Ethics and animal experimentation
All handling procedures and experiments involving the animal were approved by the Committee for the Ethical Use of Animals in Research of the State Bahia University (nº 2021.005.0018150-89). All procedures involving the animal were carried out in accordance with the ethical and biosafety guidelines.
An eight-year-old male domestic cat of undefined breed was admitted with respiratory syndrome at a veterinary clinic in Barreiras city, Bahia, Brazil. Data regarding respiratory aspects, temperature, heartbeat, weight, and other clinical aspects were kept during anamnesis. Laboratory analyses were required due to the clinical condition of the animal.
Bloods cell counts, biochemical and serological analysis
Complete blood cell counts (CBC) were performed using fresh blood samples with ethylenediaminetetraacetic acid (EDTA) at 2% (w/v). Analyses were carried out using an automated Hematoclin 2.8 VET instrument (Bioclin, Brazil), according to manufacturer`s instructions. In addition, serum levels of glucose, urea, creatinine, alanine and aminotransferase, alkaline phosphatase, and gamma glutamyl transferase were determined using a Bio-100 semi-automated analyzer (Bioclin, Brazil), according to manufacturer`s instructions. Serological tests for Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) were carried out using the FIV AC / FeLV AG COMBO VET FAST VET 013-1 (BIOCLIN, Brazil). Moreover, a serological analysis for Feline infectious peritonitis virus (FIPV) was carried out using an ImmunoComb antibody test kit (VP DIAGNOSTICO, Brazil). Those Dot ELISA analysis were carried out according to the manufacturer’s recommended protocols.
Thoracic radiography procedure was performed using a digital imaging equipment
(ECORAY, Korea) with 70 kV of potency and 1.2 milliampere seconds (mAs) as the radiographic technique. To evaluate respiratory conditions, right and left lateral and ventrodorsal projections were chosen. The entire procedure lasted 2 minutes.
Necropsy and samples collection
The postmortem examination was performed immediately after the death, and the macroscopic changes were recorded using a digital camera. At necropsy, the body was placed in the supine position for a superficial mento-pubic incision. To improve the exposure of pelvis and thorax, the hind limbs were disarticulated at the level of the hip joint and the forelegs folded down laterally dissecting the skin and subcutaneous tissue of the submandibular and cervical regions. Afterwards, costochondral disarticulation was performed in all fixation points of the ribs and the cranial and caudal pubic branches were incised. After hyoid disarticulation, the trachea and esophagus were released between the cervical muscle fascia and the entrance to the thoracic cavity, and the monobloc was pulled so that it could be detached along the entire thoracic extension to the diaphragm. Then, the diaphragm was sectioned in the dorsal semicircular portion, making a small incision in the right kidney, and continuously sectioning the abdominal set parallel to the vertebral column up to the pelvic cavity. Finally, the pelvic cavity was contoured along with the external genitalia and anus so that the monobloc was released entirely from the cadaver.
Tissue samples of kidneys, lungs, heart, trachea, liver, intestines, and spleen were stored in 10% formaldehyde at room temperature or as fresh tissues at −80°C until analysis.
Gross pathology and histopathology
Macroscopic evaluation of organs considered characteristics such as edema, congestion, discoloration, atelectasis, and consolidation. Tissues samples from kidneys, lungs, heart, trachea, liver, intestines, and spleen with standardized size of 2.0 x 1.6 x 1.2 cm were fixed overnight in a 4% formaldehyde solution and buffered with sodium phosphate 0.1 M at pH 7.2. After dehydration with ethanol, the fragments were placed in xylol and then paraffined. The samples were blocked using a TP 1020® sample blocker (LEICA, Germany) and microtomized using a RM 2255 rotary microtome (LEICA, Germany), according to manufacturer`s instructions. After that, the samples were stained with hematoxylin and eosin.
RNA extraction and reverse transcription quantitative PCR (RT-qPCR)
Nucleic acid extraction was carried out from tissue samples of the seven organs of the feline: lungs, trachea, spleen, liver, intestines, heart and kidneys. Samples were prepared by adding 1 mL of Quik-Zol Trizol reagent (LUDWIG BIOTECNOLOGIA, Brazil) for each 100 mg of tissue and homogenized by vortexing. After this process, 250 µL of each sample was loaded onto columns of Cellco-Virus RNA + DNA Preparation Kit Spin (CELLCO BIOTEC, Brazil), and RNA was purified according to manufacturer`s instructions.
