A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modeling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT-PCR, an ELISA test, viral sequencing, and virus isolation. On May 5th, 2020, the cat-owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred.

The ithree institute -University of Technology Sydney, Sydney, Australia. (B. Brito) 1 9 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.31.20220608 doi: medRxiv preprint 1 0 1 humans in the studied household -a male of around 30 years old and a female of around 60 1 0 2 years old. The residents owned three 10-years-old sibling cats, 1 male (cat 1) and 2 females (cat  Humans and cats were recruited to this case study. From humans, nasal swabs, sputum, and fresh 1 0 5 fecal samples were collected at intervals of 4 to 7 days. From cats, nasal swabs and fresh fecal 1 0 6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted November 4, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020 samples were collected at intervals of 1 to 3 days until rtRT-PCR was negative for SARS-CoV-2. At day 0 (first sampling date) a nasal swab was collected from cat 1 and fresh feces were 1 0 8 collected from all 3 cats. Cat 1 tested positive for SARS-CoV-2 by rtRT-PCR in both nasal 1 0 9 (Ct=31) and fecal sample (Ct=33), and cat 2 and 3 tested negative. In cats, at the last sampling, 1 1 0 serum samples were also collected. Nasal samples were collected using flocked swabs; fresh 1 1 1 feces were stored in ziplock bags(13-15) and deposited in viral transport media. The owners provided written consents at the time of the sample collection. For all the 1 1 3 study, human sampling was performed by nurses and animal sampling was performed by CA, USA) following the instructions recommended by the manufacturer and eluted into 50 µL. The extracted RNA was subjected to rtRT-PCR to amplify a portion of ORF1b SARS-CoV- 2(16). This PCR was previously validated at both laboratories independently, and is one of the 1 2 7 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.31.20220608 doi: medRxiv preprint RT-PCRs recommended by the WHO(17) to diagnose COVID-19. Positive and negative controls 1 2 8 for PCR and extraction were included in each run.  Sinai, New York. Cells were cultured in minimum essential medium eagle 10% fetal bovine 1 3 6 serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. Cells were seeded in 12-well culture 1 3 7 plates and cultured at 37°C with 5% CO 2 for 24-48h. Cells were rinsed and inoculated with 300 negative controls. Cells were incubated at 37°C with 5% CO 2 and monitored daily for cytopathic 1 4 0 effect (CPE) for five days. Cell cultures with no CPE were frozen, thawed, and subjected to three 1 4 1 blind passages with inoculation of fresh Vero E6 cell cultures with the lysates as described cycle threshold values and inclusion of all cats and humans. The whole genome was amplified 1 4 8 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.31.20220608 doi: medRxiv preprint with a modified version of the Artic Consortium protocol(19) with custom tiling primers as Illumina MiSeq instrument (2x150 nt). SARS-CoV-2 genomes were assembled and annotated 1 5 3 using a reference-based analysis pipeline as previously described (20). Six SARS-CoV-2 genome sequences were analyzed. These sequences were obtained 1 5 6 from the clinical nasopharyngeal specimens of two humans that reside in the household: Human 1 (hCoV-19/Chile/Santiago/op31d5/2020; Human 1) and Human 2 (hCoV- 19/Chile/Santiago/op32d10/2020; (Human 2), and four sequences from their cats -two of these 1 5 9 were collected from nasal swabs of cat 2, and a nasal sample from cat 3. Completeness of v1.26(24). Duplicate sequences were removed and non-coding regions, 5' and 3' UTR were 1 6 8 trimmed. A phylogeny was estimated using the maximum likelihood method available in IQ-1 6 9 TREE v1.6.7 employing the GTR+F+I+Γ 4 model of nucleotide substitution with 1000 bootstrap 1 7 0 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10. 1101/2020 replications. The phylogenetic tree was visualized using Figtree. SARS-CoV-2 lineages were 1 7 1 identified using the Pangolin COVID-19 Lineage Assigner web application(25). To determine the duration and dynamics of SARS-Cov-2 RNA detection in cat excretions CoV-2 RNA in nasal and fecal samples from three companion cats over a 40-day time period. The cats acquired the SARS-CoV-2 infection naturally (rather than experimentally) and were CoV-2 RNA over the time period in which the cats were sampled. All three cats had detectable  in the human samples up to 25 days after the first RNA detection. The dynamics of detectable 1 9 0 viral RNA in the cat samples were more erratic compared to that for detectable viral RNA in 1 9 1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.31.20220608 doi: medRxiv preprint humans from the same household. For example, a female cat (cat 2) presented an intermittent 1 9 2 RNA detection for at least 17 days (Supplementary information).

