The Omicron Variant BA.1.1 Presents a Lower Pathogenicity than B.1 D614G and Delta Variants in a Feline Model of SARS-CoV-2 Infection

Abstract

Omicron (B.1.1.529) is the most recent SARS-CoV-2 variant of concern, which emerged in late 2021 and rapidly achieved global predominance by early 2022. In this study, we compared the infection dynamics, tissue tropism, and pathogenesis and pathogenicity of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), and Omicron BA.1.1 (B.1.1.529) variants in a highly susceptible feline model of infection. Although D614G- and Delta-inoculated cats became lethargic and showed increased body temperatures between days 1 and 3 postinfection (pi), Omicron-inoculated cats remained subclinical and, similar to control animals, gained weight throughout the 14-day experimental period. Intranasal inoculation of cats with D614G- and the Delta variants resulted in high infectious virus shedding in nasal secretions (up to 6.3 log₁₀ TCID₅₀.Ml^-1), whereas strikingly lower level of viruses shedding (<3.1 log₁₀ TCID₅₀.Ml^-1) was observed in Omicron-inoculated animals. In addition, tissue distribution of the Omicron variant was markedly reduced in comparison to the D614G and Delta variants, as evidenced by lower in situ viral RNA detection, in situ viral immunofluorescence staining, and viral loads in tissues on days 3, 5, and 14 pi. Nasal turbinate, trachea, and lung were the main-but not the only-sites of replication for all three viral variants. However, only scarce virus staining and lower viral titers suggest lower levels of viral replication in tissues from Omicron-infected animals. Notably, while D614G- and Delta-inoculated cats presented pneumonia, histologic examination of the lungs from Omicron-infected cats revealed mild to modest inflammation. Together, these results demonstrate that the Omicron variant BA.1.1 is less pathogenic than D614G and Delta variants in a highly susceptible feline model. IMPORTANCE The SARS-CoV-2 Omicron (B.1.1.529) variant of concern emerged in South Africa late in 2021 and rapidly spread across the world causing a significant increase in the number of infections. Importantly, this variant was also associated with an increased risk of reinfections. However, the number of hospitalizations and deaths due to COVID-19 did not follow the same trends. These early observations suggested effective protection conferred by immunizations and/or overall lower virulence of the highly mutated variant virus. In this study we present novel evidence demonstrating that the Omicron BA.1.1 variant of concern presents a lower pathogenicity when compared to D614G- or Delta variants in cats. Clinical, virological, and pathological evaluations revealed lower disease severity, viral replication, and lung pathology in Omicron-infected cats when compared with D614G and Delta variant inoculated animals, confirming that Omicron BA.1.1 is less pathogenic in a highly susceptible feline model of infection.

Keywords: COVID-19; Delta; Omicron; SARS-CoV-2; cats; pathogenesis; pathogenicity; variant of concern.

FIG 1

Experimental design, body temperature and weight changes following SARS-CoV-2 inoculation in cats. Schematic representation of the experimental design of infection study in adult cats (24-40-month-old) males and females. Animals were allocated in three inoculated groups (n = 7 per group) and a control group (mock-inoculated) (n = 3). Animals were inoculated intranasally with 1 mL (0.5 mL per nostril) of virus suspension containing 5 × 10⁵ PFU of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), or Omicron BA.1.1 (B.1.1.529), or 1 mL of cell culture supernatant media (control mock-inoculated). On day 3 and 5 postinfection (pi), two cats from each group (one female and one male) were humanely euthanized and the remaining cats (one female and two males) were maintained until day 14 pi. Respiratory secretion, feces and serum were collected on the specific days as indicated (A). NS = nasal swab; OPS = oropharyngeal swab; RS = rectal swab; F = female; M = male. Body temperature (B) and body weight changes (C) following intranasal SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), or Omicron BA.1.1 (B.1.1.529) inoculation throughout the 14-day experimental period. Data are presented as means ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

FIG 2

Viral shedding dynamics in respiratory secretions and feces following SARS-CoV-2 D614G, Delta, and Omicron BA.1.1 inoculation in cats. SARS-CoV-2 RNA load assessed in nasal (A), oropharyngeal secretions (B), and feces (C), collected on days 0, 1, 3, 5, 7, 10, and 14 postinfection (pi), and in bronchoalveolar lavage fluid (BALF) collected on 3, 5, and 14 pi (D). Samples were tested for the presence of SARS-CoV-2 RNA by real-time reverse transcriptase PCR (rRT-PCR) (genomic and subgenomic viral RNA load). Infectious SARS-CoV-2 load in nasal (E) and oropharyngeal secretions (F), feces (G), and BALF (H) assessed by virus titration in rRT-PCR-positive samples. Virus titers were determined using endpoint dilutions and expressed as TCID₅₀.Ml⁻¹. Data are presented as means ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

