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.