4 DISCUSSION
Cats are popular companion animals in close contact with humans, and therefore, it is important to gain insight into their susceptibility to SARS-CoV-2 and their possible role in COVID-19 epidemiology. The longitudinal monitoring of the cats in the present study indicates that, after the owner introduced the virus in the house environment, transmission occurred first to C2 and then to C3, while C1 was not infected. No clinical signs were observed in any of the infected cats and hematology and serum biochemistry values were within normal limits. These findings support previous experimental infection/transmission studies (Gaudreault et al., 2020; J. Shi et al., 2020), as well as the limited reports of clinically affected cats around the globe, indicating that following exposure to SARS-CoV-2, cats are not invariably infected, and when infection is established, the course is mostly asymptomatic. Cats may thus be silent hosts of SARS-CoV-2, as they may not show any appreciable symptoms that might be recognized by their owners (Halfmann et al., 2020). It is possible that the outcome in companion animals may depend on several factors, such as the proximity of contact with humans shedding high viral loads, possible co-morbidities, increased susceptibility to the virus (e.g. age-dependent), or a combination of these factors (de Morais et al., 2020; J. Shi et al., 2020).
It has been reported that infected cats shed the virus for no more than 5 days following exposure (Bosco-Lauth et al., 2020; J. Shi et al., 2020). The fact that viral RNA was detected in the pharynx of both infected cats of the present study, from D7 until D13, indicates that some cats may have a more prolonged shedding period. Interestingly, serological testing of C2 against SARS-CoV-2 was negative at all sampling time-points. As previously suggested for infected humans, the absence of seroconversion may be indicative of mild infections involving only the respiratory mucosa, where secretory immune responses dominate, in conjunction with limited systemic IgG production (Staines et al., 2020). For this reason, the amount of virus-specific secretory IgA in the respiratory mucosa could serve as an indicator of immune response in humans and in susceptible animal species (Chao, Rötzschke, & Tan, 2020). Another possible explanation for this condition is that exposure to SARS-CoV-2 can induce T cell-mediated virus-specific responses, without seroconversion, similar to what has been suggested for humans (Gallais et al., 2020; Staines et al., 2020). The fact that C2 did not seroconvert to SARS-CoV-2 may account for the continued presence of the virus in its pharynx, in low titers with a slightly increasing pattern (D10-D13), after the initial decline which was observed between D7 and D10.
Fecal swabs obtained from both infected cats tested positive via real-time RT-PCR. A scenario of swallowing expectorated virus followed by a progressive tapering of SARS-CoV-2 viral load could interpret the presence of viral RNA in fecal swabs. Swallowed virus-laden sputum could either passively pass through the intestine of these animals, or the virus could replicate in it. Rapid elimination of SARS-CoV-2 from the intestinal tract of cats has been reported in a case study (Sailleau et al., 2020). This rapid clearance has been also described in an experimental infection/transmission of cats, wherein SARS-CoV-2 titers as high as ~105 viral RNA copies/g were measured in the feces of experimentally infected and exposed cats (J. Shi et al., 2020). In contrast to the aforementioned observations, the detection period of SARS-CoV-2 in the fecal swabs obtained from C3 of the present study was over 6 days long. The course of viral RNA concentrations in fecal swabs of this animal seemed to reflect the course in the oropharyngeal swabs, with a peak virus titer of ~6.5 Log10 viral RNA copies/swab being measured on D9 (i.e. 2 days after the first real-time RT-PCR-positive fecal swab). A similar SARS-CoV-2 titer kinetics pattern in stool compared to sputum has been observed symptomatic COVID-19 human patients (Wölfel et al., 2020). However, despite the fact that the observed virus titers in the stool of human patients exceeded 7 Log10viral RNA copies/swab, isolation of SARS-CoV-2 from feces was unsuccessful. Virus isolation results from viral RNA-positive small intestines of experimentally infected cats were also negative (J. Shi et al., 2020). Further research is required to thoroughly investigate the factors sustaining the presence of SARS-CoV-2 in the gastrointestinal tract of cats with or without clinical signs, as well as the infectivity of the virus shed to the environment via feces.
