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

In June–September 2021, we investigated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections in domestic dogs and cats (n = 225) in Bangkok and the vicinities, Thailand. SARS-CoV-2 was detected in a dog and a cat from COVID-19 positive households. Whole genome sequence analysis identified SARS-CoV-2 delta variant of concern (B.1.617.2). Phylogenetic analysis showed that SARS-CoV-2 isolated from dog and cat were grouped into sublineage AY.30 and AY.85, respectively. Antibodies against SARS-CoV-2 could be detected in both dog (day 9) and cat (day 14) after viral RNA detection. This study raises awareness on spill-over of variant of concern in domestic animals due to human-animal interface. Thus, surveillance of SARS-CoV-2 in domestic pets should be routinely conducted.


SARS-CoV-2 infection in dogs and cats. A cross-sectional survey for SARS-CoV-2 in dogs and cats
in Bangkok and the vicinities was conducted during June to September 2021. We collected nasal, oral, and rectal swabs from 225 animals (105 dogs and 120 cats) from 199 households. Of 225 animals, 19 animals were sampled from twelve COVID-19 positive households (Table 1). SARS-CoV-2 detection by realtime RT-PCR assay with specific primers and probes for the N2, E and RdRp genes was performed following CDC and WHO recommendations 20,21 . In this study, SARS-CoV-2 RNA could be detected from a cat (n = 1) and a dog (n = 1) from COVID-19 positive households. The positive swab samples were from a cat (CU27516) in Nonthaburi province in July 2021 and a dog (CU27791) in Bangkok in September 2021 (Table 2).
COVID-19 positive dog and cat were followed up for virological and serological investigation. A cat (CU27516) is 10 years-old, 3 kg, domestic short hair, spayed female cat. A cat was healthy and transferred to private animal hospital on 15 July 2021 (day 1) due to family members (n = 3) were COVID-19 positive and quarantined at the field hospital. All members were tested positive on 12-14 July 2021 and tested negative after 14 days. Swab (nasal, oral and rectal) and environmental samples were collected on four occasions (day 1, 3, 7, and 10). During quarantine, a cat did not show any clinical signs. Nasal and oral swabs tested positive for SARS-CoV-2 RNA at day 1 and day 3 (Ct 20.66-34.36). Low viral RNA (high Ct Value) was detected in animal hair swabs on day 3 (Ct 36.56). All samples tested negative on day 7 and day 10 ( Table 3 and Fig. 1). A dog (CU27791) is 15 years-old, 7.5 kg, Shih-Tzu breed, intact female dog. The animal was transferred to private animal hospital on 12 September 2021 due to family members (n = 4) tested positive for COVID-19 on 8-11 September 2021. Sample collection was conducted on five occasions (day 1, 3, 5, 7, and 9). SARS-CoV-2-RNA was detected in nasal and oral swabs of dogs on day 1-7 (Ct 19.06-39.87). The highest viral titers (low Ct values) were observed in nasal and oral swabs on day 3. Viral RNAs were also detected in environmental samples (hair, water container and floor) on day 3, 5, and 7 (Ct 29.87-38.13). A dog did not have fever but showed mild respiratory signs with serous nasal discharge on day 5-7 (Table 3 and Fig. 1).  In this study, we collected blood sample from SARS-CoV-2 positive animals (n = 6 serum; cat (CU27516) (n = 2 time points) and dog (CU27791) (n = 4 time points)). We tested serum samples for SARS-CoV-2 antibodies by indirect multispecies ELISA, surrogate virus neutralization test (sVNT) and pseudotyped virus neutralization (pVNT). Our result showed that a cat was positive for SARS-CoV-2 antibodies by indirect ELISA (113%-271%), sVNT (90%-95%) and pVNT (1:640) at day 14 and day 21. While a dog was positive for SARS-CoV-2 antibodies by sVNT (48.81%) at day 24 and pVNT (1:20) at day 9 and day 24 (Table 4).

