key: cord-0882559-agcq0xjs
authors: Fritz, Matthieu; Nesi, Nicolas; Denolly, Solène; Boson, Bertrand; Legros, Vincent; Rosolen, Serge G.; Briend‐Marchal, Alexandra; Ar Gouilh, Meriadeg; Leroy, Eric M.
title: Detection of SARS‐CoV‐2 in two cats during the second wave of the COVID‐19 pandemic in France
date: 2021-10-26
journal: Vet Med Sci
DOI: 10.1002/vms3.638
sha: adbe56b0d9eb088d2d6e3aa3fc36a87941e2ca97
doc_id: 882559
cord_uid: agcq0xjs

Although there are several reports in the literature of SARS‐CoV‐2 infection in cats, few SARS‐CoV‐2 sequences from infected cats have been published. In this study, SARS‐CoV‐2 infection was evaluated in two cats by clinical observation, molecular biology (qPCR and NGS), and serology (microsphere immunoassay and seroneutralization). Following the observation of symptomatic SARS‐CoV‐2 infection in two cats, infection status was confirmed by RT‐qPCR and, in one cat, serological analysis for antibodies against N‐protein and S‐protein, as well as neutralizing antibodies. Comparative analysis of five SARS‐CoV‐2 sequence fragments obtained from one of the cats showed that this infection was not with one of the three recently emerged variants of SARS‐CoV‐2. This study provides additional information on the clinical, molecular, and serological aspects of SARS‐CoV‐2 infection in cats.

SARS-CoV-2 has shown relatively generalist capacities by infecting many animal species, making it a good model in One Health research (MacLean et al., 2021) . Indeed, SARS-CoV-2 infections have been detected in numerous animal species living in close contact with infected humans. Based on RNA detection, serological studies, and experimental infections, numerous animal species have proven susceptibility to SARS-CoV-2 , including Mustelidae (ferret and mink) (Oude Munnink et al., 2020) , Canidae (dog) ,

Felidae (cat, tiger, and lion) (Sailleau et al., 2020) , and Cricetidae (hamster, rat, and mouse) (Sia et al., 2020) as well as by the highly transmissible British variant (B.1.1.7) (Ferasin et al., 2021) . On 24 March, the World Organisation for Animal Health reported cases of SARS-CoV-2 infections in cats in 17 countries (the United States, China, Belgium, Germany, Spain, France, Russia, the United Kingdom, Japan, Italy, Chile, Brazil, Greece, Canada, Argentina, Switzerland, and Latvia). In the medical and scientific literature, we found 23 papers examining natural SARS-CoV-2 infection in a total of 2242 cats Carlos et al., 2021; Chen et al., 2020; Deng et al., 2020; Ferasin et al., 2021; Fritz et al., 2021; Garigliany et al., 2020; Hamer et al., 2020; Hosie et al., 2020; Klaus et al., 2021; Michelitsch et al., 2020; Musso et al., 2020; Neira et al., 2020; Newman et al., 2020; Pagani et al., 2021; Patterson et al., 2020; Ruiz-Arrondo et al., 2020; Sailleau et al., 2020; Segalés et al., 2020; Stevanovic et al., 2020; Temmam et al., 2020; Villanueva-Saz et al., 2021; Zhang et al., 2020 

To enable cost-effective sequencing of RNA samples, we depleted the eukaryotic rRNA using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat). After rRNA depletion, double-stranded cDNA was synthesized by real time reverse-transcription PCR (RT-qPCR) using Superscript III platinum One-step Quantitative RT-PCR System (Invitrogen) as described previously with minor modifications (Corman et al., 2020) . Briefly, a 25 µl reaction contained 5 µl of RNA, 12.5 µl of 2× reaction buffer, 1 µl of reverse transcriptase/Taq mixture, 0.4 µl of a 50 mM magnesium sulphate solution (Invitrogen), 1 µl of a 10 µM primer, and 0.5l µl of a 10 µM probe. Thermal cycling was performed at 50 • C for 15 min for reverse transcription, followed by 95 • C for 2 min and then 45 cycles of 95 • C for 15 s and 60 • C for 30 s using a Light Cycler 480 (Roche).

