuperscript{1}

\textbf{Affiliations}

\textsuperscript{1}Departamento de Sanidad Animal, Facultad de
Veterinaria, Universidad de León, León, Spain.

\textsuperscript{2}IRTA, Centre de Recerca en Sanitat Animal (CReSA,
IRTA-UAB), Universitat Autònoma de Barcelona, Bellaterra, Spain.

\textsuperscript{3}Departament de Sanitat i Anatomia Animals, Facultat
de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Spain.

\textbf{Correspondence}

Héctor Puente, Departamento de Sanidad Animal, Facultad de Veterinaria,
Universidad de León, 24071 León, Spain. Email: hpuef@unileon.es. Phone
number: +34 987291306.



\textbf{Abstract}

Global emergence and re-emergence of \emph{Porcine epidemic diarrhea
virus} (PEDV), an Alphacoronavirus which causes a highly contagious
enteric disease, have led to several studies addressing its variability.
The aim of this study was to characterize the infection of weaned pigs
with \emph{Swine enteric coronavirus} (SeCoV) -a chimeric virus most
likely originated from a recombination event between PEDV
and\emph{Transmissible gastroenteritis virus,} or its mutant
\emph{Porcine respiratory coronavirus-} , and two PEDV G1b variants,
including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as
well as to determine the degree of cross-protection achieved against the
rPEDV-SeCoV.

For this purpose, forty-eight 4-week-old weaned pigs were randomly
allocated into four groups of 12 animals; piglets in groups B, C and D
were orally inoculated with a PEDV variant (B and D) or SeCoV (C), while
piglets in group A were mock inoculated and maintained as controls. At
day 20 post-infection all groups were exposed to rPEDV-SeCoV; thus,
group D was subjected to a homologous re-challenge, groups B and C to a
heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV,
respectively) and group A was primary challenged (-/rPEDV-SeCoV).
Clinical signs, viral shedding, microscopic lesions and specific humoral
and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA
and IFN-\selectlanguage{greek}γ-\selectlanguage{english}secreting cells) were monitored.

After primo-infection all three viral strains induced an
undistinguishable mild-to-moderate clinical disease with diarrhea as the
main sign and villus shortening lesions in the small intestine. In
homologous re-challenged pigs, no clinical signs or lesions were
observed, and viral shedding was only detected in a single animal. This
fact may be explained by the significant high level of
rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before
the challenge. In contrast, prior exposition to a different PEDV G1b
variant or SeCoV only provided partial cross-protection, allowing
rPEDV-SeCoV replication and shedding in feces.

\textbf{Keywords}

Porcine epidemic diarrhea virus, swine enteric coronavirus, swine
coronavirus, recombinant, immunity

\textbf{Introduction}

\emph{Porcine epidemic diarrhea virus} (PEDV) is an enveloped,
single-stranded, positive-sense RNA virus belonging to the
order\emph{Nidovirales} , family \emph{Coronaviridae} ,
subfamily\emph{Coronavirinae} , and genus \emph{Alphacoronavirus}
(Lefkowitz et al., 2017). It is the etiological agent of a highly
contagious disease known as porcine epidemic diarrhea (PED),
characterized by watery diarrhea and vomiting due to enterocyte
destruction and villous atrophy, causing up to 80-90\% mortality in
neonatal piglets (Saif, Wang, Vlasova, Jung, \& Xiao, 2019). The disease
was initially described in Europe in the 1970s (Pensaert \& de Bouck,
1978) and spread throughout Europe and Asia. Its incidence decreased
markedly in the nineties and subsequent years in Europe, while in Asia
the virus remains as a major cause of diarrhea (Carvajal et al., 2015).
In 2013, PEDV emerged in America unleashing a major epidemic that caused
substantial economic losses (Schulz \& Tonsor, 2015). Soon after,
re-emerged in Europe (Antas \& Wo\selectlanguage{polish}ź\selectlanguage{english}niakowski, 2019), becoming a major
concern for swine industry worldwide.

Two PEDV genogroups, named G1 or INDEL and G2 or non-INDEL, are
recognized based on insertions-deletions in the S1 subunit of the spike
(S) gene. Both genogroups show differences in virulence and
transmissibility (Chen et al., 2016; Gallien et al., 2018), leading to
intensive research to better understand the prevailing PEDVs in
different countries and regions. Whole genome or S gene sequencing of
isolates recovered from different European farms demonstrated that all
recent European PEDV strains are G1b (Boniotti et al., 2016; Grasland et
al., 2015; Hanke et al., 2015; Mesquita et al., 2015; Puente et al.,
2021; Theuns et al., 2015), with the only exception of a G2b isolate
from Ukraine (Dastjerdi, Carr, Ellis, Steinbach, \& Williamson, 2015).
In contrast, both genogroups have been detected on infected farms in
Asia and America (Lin et al., 2016).

In addition, a chimeric virus known as \emph{Swine enteric
coronavirus}(SeCoV) has been described across Europe (Akimkin et al.,
2016; Belsham et al., 2016; Boniotti et al., 2016; de Nova et al.,
2020). This recombinant, which causes a PED-like disease, has the S gene
from PEDV and the backbone from\emph{Transmissible gastroenteritis
virus} (TGEV) or \emph{Porcine respiratory coronavirus} (PRCV) (Belsham
et al., 2016; Boniotti et al., 2016). In Europe, SeCoV has not been
recently detected, although different PEDV variants within the G1b
genogroup have been associated with diarrhea outbreaks. Among them, a
recombinant PEDV-SeCoV (rPEDV-SeCoV) resulting from the substitution of
a ~400 nt fragment at the 5' end of the S gene with SeCoV (de Nova et
al., 2020; Valk\selectlanguage{ngerman}ó et al., 2017) is reported frequently in large producing
countries such as Spain (Puente et al., 2021) or Poland (Antas, Olech,
\& Szczotka-Bochniarz, 2021).

The aim of the present study was to characterize the infection of weaned
pigs by SeCoV and two variants of PEDV G1b, including rPEDV-SeCoV, as
well as to determine the degree of cross-protection provided in a
re-infection with rPEDV-SeCoV.

\textbf{Materials and methods}

Ethical issues

All experiments involving pigs were done under the approval of the
University of León Committee on Animal Care and Supply
(OEBA-ULE-006-2019 and OEBA-ULE-013-2020). Pigs were handled by
veterinarians and trained personnel who fulfilled the Spanish and
European Union requirements. Animals were clinically examined upon
arrival and monitored throughout the experiments.