Delta variant of SARS-CoV-2 from dog and cat in Thailand.
After SARS-CoV-2 detection, we performed whole genome sequencing from nasal swab of cat (CU27516; Ct 20.66) and dog (CU27791; Ct 19.06) by using the ARTIC multiplex PCR protocol and MinION sequencing platform (Oxford Nanopore Technolo- Table 3. Result of SARS-CoV-2 detection from cat and dog swab samples by realtime-RT-PCR. a Primers and probes specific to N gene 21 . b Primers and probes specific to E and RdRp genes of SARS-CoV-2 20 .     Table 1). Phylogenetic analysis of SARS-CoV-2 was performed by comparing complete genome of SARS-CoV-2 from dog and cat in this study and 289 SARS-CoV-2 genomes available in the GISAID and GenBank database. The sequences were aligned by using the MAFFT FFT-NS-2 algorithm and phylogenetic tree was constructed by using IQ-TREE 2 applying the GTR + Γ model of nucleotide substitution with default heuristic search options and bootstrapping with 1000 replicates. Lineage classification was performed by using the Pangolin tool. Phylogenetic analysis showed that SARS-CoV-2 of dog and cat in this study clustered with human SARS-CoV-2 of B.1.617.2 (Delta variant of concern). The cat and dog SARS-CoV-2 were grouped into sublineage AY. 30 Table 2). Characteristic mutation analysis on spike protein of SARS-CoV-2 of dog and cat showed identical mutations to those of delta variant ( Table 5). The mutations at the N-terminal-domain (NTD) (T19R, E156G, F157del, R158del), the receptor binding motif (RBM) (L452R, T478K), the subdomain 2 (D614G), S1 unit (P681R) and heptad repeat 1 (D950N) were observed. It should be noted that delta variant, AY.85 sublineage contained additional mutation at T95I on N terminal domain (NTD) and it may associate with higher viral load which promote viral transmission from human to human/animal due to close contact.

Discussion
In this study, we conducted a cross-sectional survey in dogs and cats in private animal hospitals from June-September 2021. During investigation period, we identified SARS-CoV-2 delta variant of concern (B. Virological test for SARS-CoV-2 showed that viral RNA could be detected from nasal and oral swabs of a cat and a dog at day 1 (4-5 days after the owner reported COVID-19 positive). It is interesting to note that SARS-CoV-2 RNA could also be detected in animal hair of cat at day 3 and environmental samples (hair, water container and floor) of dog at day 3 and day 7. Similarly, in previous reports, SARS-CoV-2 RNA could be found in environmental samples e.g. water container and cage floor of domestic pet as well as dust and air samples in mink farms 1,26 . Serological test for SARS-CoV-2 antibodies in this study showed that a cat developed SARS-CoV-2 antibodies since day 14 (by ELISA, sVNT and pVNT) after SARS-CoV-2 RNA detection. While a dog   www.nature.com/scientificreports/ was positive for SARS-CoV-2 antibodies at day 9 (by pVNT) and day 24 (by sVNT and pVNT). Our findings in agreement with previous studies that dogs and cats can develop antibodies against SARS-CoV-2 as early as 7 to 14 days post infection 5,27,28 . For example, our previous studies showed that SARS-CoV-2 antibodies could be detected from dogs at day 11-23 and from cats at day 6 after SARS-CoV-2 RNA detection. Notably, control dog and cat sera and pre-COVID-19 sera have been tested in our previous report 1 . However, the timing for serum sample collection when the animal might have been exposed to COVID-19 owners could influence the antibodies titer and the discrepancy between the result of ELISA, sVNT and pVNT. Phylogenetic analysis showed that the cat and dog SARS-CoV-2 were grouped into delta variant of concern (B.    29 . A previous study reported role of mutations on spike gene may not in relation to hostadapted mutations 30 . Notably, unlike previous study, M1227L on S gene was not observed in our SARS-CoV-2 from dog. It speculated that this point mutation may relate to S protein destabilization and transmission from human-to-dog 17,31 .
In conclusion, results from phylogenetic and mutation analysis suggested that the virus infecting dog and cat in this study originated from a local outbreak cluster of delta variant AY.30 and AY.85. Notably, AY.30 and AY.85 sub-lineages are predominately found in Thailand. In Thailand, most veterinary clinics and hospitals have followed COVID-19 management guideline provided by the department of livestock development, Thailand. Since most SARS-CoV-2 infected animals are asymptomatic or less symptomatic. History of close contact between owners and domestic animals in COVID-19 positive household is crucial for veterinary practitioners to monitor SARS-CoV-2 infection in domestic animals. This study is the first to report SARS-CoV-2 delta variant infection in domestic dogs and cats in Thailand. Our finding supports routine surveillance of SARS-CoV-2 in domestic animals and raises more awareness on frequent spill-over of variant of concerns due to high human-animal interface.