The detection of SARS-CoV-2 was performed using the Allplex™ 2019-nCov Assay (SEEGENE, South Korea), according to manufacturer`s instructions. Thermocycling was carried out in a QuantStudio 5 instrument (Applied Biosystems, USA) with a hold stage composed of a first step of 20 min at 50°C, followed by a second step of 15 s at 95 °C. The PCR stage was composed of a first step of 15 s at 94°C followed by a second step of 30 s at 58 °C, repeated 45 times.
SARS-CoV-2 Genome Sequencing
The RNA extracted from the seven feline samples were submitted to Next Generation Sequence. RNA extractions (8 µL) from tissue samples were submitted to reverse transcription with LunaScript® (NEB, USA), following manufacturer`s instructions. The obtained cDNA was used as template for SARS-CoV-2 genome amplification with 1200bp amplicon "midnight" primer set V5, as previously described (https://www.protocols.io/view/sars-cov2-genome-sequencing-protocol-1200bp-amplic-btsrnnd6?step=4). Two tiling-based PCR were performed using Q5 Hot Start High-Fidelity DNA Polymerase (NEB, USA) and primers as previously described (https://www.protocols.io/view/sars-cov2-genome-sequencing-protocol-1200bp-amplic-btsrnnd6?step=4). Termocycling was composed of an incubation for 30 s at 98 °C for denaturation, followed by 35 cycles of 98 °C for 15 s and 65 °C for 5 min for annealing and extension. PCR amplicons for pool 1 and pool 2 were combined for each sample and adjusted to a concentration of 5 -10 ng/µL. End-Prep reactions were performed with NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB, USA) and barcoded using ONT Native Barcoding Expansion kit (EXP-NBD104) (Oxford Nanopore Technologies, UK), according to manufacturers`s protocols. The barcoded samples were then combined and purified using AMPure XP Beads (Beckman Coulter, USA) and loaded onto Oxford Nanopore MinION SpotON Flow Cells R9.4.1 (Oxford Nanopore Technologies, UK). High accuracy base calling was carried out after sequencing from the fast5 files using the Oxford Nanopore Guppy tool (Oxford Nanopore Technologies, UK).
Mapping, primer trimming, variant calling and consensus assembly building were performed with artic-ncov2019 pipeline, using the Medaka protocol (https://artic.network/ncov-2019). The genome was assembled with at least 20x coverage. Pango lineage was attributed to the newly assembled genome using the Pangolin v3.1.11 software tool (https://pangolin.cog-uk.io/).
Study of viral genetic polymorphisms
Mutations were detected using FASTA sequences downloaded from GISAID on October 09, 2021. SARS-CoV-2 variants were called using a customized script written in bash. Bash script processing of FASTA samples produced a Variant Call Format file (VCF) for each sample. VCF files were then merged using bcftools. In the merged VCF file, we identified 813 mutations present in FASTA sequences isolated from felines (See Supplementary data-sheets 1 and 2). In order to study the potential existence of mutations characteristic of SARS-CoV-2 that infected deceased animals only, unsupervised hierarchical clustering analysis was performed using R version 4.1.0 and R library pheatmap version 1.0.12. 813 mutations identified in viral sequences isolated from animals were used to classify groups of FASTA samples using the mutation frequency of each group of interest. The groups of interest represented sequences from these 10 different origins: FASTA sequences from the Brazilian bordering states of Bahia, Tocantins and Goias; FASTA sequences from Brazil between January-February 2021 and Brazil between March-April 2021; FASTA sequences from Deceased, Alive and Unknown Deceased status animals; FASTA sequences from Manis javanica and Manis polydactila and FASTA sequences from bats of the genus Rhinolophus. All sequences were downloaded from GISAID. Frequencies of the 813 mutations detected in viruses that infected cats were then used to classify these groups using the unsupervised hierarchical clustering method. 68/813 (8.35%) mutations allowed unsupervised classification of the sequences of deceased animals and their separation from non-deceased animals.