Experimental design

Forty-eight commercial three-week-old weaned female pigs were purchased
from a PEDV-free farrow-to-wean herd. On arrival, all pigs were
confirmed to be free of PEDV, TGEV, SeCoV, \emph{Porcine
deltacoronavirus} , Rotavirus (A, B, C and H) and several
enteropathogenic bacteria (\emph{Salmonella} spp., \emph{Lawsonia
intracellularis} , enterotoxigenic \emph{E. coli} and
\emph{Brachyspira}spp.). In addition, all pigs were seronegative to PEDV
(INgezim PEDV ELISA, INGENASA). Pigs were randomly distributed into four
groups (12 animals each) and housed in separated rooms of a biosafety
level 2 animal facility. Animals were housed in a single solid floor pen
with straw bedding and fed \emph{ad libitum} with an antibiotic-free
diet. Room temperature was set at 26ºC.

After a week of acclimation, the study was carried out in two stages.
During the first stage, piglets in groups B, C and D were orally
inoculated -day post-inoculation (dpi) 0\selectlanguage{english}-, using a gastric cannula, with
3 mL of a viral inoculum
(10\textsuperscript{6}TCID\textsubscript{50}/mL) of PEDV G1b (strain
2330-Orense), SeCoV (strain 1480-Murcia-Lorca) or rPEDV-SeCoV (strain
1931-1-Valladolid-Molpeceres), respectively (Table 1). Animals in group
A were mock infected on the same day with phosphate buffered saline
solution (PBS). The second stage started twenty days later (dpi 20); all
groups were orally inoculated with 3 mL of the viral inoculum containing
rPEDV-SeCoV at 10\textsuperscript{6} TCID\textsubscript{50}/mL as
described above. Hence, during this stage group D was subjected to a
homologous re-challenge, groups B and C to a heterologous re-challenge
(PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV) and group A was primary
challenged.

Viral inoculums

Each inoculum was obtained from two three-day-old piglets that were
intragastrically inoculated with 3 mL of viral positive feces (PEDV G1b
2330-Orense, SeCoV 1480-Murcia-Lorca and rPEDV-SeCoV
1931-1-Valladolid-Molpeceres) collected from diarrheic pigs on infected
farms in Spain. After 48 hours, piglets developed severe diarrhea and
were euthanized. Small intestinal content and mucosal scrapings were
collected, diluted 1/5 in PBS, filtered through a 0.22 \selectlanguage{greek}μ\selectlanguage{english}m syringe filter
(GE Healthcare), tested by qPCR for viral quantification and
subsequently stored at -80\selectlanguage{ngerman}°C. Also, the suspensions were confirmed as
PCR negative for porcine coronavirus (PEDV, TGEV, SeCoV and PDCoV,
excluding the virus corresponding to each of the inoculums) and
Rotavirus (A, B, C and H) before being used.

Clinical monitoring and sample collection

Table 1 summarizes the animal clinical monitoring and sampling strategy.
Weight and rectal temperatures were daily measured. Clinical signs were
scored considering four relevant parameters: (a) fecal consistency (0 =
normal feces, 1 = soft stools, 2 = watery diarrhea); (b) general
condition (0 = normal, 1 = slightly depressed, 2 = depressed, 3 =
lethargic); (c) appetite (0 = hungry, 1 = partial anorexia, 2 = total
anorexia); (d) vomiting (0 = no, 1 = yes). Using these clinical scores,
a maximum value of 8 could be assigned to an individual pig on a single
day. Fecal samples were collected from all piglets at dpi 0-7, 9, 11,
13, 15, 17 and 19 as well as at dpi 20-26 -days post-re-inoculation
(dpri 0-6). Serum samples were collected weekly during the first stage
of the experiment and each 3 days during the second stage. To obtain
peripheral blood mononuclear cells (PBMC), blood samples were collected
using lithium heparin tubes immediately before re-inoculation (dpi 20)
and three days later. Finally, three animals from each group were
randomly selected and euthanized at dpi 3, 6, 23 (dpri 3) and 26 (dpri
6). Duodenum, mid jejunum, and ileum were collected at necropsy and
immediately fixed in formalin for further histological evaluation.

Virus isolation

The rPEDV-SeCoV isolate was propagated in cell culture as previously
described (Díaz et al., 2021) and used in viral neutralization test
(VNT) and ELISPOT. Briefly, a confluent monolayer of VERO cells (ATCC
CCL-81) was inoculated with a clarified and trypsin-treated (10 \selectlanguage{greek}µ\selectlanguage{english}g/mL of
Trypsin 1:250, Gibco) suspension of viral inoculum (small intestinal
content and mucosal scrapings from infected three-days-old piglets).
After 2 hours of adsorption at 37\selectlanguage{ngerman}°C, 5 mL of freshly prepared medium
including trypsin was added. After being cultured for 3 days at 37ºC and
5\% CO\textsubscript{2}, cytopathic effect characterized by round
syncytia was observed, and cultures were frozen and thawed to recover
the virus. A single virus stock was used for the immunological analysis
(4.5 log\textsubscript{10} TCID\textsubscript{50}/mL, passage 3).

Sequence analysis

Viral inoculums used for experimental challenge, together with
cell-culture adapted rPEDV-SeCoV isolate, and qPCR positive fecal
samples yielding Ct \textless{} 20 (\emph{n} = 15) were sequenced by
next generation sequencing (Cortey et al., 2019). The amino acid
sequences in neutralizing B-cell epitopes described by Okda et al.
(2017) and Kong et al. (2020) were visualized using BioEdit 7.2.5.
Strain CO13 (GenBank accession number KF272920) was used as reference
(Okda et al., 2017).

Quantification of PEDV and SeCoV in fecal samples

Feces were diluted 1:2 in sterile PBS, homogenized by vortex mixing and
centrifuged for 10 min at 20,000 g. The RNA was extracted from 140 \selectlanguage{greek}μ\selectlanguage{english}l of
the supernatant using QIAMP Viral RNA Mini Kit (QIAGEN), following the
manufacturer's instructions. RT-qPCRs with the primers and probes
targeting the M protein gene of PEDV described by Zhou et al. (2017),
and the N protein gene of TGEV described by Masuda et al. (2016) were
used for quantification of PEDV and SeCoV, respectively. Both RT-qPCRs
were carried out using a PrimeScript TM RT-PCR Kit (TAKARA) and
following the manufacturer's recommendations in a QuantStudio 1 thermal
cycler (Applied Biosystems). Cycling conditions were as follows: reverse
transcription at 42\selectlanguage{ngerman}°C for 5 min, inactivation at 95°C for 10 s, followed
by 40 cycles of denaturing at 95°C for 5 s and annealing and extension
at 60°C for 35 s. Each RNA sample was analyzed in duplicate.

Ct values were converted into viral titers using a standard curve
generated with samples of known PEDV concentration
(TCID\textsubscript{50}/mL). Thus, results were expressed as equivalent
TCID\textsubscript{50}/mL or the corresponding adjusted
TCID\textsubscript{50}/g.

