An in vitro study regarding an animal model for the attenuation of SARS-CoV-2: Are cats possible candidates?

Although the origin of the SARS-CoV-2 pandemic is not denitively known, this virus is seen that adapted to human. It is known yet, several animal species are naturally susceptible for SARS-CoV-2, including pets. In this in vitro study, it was investigated whether cats could have a host status in this infection. It was also following questioned, whether cats could be an animal model for vaccine attenuation. Felis catus whole fetus 4 (FCWF-4) and Vero cells were used in this study for propagation of SARS-CoV-2-Ank1 strain. Virus replication was controlled by immunohistochemistry and real time pcr techniques. While an increase in virus titer was detected in the serious passages made in Vero cells, no increase was observed in the FCWF-4 cell. The results were conrmed by immunohistochemistry. Due to the fact that SARS CoV 2 is a zoonotic potential, its presence in domestic animals frequently investigated in current studies. Cats can get this virus from infected owners with whom they share the same habitat. The results of this study also showed that cats are not candidates to be a natural source of infection for SARS-CoV-2.


Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which has infected 136 million people worldwide (Zhou et al., 2020;WHO, 2021), has recently been reported to cause infection in cats (Shi et  The human-to-animal transmission of SARS-CoV-2 has been proven (Pagani et  To determine the growth kinetics of the viruses in the FCWF-4 cell lines, cells were infected at a multiplicity of infection (MOI) of 0.1. After 1 h of adsorption at 37°C, cells were washed twice with the medium and incubated at 37°C with 5% CO2. After incubating for 1 h, the infectious media were replaced with fresh media (DMEM). Infected cells were incubated for 4-5 days and observed daily via an inverted microscope to inspect any virus-induced cytopathogenic effects. The infected cells were then subjected to two additional rounds of freezing and thawing at 37°C for 2-3 min. The supernatant was collected after centrifugation at 3,000 × g for 15 min at 4°C to remove any dead cells. The virus was collected from clari ed cell culture supernatants and aliquoted and frozen at − 80°C until use. The SARS-CoV-2-Ank1 strain used to infect the cells was provided by the Biosafety Level 3 Plus (BSL3+) facility of the Virology Department, Faculty of Veterinary Medicine at Ankara University (Hanifehnezhad et al., 2020).

Controlling Viral Replication
Real-time PCR and immunohistochemical methods Viral RNA was extracted using the EZ-RNA Total RNA Isolation Kit (Biological Industries, Israel) according to the manufacturer's instructions. The nal RNA pellet was resuspended in 100 µL of sterile water and used for RT-PCR. The ampli cation was performed using a probe-based Quantinova Pathogen reaction mix (Qiagen, Cat No./ID: 208654, USA) with primers (SF1 and SR1) and a probe (SPr) ( Table 1). Reaction conditions were adjusted as described by the manufacturer. Table 1 Primer used in realtime-PCR for diagnosis of SARS-CoV-2 infection.

Results
The SARS-CoV-2-Ank1 strain did not show any cytocidal effect on FCWF cells from the rst cell culture passage (Fig. 1). This was con rmed by immunohistochemical staining (Fig. 1). The virus yield in parallel cultivation studies showed signi cant (p = 0.022) variations indicating FCWF-4 cells produced fewer SARS-CoV-2-Ank1 particles when compared to those in Vero E6 cells (Fig. 2).

Discussion
Although the origin of the SARS-CoV-2 pandemic is not de nitively known, it is reported to have an animal source (a bat or pangolin) and is therefore accepted as a zoonotic infection ( Furthermore, some studies have not clari ed whether these positives are caused by SARS-CoV-2 or by cross reactions resulting from other coronaviruses that are often found in pet animals. However, the above-mentioned serological studies commonly reported that pet animals have a very low risk of infection. According to veterinarians, contact with pets does not appear to be a risk factor for SARS-CoV-2 infection. In this study, an FCWF cell line including the ACE2 receptor was infected with a human SARS-CoV-2 isolate obtained in Turkey. By performing serial cell culture passages, it was determined via real-time PCR that the viral load gradually decreased logarithmically. In addition, the infected cells were stained using an immuno uorescent conjugate. A total of 5 cell culture virus isolation studies conducted after the rst passage did not yield any positivity with the immuno uorescence test, and the positivity obtained by RT-PCR was caused by viral nucleic acid residue. Guo et al. (2008) has reported that feline tissues contain the ACE2 receptor, indicating that cats can be infected with SARS-CoVs. Indeed, SARS-CoV-2 in infected cats has also been detected. However, these few detection studies are based on the presence of the SARS-CoV-2 nucleic acid (Carlos et al., 2021). Moreover, virus isolation studies from cats, which may provide evidence of a productive viral infection, are currently limited (Segalés et al., 2020). These factors lend support to our study's hypothesis and results.
Koch's postulates, as modi ed by Rivers , have been met for the SARS virus, thus con rming the identi cation of an infectious agent. With reference to, it is necessary to isolate the agent from the original infected host, use it to infect a susceptible host and then isolate it from the second host. With this knowledge, it has been reported that domestic cats that share the same habitat with humans can acquire SARS-CoV-2 from their owners and become infected (Klaus et al., 2021). Additionally, the transmission of SARS-CoV-2 between cats sharing the same habitat has been experimentally performed.
However, in domestic environments, no naturally occurring cat-to-cat transmission has been reported to date. Although it is not possible to predict the mutations that may arise if the virus adapts to a different host, research has unambiguously shown that the currently known SARS-CoV-2 viruses are passed on to domestic cats only from humans with whom they share the same habitat. It therefore appears unlikely that the SARS-CoV-2 infection is an amphixenosis that occurs between humans and cats.
In conclusion, although the pandemic caused by SARS- Compliance with ethical standards

Con ict of interest
The authors declare no con ict of interest.