FIG 3

Tissue distribution of SARS-CoV-2 D614G, Delta, and Omicron BA.1.1 following virus inoculation in cats. Tissues were collected and processed for real-time reverse transcriptase PCR (rRT-PCR) and infectious virus titrations. Tissue distribution of SARS-CoV-2 RNA assessed by rRT-PCR. Samples from two cats per group were tested at day 3 (A) and 5 (B) postinfection (pi), and samples from three cats per group were tested at day 14 pi (C). Tissue distribution of subgenomic SARS-CoV-2 RNA (replicating RNA) assessed in tissue samples collected on days 3 (D), 5 (E), and 14 pi (F). Infectious SARS-CoV-2 in tissues assessed by virus titrations in rRT-PCR-positive samples obtained on days 3 (G), 5 (H) and 14 pi (I). Virus titers were determined using endpoint dilutions and expressed as TCID₅₀.Ml⁻¹. Data are presented as means ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

FIG 4

In situ hybridization (ISH) in tissues of SARS-CoV-2 D614G, Delta, and Omicron BA.1.1 inoculated cats. Nasal turbinate, trachea and lung samples collected from SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), and Omicron (B.1.1.529) inoculated cats on days 3, 5, and 14 postinfection (pi), and on day 14 pi from controls (mock-inoculated). Note, intensive labeling of viral RNA (SARS-CoV-2-Spike) (Red) on the tissues from D614G- and Delta-inoculated cats, while less abundant labeling on tissues from Omicron-infected animals on days 3 and 5 pi (A). In addition to above-described tissues, palate/tonsil, retropharyngeal lymph node, and heart were evaluated. ISH scores (established as described in Materials and Methods) for the tissues evaluated on days 3, 5, and 14 pi are presented in (B), (C), and (D), respectively.

FIG 5

In situ immunofluorescence (IFA) in tissues of SARS-CoV-2 D614G, Delta, and Omicron BA.1.1 inoculated cats. Tissues were subjected to IFA using a monoclonal antibody against nucleocapsid protein (NP) of SARS-CoV-2. Nasal turbinate, trachea and lung were collected from SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), and Omicron (B.1.1.529) inoculated cats on days 3, 5, and 14 postinfection (pi), and on day 14 pi from controls (mock inoculated). Note, intensive labeling of NP (Green) on the tissues from D614G-, and Delta-infected cats, while trace amount of staining on tissues from Omicron-infected animals. Nuclear counterstain was performed with DAPI (Blue) (A). In addition to above-described tissues, palate/tonsil, retropharyngeal lymph node, and heart were evaluated. IFA scores (established as described in Materials and Methods) for the tissues evaluated on days 3, 5 and 14 are presented in (B), (C), and (D), respectively.

FIG 6

Histological examination of respiratory tract of SARS-CoV-2 D614G, Delta, and Omicron inoculated cats. Histological examination of the respiratory tract from SARS-CoV-2- inoculated cats on days 3, 5, and 14 postinfection (pi), and on day 14 pi from controls (mock-inoculated). Intense inflammatory infiltrates in the nasal turbinate can be observed. Trachea and lung are less compromised, and the lesions in the lung of Omicron-inoculated cats are less intense (A). Lesion scores (established as described in Materials and Methods) for the tissues evaluated on days 3, 5, and 14 pi are presented in (B), (C), and (D), respectively.

FIG 7

Serological responses following SARS-CoV-2 D614G, Delta, and Omicron inoculation in cats. Kinetics of antibody response against SARS-CoV-2 N protein. Serum from SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), and Omicron BA.1.1 (B.1.1.529) inoculated cats collected at days 0, 7, and 14 pi were subjected to indirect ELISA analysis. y axis represents S/P ratio and different colors denotes different treatment groups. Samples were considered positive when cutoff value (S/P ratio) equal or above of 0.5. Data are presented as means ± standard error (A). Neutralizing antibodies (NAbs) responses to SARS-CoV-2 and cross-reactivity between D614G (B.1), Delta (B.1.617.2), and Omicron BA.1.1 (B.1.1.529) variants were assessed by PRNT90 (90% plaques reduction) (B) and VN (100% neutralization) (C). Data are presented as means ± standard deviation. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001.

FIG 8

Serum inflammatory cytokine response after infection with different SARS-CoV-2 variants. (A–R) Serum from SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), and Omicron BA.1.1 (B.1.1.529) inoculated cats collected at days 3, 5, 7, and 14 pi were subjected to Milliplex immunoassay to determine the protein expression levels of cytokines/chemokines. x axis represents different time points and y axis represents fold change relative to each corresponding cat at day 0 pi. Data are presented as means ± standard error. KC = neutrophil chemoattractant; RANTES (CCL5) = Regulated upon Activation, Normal T Cell Expressed and Secreted; TNF-α = tissue necrosis factor α; GM-CSF = granulocyte-macrophage colony-stimulating factor.

FIG 9

Infection dynamics and pathogenesis of SARS-CoV-2 D614G, Delta, and Omicron BA.1.1 variants in a feline model of infection. After intranasal inoculation with SARS-CoV-2 D614G or Delta, cats became lethargic, and showed increased body temperatures, while Omicron-inoculated and controls cats (mock inoculate) (gray lines in the graphs) remained subclinical and gained weight throughout the experimental period. Cats inoculated with SARS-CoV-2 D614G- and the Delta variants presented in higher levels of infectious virus shedding in nasal secretions and in tissues, whereas strikingly lower levels of virus shedding and reduced tissue distribution and histologic lesions were observed on Omicron-inoculated animals. Neutralizing antibody (NAbs) responses were higher in SARS-CoV-2 D614G, or Delta inoculated cats.