Histology

Tissue samples fixed 48 h in 10\% formalin were dehydrated, embedded,
sectioned (4 \selectlanguage{greek}µ\selectlanguage{english}m thick), mounted onto glass slides and stained with
hematoxylin-eosin. To measure villous length and crypt-depth of
duodenum, mid jejunum and ileum, three sections of each tissue were
blindly evaluated by a veterinary pathologist using a computerized image
system (Leica LAS EZ 3.4 digital imaging software).

Specific PEDV IgG and IgA

Kinetics of specific-PEDV IgG in sera were determined using a
commercially available ELISA based on the S glycoprotein (Ingezim PEDV,
INGENASA). Results were expressed as sample/positive ratio (S/P).

The same commercial kit was used to measure specific-PEDV IgA as
previously described (D\selectlanguage{ngerman}íaz et al., 2021), substituting the anti-pig IgG
conjugate by a goat anti-pig IgA HRP conjugate (Bethyl Laboratories).
Results were expressed as optical densities (ODs).

Viral neutralization test (VNT)

Neutralizing antibodies (NA) were evaluated as described by Thomas et
al. (2015), with minor modifications (Díaz et al., 2021). Mixtures (1:1)
of the cell-culture adapted rPEDV-SeCoV containing 200
TCID\textsubscript{50} and serum (dilutions 1:4 to 1:256) were
inoculated onto confluent monolayers of Vero cells. Negative controls
(mock-infected), viral infection controls (200 TCID\textsubscript{50} of
rPEDV-SeCoV) and positive controls (200 TCID\textsubscript{50} of
rPEDV-SeCoV plus positive sera) were included on each set of plates.
Plates were read after 48 h of incubation by staining with a FITC
labelled anti-PEDV monoclonal antibody (SD-1F-1 8D6-29PED-NP, Medgene
Labs) (1:200). Titres were calculated as the reciprocal of the highest
dilution resulting in \selectlanguage{english}[?] 90\% reduction of fluorescent foci compared to
viral infection controls. As previously proposed, NA titres below 8 were
not considered significant (Thomas et al., 2015).

IgA and IFN-\selectlanguage{greek}γ \selectlanguage{english}ELISPOT

rPEDV-SeCoV-specific IgA-secreting cells (SC) were measured by means of
a commercial ELISPOT kit (Pig IgA single-color ELISPOT, CTL), as
previously described (D\selectlanguage{ngerman}íaz et al., 2021; Jahnmatz et al., 2013), while
rPEDV-SeCoV-specific IFN-\selectlanguage{greek}γ-\selectlanguage{english}SC were measured using a tailor-made IFN-\selectlanguage{greek}γ
\selectlanguage{english}ELISPOT (D\selectlanguage{ngerman}íaz et al., 2021). PBMC were recovered from blood samples as
described by Diaz et al. (2021), mock-stimulated or stimulated with
rPEDV-SeCoV at a multiplicity of infection (moi) of 0.01. All tests were
run in duplicate. Results were expressed as responding cells (counts of
spots in stimulated cells minus counts of spots in unstimulated
ones)/10\textsuperscript{6} PBMC.

2.12. Statistical analysis

Proportions of diarrheic pigs and PEDV positive pigs were compared among
groups using the \selectlanguage{greek}χ\selectlanguage{english}2 test (Fisher's exact test). Numerical data were
tested for normality (Kolmogorov-Smirnov test) and statistical
differences among groups were evaluated using either ANOVA or
Kruskal-Wallis test (Connover-Imman method for multiple comparisons).
Friedman test was used for comparisons inside the same group. The area
under the curve (AUC) for viral shedding in feces was calculated using
the trapezoidal approach (Sch\selectlanguage{ngerman}äfer et al., 2001). The analyses were
carried out with IBM SPSS Statistics v26 and StatsDirect v 2.7.7 at the
5\% significance level.

\textbf{Results}

Clinical assessment

No significant differences were found in daily rectal temperatures among
groups. During the first stage (primo-infection), the highest clinical
scores were recorded in challenged groups (B, C and D) between dpi 2 and
5 (Figure 1). Significant differences were observed when the three
challenged groups were compared with the control (p\textless{}0.05), but
not when compared among them (Figure 1). Diarrhea was the main clinical
sign and the highest score or liquid diarrhea was recorded in 66.6\% of
the piglets of group B and 83.3\% of groups C and D (Appendix Figure 1).

In the second stage of the experiment, clinical scores were
significantly higher in group A (primo-infection) as compared with group
D (homologous re-challenge) -between dpri 2 and 5-, group B
(heterologous re-challenge PEDV/rPEDV-SeCoV) -between dpri 2 and 4-, and
group C (heterologous re-challenge SeCoV/rPEDV-SeCoV) -only in dpri 3-
(p\textless{}0.05) (Figure 1). No differences were observed when
clinical and diarrhea scores were compared among groups B, C and D.

Average daily gain (ADG) during the first week post-infection was
significantly lower in groups B, C and D (p\textless{}0.05) compared to
control group (A) (Appendix Table 1). In the second stage of the
experiment, ADG in group A was significantly lower than in group B and D
(p\textless{}0.05). We also observed that differences in ADG between
groups C and D and groups C and A were close to statistical significance
(p=0.059 and p=0.076, respectively).

Quantification of PEDV and SeCoV in fecal samples

Results of viral detection and quantification, as well as statistical
comparisons among groups, are shown in Figure 2. None of the
mock-inoculated pigs (group A) shed PEDV or SeCoV RNA in their feces
during the first stage of the experiment, while all pigs in the
challenged groups shed virus in their feces (Figure 2A). Maximum
shedding was reached at dpi 2 in group B (5.9
log\textsubscript{10}TCID\textsubscript{50}/g), dpi 3 in group C (6.7
log\textsubscript{10}TCID\textsubscript{50}/g) and dpi 5 in group D (5.7
log\textsubscript{10} TCID\textsubscript{50}/g) (Figure 2B). After
peaking, viral shedding in feces was progressively reduced. However, in
group C, challenged with SeCoV, a second shedding wave started at dpi 9
and extended until dpi 17. Accordingly, viral shedding measured as AUC
was significantly higher for group C from dpi 0 to dpi 20 (C
\textgreater{} B and D; p\textless{}0.05) (Figure 2C).

In the second stage of the experiment, rPEDV-SeCoV RNA was detected from
dpri 2 in all pigs of group A, reaching a maximum of 6.6
log\textsubscript{10} TCID\textsubscript{50}/g on dpri 3. On the
contrary, only one piglet of group D (homologous re-challenge) shed
virus for two consecutive days (dpri 2 and 3). Between dpri 2 and 6,
rPEDV-SeCoV was detected in 66.7\% of the piglets from group B (up to
2.2 log\textsubscript{10} TCID\textsubscript{50}/g) and 100\% from group
C (up to 4.6 log\textsubscript{10} TCID\textsubscript{50}/g). AUC was
significantly lower for group D (homologous re-challenge) as compared
with groups C (heterologous re-challenge SeCoV/rPEDV-SeCoV) and A
(primo-infection).

