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\begin{document}

\title{Prevalence and molecular characteristics of feline coronavirus in
southwest China from 2017 to 2020}



\author[1]{Qun Zhou}%
\author[1]{Yan Li}%
\author[1]{Jian Huang}%
\author[1]{Nengsheng Fu}%
\author[1]{Xin Song}%
\author[1]{Xue Sha}%
\author[1]{Bin Zhang}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Feline coronavirus (FCoV) is the causative agent of feline infectious
peritonitis and diarrhea in kittens worldwide. In this study, a total of
173 feline diarrheal fecal and ascetic fluid samples were collected from
15 catteries and six veterinary hospitals in southwest China from 2017
to 2020. FCoV was detected in 80.35\% (139/173) of the samples using the
RT-nPCR method; these included infections with 122 type I FCoV (87.8\%)
and 57 type II FCoV (41\%). Interestingly, 51 cases (36.7\%, 51/139) had
co-infection with types I and II, the first such report in mainland
China. To further analyze the genetic diversity of FCoV in southwest
China, we amplified 23 full-length spike genes, including 18 type I and
five type II FCoV. The 18 type I FCoV strains shared 85.9\%--100\%
nucleotide sequence identities between one another and the five type II
FCoV strains shared 97.4\%--98.9\% nucleotide sequence identities
between one another. This result suggests that the N-terminal domain
(NTD) of 23 FCoV strains showed a high degree of variation
(73.6\%--80.3\%). There was five type I FCoV strains with two aa
insertions (159HL160) in the NTD region. In addition, 18 strains of type
I FCoV belonged to the Ie cluster, and five strains of type II FCoV were
in the IIb cluster based on phylogenetic analysis. It is worth noting
that five type I FCoV strains also had recombination in the NTD, and the
recombination region was 135--625 nucleotides of the S gene. This study
constitutes a systematic investigation of the current infection status
and molecular characteristics of FCoV in southwest China.%
\end{abstract}%



\sloppy


\textbf{Prevalence and molecular characteristics of feline coronavirus
in southwest China from 2017 to 2020}

\textbf{Running title: FCoV in southwest China}

Qun Zhou\textsuperscript{1}, Yan Li\textsuperscript{1,2}, Jian
Huang\textsuperscript{1, 2}, Nengsheng Fu\textsuperscript{1}, Xin
Song\textsuperscript{1}, Xue Sha\textsuperscript{1}, Bin
Zhang\textsuperscript{1,2*}

\textsuperscript{1} College of Animal and Veterinary Sciences, Southwest
Minzu University, Chengdu 610041, China.

\textsuperscript{2} Key laboratory of Ministry of Education and Sichuan
Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation
and Utilization, Chengdu 610041, China.

*Correspondence:

Bin Zhang, College of Animal and Veterinary Sciences, Southwest Minzu
University, Chengdu, China.

Email: binovy@sina.com (Bin Zhang)

\textbf{Summary}

Feline coronavirus (FCoV) is the causative agent of feline infectious
peritonitis and diarrhea in kittens worldwide. In this study, a total of
173 feline diarrheal fecal and ascetic fluid samples were collected from
15 catteries and six veterinary hospitals in southwest China from 2017
to 2020. FCoV was detected in 80.35\% (139/173) of the samples using the
RT-nPCR method; these included infections with 122 type I FCoV (87.8\%)
and 57 type II FCoV (41\%). Interestingly, 51 cases (36.7\%, 51/139) had
co-infection with types I and II, the first such report in mainland
China. To further analyze the genetic diversity of FCoV in southwest
China, we amplified 23 full-length spike genes, including 18 type I and
five type II FCoV. The 18 type I FCoV strains shared 85.9\%--100\%
nucleotide sequence identities between one another and the five type II
FCoV strains shared 97.4\%--98.9\% nucleotide sequence identities
between one another. This result suggests that the N-terminal domain
(NTD) of 23 FCoV strains showed a high degree of variation
(73.6\%--80.3\%). There was five type I FCoV strains with two aa
insertions (159HL160) in the NTD region. In addition, 18 strains of type
I FCoV belonged to the Ie cluster, and five strains of type II FCoV were
in the IIb cluster based on phylogenetic analysis. It is worth noting
that five type I FCoV strains also had recombination in the NTD, and the
recombination region was 135--625 nucleotides of the S gene. This study
constitutes a systematic investigation of the current infection status
and molecular characteristics of FCoV in southwest China.

