First description of a multisystem and lethal SARS-CoV-2 variant of a P.1 (Gamma) infection of concern in a FeLV-positive cat | Journal of Virology
Ethics and animal experimentation
All handling procedures and experiments involving the animal have been approved by the Committee for the Ethical Use of Animals in Research of the State University of Bahia (no. 2021.005.0018150-89). Procedures involving the animal were performed in accordance with ethical and biosafety guidelines.
An eight-year-old male domestic cat of an undefined breed was admitted on April 10, 2021, with respiratory syndrome to a veterinary clinic in Barreiras, Bahia, Brazil. Data regarding respiratory aspects, temperature, heart rate, weight and other clinical aspects were kept during the anamnesis. In addition, laboratory analyzes were necessary due to the animal’s clinical condition.
Blood count, biochemical and serological analyzes
The blood sample was obtained by jugular venipuncture and collected in test tubes with or without 2% (w/v) ethylenediaminetetraacetic acid (EDTA) (EDTA), for hematological and biochemical/serological analyses, respectively. Complete blood counts (CBC) were performed using fresh blood samples with 2% (w/v) EDTA. Analyzes were performed using an automated Hematoclin 2.8 VET instrument (Bioclin, Brazil), according to the manufacturer’s instructions. Additionally, serum levels of glucose, urea, creatinine, alanine and aminotransferase, alkaline phosphatase and gamma-glutamyl transferase were determined using a semi-automated Bio-100 analyzer. (Bioclin, Brazil), according to the manufacturer’s instructions. Serological immunochromatographic tests for Feline immunodeficiency virus (IVF) and Feline leukemia virus (FeLV) were performed using FIV AC / FeLV AG COMBO VET FAST VET 013-1 (BIOCLIN, Brazil), according to the protocols recommended by the manufacturer. Additionally, Dot ELISA-based serologic analysis for Feline Infectious Peritonitis Virus (FIPV) was performed using an ImmunoComb antibody test kit (VP DIAGNOSTICO, Brazil). The Dot ELISA assay was performed according to the protocols recommended by the manufacturer.
The chest radiography procedure was performed using digital imaging equipment (ECORAY, Korea) with 70 kV power and 1.2 milliampere seconds (mAs) as the radiographic technique. To assess respiratory conditions, right and left lateral and ventrodorsal projections were chosen. The whole procedure lasted 2 min.
Necropsy and sample collection
The autopsy the examination was performed immediately after death and macroscopic changes were recorded using a digital camera. The body was placed in the supine position and the abdominal cavity was opened by medial incision, using the White line for reference. To improve exposure of the pelvis and thorax, the hindlimbs were disarticulated at the hip joint and the forelimbs bent laterally, dissecting the skin and subcutaneous tissue from the submandibular and cervical regions. Then, costochondral disarticulation was performed in all rib attachment points, and the cranial and caudal pubic rami were incised. After hyoid disarticulation, the trachea and esophagus were released between the cervical muscle fascia and the entrance to the chest cavity, and the one-piece was pulled so that it could be detached with full chest extension up to the diaphragm . Next, the diaphragm was transected in the dorsal semicircular portion by making a small incision in the right kidney and continuously transecting the abdominal assembly parallel to the spine down to the pelvic cavity. Finally, the pelvic cavity was profiled with the external genitalia and anus so that the one-piece was completely freed from the cadaver.
Kidney, lung, heart, trachea, liver, intestine, and spleen tissue samples were preserved in 10% formaldehyde at room temperature or as fresh tissue at -80°C until cold. to analysis.
Gross pathology and histopathology
Gross organ evaluation took into account features such as edema, congestion, discoloration, atelectasis, and consolidation. Tissue samples from kidney, lung, heart, trachea, liver, intestine and spleen with a standardized size of 2.0 × 1.6 × 1.2 cm were fixed during the overnight in 4% formaldehyde solution and buffered with 0.1 M sodium phosphate at pH 7.2. After dehydration with ethanol, the fragments were placed in xylol and then paraffined. Then, the samples were blocked using a TP 1020® sample blocker (LEICA, Germany) and microtomized using a rotary microtome RM 2255 (LEICA, Germany), according to the manufacturer’s instructions. . After that, the samples were stained with hematoxylin and eosin.
RNA extraction and quantitative reverse transcription PCR (RT-qPCR)
The extraction of nucleic acids 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 every 100 mg of tissue and homogenized by vortexing. After this process, 250 µL of each sample was loaded onto Cellco-Virus RNA + DNA Preparation Kit Spin columns (CELLCO BIOTEC, Brazil) and the RNA was purified according to the manufacturer’s instructions.
Detection of SARS-CoV-2 was performed using the Allplex™ 2019-nCov test (SEEGENE, South Korea), according to the manufacturer’s instructions. Thermocycling was performed in a QuantStudio 5 instrument (Applied Biosystems, USA) with a holding step consisting of a first step of 20 min at 50°C, followed by a second step of 15 s at 95°C. The PCR step consisted 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 SARS-CoV-2 genome from feline samples was retrieved by a multiplex amplicon tiling approach using nanopore sequencing. Briefly, RNA extractions (8 µL) from tissue samples were reverse transcribed with LunaScript® (NEB, USA), following the manufacturer’s instructions. The resulting cDNA was used as template for SARS-CoV-2 genome amplification using Q5 Hot Start High-Fidelity DNA Polymerase primer set (NEB, USA) 1200 bp amplicon “midnight ”  Thermocycling consisted of a 30 s incubation 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 the NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB, USA) and coded using the ONT Native Barcoding Extension Kit (EXP-NBD104 ) (Oxford Nanopore Technologies, UK), following manufacturers’ protocols. . The barcoded samples were then combined and purified using AMPure XP beads (Beckman Coulter, USA) and loaded onto Oxford Nanopore MinION SpotON R9.4.1 flow cells (Oxford Nanopore Technologies, UK United). High-precision baseline calling was performed using the Oxford Nanopore Guppy tool (Oxford Nanopore Technologies, UK).
Mapping, primer trimming, variant calling, and consensus assembly were performed with the artic-ncov2019 pipeline, using the Medaka protocol (https://artic.network/ncov-2019). The genome was assembled with at least 20× coverage. The Pango lineage was assigned to the newly assembled genome using the Pangolin software tool v3.1.11 (https://pangolin.cog-uk.io/).
Phylogenetic inferences were made by comparing the SARS-CoV-2 genome obtained from the feline sample with a high-quality SARS-CoV-2 genome dataset available through GISAID sampled from December 2019 to February 2022 (supplementary file 1: table S1). Closely related sequences (no more than five mutations) were selected using AudacityInstant (GISAID) searches of the entire EpiCoV database. The final dataset consisted of 2334 human and cat samples from all major WHO SARS-CoV-2 clades collected primarily from Brazil and South America. Genomes were analyzed using the NextStrain pipeline . Briefly, the genome sequences were aligned using MAFFT  and a maximum likelihood tree was inferred using IQ-Tree . Ancestral reconstruction and timescale were estimated using augury and tree . The tree was rooted at the ancestor Wuhan/WH01/2019 and Wuhan/Hu-1/2019.