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

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.

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 analyses were required due to the clinical condition of the animal.

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). Analyses 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 (FIV) 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.

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 min.

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 linea alba as a reference. To improve the exposure of 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.

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 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.

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 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 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 analysed 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.

An eight-year-old male domestic cat of undefined breed was admitted at a veterinary clinic in Barreiras, Bahia, Brazil, presenting intense dyspnea, cyanotic mucous, and wheezing in the right hemithorax auscultation. The animal was previously exposed to its owner and more two persons, who were confirmed to be infected by SARS-CoV-2 (data not shown). Pet`s body temperature was considered normal for cats (38.6 °C). The oxygen saturation was shown to be 87%. In addition, thoracic radiography showed increased radiopacity in the cranial and caudal lobes of lungs, as well as peribronchial infiltrates compatible with incipient bronchitis, as shown by radiographic views of ventrodorsal (Fig. 1a) and right (Fig. 1b) and left (Fig. 1c) sides, indicating acute pneumonia. The animal presented severe respiratory failure four hours later and died. Altogether, these results indicate that the cat suffered severe acute respiratory syndrome. Blood samples were collected before death for hematological, serological, and biochemical analyses.

Regarding haematological findings, we found lymphocytopenia (2,2 lymphocytes/mm³ in WBC), anemia (RBC = 1, 41 × 10¹² erytrocytes/L; Haemoglobin = 3,0 g/dL) and severe thrombocytopenia (17 × 10⁹ platelets/L). Regarding serum biochemical markers, increased levels of urea (71.20 mg/dL) and glucose (176 mg/dL) were found. Furthermore, the diagnosis of FIV/FeLV infections performed by serological tests showed a positive result for FeLV, which has been associated with immunosuppression and the hematological disturbances observed.

Viral RNA was detected in all collected fresh tissues (see Table 1). According to cycle threshold (CT) values, the highest viral load was found in the trachea, followed by the liver, spleen, heart, lungs, kidneys, and intestines. The presence of viral RNA in all these tissue samples indicates a multisystemic viral infection. In addition, the virus was confirmed to be a variant of concern P.1 (Gamma), as shown by genome sequencing. The sequence was deposited at GISAID (EPI_ISL_4565991).

As shown in Fig. 2, the newly determined genome clustered within the VOC Gamma. To our knowledge, this is the first description of the Gamma variant lineage sequenced from cats globally. After genome comparison and ancestral mutation reconstruction, no signal of host adaptation was found in EPI_ISL_4565991. All the non-synonymous mutations observed were previously described as the Gamma variant lineage-defining mutations: ORF1a (S1188L, K1795Q), ORF1a (S3675-, G3676-, F3677-), ORF1b (P314L, E1264D), S (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F), ORF3a (S253P), ORF8 (E92K), N (P80R, R203K, G204R), ORF9b (Q77E).

Collapsed and emphysematous areas with air distending the interlobular septa of the lungs were seen in gross pathology (Fig. 3a, b). In addition, the kidneys were shown to be pallid, shorter, irregular, and firm. With a cross-section, the cortical region had a reduced thickness, and there was a secretion with a catarrhal aspect in the calyxes and pelvis (Fig. 3c). These results collectively indicate severe damages in the lungs and kidneys of the feline co-infected by SARS-CoV-2 and FeLV.

Histopathological investigation showed specific microscopic changes in the trachea, lungs, liver, and kidneys. Histological sections of the trachea and kidneys revealed multifocal lymphoplasmacytic inflammatory infiltrate, which indicates tracheitis (Fig. 4a–d) and moderate lymphoplasmacytic interstitial nephritis (Fig. 4e–h), respectively (Fig. 4). The lung sections, in turn, revealed multiple areas of atelectasis (Fig. 5a). In addition, compensatory emphysema foci (Fig. 5b) and sparse inflammatory infiltrate associated with the peri bronchial mucous glands were observed (Fig. 5a–g). Furthermore, liver microscopy revealed vacuolar degeneration of hepatocytes, seen in periportal hepatitis. Hepatic lymphoplasmacytic infiltrations were mild (Fig. 6). Collectively, these histopathologic results reinforce that trachea, lungs, liver, and kidneys were severely damaged by SARS-CoV-2 infection.