\textbf{KEYWORDS}

feline coronavirus, detection; S gene; NTD; recombination

\textbf{INTRODUCTION}

Feline coronavirus (FCoV) is a \emph{Coronaviridae} family member
(genus\emph{Alphacoronavirus} ), first identified in America in 1968.
This identification was followed by outbreaks of the disease in many
countries, severely endangering kitten health (Chang, Egberink, Halpin,
Spiro, \& Rottier, 2012; Pedersen, 2009; Ward, Munn, Gribble, \&
Dungworth, 1968). FCoV includes two pathogenic biotypes: feline enteric
coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV
infection is characterized by mild diarrhea. In contrast, FIPV
efficiently replicates in macrophages/monocytes and can lead to feline
infectious peritonitis (FIP), a highly lethal systemic granulomatous
disease (Ehmann et al., 2018; Oguma, Ohno, Yoshida, \& Sentsui, 2018).

FCoV is an enveloped, single-stranded, positive-sense RNA virus of
approximately 29 kb in size, containing 11 open reading frames encoding
four structural proteins: spike (S), envelope (E), membrane (M),
nucleocapsid (N), and seven nonstructural proteins (Dye \& Siddell,
2005; Pedersen, 2009). The S protein of FCoV is a membrane glycoprotein
thought to be a viral regulator of binding and entry to the cells
(Bosch, van der Zee, de Haan, \& Rottier, 2003; Jaimes \& Whittaker,
2018; Millet \& Whittaker, 2015). This protein is also involved in FCoV
tropism and virulence and is the switch from enteric disease to FIP
(Belouzard, Millet, Licitra, \& Whittaker, 2012; Jaimes \& Whittaker,
2018; B. N. Licitra et al., 2013). The S protein comprises two amino
acid (aa) subunits, S1 and S2, with the S1 being more genetically
diverse than S2 (Yang et al., 2020). S1 contains a receptor-binding
domain and is responsible for the initial attachment of the virus to the
surface of host cells (Belouzard et al., 2012; Jaimes \& Whittaker,
2018; Wu, Li, Peng, \& Li, 2009). Based on the amino acid sequence of
the S protein, FCoV is classified into two genotypes, types I and II
(Motokawa, Hohdatsu, Hashimoto, \& Koyama, 1996; Takano, Satomi, Oyama,
Doki, \& Hohdatsu, 2016). Type II FCoV emerged via double recombination
between type I FCoV and type II canine coronavirus (CCoV) (Herrewegh,
Smeenk, Horzinek, Rottier, \& de Groot, 1998). As a result of this
recombination, the spike (S) gene and adjacent regions of type I FCoV
were replaced by the corresponding part of the CCoV genome (Herrewegh et
al., 1998; Lin, Chang, Su, \& Chueh, 2013; Terada et al., 2014).

FCoV has a worldwide distribution (Amer et al., 2012; An et al., 2011;
Klein-Richers et al., 2020; Li et al., 2019; Luo, Liu, Chen, \& Chen,
2020; McKay et al., 2020; Soma, Wada, Taharaguchi, \& Tajima, 2013).
Infections with FCoV are mostly type I FCoV, while type II FCoV occurs
primarily in southeast Asia (An et al., 2011; Li et al., 2019; Luo et
al., 2020). With a gradual increase in the number of cats raised in
China, coronavirus-related diseases have become more and more complex
and diverse in cats. Nevertheless, the prevalence and genetic diversity
of FCoV in southwest China remain unclear. Therefore, in this study, we
recorded the current infection status of FCoV in southwest China and
analyzed the sequences of currently circulating FCov strains.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1. Samples collection}

One hundred seventy-three samples were collected from cats under one
year of age from 15 catteries and six veterinary hospitals in southwest
China from 2017 to 2020, of which 93 samples were from catteries, and 80
samples were from veterinary hospitals. Of the 173 samples, 150 fecal
samples were obtained from cats with diarrhea, and 23 samples of ascetic
fluid were obtained from FIP-suspected cats. The cattery samples were
collected from pet breeding centers and stray animal shelters in
southwest China, both considered multi-cat environments. The samples
collected at the veterinary hospital were individual, home-owned pets
considered to come from single-cat environments. The cat breeds included
British Shorthair, Nulla luctus felis, Ragdoll, Garfield, and Persian.
The samples were shipped on drikold and stored at --80 \selectlanguage{ngerman}°C.

