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.
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.