Sequence comparison

A total of 25 RT-qPCR positive fecal samples (Ct\textless{} 20)
recovered throughout the experiment (n=4, n=7, n=8 and n=6 for groups A,
B, C and D, respectively) were sequenced, together with the cell-culture
adapted virus used for the immunological assays. Whole genome nucleotide
identity was higher than 99.6\% compared to the original inoculum for
all samples.

Among the five known neutralizing B-cell epitopes described by Okda et
al. (2017), 14 changes were observed between rPEDV-SeCoV and SeCoV,
while only 3 were observed when PEDV was compared to rPEDV-SeCoV (Table
2).

Histopathology and morphometry

Microscopic lesions consisting of shorted and fused villi were observed
in all challenged animals euthanized in the first stage of the
experiment, particularly in the duodenum and mid jejunum at dpi 3
(Figure 3). During the second stage, piglets of group A showed more
evident microscopic lesions, followed by group C.

Mean villous height to crypt-depth ratios for each intestinal segment
and group were compared (Table 3). At dpi 3, piglets in groups B, C and
D showed lower ratios than mock-infected animals for all small intestine
segments. During the second stage of the study (dpri 3 and 6), villous
shortening was more evident in primo-infected pigs (group A), which
showed a significant reduction in these ratios, compared to groups B and
D (heterologous and homologous challenge, respectively). A reduction of
villous height to crypt-depth ratio was also evident in duodenum and mid
jejunum in group C, although significant differences with groups A or D
were not observed.

Detection of specific IgGs and IgAs

No IgG antibodies against PEDV S glycoprotein were detected in any of
the pigs at dpi 0. Mock-infected pigs (group A) remained negative during
the first stage of the experiment. At dpi 6, one piglet in group C
(11\%) and two from group D (22\%) seroconverted (Figure 4A), while at
dpi 13, the percentage of seropositive piglets increased to 83\% in
groups B and C (5 out of 6) and 100\% in group D (6 out of 6). In the
second stage of the experiment, 2 out of 3 piglets in group A (66.7\%)
were seropositive at dpri 6. Once seroconverted, all piglets remained
positive by ELISA during the remaining days of the study.

IgG kinetics based on mean S/P ratios are shown in Figure 4B. An
increase was observed in groups B, C and D when comparing S/P ratio
before (dpi 20) and after (dpri 6) re-challenge (booster effect), with
no statistical differences.

IgA kinetics based on mean OD values are shown in Figure 4C. A
significant booster effect was observed in groups B and C
(p\textless{}0.05), when results obtained before (dpi 20) and after
(dpri 6) heterologous challenge were compared. In contrast no booster
effect was observed in group D (homologous challenge).

Detection of specific neutralizing antibodies (NA)

NA against rPEDV-SeCoV were detected in all challenged pigs (groups B, C
and D) at dpi 20, reaching group D the highest values (D \textgreater{}
B and C; p\textless{}0.05) (Table 4). NA dropped in all infected groups
after re-challenge. Thus, at dpri 3 only two animals in groups B and C
(33.3\%) and five in group D (83.3\%) showed NA titers \selectlanguage{english}[?] 8. Finally, NA
increased again at dpri 6 in all re-challenged groups, being all animals
positive.

IgA and IFN-\selectlanguage{greek}γ \selectlanguage{english}ELISPOT

Mean numbers of specific-rPEDV-SeCoV IgA-SC were significantly higher in
challenged pigs when compared to group A at dpi 20 (p\textless{}0.05)
(Table 4). Moreover, a significant booster was observed at dpri 3 in
groups B, C and D (p\textless{}0.05), without differences among them.

Also, mean numbers of specific-rPEDV-SeCoV IFN-\selectlanguage{greek}γ-\selectlanguage{english}SC were higher in
groups B, C and D when compared to group A at dpi 20 and dpri 3
(p\textless{}0.05) (Table 4). Moreover, group D showed higher values for
both time points when compared with groups B and C (p\textless{}0.05).
Again, a significant booster was observed at dpri 3 for groups B, C and
D (p\textless{}0.05).

\textbf{Discussion}

PEDV genetic variability through mutations and recombinations has been
demonstrated (Wang et al., 2019). Also, clinical and epidemiological
differences, in terms of virulence and transmissibility, have been
described among PEDV G1b and G2b strains (Chen et al., 2016; Gallien et
al., 2018). Nonetheless, potential differences in clinical signs, viral
shedding, lesions, or intensity of the induced immunity by different
variants of PEDV G1b or SeCoV, a PEDV/TGEV chimeric virus, have not been
well characterized. In this sense, although SeCoV has been identified in
pig fecal samples from several European countries (Akimkin et al., 2016;
Belsham et al., 2016; Boniotti et al., 2016; de Nova et al., 2020), its
virulence is not yet experimentally assessed. The present study is the
first comparative characterization of two PEDV G1b experimental
infections, including a rPEDV-SeCoV isolate that has recently reported
as predominant in Europe (Antas et al., 2021; Puente et al., 2021), plus
a SeCoV strain. Cross-protection provided by these PEDV variants or
SeCoV against the challenge with the rPEDV-SeCoV strain was also
investigated.

In agreement with previous reports in weaned pigs infected by PEDV G1b
(D\selectlanguage{ngerman}íaz et al., 2021; Gallien et al., 2018) or G2b (Crawford et al., 2015;
Gerber et al., 2016; Jung, Annamalai, Lu, \& Saif, 2015; Krishna et al.,
2020; Madson et al., 2014), the clinical disease induced in
primo-infected animals was mild-to-moderate. This fact was probably
associated to the already described age-dependent disease severity
(Carvajal et al., 2015; Stevenson et al., 2013), irrespective of the
high dose used for the challenge. Despite signs were not severe, the
infection clearly impacted animal growth as observed by weight daily
gain during the first week, as previously described in pigs exposed to
both PEDV genogroups (Gallien et al., 2018; Madson et al., 2014).
Clinical course was indistinguishable among all viruses, although a
slightly prolonged duration of clinical illness was observed in SeCoV
infected piglets that were already affected at dpi 1 and showed diarrhea
until dpi 11.