\textbf{2.2. RNA extraction and cDNA synthesis}

The fecal and ascetic fluid samples were fully resuspended in
phosphate-buffered saline (1:5 w/v) and centrifuged at 10,000 × g for 10
min. According to the manufacturer's instructions, viral RNA was
extracted from 300 \selectlanguage{greek}μ\selectlanguage{english}L of the fecal suspension using RNAios Plus (TaKaRa
Bio Inc). The cDNA was synthesized using the PrimeScript RT Reagent Kit
according to the manufacturer's instructions (TaKaRa Bio Inc) and then
stored at --20 \selectlanguage{ngerman}°C until required.

\textbf{2.3. Detection and typing of feline coronavirus}

The 3'UTR of FCoV was amplified using RT-nested PCR as described by
Herrewegh et al. (Herrewegh et al., 1995), and a positive 3'UTR produced
a 177 bp amplicon on electrophoresis. Furthermore, for samples in which
the 3'UTR gene was detected, genotyping was performed with RT-nPCR as
reported by Addie et al. (DD Addie, Schaap, Nicolson, \& Jarrett, 2003),
providing 360 and 218 bp amplicons of FCoV types I and II, respectively.
The PCR detection was performed in a 25 \selectlanguage{greek}μ\selectlanguage{english}L volume, including 1 \selectlanguage{greek}μ\selectlanguage{english}L of
template, 1 \selectlanguage{greek}μ\selectlanguage{english}L of each primer (10 pmol), 8 \selectlanguage{greek}μ\selectlanguage{english}L of ddH2O, and 12.5 \selectlanguage{greek}μ\selectlanguage{english}L of
Quick Taq HS DyeMix at 2 \selectlanguage{ngerman}× concentration (Toyobo, Japan). The PCR
conditions were set at 94 °C for 2 min, 35 repeats of denaturing at 94
°C for 30 s, annealing at 51 °C for 30 s, extension at 68 °C for 1 min,
and a final extension at 72 °C for 8 min. The RT-PCR products were
analyzed using 1.5\% agarose gel electrophoresis and visualized using
ultraviolet illumination. Finally, the PCR products consistent with the
expected size were sent to Sangon Biotech (Shanghai, China) for
sequencing.

\textbf{2.4. PCR amplification of S gene}

The complete S gene was PCR-amplified from samples already known to be
FCoV-positive according to the primers in Table S1. All PCR products
were purified using the Omega Gel Kit following the manufacturer's
instructions. They were ligated to the pMD19-T vector (TaKaRa Bio Inc.)
and transformed into DH5\selectlanguage{greek}α \selectlanguage{english}competent \emph{Escherichia coli} cells for
sequencing. The S sequences were assembled using SeqMan software
(version 7.0; DNASTAR).

\textbf{2.5. Sequence, phylogenetic, and recombination analyses}

The homologies of the nucleotide and deduced amino acid (aa) sequences
were determined using the MegAlign program in DNASTAR 7.0 software, and
a heat map was constructed based on sequence homology using HemI
software (Heatmap Illustrator, version 1.0.3.7); MEGA X 10.2.4
(https://www.megasoftware.net/) was used for multiple sequence alignment
and to subsequently build neighbor-joining phylogenetic trees with
bootstrap testing (1,000 replicates). Phylogenetic trees were pruned and
re-rooted using the Interactive Tree Of Life software, version 5.6.3
(https://itol.embl.de/), an online tool for displaying the circular tree
annotation. Recombination events were assessed using the recombination
detection program RDP 4.0 (version 4.9.5) with the RDP, GeneConv,
Chimaera, MaxChi, BootScan, SiScan, and 3Seq methods. The results were
used for Simi larity Plotting analysis using the SimPlot program version
3.5.1 with a window size of 200 bp and a step size of 20 bp.

