Myocarditis in naturally infected pets with the British variant of COVID-19

Luca Ferasin, DVM, Ph.D, Matthieu Fritz, Ph.D, Heidi Ferasin, DVM, Pierre Becquart, Ph.D, Vincent Legros, DVM, Ph.D and Eric M. Leroy
Original article: Myocarditis in naturally infected pets with the British variant of COVID-19; 3/18/2021; Translation 1.4.2021

Abstract

Pets may become infected with SARS-CoV-2, but based on the limited information available so far, it is not known whether the new UK variant B.1.1.7 can more easily infect certain animal species or increase the possibility of human-to-animal transmission. In this study, we report the first cases of infection of domestic cats and dogs by British variant B.1.1.7 SARS-CoV-2 diagnosed at a specialized veterinary hospital in the south-east of England. We further found that many owners and caregivers of these pets developed respiratory symptoms in Covid-19 3-6 weeks before their pets became ill, and also had a positive PCR test for Covid-19. Interestingly, all of these B.1.1.7 infected pets developed atypical clinical manifestations, including severe myocarditis-related cardiac abnormalities and severe deterioration of the patient's overall health, but without any primary respiratory symptoms. Our findings demonstrate for the first time the ability of pet animals to be infected with variant B.1.1.7 SARS-CoV-2 and raise questions about its pathogenicity in these animals. In addition, given the increased infectivity and transmissibility of variant B.1.1.7 for humans, these findings also highlight, more than ever, the risk that pets may potentially play a more important role in the dynamics of SARS-CoV-2 outbreaks than previously thought.

Introduction

The COVID-19 pandemic due to severe acute respiratory syndrome caused by type 2 coronavirus (SARS-CoV-2), characterized by a change in the amino acid spike protein (S) D614G (referred to as variant B. 1), has affected several million cases worldwide. This global situation has favored the occurrence of many genomic mutations, some of which have created variants with selective advantages. ¹. At the end of autumn 2020, three notable variants emerged in several countries, which then spread rapidly around the world, including B.1.1.7 (also known as 20I / N501Y.V1), which was first identified in England. ², B.1.351 (20J / N501Y.V2) first detected in South Africa and recently identified "Brazilian" variants of P.1 (20I / N501Y.V3). These three variants carry a constellation of genetic mutations, including those at the level of the protein S receptor binding domain (RDB), which is necessary for binding to the ACE-2 receptor of the host cell to facilitate virus entry.

It is estimated that variant B.1.1.7, also referred to as the dreaded variant (VOC) 202012/01 or 20I / 501Y.V1, appeared in September 2020 in Kent, a county in the south-east of England, and quickly surpassed other existing ones in England. variants due to increased portability and infectivity ². Multiple evidence suggests that its increased portability is driven by the N501Y mutation and the Δ69 / 70 amino acid deletion in RDB ³. Subsequently, the incidence of B.1.1.7 increased during the national lockdown introduced by the UK Government from 5 November to 2 December 2020, despite severe restrictions, which led to an extraordinary increase in COVID-19 cases, which particularly affected the Greater London area. . As of February 7, 2021, VOC 202012/01 represented approximately 95% new SARS-CoV-2 infections in the United Kingdom and has currently been identified in at least 86 countries.

There have been several cases of SARS-CoV-2 infection in pets (especially cats and dogs) worldwide, and these animals are thought to have been infected by their owners or caregivers. Pet infections have mostly led to unnoticeable to mild digestive and respiratory symptoms such as cough, rhinitis, sneezing and conjunctivitis. ^4-6. Interestingly, despite the uncontrolled increase in COVID-19 cases in the UK since November 2020, no SARS-CoV-2 infections have been reported in pets. More surprisingly, to the best of the authors' knowledge, no natural infection of any B.1.1.7 animal has ever been documented, either in England or anywhere else.