Although extended viral shedding, up to 42 days, has been described in
weaned pigs exposed to PEDV (Crawford et al., 2015; Díaz et al., 2021;
Gallien et al., 2018), the design of our experiment did not allow to
monitor prolonged shedding. However, viral shedding was still detected
in a single animal (16.6\%) from both PEDV exposed groups at dpi 15. At
that time both pigs were asymptomatic, fact that could facilitate the
maintenance and transmission of the infection on swine farms. This fact
was even more obvious in SeCoV infected animals, which showed a clear
viral shedding reactivation, with all pigs positive in feces at dpi 13
and 15. A similar shedding profile was described in PEDV infected
piglets challenged at 3-4 days of age (Lin et al., 2015) or at weaning
(Madson et al., 2014; Thomas et al., 2015), which has been associated
with PEDV replication in new regenerated enterocytes (Lin et al., 2015).
Both maximal and total shedding load (AUC) were higher in SeCoV compared
to PEDV infected pigs. This result suggests an increased ability of this
chimeric virus for replication in the enterocytes of the intestinal
villi, compared to PEDV. Further studies based on immunohistochemistry
assays are required to elucidate the differences in intestinal PEDV and
SeCoV replication.

Microscopic lesions characterized by villous atrophy and fusion were
also identical among the three infected groups. Villus height to crypt
depth ratio was used to evaluate the degree of microscopic lesions as
previously described (Jung et al., 2015; Madson et al., 2014; Thomas et
al., 2015). Although this ratio can vary depending on several factors
such as pig genetics or diet, usually it is about 3:1 in weaned piglets
(Moon, 1971). In our work, this ratio varied between 1.58 and 2.71 in
control pigs, but was significantly reduced in PEDV or SeCoV infected
pigs (range 0.88 to 1.19). To the best of our knowledge, this is the
first research which evaluates microscopic lesions in weaned pigs
exposed to PEDV G1b or SeCoV. Our results suggest that, both the
location within the small intestine and the degree of villi shortening,
are like previously reported lesions in weaned pigs exposed to PEDV G2b
isolates (Jung et al., 2015; Madson et al., 2014).

In the evaluation of the serological response, PEDV specific IgG and IgA
antibodies were detected in pigs exposed to SeCoV using a commercial
ELISA based on the S-protein, confirming that indirect diagnostic
methods based on this particular protein can lead to misidentification
of SeCoV, as it occurs with direct detection (de Nova et al., 2020).
According to previous reports (Lin et al., 2015; Thomas et al., 2015;
Gerber et al., 2016; Krishna et al., 2020; Díaz et al., 2021), specific
IgG antibodies were detected in most PEDV infected pigs by dpi 14, with
the highest increase in S/P ratio between dpi 7 and 14. SeCoV infected
pigs showed a more intense and slightly delayed specific IgG response,
reaching its maximum at dpi 20. Particularities of SeCoV infection which
could affect the time-lapse to specific IgG response establishment
should be further studied.

Despite the amino acid substitutions observed in known neutralizing
B-cell epitopes between SeCoV versus rPEDV-SeCoV or PEDV versus
rPEDV-SeCoV, it is worth noting that rPEDV-SeCoV-specific NA were
detected in 100 \% of the challenged pigs at dpi 20. As expected, mean
NA titer was significantly higher in rPEDV-SeCoV as compared to PEDV or
SeCoV infected pigs.

The degree and duration of cross-protection against subsequent
infections, particularly heterologous, are aspects of great practical
interest to design PEDV control strategies (Gerdts \& Zakhartchouk,
2017). Thus, full protection against disease and sterilizing immunity
have been reported in the short-term (few weeks after primo-infection),
for both homologous (Crawford et al., 2015; Gerber et al., 2016) and
heterologous PEDV challenges (Krishna et al., 2020). On the contrary,
only partial protection has been described in the long-term (Díaz et
al., 2021). In our study, as it would be expected, piglets subjected to
a homologous challenge three weeks after primo-infection did not show
any clinical signs or lesions. Also, only one animal shed a low amount
of virus (1.37 log\textsubscript{10} TCID\textsubscript{50}/g) in feces
on dpri 2 and 3. In contrast, heterologous PEDV challenge led to fecal
shedding in 66\% of the piglets for 4 days, with no relevant clinical
disease or lesions. Considering the minimal infectious dose proposed for
PEDV (Thomas et al., 2015), the PEDV titers observed in the heterologous
infected animals suggests their potential to transmit the infection. On
the contrary, the single animal shedding virus in the homologous
challenge, despite being positive, would not be considered infectious.
Finally, some degree of diarrhea, shortening of the villi and reduction
in daily weight gain were observed in the piglets primo-infected with
SeCoV, suggesting a lower level of protection. Among these piglets,
100\% shed rPEDV-SeCoV during heterologous challenge, although viral
titers were reduced up to 2 log\textsubscript{10} compared to
primo-infected animals (group A). Also, shedding titers were clearly
above the minimal infectious dose for PEDV. Altogether, our results
suggest that there is only a partial level of cross-protection, against
clinical disease and viral shedding, after heterologous infection. From
a practical point of view, recurrent PEDV infections in farms can occur,
even when the introduction takes place few weeks apart. This phenomenon
emphasizes the need to maintain high levels of external biosecurity on
swine farms.

Serum titers of IgG and IgA antibodies against PEDV S glycoprotein were
similar in the three exposed groups before second challenge. This result
suggests a lack of correlation with protection. In agreement with
previous studies (Gerber et al., 2016; Krishna et al., 2020), no
significant increase in serum PEDV-specific IgG, IgA and NA levels after
homologous short-term re-challenge were observed. On the contrary, Diaz
et al. (2021) demonstrated a strong serological anamnestic response
(IgG, IgA and NA) after homologous re-challenge 5 months apart. A high
titer of specific NA in the gut will probably be able to limit viral
replication and will not allow for a significant booster after a
short-term re-exposition (Krishna et al., 2020). In our study, minimal
viral shedding observed in a single piglet among those animals subjected
to homologous infection supports this hypothesis. Moreover, piglets
previously exposed to PEDV or SeCoV showed a significant lower NA
response at dpi 20. These animals had higher viral shedding after
re-infection and showed an anamnestic response for IgA and NA in serum.
A similar pattern was observed for rPEDV-SeCoV-specific IgA-SC, also in
the homologous challenge group, pointing to the presence of effector or
memory cells.

Concerning rPEDV-SeCoV-specific IFN\selectlanguage{greek}γ-\selectlanguage{english}SC, a significant booster was
detected in all groups, increasing 3-4 times after the homologous or
heterologous challenge. Before the challenge, the highest value was
observed in rPEDV-SeCoV. The leading role of NA regarding protection has
been stablished (D\selectlanguage{ngerman}íaz et al., 2021; Krishna et al., 2020), in agreement
with the results of this study. However, since the precise role of
cell-mediated immunity, measured as IFN\selectlanguage{greek}γ-\selectlanguage{english}SC, is not well known, it may
not be ruled out a certain involvement of cellular response in
protection.