\textbf{2.6. Statistical analysis}

The collected data were coded and entered into a computer using Epi
Info 7 version 7.2.0.1 and were then exported to SPSS version 23.0 for
analysis. The chi-square test was used to assess the association of
variables with the primary outcome. \emph{P} -values \textless{}0.05
were considered to indicate statistical significance.

\textbf{3. RESULTS}

\textbf{3.1. Prevalence of FCoV in southwest China}

From May 2017 to December 2020, 23 FIP-suspected samples of ascites
fluid and 150 diarrheal fecal samples from veterinary hospitals and
catteries in southwest China were tested using RT-nPCR amplification. Of
the 173 samples, 139 (80.35\%, 95 \% CI; 73.6\%--86\%) were positive for
FCoV (Table 1), including the positive rates of FCoV in FIP-suspected
cat; the rates in diarrheal cats were 95.65\% (22/23, 78.1\%--99.9\%)
and 78\% (117/150, 95\% CI; 70.5\%--84.3\%), respectively. As shown in
Table 2, the positive rates of FCoV in multi-cat environments and
single-cat households were 85\% (79/93, 95\% CI: 76\%--91.5\%) and
66.67\% (38/57, 95\% CI: 52.9\%--78.6\%), respectively. These results
suggest that FCoV is highly prevalent in southwest China. The detection
rate of FCoV was significantly associated with the cat's living
environment (\emph{p} = 0.014) and clinical symptoms (\emph{p} = 0.05).

The 139 positive samples were typed using type I and II FCoV typing
primers. Of the 127 successfully typed FCoV-positive samples, type I
FCoV was predominant, accounting for 87.77\% (122/139, 95\% CI:
81.1\%--92.7\%), and type II FCoV accounting for 41.01\% (57/139, 95\%
CI: 32.7\%--49.7\%). Eleven other samples failed typing, possibly due to
the low viral load in samples or the potential mismatching between
primers and templates. Interestingly, co-infection with type I and II
FCoV were detected in 51 samples (36.69\%, 95\% CI: 28.7\%--45.3\%), the
first such report in mainland China, and the detection rate of
co-infection with types I and II FCoV were significantly higher in the
multi-cat environment than in the single-cat environment
(\emph{p}\textless{} 0.01; Table 2).

\textbf{3.2 Amplification and sequence analysis of FCoV S gene}

The S protein, encoded by the FCoV S gene, is considered the primary
viral regulator in host cell entry; it possesses a receptor-binding
domain, while mutations in amino acids of the FCoV S protein are
associated with the conversion of FECV to FIPV (Bosch et al., 2003;
Jaimes \& Whittaker, 2018; Millet \& Whittaker, 2015). To further
understand the molecular characteristics and genetic variation of the
FCoV S gene in southwest China, 18 type I FCoV and five type II FCoV
strains were selected to amplify the whole gene of FCoV S according to
various years, living environments, and regions. The 18 type I FCoV S
genes were 4395--4419 bp in length, each encoding a protein of
1465--1473 aa residues (GenBank accession number MW316830-MW316847).
Five type II FCoV S genes were 4353--4362 bp in length, each encoding a
protein of 1451--1454 aa residues (GenBank accession number
MW316848-MW316852).

A heatmap was constructed using HemI software based on the sequence
homology of type I FCoV with classical strain Black and type II FCoV
with classical strain 79--1146, respectively (Figure 1). The results
showed that the NTD variation in S1 subunit was the highest. The NTD
homology between 23 strains and the reference strain was 73.6\%--80.3\%;
the S2 subunit was relatively conserved, the S2 subunit homology between
23 strains and the reference strain was 93.9\%--94.2\%. The 18 type I
FCoV strains shared 85.9\%--100\% nucleotide sequence identities and
89.4\%--100\% aa identities. They shared 87.7\%--93.8\% nucleotide
sequence identity and 87.9\%--93.1\% aa sequence identities with all 64
of the complete type I FCoV S genes available in the GenBank database.
Sequence analysis of 105 FCoV strains showed that seven type I FCoV
strains (SMU-CD86, SMU-CD77, SMU-CD9, SMU-CD8, SMU-CD60, SMU-CDF12,
SMU-CDF19) identified in this study had two aa insertions in the NTD
(159HL160), and strains with this insertion pattern also appeared in
strains that have been reported in China and the Netherlands (JN183882,
KF530123, KY566209, KY566210, KY566211). The five type II FCoV strains
shared 97.4\%--98.9\% nucleotide sequence identities and 95\%--98.8\% aa
identities between one another and shared 86.9\%--97.8\% nucleotide
sequence identity 84.9\%--97.7\% aa sequence identities with the 18 type
II FCoV reference strains. The type II FCoV strain SMU-CQ18 showed three
discontinuous unique aa deletions (209N, 214M/L, 217R) in the NTD. These
data showed that the nucleotide of the carbohydrate-bound NTD in the 23
S genes had significant mutations, and there were also insertions and
deletions of aa in the NTD.

