SARS-CoV-2 delta variant infection in domestic dogs and cats, Thailand

Collecting specimens from domestic dogs and cats for SARS-CoV-2

From June to September 2021, we conducted a survey for SARS-CoV-2 in domestic dogs and cats living in and around Bangkok. Samples from dogs and cats were collected from participating veterinary clinics and hospitals. In total, we collected 225 samples from dogs (n=105) and cats (n=120) from COVID-19 positive households (n=12) and households with unknown status (n=187) (tables 1 and 2). We collected nasal (dog; n=8; cat; n=16), oral (dog; n=102, cat; n=117) and rectal (dog; n=104, cat; n=120) swabs from animals using a flocked nylon swab (Copan®, CA, USA). Swab specimens were placed in RNA protect® (Qiagen LLC, Maryland, USA) and transported to the laboratory within 24 h. In this study, we followed a dog (n = 1) and a cat (n = 1) positive for SARS-CoV-2. During follow-up, we collected nasal swabs (n=9), oral swabs (n=9), rectal swabs (n=9) from the animals. Additionally, environmental swabs, hair (n=7), water tank (n=7), and soil (n=7) were also collected (Table 3). We collected a blood sample (n = 6 serum; 2 time points from a cat and 4 time points from a dog) from SARS-CoV-2 positive animals (Table 4). This study was conducted under the approval of the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Veterinary Science, Chulalongkorn University, Thailand (Approval No. 2031035). All methods were performed in accordance with current guidelines and regulations.

Detection of SARS-COV-2 RNA

Viral RNA extraction was performed using the GENTi automated nucleic acid extraction system (GeneAll® Seoul, Korea). For detection of SARS-CoV-2 RNA, real-time RT-PCR assays based on specific primers and probes (N2, E, and RdRp) were performed according to CDC and WHO recommendations^20.21. Briefly, a 25 µl total reaction contained 2 µl of RNA, 12.5 µl of SuperScript® III Platinum® One-Step Quantitative RT-PCR System 2X Reaction Buffer (Invitrogen, CA, USA), 1 µl Reverse Transcriptase/Platinum Taq, 0.8 mM MgSO4, 0.8 µM each primer and probe, and RNase-free water. The real-time RT-PCR reaction was set up at 50°C for 15 min, followed by 95°C for 2 min and 45 cycles of 95°C for 15 s and 58°C for 30 s (E and RdRP genes ) or 60°C for 30 s (N2 gene). Samples with a Ct value

Characterization of SARS-CoV-2

RNA samples from a SARS-CoV-2 positive cat nasal swab (C27516) collected July 15, 2021 (Ct value 20.66) and a dog nasal swab (CU27791) collected on September 12, 2021 (Ct value 19.06) were subjected to whole genome sequencing using Oxford Nanopore. We used the ARTICS nCoV-2019 V3 (LoCost) sequencing protocol to amplify the viral genome. Briefly, the diluted RNA (8 µl) was mixed with 2 µl of LunaScript® RT SuperMix (NEB, Ipswich, MA, USA). cDNA synthesis was performed at 25°C for 2 min, 55°C for 10 min and 95°C for 1 min. The ARTIC protocol using two SARS-CoV-2 primer pools for multiplex PCRs was performed using Q5® Hot Start High-Fidelity DNA Polymerase (NEB, MA, USA) with a PCR reaction at 98°C for 30 s and 35 cycles of 98°C for 15 s and 65°C for 5 min. After PCR amplification, library preparation was performed following the Oxford Nanopore Rapid Sequencing Kit (SQK-RAD004) with ARTIC SARS-CoV-2 Genome Sequencing Protocol^32.33. Briefly, 7.5 µl of pooled PCR products (10 µl of pool 1 and 10 µl of pool 2) were added to 2.5 µl of fragmentation mix. Then the mixture was incubated at 30°C for 1 min, 80°C for 1 min and 4°C for 30 s. The mixture was cleaned by AMPure XP Bead Cleanup (Beckman Coulter, CA, USA) and eluted with 15 µl of 10 mM Tris–HCl pH 8.0. The mixture was loaded into the Oxford Nanopore MinION device under MinKNOW software version 19.12.5 (Oxford nanopore technologies, Oxford, UK) (https://nanoporetech.com/nanopore-sequencing-data-analysis)³⁴. After sequencing, nucleotide sequences were filtered using the sequencing summary file under the following parameters: minimum read length ≥ 500 nt and read quality ≥ 7. Qualified reads were converted from “Fast5” format in “Fastq” format using the GPU version of the Nanopore Guppy basecaller tool (v3.4.4). Sequences in Fastq format were assembled using the genome detection program³⁵ and de novo approach with Qiagen CLC Genomics Benchwork software version 20.0.4 (QIAGEN, CA, USA) (https://digitalinsights.qiagen.com/ products/qiagen-clc-main-workbench/). Whole genome sequences of SARS-CoV-2 have been deposited in GenBank (OK555092 and OK539641) and GISAID (EPI_ISL_5320246 and 5315539).

