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.