Pattern assortment from home canines and cats for SARS-CoV-2
Throughout June–September 2021, we performed SARS-CoV-2 survey in home canines and cats dwelling in Bangkok and the vicinities. Samples from canines and cats have been collected from collaborating veterinary clinics and hospitals. In complete, we collected 225 samples from canines (n = 105) and cats (n = 120) from COVID-19 constructive family (n = 12) and unknown standing households (n = 187) (Tables 1 and 2). We collected nasal swabs (canine; n = 8; cat; n = 16), oral swabs (canine; n = 102, cat; n = 117) and rectal swabs (canine; n = 104, cat; n = 120) from the animals by utilizing flocked nylon swab (Copan®, California, USA). The swab samples have been positioned in RNA shield® (Qiagen LLC, Maryland, USA) and transported to the laboratory inside 24 h. On this examine, we adopted up SARS-CoV-2 constructive canine (n = 1) and cat (n = 1). Throughout comply with up, we collected nasal swabs (n = 9), oral swabs (n = 9), rectal swabs (n = 9) from the animals. As well as, environmental swabs, hair (n = 7), water container (n = 7), and flooring (n = 7) have been additionally collected (Desk 3). We collected blood pattern (n = 6 serum; 2 time factors from a cat and 4 timepoints from a canine) from SARS-CoV-2 constructive animals (Desk 4). This examine was performed below the approval of the Institutional Animal Care and Use Committee (IACUC) of the School of Veterinary Science, Chulalongkorn College, Thailand (approval No. 2031035). All strategies have been carried out in accordance with related pointers and laws.
Detection of SARS-COV-2 RNA
Viral RNA extraction was carried out by utilizing GENTi—Automated Nucleic Acid Extraction System (GeneAll® Seoul, Korea). For the detection of SARS-CoV-2 RNA, realtime RT-PCR assays based mostly on particular primers and probes (N2, E and RdRp) have been carried out following CDC and WHO suggestions20,21. Briefly, a complete 25 µl response contained 2 µl of RNA, 12.5 µl of 2X response buffer of the SuperScript® III Platinum® One-Step Quantitative RT-PCR System (Invitrogen, California, USA), 1 µl of reverse transcriptase/Platinum Taq, 0.8 mM MgSO4, 0.8 µM every primer and probe and RNase-free water. Realtime RT-PCR response was setup at 50 °C for 15 min, adopted by 95 °C for two 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 worth of < 40 have been thought of constructive. As a result of limitation of institute and IACUC approval, virus isolation didn’t carry out within the examine.
Characterization of SARS-CoV-2
The RNA samples from the nasal swab of a cat (C27516) with SARS-CoV-2 constructive collected on 15 July 2021 (Ct worth 20.66) and the nasal swab of a canine (CU27791) collected on 12 September 2021 (Ct worth 19.06) have been subjected to complete genome ssequencing by utilizing Oxford Nanopore. We used the ARTICS nCoV-2019 sequencing protocol V3 (LoCost) to amplify viral genome. Briefly, diluted RNA (8 µl) was blended with 2 µl of LunaScript® RT SuperMix (NEB, Ipswich, MA, USA). The cDNA synthesis was carried out at 25 °C for two min, 55 °C for 10 min and 95 °C for 1 min. The ARTIC protocol utilizing two swimming pools of the SARS-CoV-2 primers for multiplex PCRs was carried out by utilizing Q5® Sizzling Begin Excessive-Constancy DNA polymerase (NEB, MA, USA) with PCR response at 98 °C for 30 s and 35 cycles of 98 °C for 15 s and 65 °C for five min. After PCR amplification, library preparation was carried out following the Oxford Nanopore fast sequencing equipment (SQK-RAD004) with ARTIC SARS-CoV-2 genome sequencing protocol32,33. Briefly, 7.5 µl of pooled PCR merchandise (10 µl of pool 1 and 10 µl of pool 2) was added to 2.5 µl of fragmentation combine. Then the combination was incubated at 30 °C for 1 min, 80 °C for 1 min, and 4 °C for 30 s. The combination 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 combination was loaded into Oxford Nanopore MinION machine below MinKNOW model 19.12.5 software program (Oxford nanopore applied sciences, Oxford, UK) (https://nanoporetech.com/nanopore-sequencing-data-analysis)34. After sequencing, nucleotide sequences have been filtered utilizing the sequencing abstract file below the next parameters: minimal learn size ≥ 500 nt and skim high quality ≥ 7. The qualify reads have been conversed from “Fast5” into “Fastq” format by utilizing the GPU model of the Nanopore Guppy basecaller (v3.4.4) software. The Fastq format sequences have been assembled utilizing the genome detective program35 and de-novo strategy with Qiagen CLC Genomics Benchwork model 20.0.4 software program (QIAGEN, CA, USA) (https://digitalinsights.qiagen.com/ merchandise/qiagen-clc-main-workbench/). Complete genome sequences of the SARS-CoV-2 have been deposited into the GenBank (OK555092 and OK539641) and GISAID (EPI_ISL_5320246 and 5315539).