\textbf{3.3 Phylogenetic analysis of FCoV S Gene}

To track the evolution of FCoV strains identified in southwest China,
all FCoV strains in the GenBank database were downloaded, the S genes of
82 FCoV strains, 14 CCoV strains, and one transmissible gastroenteritis
virus (TGEV) strain in the GenBank database. The 23 FCoV strains S genes
identified in this study were used to construct a phylogenetic tree. The
82 type I FCoV strains were formed five clusters in the S gene-based on
the phylogenetic tree (Figure 2), which were tentatively named type Ia,
Ib, Ic, Id, and Ie in this study. Type Ia, Ib, and Ic clusters were
composed of strains from Europe and North America. The Id cluster
consists of strains from Europe and America. All 18 type I FCoV strains
belonged to the type Ie cluster, including strains from countries such
as Japan, the Netherlands, and Belgium. These results indicate that the
type Ie is a mixed cluster consisting of strains from Asia and Europe
and is more complex than the other clusters' genetic diversity.

All type II FCoV in the GenBank database form two distinct clusters in
the phylogenetic tree (Figure 2), tentatively named type IIa FCoV and
type IIb FCoV, respectively. Type IIa FCoV consists of strains from
China and the USA. The five type II FCoV in this study clustered with
four other type II FCoV strains and five CCoV strains as type IIb FCoV
and were most closely related to the isolated CCoV strains in China in
2019. In the present study, 18 type I FCoV belonged to type Ie FCoV, and
five type II FCoV belonged to type IIb FCoV.

\textbf{3.4 Recombination analysis of type I FCoV S gene}

Recombination analysis of the 23 complete S genes showed that
recombination events had occurred in five of the type I FCoV strains
(SMU-CDF19, SMU-CDF12, SMU-CD8, SMU-CD77, SMU-CD9) based on the RDP 4.0
(six methods) and SimPlot 3.5.1. The recombination breakpoint based on
RDP 4.0 identified the beginning of the breakpoint at nucleotide 180 in
the fragment (breakpoint 99\% confidence intervals: nucleotide positions
1--295 in the fragment) and nucleotide 606 at the end of the breakpoint
(breakpoint 99\% confidence intervals: nucleotide position 557--1050 in
the fragment). For example, the major parental strain of SMU-CD9 was the
Belgian strain UG-FH8 (KX722529), and the minor parental strain was the
Chinese HLJ/HRB/13 (KY566211), with a recombination score of 0.535 and a
predicted recombination breakpoint of nucleotides 135 and 625 (Figure
3). Although the recombination breakpoints predicted by RDP 4.0 and
SimPlot differ, both programs showed that the recombination region was
located in the NTD of S gene (at nucleotide region 45--804 aa in the
full-length FCoV S gene). To further verify this recombinant event,
phylogenetic trees of the regions 1--135, 135--625, and 626--4410
nucleotides were constructed. A discrepancy was found between the
phylogenetic trees, further confirming the recombination events.