Coronaviruses are capable of adapting quickly to new hosts through the processes of genetic recombination and mutation in vivo (Boni et al., 2020; Z. Shi & Hu, 2008). Through the combined analysis of SARS-CoV-2 whole genome sequences obtained from the cats and their owner, changes in the viral genome indicating cross-species adaptation of the virus were investigated. Our results support the notion that human SARS-CoV-2 strains are relatively well-adapted to feline hosts and cross-species adaptation is not initially required for the efficient virus replication. Whole genome sequencing of the detected SARS-CoV-2 strains revealed a T-to-C SNP in the nucleotide position 24544 in the oropharyngeal swab from C3. The fact that the difference between the C variant ratio in the oropharyngeal and in the fecal swab of the same cat was statistically significant is indicative of SARS-CoV-2 proliferation in this animal’s intestine. Additionally, the presence of a significant fraction of 24544T reads in the oropharyngeal sample of C3 supports the notion that the transition from T to C was in the course to become dominant in the pharynx of this animal. Due to the high similarity among the viral genomes obtained from the owner and the cats, it cannot be ascertained whether the virus was transmitted directly from the owner to both cats, or that transmission from C2 to C3 took place. However, taking into consideration the chronological order of the real-time RT-PCR positivity in the two cats, as determined by the kinetics in virus titers, and the fact that the C variant in position 24544 was not observed in C2, it is suggested that the owner was the source of infection for C3 as well. This hypothesis is also supported by the very close contact of each cat with their owner, and is in agreement with reports available so far that denote the human-to-feline transmission of the virus. Evidence-based support in favor of a cat-to-human SARS-CoV-2 transmission is not currently available, and this specific report does not match the essential criteria to demonstrate cat-to-human transmission (de Morais et al., 2020; Totton, Sargeant, & O’Connor, 2020).
The absolute overlap of amino-acid sequences among the sequenced viral strains signifies the absence of emergence of cross-species adaptive mutations. In a previous experimental infection study, SARS-CoV-2 variants possessing the S-H655Y amino-acid substitution have been reported to rapidly arise and become fixed following transmission between cats, suggesting that this site may be under positive selection in feline hosts (Braun et al., 2020). This substitution was not found in any of the cat samples which were sequenced in the present study. The S-D614G amino-acid substitution was present, which is characteristic of the GISAID G and G-related clades and has been reported extensively in the past, as linked to increased SARS-CoV-2 infectivity and transmissibility (Hou et al., 2020). This substitution, along with the amino-acid substitution NSP12-P323L, which was also identified herein, have been previously retrieved from human isolates originating from all continents of the earth and seem to co-evolve (Coppée, Lechien, Declèves, Tafforeau, & Saussez, 2020). The rare variant S-V1104L was also identified in the present report. This amino-acid substitution has occurred independently before, in isolates belonging to three other B1 lineages (GISAID EPI ISL 539372 - Switzerland, B.1.160; GISAID EPI ISL 722879 - Italy, B.1.177; GISAID EPI ISL 582648 - United Arab Emirates, B.1.1.1). The E19G amino-acid substitution in ORF3a is possibly indicative of the phylogenetic origin of the detected strains, as it was present only in one cluster of isolates within B1.1 lineage.
Regarding C1, the mild diarrhea signs were transient and completely subsided within a few days. This condition was reliably noticed by the owner, a veterinary medicine student, who was worried about the possibility that the diarrhea could be due to SARS-CoV-2 infection, and thus notified us. The findings from the virological and serological testing showcase that the diarrhea observed in C1 cannot be attributed to SARS-CoV-2. The cause of the diarrhea signs could be linked to factors associated with the animal’s diet, or less likely, to other infectious or parasitic agents. Results further support previous observations indicating that companion animals are not easily infected by SARS-CoV-2, even when they are in close contact with infected owners (Temmam et al., 2020). Additionally, our findings highlight difficulties in spontaneous cat-to-cat SARS-CoV-2 transmission, given that C1 was in continuous proximity to C2 and C3 during the peak of the viral shedding from them. The difficulty in cat-to-cat transmission is also supported by the findings of the previously discussed experimental infection/transmission study (J. Shi et al., 2020). The S-D614G amino-acid, which was identified in the viral strains of the present study, has been linked with increased SARS-CoV-2 replication in the upper airway of hamsters, suggesting its possible role in viral transmissibility (Plante et al., 2020). However, despite the prolonged contact of C1 with the other two infected cats and their owner, this cat remained uninfected.
In conclusion, the present study comprises the first report of SARS-CoV-2 natural infection of cats in Greece. The owner acted as a source of the virus for the animals, as COVID-19 symptoms started one week before the first detection of the virus in the cats, confirming once again the reverse zoonotic character of SARS-CoV-2 transmission. Our results support the notion that human SARS-CoV-2 strains are relatively well-adapted to cats, despite the absence of emergence of cross-species adaptive mutations. It is still unclear whether the asymptomatic animals could play a role in COVID-19 epidemiology, in case of interaction with naïve animals and/or people. Both infected cats and their owner were not able to transmit the virus to a third cat living in the same place, despite their very close contact and the high SARS-CoV-2 titers measured in the pharynx of the infected cats on the first sampling time-point. It remains to be elucidated whether infected cats shed infective virus to the environment, and until then, keeping infected cats inside is a reasonable suggestion. Besides the risk of possible transmission of SARS-CoV-2 from infected cats to humans, it is advisable for COVID-19 patients or asymptomatic virus carriers to avoid contact with cats, in order to minimize the possibilities of infecting them.