Sample collection from domestic dogs and cats for SARS-CoV-2. During June-September 2021,
we conducted SARS-CoV-2 survey in domestic dogs and cats living in Bangkok and the vicinities. 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 household (n = 12) and unknown status households (n = 187) (Tables 1 and 2). We collected nasal swabs (dog; n = 8; cat; n = 16), oral swabs (dog; n = 102, cat; n = 117) and rectal swabs (dog; n = 104, cat; n = 120) from the animals by using flocked nylon swab (Copan®, California, USA). The swab samples were placed in RNA protect® (Qiagen LLC, Maryland, USA) and transported to the laboratory within 24 h. In this study, we followed up SARS-CoV-2 positive dog (n = 1) and cat (n = 1). During follow up, we collected nasal swabs (n = 9), oral swabs (n = 9), rectal swabs (n = 9) from the animals. In addition, environmental swabs, hair (n = 7), water container (n = 7), and floor (n = 7) were also collected (Table 3). We collected blood sample (n = 6 serum; 2 time points from a cat and 4 timepoints 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 carried out in accordance with relevant guidelines and regulations. Table 5. Characteristic mutations of SARS-CoV-2 delta variant from cat (AY.30) and dog (AY.85) and reference viruses. a SARS-CoV-2 characterized in this study. b SARS-CoV-2 characterized in this study. 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 . In brief, 7.5 µl of pooled PCR products (10 µl of pool 1 and 10 µl of pool 2) was added to 2.5 µl of fragmentation mix.  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. The qualify reads were conversed from "Fast5" into "Fastq" format by using the GPU version of the Nanopore Guppy basecaller (v3.4.4) tool. The Fastq format sequences were assembled using the genome detective program 35

Phylogenetic analysis of SARS-CoV-2.
Whole genome sequences of SARS-CoV-2 were subjected to lineage classification by using the COVID-19 sequences of the Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) (https:// cov-linea ges. org/ resou rces/ pango lin. html). Phylogenetic analysis of SARS-CoV-2 was performed by comparing nucleotide sequences of 289 SARS-CoV-2 genomes available in the GISAID and GenBank database. The 5′ and 3′ untranslated regions were trimmed with at least 95% reference genome coverage (Wuhan-Hu-1) (at least 29,000 bp in length). The dataset alignment was performed by using the MAFFT FFT-NS-2 algorithm with default parameter settings 36 . The maximum likelihood tree was constructed by using IQ-TREE version 2.1.3 (http:// www. iqtree. org/) 37 using the GTR + Γ model of nucleotide substitution 38 , default heuristic search options, and ultrafast bootstrapping with 1000 replicates 39 . Tree was visualized by iTOL version 6.0 (https:// itol. embl. de/) 40 . Lineage classification was performed by using the Pangolin tool 41 . Genetic mutation analysis of the SARS-CoV-2 was performed by comparing deduced amino acids of each gene of the viruses based on variant classifications and definitions 42,43 . The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript Biotech, Jiangsu, China) was used to detect neutralizing antibodies. The assay detects SARS-CoV-2 antibodies by measurement of antibodymediated inhibition of SARS-CoV-2 RBD-ACE2 interaction. In brief, 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 treated serum then added to ACE2-coated ELISA plate and incubated at 37 °C for 15 min. Then the uncaptured substrate was washed out by using 260 µL of washing solution for four times. Colorimetric signal was developed by using TMB substrate at 25 °C for 15 min. Absorbance reading at 450 nm was acquired using a microplate reader immediately after adding stop solution. The percentage inhibition was calculated. The sample with % inhibition ≥ 20% indicate the presence of SARs-CoV-2 neutralizing antibody, otherwise are negative 44 .