The results

We report a sudden increase in the number of domestic dogs and cats with myocarditis at The Ralph Veterinary Referral Center (RVRC) on the outskirts of London (UK) between December 2020 and February 2021, with an unexpected increase in incidence from 1.4% to 12.8% (8, 5% in cats and 4.3% in dogs). This sudden increase in cases appears to have followed the curve and timeline of the COVID-19 human pandemic in the UK as a result of option B.1.1.7, which began in mid-December 2020 and culminated in late January 2021 before returning to historical levels in mid-February. 2021

None of these myocarditis patients had a history of heart disease and their clinical picture was similar and characterized by acute onset of lethargy, anorexia, tachypnoea / dyspnoea (secondary to congestive heart failure) and in some cases syncopal events. Diagnostic examinations revealed the presence of elevated cardiac troponin-I (median 6.8; range 0.68 to 61.1 ng / ml [normal reference range 0.0-0.2 ng / ml]) accompanied by echocardiographic evidence of myocardial remodeling and / or signs of pleural effusion and / or pulmonary edema, often confirmed on chest radiographs, and / or severe ventricular arrhythmias on electrocardiography (see Additional Figure S1). All affected animals achieved remarkable improvement after cage rest, oxygen therapy, acute diuresis, and in some cases antiarrhythmic treatment with sotalol and fish oil before being discharged for oral treatment after several days of intensive care. Notably, most owners and caregivers of these pets with myocarditis developed respiratory symptoms in Covid-19 within 3-6 weeks before their pets became ill, and many of these owners had a positive PCR test for Covid-19. Due to this coincidence and the interesting simultaneous development of myocarditis in these pets and the outbreak of B1.1.7 COVID-19 in the UK, we decided to investigate SARS-CoV-2 infection in these animals. For this purpose, serum samples as well as oro / nasopharyngeal and rectal swabs were taken from seven animals (six cats and one dog) at the initial presentation at the RVRC between 22 January and 10 February 2021 (Table 1). During the same period, we took blood samples from four other pets (two cats and two dogs) during their recovery, 2-6 weeks after they developed symptoms of myocarditis. None of the 11 myocarditis animals developed any influenza-like symptoms and all improved clinically over several days of intensive care, although one cat (LL) had a relapse of her clinical symptoms characterized by severe lethargy and uncontrolled ventricular tachycardia one week after discharge. its owners to opt for euthanasia. All cats and dogs were neutered and aged from one to 12 years. After collection, all samples were stored at -20 ° C until transport on ice to the MIVEGEC laboratory in Montpellier, France for serological and virological examinations.

Kind of	Tribe	Age	Gender	Days	General symptoms	T ° C	Cardiac abnormalities	Troponin ng / ml	Covid-19 + contact	SN	SARS-CoV-2 ddPCR	Serology
?	DSH	9	M	2	Lethargy Anorexia	37.1	CHF	7.9	✔️	-	-	-
?	DSH	9	M	2	Lethargy Anorexia	37.0	CHF, VA	0.68	✔️	-	33 copies of RNA / µL	-
?	Manx	12	F	2	Lethargy	35.2	CHF, VA	6.8	❓	-	-	-
?	Sphynx	10	F	3	Syncope	37.9	CHF, VA	45.6	❓	-	12 copies RNA / µL	-
?	Labrador	9	F	4	Lethargy Anorexia Hemorrhagic diarrhea	37.6	VA	43.5	✔️	-	13 copies RNA / µL	-
?	DSH	9	M	8	Lethargy	37.8	CHF	1.31	❓	-	-	N protein
?	Scottish Fold	1	M	10	Lethargy Anorexia	37.3	CHF	12.1	❓	-	-	-
?	Mastiff	8	F	14	Syncope	38.0	CHF, VA	2.5	❓	-	ON THE	-
?	Siberian	1	F	28	Lethargy	38.5	CHF	4.92	✔️	-	ON THE	N protein RBD S trimmer
?	Dalmatian	8	M	37	Syncope	38.2	VA	61.1	✔️	+	ON THE	RBD S trimmer
?	Persian	1	M	64	Lethargy	37.5	CHF, VA	0.83	❓	-	ON THE	-

Oro / nasopharyngeal and rectal swabs were tested by droplet digital RT-PCR (ddPCR) targeting one region specific for the SARS-CoV-2 N gene and two Spike protein regions specific for the three current predominant SARS-CoV-2 variants, namely 20I / N501Y.V1, 20J / N501Y.V2 and 20I / N501Y.V3. One target region containing the N501Y mutation is common to three variants and the other target region containing the Δ69-70 deletion is specific to variant B.1.1.7. Sera were tested for SARS-CoV-2 specific IgG using three microsphere immunoassays (MIA) that detected IgG binding to N protein, S1-RBD protein or S trimeric protein, as well as a retrovirus-based assay to detect SARS-CoV-2. 2 neutralizing antibodies (see Materials and Methods).