To sum up, an experimental challenge of 4-week-old pigs with two PEDV
G1b variants and one SeCoV strain induced an undistinguishable
mild-to-moderate clinical disease, characterized by diarrhea and
microscopic lesions of shorter and fused villi. Viral shedding was
slightly higher on SeCoV infected pigs and exceeds clinical disease
recorded in the three viral strains tested. This could explain the
ability of these enteric coronaviruses to easily spread. Protection
against clinical disease and viral shedding after a short-term
re-challenge was strain-dependent, a fact which should be taken into
consideration when immunizing pigs against PEDV. Finally, great
diversity of PEDV isolates, together with this limited cross-protection,
makes necessary a continuous monitoring of novel PEDV variants that may
emerge locally or globally.

\textbf{Declaration of competing interest}

None of the authors of this study has a financial or personal
relationship with other people or organizations that could
inappropriately influence or bias the content of the paper.

\textbf{Ethics approval and consent to participate}

All procedures involving animals were approved by the institutional
bioethical committee (Reference Number OEBA-ULE-006-2019 and
OEBA-ULE-013-2020), and performed according to European regulations
regarding animal welfare and protection of animals used for experimental
and other scientific purposes.

\textbf{Acknowledgments}

This work was supported by the program of the National Institute of
Agricultural and Food Research and Technology (INIA project
E-RTA2015-0003-C02-01 and E-RTA2015-0003-C02-02) of Spanish Government.
H. Puente, O. Menc\selectlanguage{ngerman}ía-Ares, L. Pérez-Pérez, M. Cortey and H. Argüello
were supported by Spanish Government (FPU17/00466, FPU16/03485,
PRE2020-093762, RYC-2015-1715-4 and BEAGAL-18-106, respectively) and M.
Gómez-García by Junta de Castilla y León (LE131-18).

\textbf{Data availability statement}

Data are available in the GenBank database and by direct contact with
the correspondence author.

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, 36--41. https://doi.org/10.1016/j.mcp.2017.02.002

Table 1. Experimental design, clinical evaluation and sampling
throughout the experiment.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{1\textsuperscript{st} STAGE}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{1\textsuperscript{st} STAGE}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{2\textsuperscript{nd} STAGE}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{2\textsuperscript{nd} STAGE}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Days post-infection (dpi) 0-20\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Days post-infection (dpi) 0-20\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Days post-infection (dpi) 20-26 or days post-reinfection (dpri)
0-6\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Days post-infection (dpi) 20-26 or days post-reinfection (dpri)
0-6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4-weeks-old piglets (n=48)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Inoculation\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7-weeks-old piglets (n=24)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Inoculation with rPEDV-SeCoV\textsuperscript{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Group A (n=12)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Mock\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Group A (n=6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
First challenge\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Group B (n=12)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
PEDV G1b\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Group B (n=6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Heterologous re-challenge (PEDV/rPEDV-SeCoV)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Group C (n=12)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
SeCoV\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Group C (n=6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Heterologous re-challenge (SeCoV/rPEDV-SeCoV)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Group D (n=12)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Recombinant PEDV G1b-SeCoV\textsuperscript{3} (rPEDV-SeCoV)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Group D (n=6)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Homologous re-challenge (rPEDV-SeCoV/rPEDV-SeCoV)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Clinical evaluation, weighing and viral detection (all animals)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
dpi 0-7, 9, 11, 13, 15, 17 and 19\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Clinical evaluation, weighing and viral detection (all animals)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
dpi 20-26 (dpri 0-6)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Serum sample (all animals)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
dpi 0, 6, 13 and 20\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Serum sample (all animals)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
dpi 23 and 26 (dpri 3 and 6)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Heparin blood sample (all animals)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
dpi 20\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Heparin blood sample (all animals)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
dpi 23 (dpri 3)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Euthanasia and tissue sampling (3 animals/group/day)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
dpi 3 and 6\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Euthanasia and tissue sampling (3 animal/group/day)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
dpi 23 and 26 (dpri 3 and 6)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{1} Strain 2330-Orense, GenBank accession nr. MN692791.

\textsuperscript{2} Strain 1480-Murcia-Lorca, GenBank accession nr.
MN692770.

\textsuperscript{3} Strain 1931-1-Valladolid-Molpeceres, GenBank
accession nr. MN692784.

Appendix Table 1. Average daily gain (ADG): mean and standard deviation
for each of the groups throughout the experiment. At day 0, pigs from
group A were mock-infected while pigs from groups B, C and D were
challenged with PEDV, SeCoV and rPEDV-SeCoV, respectively. At the start
of the 4\textsuperscript{th} week (day 20) all pigs were challenged with
rPEDV-SeCoV. Letters show significant differences between groups for a
particular week (p\textless{}0.05).\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
ADG (Kg) & A (mock-infected) & B (PEDV) & C (SeCoV) & D
(rPEDV-SeCoV)\tabularnewline
\midrule
\endhead
& Mean \selectlanguage{ngerman}± standard deviation & Mean \selectlanguage{ngerman}± standard deviation & Mean \selectlanguage{ngerman}±
standard deviation & Mean \selectlanguage{ngerman}± standard deviation\tabularnewline
1\textsuperscript{st} week & 0.198\selectlanguage{ngerman}±0.060\textsuperscript{a} &
0.065\selectlanguage{ngerman}±0.049\textsuperscript{b} & 0.075\selectlanguage{ngerman}±0.076\textsuperscript{b} &
0.098\selectlanguage{ngerman}±0.057\textsuperscript{b}\tabularnewline
2\textsuperscript{nd} week & 0.338\selectlanguage{ngerman}±0.092 & 0.276\selectlanguage{ngerman}±0.051 & 0.248\selectlanguage{ngerman}±0.075 &
0.257\selectlanguage{ngerman}±0.097\tabularnewline
3\textsuperscript{rd} week & 0.367\selectlanguage{ngerman}±0.058 & 0.331\selectlanguage{ngerman}±0.070 & 0.357\selectlanguage{ngerman}±0.084 &
0.262\selectlanguage{ngerman}±0.088\tabularnewline
4\textsuperscript{th} week & 0.029\selectlanguage{ngerman}±0.038\textsuperscript{b} &
0.338\selectlanguage{ngerman}±0.022\textsuperscript{a} & 0.190\selectlanguage{ngerman}±0.128\textsuperscript{ab} &
0.362\selectlanguage{ngerman}±0.016\textsuperscript{a}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 2. Amino acid substitutions (red) determined in neutralizing
B-cell epitopes (NE) (Okda et al., 2017) in the isolates used in the
experiment. The corresponding amino acid positions are detailed beside
the amino acid code.