To increase the amount of genetic material available for sequencing, whole transcriptome amplification (WTA) was performed with Quan-tiTect Whole Transcriptome kit (QIAGEN) according to the manufacturer's instructions. Amplified DNA reactions was then purified using AMPure XP beads and quantified using Qbit dsDNA BR Assay Kit on a Qubit 3.0 fluorimeter (Invitrogen).

In complement to the de novo approach, we also used an adapted version of the published protocol from the ARTIC Network using ARTIC (Quick, 2020) primer scheme version 3, which produces ∼400 bp overlapping amplicons over the SARS-CoV-2 genome.

Oxford Nanopore Technology library preparation and MinION sequencing Libraries were prepared without shearing to maximize sequencing read length. The Oxford Nanopore Technology (ONT) protocol for native barcoding genomic DNA sequencing was followed using the barcoded ligation sequencing kit SQK-LSK108 and the EXP-NBD104 Native 

Following the MinION run, reads generated were basecalled and subsequently demultiplexed using Guppy GPU basecaller and barecoder (Oxford Nanopore Technologies). Reads were then mapped against a custom reference of SARS-CoV-2 genome comprising four Chinese and 70 early French sequences using Bowtie2 (Langmead & Salzberg, 2012) and minimap2 (Li, 2018) . Finally, a consensus genome sequence based on mapped reads was generated with BCFtools consensus (Li, 2011) .

SARS-CoV-2 sequences were deposited on GISAID (EPI_ISL_1328819; EPI_ISL_1328821; EPI_ISL_1328824; EPI_ISL_1328826).

Cat serum samples were tested using a multiplex microsphere immunoassay (MIA). Note that 10 µg of three recombinant SARS-CoV-2 antigens nucleoprotein (N), receptor-binding domain (RBD), and trimeric spike (tri-S) were used to capture specific serum antibodies, whereas a human protein (O6-methylguanine DNA methyltransferase) was used as a control antigen in the assay. Distinct MagPlex microsphere sets (Luminex Corporation) were respectively coupled to viral antigens using the amine coupling kit (Bio-Rad Laboratories) according to the manufacturer's instructions. The MIA procedure was performed as described previously . Briefly, microsphere mixtures were successively incubated with serum samples (1:400) biotiny-lated protein A and biotinylated protein G (4 µg/ml each) (Thermo Fisher Scientific), and Streptavidin-R-Phycoerythrin (4 µg/ml) (Life Technologies) on an orbital shaker and protected from the light. Measurements were performed using a Magpix instrument (Luminex). To account for non-specific binding of antibodies to beads, relative fluorescence intensities (RFI) were calculated for each sample by dividing the median fluorescence intensity (MFI) signal measured for the antigen-coated microspheres by the MFI signal obtained for the control microspheres. Specific seropositivity cut-off values for each antigen were set at three standard deviations above the mean RFI of the 18 dogs and 14 cat serum samples sampled before 2019. Based on a pre-pandemic population, MIA specificity was set at 100% for dogs and cats.

An MLV-based pseudoparticle carrying a GFP reporter pseudotyped with SARS-CoV-2 spike protein (SARS-CoV-2pp) was used to measure the neutralizing antibody activity in cats' sera. Each SARS-CoV-2 positive sample detected by MIA was processed according to a neutralization procedure as previously described . The level of infectivity is expressed as the percentage of GFP-positive cells and compared to cells infected with SARS-CoV-2pp incubated without serum. Pre-pandemic cats' sera from France was used as negative controls, and a commercial anti-SARS-CoV-2 RBD antibody (Sino Biological) was used as a positive control.

Cat 1 was a solitary and sedentary 5-year-old female of European origin whose only contact was with her owner. Her last vaccination was 3 years prior and she was presented with no previous medical history.