Phylogenetic evaluation of SARS-CoV-2
Complete genome sequences of SARS-CoV-2 have been subjected to lineage classification by utilizing the COVID-19 sequences of the Phylogenetic Project of Named International Outbreak Lineages (PANGOLIN) (https://cov-lineages.org/sources/pangolin.html). Phylogenetic evaluation of SARS-CoV-2 was carried out by evaluating nucleotide sequences of 289 SARS-CoV-2 genomes obtainable within the GISAID and GenBank database. The 5′ and three′ untranslated areas have been trimmed with at the very least 95% reference genome protection (Wuhan-Hu-1) (at the very least 29,000 bp in size). The dataset alignment was carried out by utilizing the MAFFT FFT-NS-2 algorithm with default parameter settings36. The utmost probability tree was constructed by utilizing IQ-TREE model 2.1.3 (http://www.iqtree.org/)37 utilizing the GTR + Γ mannequin of nucleotide substitution38, default heuristic search choices, and ultrafast bootstrapping with 1000 replicates39. Tree was visualized by iTOL model 6.0 (https://itol.embl.de/)40. Lineage classification was carried out by utilizing the Pangolin software41. Genetic mutation evaluation of the SARS-CoV-2 was carried out by evaluating deduced amino acids of every gene of the viruses based mostly on variant classifications and definitions42,43.
Detection of SARS-CoV-2 antibodies
The ID Display® SARS-CoV-2 Double Antigen Multi-species ELISA equipment (ID VET, Montpellier, France) was used to detect IgG antibodies in opposition to N protein of the SARS-CoV-2 virus in animal sera. We carried out the ELISA take a look at in accordance with the manufacture’s advice. Briefly, 25 µl of every serum pattern was diluted at 1:1 ratio with the dilution buffer and added to every nicely. The 96-well plate was incubated at 37 °C for 45 min. The plate was washed with 300 µl of washing resolution and 100 µl of N protein recombinant antigen horseradish peroxidase (HRP) conjugate was added to every nicely and incubated at 25 °C for 30 min. The plate was washed 3 instances with 300 µl of washing resolution. After washing, 100 µl of the substrate resolution was added into every nicely and incubated at 25 °C for 20 min. Then, 100 µl of the cease resolution was added. The response was learn and recorded for the optical densities (O.D.) at 450 nm. The O.D. was calculated because the S/P proportion (S/P%). If S/P% worth larger than or equal to 60% is taken into account constructive, whereas pattern with S/P% between 50 and 60% is taken into account as uncertain, and pattern with S/P% < 50 is destructive.
The cPass SARS-CoV-2 Neutralization Antibody Detection Package (GenScript Biotech, Jiangsu, China) was used to detect neutralizing antibodies. The assay detects SARS-CoV-2 antibodies by measurement of antibody-mediated inhibition of SARS-CoV-2 RBD-ACE2 interplay. Briefly, 50 µl of diluted 1:10 serum was incubated with 50 µl of HRP-conjugated RBD and incubated at 37 °C for 30 min. The 100 µl of handled serum then added to ACE2-coated ELISA plate and incubated at 37 °C for 15 min. Then the uncaptured substrate was washed out by utilizing 260 µL of washing resolution for 4 instances. Colorimetric sign was developed by utilizing TMB substrate at 25 °C for 15 min. Absorbance studying at 450 nm was acquired utilizing a microplate reader instantly after including cease resolution. The share inhibition was calculated. The pattern with % inhibition ≥ 20% point out the presence of SARs-CoV-2 neutralizing antibody, in any other case are destructive44.
Pseudotype virus neutralization take a look at was carried out by utilizing a SARS-CoV-2 lentiviral pseudotype and HEK293T expressing human ACE2. The hACE2 expressing goal cells was produced by secure transduction of HEK293T cell with lentiviral vector harbouring hACE2 gene (pHAGE-EF1alphaInt-ACE2-WT, BEI Sources, VA, USA; NR-52512) and enriched by magnetic cell sorting utilizing mouse anti-hACE2 (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 plasmids pSPAX2 (Addgene, MA, USA; plasmid # 12,260), pCCGW and pHDM-SARS-CoV-2 spike (BEI Sources, VA, USA; NR-53742) into HEK293T cell. The pHDM-SARS-CoV-2 spike vector encodes the codon optimized spike gene of SARS-CoV-2 pressure Wuhan-Hu-1 (GenBank NC_045512). All serum samples have been heat-inactivated in a biosafety cupboard at 56 °C for 60 min and twofold serially-diluted protecting 1:20 to 1:40,960 in DMEM supplemented with 10% fetal bovine serum. The sera have been 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 into the combination and incubated at 37 °C for 48 h. A dilution at which the 50% of an infection as in comparison with anti-SARS-CoV-2-negative serum is inhibited (IC50) was used as take a look at cut-off. The plant-based anti-SARS-CoV-2 antibodies; H4 was used as antibody constructive management45,46.
The Institutional Animal Care and Use Committee of the School of Veterinary Science, Chulalongkorn College, Thailand accepted animal examine (IACUC No. 2031035). All strategies have been carried out in accordance with related pointers and laws. The examine complies with the ARRIVE pointers. The IACUC committee of the School of Veterinary Science, Chulalongkorn College, Thailand accepted the knowledgeable consent. The knowledgeable verbal consent was obtained from all pet house owners and personal animal hospital workers after explaining the goals and advantages of the examine throughout pattern assortment.