\textbf{Amino acid substitution and its position in each strain}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{PEDV\textsuperscript{1}} & \textbf{SeCoV\textsuperscript{2}} &
\textbf{rPEDV-SeCoV\textsuperscript{3}}\tabularnewline
\textbf{NE 499-600} & & &\tabularnewline
& Ser517 & Ala517 & Ser517\tabularnewline
& Ile527 & Val527 & Ile527\tabularnewline
& Leu536 & Phe536 & Phe536\tabularnewline
& Thr537 & Ser537 & Ser537\tabularnewline
& Asp542 & Glu542 & Asp542\tabularnewline
& Ser549 & Thr549 & Ser549\tabularnewline
& Asp566 & Thr566 & Asp566\tabularnewline
& Ser583 & Asn583 & Ser583\tabularnewline
& Val587 & Ile587 & Val587\tabularnewline
& Gly594 & Gly594 & Ser594\tabularnewline
\textbf{NE 722-731} & & &\tabularnewline
& Ser719 & Asn179 & Ser719\tabularnewline
& Ser724 & Asn724 & Ser724\tabularnewline
\textbf{NE 744-759} & & &\tabularnewline
& Lys755 & Thr755 & Lys755\tabularnewline
\textbf{NE 747-774} & & &\tabularnewline
& Lys755 & Thr755 & Lys755\tabularnewline
& Ser764 & Tyr764 & Ser764\tabularnewline
& Ser766 & His766 & Ser766\tabularnewline
\textbf{NE 1371-1377} & \textbf{No changes} & \textbf{No changes} &
\textbf{No changes}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{1} Strain 2330-Orense, GenBank accession nr. MN692791.

\textsuperscript{2} Strain 1480-Murcia-Lorca, GenBank accession nr.
MN692770.

\textsuperscript{3} Strain 1931-1-Valladolid-Molpeceres, GenBank
accession nr. MN692784.

Table 3. Villous height to crypt depth ratio (\selectlanguage{greek}µ\selectlanguage{english}m/\selectlanguage{greek}µ\selectlanguage{english}m): mean and standard
deviation (SD). At day post-inoculation (dpi) 0, pigs from group A were
mock-infected while pigs from groups B, C and D were challenged with
PEDV, SeCoV and rPEDV-SeCoV, respectively. At dpi 20, all pigs were
challenged with rPEDV-SeCoV. Three animals were euthanized per group at
dpi 3, 6, 23 and 26. Letters show significant differences between groups
for each particular day (p\textless{}0.05).\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
dpi & Group & Duodenum (mean \selectlanguage{ngerman}± SD) & Mid jejunum (mean \selectlanguage{ngerman}± SD) & Ileum
(mean \selectlanguage{ngerman}± SD)\tabularnewline
\midrule
\endhead
3 & A & 2.36\selectlanguage{ngerman}±0.07\textsuperscript{a} & 2.20\selectlanguage{ngerman}±0.26\textsuperscript{a} &
2.06\selectlanguage{ngerman}±0.40\textsuperscript{a}\tabularnewline
& B & 1.01\selectlanguage{ngerman}±0.17\textsuperscript{b} & 0.90\selectlanguage{ngerman}±0.17\textsuperscript{b} &
0.96\selectlanguage{ngerman}±0.14\textsuperscript{b}\tabularnewline
& C & 0.92\selectlanguage{ngerman}±0.38\textsuperscript{b} & 0.96\selectlanguage{ngerman}±0.38\textsuperscript{b} &
0.99\selectlanguage{ngerman}±0.51\textsuperscript{b}\tabularnewline
& D & 0.99\selectlanguage{ngerman}±0.23\textsuperscript{b} & 0.83\selectlanguage{ngerman}±0.18\textsuperscript{b} &
0.85\selectlanguage{ngerman}±0.43\textsuperscript{b}\tabularnewline
6 & A & 2.57\selectlanguage{ngerman}±0.28\textsuperscript{a} & 2.72\selectlanguage{ngerman}±0.14\textsuperscript{a} &
1.58\selectlanguage{ngerman}±0.11\textsuperscript{a}\tabularnewline
& B & 1.20\selectlanguage{ngerman}±0.19\textsuperscript{b} & 1.32\selectlanguage{ngerman}±0.54\textsuperscript{b} &
1.24\selectlanguage{ngerman}±0.41\textsuperscript{ab}\tabularnewline
& C & 1.32\selectlanguage{ngerman}±0.01\textsuperscript{b} & 1.56\selectlanguage{ngerman}±0.20\textsuperscript{b} &
1.31\selectlanguage{ngerman}±0.12\textsuperscript{ab}\tabularnewline
& D & 1.36\selectlanguage{ngerman}±0.39\textsuperscript{b} & 1.25\selectlanguage{ngerman}±0.42\textsuperscript{b} &
0.96\selectlanguage{ngerman}±0.05\textsuperscript{b}\tabularnewline
23 & A & 0.91\selectlanguage{ngerman}±0.14\textsuperscript{b} & 0.93\selectlanguage{ngerman}±0.01\textsuperscript{b} &
0.89\selectlanguage{ngerman}±0.10\textsuperscript{b}\tabularnewline
& B & 1.98\selectlanguage{ngerman}±0.19\textsuperscript{a} & 2.05\selectlanguage{ngerman}±0.09\textsuperscript{a} &
1.97\selectlanguage{ngerman}±0.26\textsuperscript{a}\tabularnewline
& C & 1.66\selectlanguage{ngerman}±0.11\textsuperscript{ab} & 1.32\selectlanguage{ngerman}±0.05\textsuperscript{ab} &
1.46\selectlanguage{ngerman}±0.06\textsuperscript{a}\tabularnewline
& D & 2.12\selectlanguage{ngerman}±0.55\textsuperscript{a} & 2.25\selectlanguage{ngerman}±0.31\textsuperscript{a} &
1.86\selectlanguage{ngerman}±0.14\textsuperscript{a}\tabularnewline
26 & A & 1.16\selectlanguage{ngerman}±0.20\textsuperscript{b} & 1.21\selectlanguage{ngerman}±0.16\textsuperscript{b} &
1.27\selectlanguage{ngerman}±0.14\textsuperscript{b}\tabularnewline
& B & 1.99\selectlanguage{ngerman}±0.03\textsuperscript{a} & 2.04\selectlanguage{ngerman}±0.35\textsuperscript{a} &
1.85\selectlanguage{ngerman}±0.12\textsuperscript{a}\tabularnewline
& C & 1.51\selectlanguage{ngerman}±0.16\textsuperscript{ab} & 1.57\selectlanguage{ngerman}±0.25\textsuperscript{ab} &
1.80\selectlanguage{ngerman}±0.12\textsuperscript{a}\tabularnewline
& D & 1.81\selectlanguage{ngerman}±0.29\textsuperscript{a} & 2.30\selectlanguage{ngerman}±0.05\textsuperscript{a} &
1.74\selectlanguage{ngerman}±0.08\textsuperscript{a}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 4. Detection of specific neutralizing antibodies (NA), IgA
secreting cells (SC) and IFN-\selectlanguage{greek}γ-\selectlanguage{english}SC against rPEDV-SeCoV. Letters show
significant differences between groups. Booster effect shows the
comparison of results obtained immediately before and after the
re-challenge (dpi 20 versus dpri 3 or dpri 6) within each group (*
indicates statistically significant differences).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Group}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{dpi 0}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{dpi 20}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{dpri 3}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{dpri 6}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Booster}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{NA: Percentage of positive animals (mean titer ± standard
deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{NA: Percentage of positive animals (mean titer ± standard
deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{NA: Percentage of positive animals (mean titer ± standard
deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{NA: Percentage of positive animals (mean titer ± standard
deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{NA: Percentage of positive animals (mean titer ± standard
deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{NA: Percentage of positive animals (mean titer ± standard
deviation)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{A}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0\%\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0\% (0/6)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0\% (0/6)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
100\% (3/3) 18.7 \selectlanguage{ngerman}± 4.6\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{B}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0\%\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
100\% (6/6) 19.3 \selectlanguage{ngerman}± 7.4\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
33.3\% (2/6) 8.0 \selectlanguage{ngerman}± 0.0\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
100\% (3/3) 30.7 \selectlanguage{ngerman}± 28.9\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{C}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0\%\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
100\% (6/6) 24.7 \selectlanguage{ngerman}± 13.5\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
33.3\% (2/6) 8.0 \selectlanguage{ngerman}± 0.0\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
100\% (3/3) 56.0 \selectlanguage{ngerman}± 36.6\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{D}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0\%\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
100\% (6/6) 48.0 \selectlanguage{ngerman}± 14.3\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
83.3\% (5/6) 13.6 \selectlanguage{ngerman}± 6.7\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
100\% (3/3) 21.3 \selectlanguage{ngerman}± 9.2\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{IgA SC (mean ± standard deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{IgA SC (mean ± standard deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{IgA SC (mean ± standard deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{IgA SC (mean ± standard deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{IgA SC (mean ± standard deviation)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{IgA SC (mean ± standard deviation)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{A}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4.0 \selectlanguage{ngerman}± 0.0\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.6 \selectlanguage{ngerman}± 2.1\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{B}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
18.1 \selectlanguage{ngerman}± 11.8\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
32.1 \selectlanguage{ngerman}± 14.2\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
*\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{C}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
16.9 \selectlanguage{ngerman}± 8.3\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
33.3 \selectlanguage{ngerman}± 16.7\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
*\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{D}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
16.2 \selectlanguage{ngerman}± 7.7\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
25.0 \selectlanguage{ngerman}± 5.8\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{-}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{*}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{IFN-γ-SC (mean ± standard deviation)}\selectlanguage{ngerman}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{IFN-γ-SC (mean ± standard deviation)}\selectlanguage{ngerman}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{IFN-γ-SC (mean ± standard deviation)}\selectlanguage{ngerman}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{IFN-γ-SC (mean ± standard deviation)}\selectlanguage{ngerman}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{IFN-γ-SC (mean ± standard deviation)}\selectlanguage{ngerman}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{IFN-γ-SC (mean ± standard deviation)}\selectlanguage{ngerman}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{A}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.3 \selectlanguage{ngerman}± 2.3\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3.0 \selectlanguage{ngerman}± 2.1\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{B}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8.1 \selectlanguage{ngerman}± 4.7\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
25.3 \selectlanguage{ngerman}± 10.6\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
*\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{C}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
11.1 \selectlanguage{ngerman}± 6.0\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
41.4 \selectlanguage{ngerman}± 25.0\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
*\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{D}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
26.4 \selectlanguage{ngerman}± 10.5\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
103.3 \selectlanguage{ngerman}± 53.6\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
*\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Pigs from group A were mock-infected while pigs from groups B, C and D
were challenged with PEDV, SeCoV and rPEDV-SeCoV, respectively. At day
post-inoculation (dpi) 20, all pigs were challenged with rPEDV-SeCoV.