On 24 by RT-qPCR targeting gene E with a Ct-value of 21.43 (Table 1) . We did not obtain a SARS-CoV-2 sequence due to the sample's poor conservation condition prior to its arrival at the lab. To detect anti-SARS-CoV-2 IgG antibodies, the serum of cat 1 was analyzed using MIA and retrovirus-based pseudoparticle assay. Antibodies against N, RBD, and tri-S SARS-CoV-2 proteins were detected as well as a robust neutralization, allowing over 90% neutralization in the SARS-CoV-2pp assay confirming productive infection in cat 1 (Table 1) .

Cat 2 (Table 1) .

No viral RNA was detected in the rectal swabs. Again, due to poor conservation of the swabs prior to their arrival in our laboratory, only five partial fragments of the SARS-CoV-2 genome could be obtained by high-throughput sequencing on RNA derived from the oropharyngeal swabs. In fragment 2, we did not observe the 11288-11296 deletion characterizing three variants of concern (English B1. 

Here, we report SARS-CoV-2 infection in two cats, diagnosed by molecular and serological assays and with mild clinical manifestations, including sneezing for one cat. Although we cannot definitively rule out infection of the cats by an individual outside the household, the information given by COVID-19-positive owners, including the exclusive and unique contact with its owner for cat 1 and the general deterioration in the condition of all cats of the cat 2 owner, strongly suggests a transmission from owners to cats.

Human-to-cat transmission is now reported worldwide, and while certainly not insignificant in COVID-19-positive households, precise estimates of the frequency of such transmission has not been established Hamer et al., 2020) . To our knowledge, among the 23 papers investigating possible SARS-CoV-2 infection in cats, only 14 reported PCR results confirming infection in 27 cats Carlos et al., 2021; Ferasin et al., 2021; Garigliany et al., 2020; Hamer et al., 2020; Hosie et al., 2020; Klaus et al., 2021; Musso et al., 2020; Neira et al., 2020; Newman et al., 2020; Pagani et al., 2021; Ruiz-Arrondo et al., 2020; Sailleau et al., 2020; Segalés et al., 2020) . The present study is only the second showing molecular results of cat infection in France, 8 months after the first case reported by Sailleau et al. (2020) . It appears that the majority of infections in cats are asymptomatic, but can, at times, lead to lethargy, mild respiratory or digestive disease and, rarely, acute respiratory clinical signs (as observed in the two cats) (Garigliany et al., 2020; Musso et al., 2020) . We report here one case of mild respiratory clinical signs (sneezing) in a cat, an observation consistent with symptomatic infections sporadically observed in other studies. We did not observe any digestive clinical signs, as previously reported in a French cat (Sailleau et al., 2020) . In accordance with other recent studies, our results suggest that since the beginning of SARS-CoV-2 pandemic, almost 1 year after the first report of infection in a cat, the pathology in cats has not changed globally, with only a relatively small proportion of cases reported as a result of clinical investigation by a veterinarian. This low pathogenicity can explain the paucity of studies reporting SARS-CoV-2 infection in cats in the absence of a global pet detection policy.

The two cats in the study were sampled during the second wave of infection in France and at the beginning of the ongoing emergence of multiple novel variants. Although we did not find evidence of infection by one of the three novel variants (B.1.1.7, B.1.351, and P.1) in cat 2, the emergence of these new variants raises the question of potential changes in pathogenicity or transmissibility in domestic animals. This question will become rapidly crucial in a very near future as the British variant, known to be much more infectious, is currently removing the ancestral variant of SARS-CoV-2 in France as well in other countries of Europe. Therefore, it is becoming more and more important to implement a One Health approach to face SARS-CoV-2 epidemic that takes into account infection and viral circulation in pets.

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We are grateful to the pet owners for giving us their permission to sample their pets. We thank veterinarians that helped us with sampling.We thank Estelle Leperchois, Simon Thierry, and Trung Thanh Nguyen for technical support and assistance. We are grateful to François-Loïc