Phylogenetic analysis of SARS-CoV-2

SARS-CoV-2 whole genome sequences were lineage classified using the Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) COVID-19 sequences (https://cov-lineages.org/resources/pangolin.html). The phylogenetic analysis of SARS-CoV-2 was carried out by comparing the nucleotide sequences of 289 SARS-CoV-2 genomes available in the GISAID database and GenBank. The 5′ and 3′ untranslated regions were cut with at least 95% (Wuhan-Hu-1) reference genome coverage (at least 29,000 bp in length). Dataset alignment was performed using the MAFFT FFT-NS-2 algorithm with default settings³⁶. The maximum likelihood tree was constructed using IQ-TREE version 2.1.3 (http://www.iqtree.org/)³⁷ using the GTR+Γ model of nucleotide substitution³⁸default heuristic search options and super-fast startup with 1,000 replicates³⁹. The tree was visualized by iTOL version 6.0 (https://itol.embl.de/)⁴⁰. Lineage classification was performed using the Pangolin tool⁴¹. SARS-CoV-2 genetic mutation analysis was performed by comparing the inferred amino acids of each gene of the viruses based on classifications and variant definitions^42.43.

Detection of SARS-CoV-2 antibodies

The ID Screen® SARS-CoV-2 Double Antigen Multi-species ELISA kit (ID VET, Montpellier, France) was used to detect IgG antibodies directed against the N protein of the SARS-CoV-2 virus in animal serum. We performed the ELISA test according to the manufacturer’s recommendation. Briefly, 25 µl of each serum sample was diluted 1:1 with dilution buffer and added to each well. The 96-well plate was incubated at 37°C for 45 min. The plate was washed with 300 µl of wash solution and 100 µl of horseradish peroxidase (HRP) conjugate of protein N recombinant antigen was added to each well and incubated at 25°C for 30 min. The plate was washed 3 times with 300 µl wash solution. After washing, 100 µl of the substrate solution was added to each well and incubated at 25°C for 20 min. Then 100 µl of the stop solution was added. The reaction was read and recorded for optical densities (OD) at 450 nm. The OD was calculated as the percentage S/P (S/P%). If the S/P% value greater than or equal to 60% is considered positive, while the sample with S/P% between 50 and 60% is considered doubtful, and the sample with S/P%

The cPass SARS-CoV-2 Neutralizing Antibody Detection Kit (GenScript Biotech, Jiangsu, China) was used to detect neutralizing antibodies. The test detects SARS-CoV-2 antibodies by measuring antibody-mediated inhibition of the SARS-CoV-2 RBD-ACE2 interaction. Briefly, 50 µl of serum diluted 1/10 was incubated with 50 µl of HRP-conjugated RBD and incubated at 37°C for 30 min. The 100 μl of treated serum was then added to an ELISA plate coated with ACE2 and incubated at 37°C for 15 min. Then, uncaptured substrate was washed using 260 µL wash solution four times. The colorimetric signal was developed using TMB substrate at 25°C for 15 min. Absorbance reading at 450 nm was acquired using a microplate reader immediately after addition of Stop Solution. The percent inhibition was calculated. Sample with % inhibition ≥ 20% indicates the presence of SARs-CoV-2 neutralizing antibodies, otherwise are negative⁴⁴.

The pseudotype virus neutralization assay was performed using a SARS-CoV-2 lentiviral pseudotype and HEK293T expressing human ACE2. Target cells expressing hACE2 were produced by stable transduction of HEK293T cells with a lentiviral vector harboring the hACE2 gene (pHAGE-EF1alphaInt-ACE2-WT, BEI Resources, VA, USA; NR-52512) and enriched by magnetic cell sorting at the mouse anti-hACE2 help. (Sino-biological, China) and goat anti-mouse IgG microbeads with MACS LS column (Miltenyi Biotec Asia Pacific, Singapore). The SAS-CoV-2 lentiviral pseudotype was produced by co-transfecting the plasmids pSPAX2 (Addgene, MA, USA; plasmid #12,260), pCCGW and pHDM-SARS-CoV-2 spike (BEI Resources, VA, USA; NR -53742) in the HEK293T cell. The spike vector pHDM-SARS-CoV-2 encodes the codon-optimized spike gene of SARS-CoV-2 strain Wuhan-Hu-1 (GenBank NC_045512). All serum samples were heat inactivated in a biological safety cabinet at 56°C for 60 min and serially diluted twice spanning 1:20 to 1:40 960 in DMEM supplemented with 10% fetal bovine serum . Sera were incubated with 50 µL of 100 TCID₅₀ of the SARS-CoV-2-lentiviral pseudotype at 37°C for 1 h. Then 50 µL of 1 × 10⁴ HEK293T-hACE2 cell was added to the mix and incubated at 37°C for 48 h. A dilution at which 50% infection relative to negative anti-SARS-CoV-2 serum is inhibited (IC50) was used as the test threshold. anti-SARS-CoV-2 antibodies of plant origin; H4 was used as a positive antibody control^45.46.

Ethics statement

The Institutional Animal Care and Use Committee of the Faculty of Veterinary Science, Chulalongkorn University, Thailand, approved the animal study (IACUC No. 2031035). All methods were performed in accordance with current guidelines and regulations. The study complies with the ARRIVE guidelines. The IACUC Committee of the Faculty of Veterinary Science, Chulalongkorn University, Thailand, approved the informed consent. Informed verbal consent was obtained from all animal owners and private veterinary hospital staff after explaining the aims and benefits of the study during sample collection.