1. Introduction
The Feline coronavirus (FCoV) is ubiquitous in feline populations worldwide, and despite being first described in 1963 [
1], it remains one of the most poorly understood feline viruses. The virus belongs to the order
Nidovirales, family
Coronaviridae, subfamily
Orthocoronavirinae, and genus
Alphacoronavirus 1. Two primary forms (biotypes) of FCoV are recognized: the avirulent enteric form (Feline Enteric Coronavirus (FECV)) and the virulent form (Feline Infectious Peritonitis Virus (FIPV)) causing a fatal clinical disease, feline infectious peritonitis (FIP). Infection with FCoV is a major problem in multiple-cat households and, to a lesser extent, in free-roaming cats. The virus is endemic especially in multi-cat environments, where many cats are kept together in a small space, such as catteries and shelters. Infection with FCoV is common in domestic cats, with up to 90% of cats within multi-cat households being seropositive [
2]. The majority of FCoV infections are asymptomatic or are associated with mild intestinal disease; however, approximately 1–5% of infected cats develop FIP [
3,
4], which is characterized by the development of a variable combination of pyogranulomatous polyserositis, vasculitis and granulomatous lesions in organs, and an extremely high mortality rate [
5,
6].
The FECV infection is spread mostly by the orofecal route [
7,
8], while other modes of transmission are rather rare [
4,
9]. Fecal shedding of the virus usually begins within one week of exposure [
10]. The primary stage of infection after exposure to FCoV can last from 7 to 18 months. During this period, the highest levels of viral shedding are observed [
8]. In naturally infected cats, the initial shedding levels decrease over two years [
11]. Cats may shed FECV with low, moderate or high frequency. Shedding usually persists for 4 to 6 months, followed by intermittent shedding and eventual clearance [
12]. In some cats, FECV shedding may persist for more than 18 months [
8].
The patterns of fecal shedding of FECV may differ between single-cat and multi-cat households [
13]. A large proportion of cats in multiple-cat environments undergo cycles of infection and shedding, recovery, and reinfection, shedding the virus the entire time [
7]. More than 80% of cats living in catteries shed FECV while being clinically asymptomatic [
14]. In multi-cat households, persistent (10–15%), intermittent (70–80%) or self-limiting FECV shedding patterns were observed; about 5% of cats were identified as non-shedders [
15]. Based mostly on unpublished data, Addie et al. [
16] recently discussed the possibility that intermittent FECV shedding may in fact be false negativity due to technical reasons. It was suggested that identification of long-term carriers requires multiple positive results for at least eight consecutive months, while a cat could be considered negative with multiple negative results over a period of 5 months [
15]. Persistent shedders can be infected with the same strain of the virus the whole time, but reinfection after recovery with the same or a different virus strain has also been reported [
11,
17].
Asymptomatic persistent shedders represent the most important source of infection. They were identified as a source of infection for susceptible kittens [
4]. Host factors such as age, environmental stress, concurrent disease and especially various types of immune responses play an important role in the development of FIP [
18]. Genetics represents one of the factors contributing to the observed variation in the course of coronavirus infections, including the shedding status in humans [
19]. In cats, GWAS candidate regions and several individual candidate genes were observed to be associated with FIP [
20,
21]. Much less is known about the role of the host in FECV fecal shedding. Interbreed differences in the presence of anti-FCoV antibodies in healthy cats have been reported repeatedly [
13,
22,
23]. However, as some of these differences were observed between purebred and crossbred cats from different environments, it is not clear to what extent they might be due to non-genetic factors, such as the intensity of close contacts. Addie et al. [
17] genotyped the feline Major Histocompatibility Complex (
MHC)
FLA class II
DRB genes in 25 cats for which fecal shedding status was determined. They found no statistical association with the MHC genes analyzed.
Studies of the variation in the shedding status of FECV can contribute to the understanding of its role in the spread of coronavirus infections among domestic cats. Identification of genes involved in phenotypic variation of complex traits including infections may contribute to the identification of mechanisms underlying the phenotypes observed [
24]. Finally, strong phenotype/genotype associations may be considered for identification of cats susceptible to massive virus shedding.
In a broader context, the current COVID-19 pandemic raises questions about similarities between coronaviruses infecting different mammalian species including pets. As non-symptomatic SARS-CoV-2 shedders represent a serious epidemiological threat, mechanisms of coronavirus shedding merit attention. Cats have been reported to be susceptible to subclinical infection with SARS-CoV-2, being able to shed the virus through oral and nasal secretions without any clinical signs and to transmit the virus to other cats by direct contact [
25,
26]. Considering our so far rather fragmented knowledge about SARS-CoV-2 infection, it is not clear to what extent human and cat coronaviruses are similar or differ. Information retrieved from animal models including cats can be helpful for the current COVID-19 research [
27].