\textbf{DISCUSSION}

FCoV is a lethal infectious agent that causes effusions in the pleural
and abdominal cavities in domestic cats. There are no effective
vaccines, and management is based on biosafety prevention and control
(Haake, Cook, Pusterla, \& Murphy, 2020; Pedersen, 2009). FCoV is
ubiquitous in cat colonies, with detection rates ranging from
6.6\%--95\% (Amer et al., 2012; An et al., 2011; Klein-Richers et al.,
2020; Li et al., 2019; Luo et al., 2020; McKay et al., 2020). Few
epidemiological studies concerning FCoV have been conducted in southwest
China. In our study, 173 samples from sick cats were included for FCoV
detection, and the positive rate of these samples was 80.35\% (139/173),
much higher than the prevalence reported in other regions of China and
other countries in recent years (Klein-Richers et al., 2020; Li et al.,
2019; Luo et al., 2020; McKay et al., 2020). The results suggest that
the prevalence of FCoV in cats with diarrhea and FIP is high in
southwest China. Several studies reported that living environments are
associated with FCoV infection (D Addie et al., 2009; Drechsler,
Alcaraz, Bossong, Collisson, \& Diniz, 2011; Sharif et al., 2009). In
the present study, the infection rate of FCoV in the multi-cat
environments was significantly higher than that of the single-cat
environment, in line with previous studies (Drechsler et al., 2011; Li
et al., 2019).

Type I FCoV is the dominant genotype in most countries, including
Austria (86\%) (Benetka et al., 2004), Malaysia (97.5\% )(Amer et al.,
2012), and China (72.2\%) (Li et al., 2019), while type II is less
common and is mainly found in southeast Asian countries. Co-infection
with types I and II were reported sporadically in Europe a decade ago
and in southeast Asia in recent years but has not been reported in
mainland China (An et al., 2011; Benetka et al., 2004; Luo et al., 2020;
Soma et al., 2013). In this study, in accord with reports from other
countries (Amer et al., 2012; Benetka et al., 2004; Li et al., 2019),
the dominant genotype of FCoV was also type I FCoV in southwest China
(87.77\%, 122/139), and the prevalence of type II FCoV was significantly
higher than that in other regions (41\%, 57/139). We found co-infection
of types I and II in southwest China with high prevalence (36.7\%,
51/139); this is the first report of co-infection of types I and II FCoV
in mainland China.

Point mutations, insertions, deletions, and recombination are common in
the coronavirus genome, and they are responsible for the emergence of
new coronavirus strains. The FCoV S protein is implicated as a regulator
of viral binding and entry into cells. Mutations in the S protein of
FCoV may cause changes in virus tropism, leading to a switch from
enteric disease to FIP (Millet \& Whittaker, 2015; Yang et al., 2020).
Consistent with previously reported strains, the highly mutated region
of the FCoV S gene was the NTD in 23 strains in our study, and the
degree of mutation was inconsistent across strains (59.3\%--87\%).
Furthermore, two aa (159HL160) insertions were present in the NTD
between ten FCoV strains from China and two strains from the Netherlands
(KF530123, JN183882). Previous studies showed that the NTD in the FCoV
S1 subunit has sugar-binding functions and aids in host receptor-binding
(Belouzard et al., 2012; Jaimes \& Whittaker, 2018); however, it remains
unclear whether the NTD mutation and insertions of the strains in this
study affect the pathogenicity of FCoV and the adaptability of the virus
to host receptor.

Genome sequences and subsequent phylogenetic analysis showed that FCoV
isolates form geographical clusters (Kipar \& Meli, 2014). In Italy and
Brazil, investigators reconstructed the origin and spatiotemporal
distribution of type I FCoV by phylogeographic analysis and found that
from the USA, the virus likely entered Germany and spread to other
European countries (Lauzi et al., 2020; Myrrha et al., 2019). In the
present study, 81 type I FCoV strains were clustered into five distinct
clusters in the phylogenetic tree (types Ia, Ib, Ic, Id, and Ie), with
significant geographic differences, and all type I FCoV identified in
Asia belonged to Ie FCoV, including some strains identified in Europe.
The information from limited samples suggests that the origin of FCoV
strains in China may be closely related to strains in Europe.

Type II CCoV is divided into two sub-genotypes, types IIa and IIb, based
on differences in the S protein amino acid sequence (Decaro et al.,
2010; B. Licitra, Duhamel, \& Whittaker, 2014). The amino acid sequence
of type IIb CCoV S protein is highly similar to TGEV (Decaro et al.,
2010; B. Licitra et al., 2014). The existing type II FCoV strains can be
divided into two different subtypes, types IIa and IIb. Using this
classification method, five type II FCoV strains belonged to type IIb
FCoV. These data suggest that the type I FCoV strains identified in
China are more genetically diverse than the type II FCoV strains, in
line with previous studies (Li et al., 2019; Luo et al., 2020).