9 3
To assess the infection of the cats and humans, we made clinical observations for 1 9 4 symptoms of SARS-CoV-2 infection and performed serological testing. Cat 1 presented with 1 9 5 mild clinical signs including dullness, lethargy, and coughing without fever. Cats 2 and 3 were 1 9 6 asymptomatic. On the last sampling on day 33 after first detection on cats, cat 2 tested positive 1 9 7 for SARS-CoV-2 antibodies by ELISA. Both humans displayed COVID-19 symptoms including 1 9 8 fever, lethargy, and coughing. However, the female human was more clinically affected and was  Multiple attempts to isolate the virus from low Ct (high levels of SARS-CoV-2 RNA; 2 0 2 21.9 -24.1) samples were made without success. Of the specimens sent for genome sequencing one had coverage of 69·3% (human 2, day 2 0 5 10, nasopharyngeal) and five had a genome coverage >98% (three nasal swabs from cat 2, one 2 0 6 nasal swab from cat 3, and one nasopharyngeal from human 1) (Appendix Figure). The 2 0 7 remaining sequenced samples had a coverage <50% and were not included in the analyses.  CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10. 1101/2020 We conducted a phylogenetic analysis to investigate any relationship between the SARS-2 1 3 CoV-2 RNA excreted by different cats and humans within the household. All six sequences 2 1 4 obtained fell into a monophyletic group defined by a C25344T (S1261F/Spike) nucleotide 2 1 5 substitution ( Figure 2). Notably, there were no nucleotide differences between the viral 2 1 6 sequences obtained from cats and one of the household humans (hCoV-2 1 7 19/Chile/Santiago/op31d5/2020; Human). The viral sequence from the female human (hCoV- of the sequences (C11511T/T3749I/nsp6) and (C13740T/syn/RdRp) but fell in the same group. An epidemiologically unrelated Chilean SARS-CoV-2 sequence (GISAID accession number 2 2 1 EPI_ISL_468748) collected in the same city on April was also grouped within this cluster of 6 2 2 2 sequences. CoV-2 antibodies detected by ELISA confirmed the infection in cat 2. Our household study thus 2 3 1 provides preliminary guidance for managing the care and quarantine of cats in households in 2 3 2 which SARS-CoV-2 is present. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted November 4, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020 The results, particularly the similarity of genome sequences depicted in the phylogenetic 2 3 4 analysis, strongly support the idea that SARS-CoV-2 can be transmitted between humans and 2 3 5 cats living in the same household. It is suspected that the humans were infected following 2 3 6 exposure to SARS-CoV-2-positive neighbors days before. We think it likely that cat 1 was the 2 3 7 first infected of the three cats because this animal had closer contact than the other two cats with 2 3 8 human 1 (male), sharing his bed. This is supported by our observation that viral RNA from Whether all cats in the study household were infected from humans, or whether some 2 5 2 transmission occurred between animals, cannot be determined. Nevertheless, the possibility of 2 5 3 some transmission between cats in the household is suggested by the 4-day delay in cats 2 and 3 2 5 4 testing positive to SARS-CoV-2 by rtRT-PCR compared to cat 1. All three cats had direct 2 5 5 contact with each other. Transmission between cats in the household would be consistent with 2 5 6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.31.20220608 doi: medRxiv preprint the findings of Shi et al. (2020) who studied transmission between cats in an experimental 2 5 7 setting 7 . In that study, inoculated and infected sub-adult cats were placed next to susceptible cats 2 5 8 in an adjacent cage (non-direct contact). Of the three replicates, one exposed cat gave rise to a 2 5 9 positive rtRT-PCR on the 5th day post-exposure(7). To date, however, we have only identified 2 6 0 three rtRT-PCR-positive cats, with one of these also positive by ELISA, and one cat of another 2 6 1 household similarly positive by ELISA. Consequently, it is likely that cat-to-cat transmission 2 6 2 events are not as common as human-to-human transmission. In summary, at present, there is insufficient data to identify and prioritize the drivers for shorter patterns of excretion for SARS-CoV-2 RNA compared to humans. These results