Therefore, the objectives of this study were (i) to determine patterns of one-year fecal shedding of FECV in a cohort of cats in multi-cat households with different pure breeds, (ii) to define phenotypes suitable for analyzing the genetic variation underlying the variation in the shedding status, (iii) to identify and characterize candidate genes and their polymorphic markers potentially associated with the shedder status and (iv) to test associations between candidate polymorphic markers and FECV shedding in the cohort analyzed.
3. Discussion
The results of association analyses primarily depend on the phenotypes defined. Based on the parameters of specificity and sensitivity, we are confident that our qPCR assay detected cats shedding FECV with sufficient accuracy. All cats studied were from multi-cat households with at least two cats in close contact, sharing the same toilet. Within all groups, at least one cat in each household was shedding FECV. Therefore, we believe that all cats were exposed to infection. The observed dynamics of shedding patterns are in agreement with this assumption, although the infection pressure was necessarily not the same between different catteries. We have observed similar shedding patterns to those identified by Addie et al. [
15] in a similar type of study. In terms of persistency of shedding, different types of shedders along with non-shedders were observed. For cats with three or fewer consecutive samplings positive for FECV (37%), it was not possible to determine the shedder status unequivocally, and therefore, this group was analyzed separately, as a special phenotype. Despite possible methodological variables during sample preparation and processing, the estimated numbers of virus particles in individual samples provided by the qPCR allowed us to distinguish non-overlapping groups of cats, which we analyzed as distinct semi-quantitative phenotypes (
Supplementary Table S8).
Significant associations of candidate genes with both persistency and intensity of shedding were observed. An overall comparison across the two categories of phenotypes showed that a vast majority of the most significant associations observed for individual SNPs and haplotypes as well as for intragenic and intergenic composed genotypes involved single extreme phenotypes, i.e., persistent shedders (PS) and/or high-intensity shedders (HS), on one hand, and non-shedders (NS), on the other hand, in comparison with various other phenotypes or their combinations (
Table 3,
Table 4,
Table 5,
Table 6 and
Table 7). These single phenotypes often shared alleles/genotypes (individual data are available in the Mendeley repository). Based on these results, it seems that the candidate genes studied here influence especially host susceptibility to virus infection expressed by the phenotypes PS and HS, and host resistance manifested as the NS phenotype. Only rarely were significant associations with the candidate genes observed for the remaining “intermediate” phenotypes defined as single phenotypes (as opposed to when merged with other phenotypes). We thus may speculate that the observed variation in shedding patterns could primarily result from a quantitative variation in the intensity of shedding and that there are two major phenotypes associated with different genotypes, i.e., resistant non-shedders and highly susceptible cats permanently shedding high amounts of viral particles. Interestingly, for some SNPs within the
SLX4IP gene, heterozygotes shed fewer virus particles than homozygotes (
Supplementary Table S7), indicating a possible advantage of heterozygosity of the corresponding GWAS candidate genomic region. Cats of average resistance/susceptibility to FECV infection would then represent a range of variation between these two extreme phenotypes, influenced not only by the underlying genetic variation but also by a variety of non-genetic factors, as was observed for infection with FIPV and for clinical FIP [
18]. This assumption is in agreement with the idea of non-existence of intermittent shedders [
16].
Biological interpretations of statistical association studies have several limitations. The cohort analyzed here is a heterogeneous multi-breed group with two prevailing breeds, Maine Coon and British Shorthair, and composed of cats of different ages, some of them related. Pure breeds are under strong pressure of artificial selection and are generally more homozygous than for example crossbred stray cats (
Supplementary Materials Table S1). As such, the group studied is structured, which may have led to false positive results. However, it proved to be impossible to establish a sufficiently large group matching exactly the purposes of this experiment. The composition of the group analyzed was primarily established by breeders willing to participate in such a long-term study. We thus had only limited chances to control breed, age and other factors important for an ideal statistical design. On the other hand, a comparison with a group of stray cats, which are supposed to represent a wide range of intraspecies variation, showed that for associated markers, their minor allele frequencies (MAFs) were mostly similar in both groups, including the genes involved in the most significant associations. Nevertheless, we cannot exclude that some of the associations presented here are false positives. Although most of the associated markers were primarily in Hardy–Weinberg equilibrium (HWE), some of them were not. However, these differences in HWE concerned different SNPs within the same genes; most associated SNPs were in HWE except
NCR2 (
Supplementary Tables S2 and S3). Although no significant associations were observed for this gene after corrections, in composed genotypes, it clearly contributed to associations, increasing the significance of the
p values of its respective partners by one order of magnitude (
Table 7,
Supplementary Table S6).
