Detection of SARS-CoV-2 and a possible variant in shelter cats

Abstract

SARS-CoV-2 is the cause of mild to severe acute respiratory disease that led to significant loss of human lives worldwide between 2019 and 2022. The virus has been detected in various animals including cats and dogs making it a major public health concern and a One Health issue. In this study, conjunctival and pharyngeal swabs (n = 350) and serum samples (n = 350) were collected between July and December 2020 from cats that were housed in an animal shelter and tested for the infection of SARS-CoV-2 using real time reverse-transcription polymerase chain reaction (rRT-PCR) that targeted the N1 and N2 genes, and a SARS-CoV-2 surrogate virus neutralization Test (sVNT), respectively. 203 (58%) swab samples were negative (N1 and N2 not detected), 2 (0.6%) were positive (N1 and N2 detected) and 145 (41%) were inconclusive (only N1 detected). Analysis of the N2 region and multiple sequence alignment revealed base-pair deletions and substitutions in the N2 probe binding region of the feline samples RNA extracts in comparison with the positive control and human SARS-CoV-2 sequences in the GenBank database. Substituting the N2 probe with a probe derived from the cat sample amplicon sequences, 123 of 127 (96.9%) of the N2 negative samples returned positive. All but one of the 350 serum samples were negative for SARS-CoV-2 antibody. These observations indicated that although detection of SARS-CoV-2 infection was low in the samples tested, pet cats can harbor the virus and serve as potential source for virus spread that may lead to human infections. Additionally, cats may harbor a yet-to-be described virus that is somewhat related to SARS-CoV-2.

Fig 1. Gel-Red stained agarose gel of PCR products after conventional PCR using the 2019-nCoV_N2-F and 2019-nCoV_N2-R primers (Table 1) and the cDNA generated from first round amplification by rRT-PCR as template.

The assay produced the expected 67-bp product of SARS-CoV-2 (lanes 2 and 6) in addition to a second product of 60-bp (lanes 3–5; 7–10) respectively. Lanes: M, HaeIII-digested øx174 DNA molecular weight standards; 1, negative control (no template); 2; positive control (ATCC VR-1986D); 3–10, feline pharyngeal and conjunctival samples.

Fig 2. SARS-CoV-2 N2 gene region sequence alignment.

Alignment of the nucleotide sequences of the N2 gene region of SARS-CoV-2 from human source (GenBank accession number OR855668.1) with sequences derived from nucleic acid extracts from cat samples. The deletion is boxed and marked with hashes. Base-pair substitutions are boxed. The 2019-nCoV_N2-P and N2-CPR probes are indicated on top of the figure.

Fig 3. Phylogenetic analysis.

Analysis of the nucleotide sequences of the N2 gene region of SARS-CoV-2 and sequences derived from nucleic acid extracts from cat samples using the neighbor joining method showed that the sequences clustered into two distinct groups according to sequence types and are separated from each other with an inter-cluster distance of 0.167. One group consisted of the sequences of SARS-CoV-19 from human at the GenBank database (accession number OR855668.1) and a confirmed positive cat case (643464). The second group consisted of sequences obtained from feline samples RNA extracts consisting of deletion and substitutions compared with the CDC N2 primers probe binding region. The feline sequences were indistinguishable and somewhat related to SARS-CoV-2.