Recombination events are often reported in human and animal
coronaviruses, and they play pivotal roles in the evolution of
coronaviruses. The S protein is the most common recombination region,
and recombination of this protein allows the emergence of new strains
with altered virulence and potentially broader host ranges (Boniotti et
al., 2016; Decaro et al., 2010; Herrewegh et al., 1998; Na, Moon, \&
Song, 2021; Qin et al., 2019). Here, we show for the first time
recombination in type I FCoV with the recombination region located at
the NTD of the S protein. Similar recombination events have been
reported in CCoV, porcine epidemic diarrhea virus and infectious
bronchitis virus (Ovchinnikova et al., 2011; Qin et al., 2019; Regan et
al., 2012). This novel recombination event occurred in five S genes and
contributed to the emergence of new variants of FCoV or led to changes
in FCoV tropism. The recombination event in S genes of type I FCoV
further contributes to our understanding of the evolution and genetic
diversity of FCoV from cats.

In conclusion, Type I FCoV strains are the dominant genotype of FCoV in
southwest China. The presence of co-infection of types I and type II in
feline populations in mainland China were reported for the first time.
Genomic analysis indicated that type I FCoV strains showed significant
mutations, and there were recombination events. This study helps develop
a profile of the current FCoV status and might provide an outline for
future research on the FCoV spike protein gene.

\textbf{ACKNOWLEDGEMENTS}

This study was supported by funding from the Applied Basic Research
Project of Sichuan Province, China (grant no. 2020YJ0247), the National
Natural Science Foundation of China (grant no.31772766), and the
Fundamental Research Funds for the Central Universities of Southwest
Minzu University (grant no. 2021NYYXS85).

\textbf{ETHICAL APPROVAL}

In this study, all the experiments were preformed according to the
permit guidelines established by Southwest Minzu University, China. The
experimental protocols were approved by the Animal Care and Use
Committee of Southwest Minzu University. The sampling and data
publication were also approved by the cats' owners.

\textbf{CONFLICT OF INTEREST}

The authors declare that there is no conflict of interest.

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\textbf{Figure and table legends}

\textbf{Table 1.} Detection and typing results of FCoV

\textbf{Table 2.} Correlation of the FCoV prevalence with clinical
symptoms and living environments

\textbf{Figure 1.} Heatmap based on the nucleotide sequence homology
between 23 FCoV strains and reference strains (window size, 100
nucleotides). 1a, based on the nucleotide sequence homology between the
18 type I FCoV strains in this study and the classical strain Black. 1b,
based on the nucleotide sequence homology between the five type II FCoV
strains in this study and the classical strain 79-1146. The colors
represent sequence identity (blue for lower identities and red for
higher identities) among different strains. The bar on the right
represents the hierarchical similarity among the 43 or 44 meta-clusters.

\textbf{Figure 2.} Phylogenetic tree based on the nucleotide sequences
of the entire S genes of 23 FCoV strains and other 82 FCoV strains, 14
CCoV strains, and one TGEV strain. The tree was constructed using the
neighbor-joining method. The red font indicates the S gene sequences of
type I FCoV, and the blue font indicates the S gene sequences of type II
FCoV in this study. The reference sequences from GenBank are indicated
by strain abbreviations, including GenBank accession number, genotype,
country, and collection year.

\textbf{Figure 3.} Recombination analysis of the FCoV S gene. (a).
SimPlot analysis of the complete genome represented by
M/SMU-CD9/2017/FCoV1 strain; window size, 200 bases; step, 20 bases. The
vertical axis indicates nucleotide sequence similarity (\%) between the
query strain and reference strains. (b) Phylogenetic trees for region A
(nucleotides 1--135), region B (nucleotides 136--625), and region C
(nucleotides 626--4410) of the FCoV strains were constructed using the
neighbor-joining method. Red circles indicate the S gene in this study.
Black squares indicate major and minor parental strains.

\textbf{Supporting information legends}

\textbf{Table S1.} FCoV S gene amplification primers\selectlanguage{english}
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