Figure 1. Clinical score (fecal consistency: 0-2 + general condition:
0-3 + appetite: 0-2 + vomiting: 0-1): mean and standard deviation (error
bars) for each group throughout the experiment. Letters show significant
differences between groups for each particular day (p\textless{}0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

At day post-inoculation (dpi) 0, pigs from group A were mock-infected
while pigs from groups B, C and D were challenged with PEDV, SeCoV and
rPEDV-SeCoV, respectively. At dpi 20, all pigs were challenged with
rPEDV-SeCoV.

Appendix Figure 1. Fecal consistency: percentage of animals with normal
feces (grey), soft stools (orange) and liquid feces (yellow) for each
group. Letters show significant differences between groups for each
particular day (p\textless{}0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

At day post-inoculation (dpi) 0, pigs from group A were mock-infected
while pigs from groups B, C and D were challenged with PEDV, SeCoV and
rPEDV-SeCoV, respectively. At dpi 20, all pigs were challenged with
rPEDV-SeCoV.

Figure 2. Viral detection in fecal samples throughout the experiment.
(A) Percentage of RT-qPCR positive animals. (B) Average viral
quantification (log\textsubscript{10} TCID\textsubscript{50}/g). (C)
Area under the curve (AUC) for RNA viral shedding. Letters show
significant differences between groups for each particular day
(p\textless{}0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

At day post-inoculation (dpi) 0, pigs from group A were mock-infected
while pigs from groups B, C and D were challenged with PEDV, SeCoV and
rPEDV-SeCoV, respectively. At dpi 20, all pigs were challenged with
rPEDV-SeCoV.

Figure 3. Photomicrographs revealing severe villous atrophy and fusion
at days post-infection (dpi) 3 and 6 in the duodenum of pigs challenged
with PEDV (groups B and D) and SeCoV (group C). After re-challenge,
lesions were also evident in pigs challenged for the first time (group
A) and in those previously exposed to SeCoV (group C), but were absent
in pigs from groups B and D previously exposed to two variants of PEDV.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 4. PEDV-specific IgG and IgA kinetics determined using a
commercial ELISA. (A) Percentage of IgG positive animals. (B) Mean S/P
ratios and standard deviation (error bars) of IgG detection per group.
The dotted line shows the cut-off value of the test (0.3). (C) Mean ODs
and standard deviation (error bars) of IgA detection. The dotted line
shows the cut-off value of the test (0.05). Letters show significant
differences between groups for each particular day (p\textless{}0.05).
Booster effect shows the comparison of results obtained immediately
before and after the re-challenge (dpi 20 versus dpri 3 or dpri 6)
within each group (* indicates statistically significant differences).

At day post-inoculation (dpi) 0, pigs from group A were mock-infected
while pigs from groups B, C and D were challenged with PEDV, SeCoV and
rPEDV-SeCoV, respectively. At dpi 20, all pigs were challenged with
rPEDV-SeCoV.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Appendix-Figure-1/Appendix-Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