All oro / nasopharyngeal swabs were found to be negative for SARS-CoV-2 ddPCRs. However, positive ddPCR signals were obtained for three areas from rectal swabs from three of the seven animals (two cats and one dog), indicating infection with British variant B.1.1.7. The RNA concentration ranged from 12 to 33 copies / μL of sample, indicating a low viral load (Table 1). In addition, samples from one animal taken during the acute phase of the disease, which showed a negative ddPCR result, as well as from two of the four animals taken during the recovery period, were found to contain SARS-CoV-2 antibodies. Thus, in total, six of our 11 animals tested were shown to be SARS-CoV-2 positive, either ddPCR or serologically. More interestingly, considering only five animals whose owners or caregivers were laboratory confirmed for Covid-19, four showed SARS-CoV-2 positive (Table 1).

Discussion

As far as we know, this is the first report of an infection of cats and dogs by the British variant SARS-CoV-2 B.1.1.7. Given the increased infectivity and transmissibility of human B.1.1.7, the discovery of B.1.1.7 infected cats and dogs emphasizes more than ever the risk that pets may potentially play a significant role in the dynamics of SARS-CoV-2 outbreaks, as previously thought. Therefore, further studies are urgently needed to examine the likelihood of transmission of animal-animal as well as animal-human variant B.1.1.7 and to demonstrate whether the N501Y mutation and the Δ69-70 deletion cause the virus to be more infectious to these animals.

Another remarkable and unexpected finding of our study is the development of unusual clinical manifestations in cats and dogs infected with B.1.1.7, including severe cardiac abnormalities secondary to myocarditis and severe deterioration of the patient's overall health, but without any primary respiratory symptoms. With the exception of the only cat in Spain who experienced cardiorespiratory failure leading to severe respiratory distress ⁷, both natural and experimental SARS-CoV-2 infections have been reported in cats and dogs, either asymptomatic or with mild upper respiratory disease. Although B.1.1.7 infection in humans appears to be associated with higher COVID-19 mortality or clinical severity, the association between myocarditis and B.1.1.7 infection in pets should be recognized and addressed. ⁸ In this context, it is important to emphasize the fact that myocarditis associated with multisystem inflammatory syndrome is also a well-known complication of COVID-19 in humans (both adults and children), probably due to an exaggerated immune response of the host. ^9,10.

Our findings demonstrate the ability of pet animals to become infected with SARS-CoV-2 B.1.1.7 and raise questions about its pathogenicity in these animals. Therefore, there is an urgent need to significantly accelerate and strengthen the investigation and surveillance of animal infections with highly transmissible variants such as British B.1.1.7, South African B1.351 and Brazilian P.1 as part of the global response to the ongoing multi-species pandemic of COVID-19.

Materials and methods

RNA extraction

Rectal and oro / nasopharyngeal swabs were resuspended by vortexing in 300 μL PBS. Total RNA was extracted from 200 μL of rectal swab supernatant and 200 μL of nasopharyngeal swab virus transport medium. Extraction was performed on an IndiMag 48 extraction system (Indical Bioscience) using magnetic bead technology with the IndiMag Pathogen kit according to the manufacturer's instructions. The elution volume was 100 μL.

One-step dRT-PCR

The RT-dPCR procedure was performed according to the manufacturer's instructions using a QIAcuity 8.5 plexus (Qiagen, Germany), a QIAcuity One-Step Viral RT-PCR kit (cat. No. 1123145, Qiagen, Germany) and a 24-well 26K nanoplate ( Cat. No. 250001, Qiagen, Germany). The ddRT-PCR technique showed higher sensitivity and specificity for the diagnosis of COVID-19 compared to RT-qPCR. ¹¹