We were confronted with a rather complex situation for MHC class II genes, which did not allow us to analyze associations of
DRA-DRB haplotypes. There are at least two
DRA loci and several
DRB loci in cats. Non-distinction of different
DRB loci, undetermined
DRB genotypes, and low numbers of cats of several different breeds were probably reasons why Addie et al. [
17] found no associations of FECV shedding with MHC. Similarly, our primers proved to amplify two different
DRA loci, with one, two, three or four alleles observed per individual, and we did not succeed in designing
DRA locus-specific primers. On the other hand, we are confident that our
DRB primers were locus-specific, with one or two alleles observed per individual. In this situation, composed MHC
DRA/DRB genotypes were not informative.
Despite all of these limitations, we are confident that it is reasonable to suggest that at least some of these SNP associations indicate biologically plausible effects of specific genes on the shedder phenotypes. All of them remained highly significant even after overconservative Bonferroni corrections, some of them at
p values exceeding 0.01 by orders of magnitude. Some of them could be confirmed on even smaller numbers of cases by a within-breed analysis, reducing some of the biases. The most significant association (
p < 0.0061) was found in the British Shorthair subgroup of cats for
NCR1, where a SNP (#3093) was associated with differences between non-shedders/low shedders and medium/high shedders (
Supplementary Materials Table S2).
Associations observed for composed SNP genotypes showing functional synergies, especially between the NCR1 and NCR2 genes, as well as strong associations of physically linked genes also support the hypothesis of their biological plausibility.
This hypothesis is compatible with known biological roles of functional candidates. MHC class I and II loci were associated with different coronavirus infections and their outcomes in poultry [
28]. Associations with MHC loci and mortality in COVID-19 was recently reported [
29].
The natural cytotoxicity receptors on NK cells have different functions in important biological processes, such as virus immunity, cancer immunity and human pregnancy [
30]. The product of the human
NCR1 gene (NKp46) was shown to recognize viral hemaglutinins of various virus families, including influenza, Sendai virus, poxvirus and Newcastle disease virus [
31]. NK cells seem to play important roles in human coronavirus infections of zoonotic origin [
32], and they also seem to be important for FIPV infection of cats [
33,
34]. Our data suggest that NK cells might be important for controlling the process of FECV shedding as well.
On the other hand, the positional/functional candidates represented a kind of positive control of this experiment; the results obtained indicate that they are not false positives. Although they were selected based on previously reported associations of the chromosome regions, where they are located, with clinical FIP [
21], the positional candidates
PCSK2, SNX5 and
SLX4IP showed significant associations with FECV shedder phenotypes as well (
Table 1,
Table 2,
Table 3,
Table 4 and
Table 5,
Supplementary Tables S4 and S5). Most of these loci were in HWE (
Supplementary Tables S4 and S5, information about all SNPs are in Mendeley repository). Based on their physical positions in the cat genome assembly FelisCatus v. 9.0 [
35] and on the
p values of their associations with the phenotypes studied, we could tentatively outline the candidate region on chromosome A3 (
Figure 1).
The physiological functions of these primarily positional candidates do not contradict the hypothesis of their involvement in mechanisms of response to a viral infection. It was shown that the activation of the SLX4 complex during HIV infection may modify innate immune sensing and interferon production [
36]. SNX5 is involved in macropinocytosis and antigen processing in macrophages [
37]. The role of PCSK2 is unclear; it is a serine protease expressed in enterocytes, and coronavirus spike proteins require cleavage when entering the cell [
38].
In summary, we have observed similar patterns of fecal FECV shedding in multi-cat catteries to those previously reported by other authors. So far, only genes associated with clinical disease induced by FIPV have been identified, and no information about genes underlying the variation in FECV shedding has been available. For this purpose, we have developed polymorphic functional and positional candidate gene markers, and for some of them, we have observed their non-random distribution amongst different categories of FECV shedders and non-shedders. The majority of highly significant associations were observed for extreme phenotypes. However, the phenotypes analyzed in this study are complex, as they are influenced by multiple factors, including re-infections; nonetheless, differences between high-intensity shedders, persistent shedders and non-shedders compared to various combinations of other phenotypes were distinct for several markers.
The data presented here contributed to a better characterization of shedders, the most important source of infection in multi-cat households. Known biological functions of genes associated with FECV shedding in this study support the hypothesis of the biological plausibility of the selected functional candidates. The results suggest that innate immunity and especially the role of NK cells in cat coronavirus infections, not yet studied in this context, merit further attention. Associations of positional candidate markers, i.e. SNPs located within a genomic region identified by a GWAS as a candidate for FIPV susceptibility, suggest that at least some of the immune mechanisms involved in the control of infection with FIPV also operate in the control of FECV shedding, which is a hypothesis that we are currently testing.