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First description of a multisystemic and lethal SARS-CoV-2 variant of concern P.1 (Gamma) infection in a FeLV-positive cat | VirologyJournal


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). The procedures involving the animal were carried out in accordance with the ethical and biosafety guidelines.

clinical assessment

An eight-year-old male domestic cat of an undefined breed was admitted on April 10, 2021, with respiratory syndrome at a veterinary clinic in Barreiras, Bahia, Brazil. Data regarding respiratory aspects, temperature, heartbeat, weight, and other clinical aspects were kept during anamnesis. In addition, laboratory analyzes were required due to the clinical condition of the animal.

Blood cell count, biochemical and serological analysis

The blood sample was obtained by the jugular venous puncture and collected in test tubes with or without ethylenediaminetetraacetic acid (EDTA) at 2% (w/v), for hematological and biochemical/serological analyses, respectively. Complete blood cell counts (CBC) were performed using fresh blood samples with EDTA at 2% (w/v). Analyzes were carried out using an automated Hematoclin 2.8 VET instrument (Bioclin, Brazil), according to the 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 immunochromatographic tests for Feline immunodeficiency virus (IVF) and feline leukemia virus (FeLV) were carried out using the FIV AC / FeLV AG COMBO VET FAST VET 013-1 (BIOCLIN, Brazil), according to the manufacturer’s recommended protocols. Moreover, a serological analysis based on Dot ELISA for the Feline infectious peritonitis virus (FIPV) was carried out using an ImmunoComb antibody test kit (VP DIAGNOSTICO, Brazil). The Dot ELISA analysis was carried out according to the manufacturer’s recommended protocols.

Imaging diagnostics

Thoracic radiography procedure was performed using a digital imaging equipment (ECORAY, Korea) with 70 kV of power 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 min.

Necropsy and samples collection

the postmortem examination was performed immediately after the death, and the macroscopic changes were recorded using a digital camera. The body was placed in dorsal decubitus and the abdominal cavity was opened by medial incision, using the dawn line as a reference. To improve the exposure of the pelvis and thorax, the hind limbs were disarticulated at the hip joint level and the forelegs folded down laterally, dissecting the skin and subcutaneous tissue of the submandibular and cervical regions. Afterward, 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 with 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. Tissue samples from kidneys, lungs, heart, trachea, liver, intestines, and spleen with a standardized size of 2.0 × 1.6 × 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. Next, the samples were blocked using a TP 1020® sample blocker (LEICA, Germany) and microtomized using a RM 2255 rotary microtome (LEICA, Germany), according to the 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 the manufacturer`s instructions.

The detection of SARS-CoV-2 was performed using the Allplex™ 2019-nCov Assay (SEEGENE, South Korea), according to the 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

SARS-CoV-2 genome from feline samples was recovered by amplicon tiling multiplex approach using nanopore sequencing. Briefly, RNA extractions (8 µL) from tissue samples were submitted to reverse transcription with LunaScript® (NEB, USA), following the manufacturer`s instructions. The obtained cDNA was used as template for SARS-CoV-2 genome amplification using Q5 Hot Start High-Fidelity DNA Polymerase (NEB, USA) 1200 bp amplicon “midnight” primer set [6] Thermocycling was composed of 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’ 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 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. Pango lineage was attributed to the newly assembled genome using the Pangolin v3.1.11 software tool (https://pangolin.cog-uk.io/).

Phylogenetic analysis

Phylogenetic inferences were carried out comparing the SARS-CoV-2 genome obtained from the feline sample with a dataset of high-quality SARS-CoV-2 genomes available through GISAID sampled from December 2019 to Feb 2022 (Additional file 1: Table S1). Closely related sequences (no more than five mutations) were selected using AudacityInstant (GISAID) searches against the entire EpiCoV database. The final dataset consisted of 2334 human and cat samples from all the major WHO SARS-CoV-2 clades sampled mainly from Brazil and South America. The genomes were analyzed using the NextStrain pipeline [7]. Briefly, the genomes sequences were aligned using MAFFT [8] and a maximum likelihood tree was inferred using IQ-Tree [9]. The ancestral reconstruction and timescale were estimated using augur and treetime [10]. The tree was rooted at Wuhan/WH01/2019 and Wuhan/Hu-1/2019 ancestor.



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