Briefly, the RT-dPCR reaction mixture was assembled as follows: 4x one-step viral RT-PCR Master Mix 10 μl, 100x multiplex reverse transcription mixture 0.4 μl, 20x primer and probe set 0149, 0130, 0150 (ref IAGE) 2 μl x3 (6 μL), RNase-free water 22.6 μl and RNA template 1 μl, in a final volume of 40 μl. Target 0130 target 2019-nCoV_N2 NC_045512v2 fluorophore HEX, amplicon length 67 bp. Target region 0149: deletion of mutations 69-70, lines B1.1 and B1.258, fluorophore HEX, amplicon length 100 bp. Target region 0150 S: mutation N501Y, line B1.1.7, fluorophore Cy5, amplicon length 133 bp. The sequences are confidential and are stored under number EP20306715.2. The mixture was prepared on a pre-plate and then transferred to a 24-well 26K nanoplate. It was then introduced into the QIAcuity 8, a fully automated system. The procedure included i) a priming and rolling step to generate and isolate the ventricular compartments, ii) an amplification step according to the following cyclic protocol: 50 ° C for 40 minutes for reverse transcription, 95 ° C for 2 minutes for enzyme activation, 95 ° C after for 5 s for denaturation and 60 ° C for 30 s for annealing / extension for 40 cycles and iii) the imaging step was performed by reading in the following channels FAM, HEX and CY5. The entire workflow time for these three steps was approximately 2 hours. Experiments were performed using a negative control (no template control, NTC) and a positive control (the patient sample was confirmed as positive by RT-PCR using our routine diagnostic testing). All reactions had at least 25,400 compartments. Data were analyzed using QIAcuity Suite Software V1.1.3 (Qiagen, Germany) and expressed as copies / μl.

Microsphere immunoassay

Serum samples from cats and dogs were tested by multiplex microscopic immunoassay (MIA). 10 μg of three recombinant SARS-CoV-2 antigens were used to capture specific serum antibodies: nucleoprotein (N), receptor binding domain (RBD) and trimeric spike (tri-S). Separate MagPlex microsphere kits (Luminex Corp) were appropriately coupled to viral antigens using an amine coupling kit (Bio-Rad Laboratories) according to the manufacturer's instructions, while the recombinant human protein-coupled microsphere kit (O6-methylguanine DNA methyltransferase) was used as a control in the assay. . The MIA procedure was performed as described in ¹². Briefly, microsphere mixtures were sequentially incubated protected from light on an orbital shaker with serum samples (1: 400), biotinylated protein A and biotinylated protein G (4 μg / ml each) (Thermo Fisher Scientific) and Streptavidin-R-phycoerythrin (4 μg / ml) (Life technologies). Measurements were performed using a Magpix instrument (Luminex). Relative fluorescence intensities (RFI) were calculated for each sample by dividing the MFI signal measured for the antigen-coated sets of microspheres by the MFI signal obtained for the set of control microspheres to take into account the non-specific binding of the antibodies to the beads. Specific seropositivity limits for each antigen were set to three standard deviations above the mean RFI value of serum samples from 29 dogs and 30 cats collected before 2019. Based on the pre-pandemic population, the MIA specificity for N protein was set at 96.6%. for dogs and cats, 96.6% for RBD for cats and 100% for dogs and 100% for tri-S for cats and 96.6% for dogs.

Measurement of neutralizing activity

An MLV-based pseudo-particle carrying a SARS-CoV2 tip pseudotyped (SARS-CoV-2pp) was used to measure neutralizing activity in cat and dog sera. Each SARS-CoV2 positive sample detected by MIA was treated by a neutralization procedure as described in ¹³. The level of infectivity was expressed as % GFP positive cells and compared to SARS-CoV-2pp infected cells incubated without serum. Pre-pandemic sera from France were used as negative controls and anti-SARS-CoV-2 RBD antibody was used as a positive control.

Declaration of conflict of interest

None of the authors in this study has any conflict of interest (financial or personal).

Financing

The study was funded by the French National Research Agency (ANR-RA-COVID-19; Geographical and temporal serological study of SARS-CoV-2 infection of companion animals during the second wave of COVID-19 in France, CoVet) and the IDEXLYON project of the Université de Lyon as part of the "Programme Investissements d'Avenir" (ANR-16-IDEX-0005) and the Institut de Recherche pour le Développement (IRD).

Acknowledgement

We are grateful to the pet owners for giving us permission to collect samples from their animals. We also thank Dr. Laurent Locquet and Dr. Altin Cala for contributing to the clinical management of these patients. We also thank Franz Durandet (IAGE – Ingénierie et Analyze en Génétique Environnementale, Grabels, France), Alix de Mont-Marin (Innovative Diagnostics, France) and Afif M. Abdel Nour (Qiagen, France) for help with molecular biology. We also thank François Loïc Cosset, Solène Denolly, Bertrand Boson from CIRI – Center International de Recherche en Infectiologie, Team EVIR, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U111, UMR5308, ENS Lyon, for the development of the seroneutralization technique. Finally, we thank Dr. Thierry Buronfosse for the kind donation of pre-pandemic sera.

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