In a recent study posted to the bioRxiv* preprint server, researchers evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure among rats inhabiting New York City (NYC) during the fall of 2021.
Studies have reported that SARS-CoV-2 VOCs (variants of concern) such as the Alpha VOC, Beta VOC, and Gamma VOC comprise infectivity-enhancing mutations in their spike (S) protein RBD (receptor-binding domain). SARS-CoV-2 tropism expansion has raised probable risks of reverse SARS-CoV-2 transmission into rodents. Studies have indicated probable SARS-CoV-2 exposure in animals; however, SARS-CoV-2 ribonucleic acid (RNA) has not been detected in rodents. Whether rats can be infected by the relatively recent Delta VOC and Omicron VOC remains unknown.
About the study
In the present study, researchers evaluated the ability of Omicron and Delta VOCs to infect Rattus norvegicus species of rats and investigated SARS-CoV-2 exposure among Norwegian rats.
To determine rat exposure to SARS-CoV-2, coronavirus disease 2019 (COVID-19), surveillance was conducted in Norway Rattus norvegicus rats between 13 September and 16 November 2021, during the period of Delts VOC predominance. Enzyme-linked immunosorbent assays (ELISA) were performed to measure immunoglobulin M (IgM) or IgG antibody titers. Microneutralization assays analyzed seropositive rat sera for responses against the SARS-CoV-2 B.1 strain, Alpha VOC and Delta VOC. For control experiments, sera from uninfected SD (Sprague Dawley) rats and those infected by the Sialodacryoadenitis virus, Parker’s rat coronavirus (CoV), or other rat CoVs were analyzed.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed. The qRT-PCR-positive samples were subjected to SARS-CoV-2 genome sequencing analysis, following which phylogenetic and molecular characterization analyses were performed. In addition, rat samples were subjected to pan-viral target hybridization enrichment sequencing analysis. Further, to investigate whether the Delta and Omicron SARS-CoV-2 VOCs could infect rats, wild-type SD rats were challenged intranasally with Alpha, Delta, or Omicron VOCs, and their samples were obtained at two- and four-days post-infection (dpi).
To evaluate the adaptive and innate immunological responses induced by SARS-CoV-2 in the rodents, the pulmonary chemokine/cytokine expression was determined at two dpi and four dpi, and antibody titers were assessed at 21 dpi. To detect probable host-adapted mutations, rodent lung tissues were challenged with Alpha, Delta, or Omicron VOCs and sequenced. To evaluate SARS-CoV-2 replication efficiency among SD rats, the interactions between rat angiotensin-converting enzyme 2 (ACE2) and S RBD of Alpha, Delta, and Omicron VOCs were modeled computationally.
A total of 79 rats captured from three NYC sampling locations were sampled, of which 13 (17%) rats demonstrated positive IgM or IgG titers. Alpha, Delta, and Omicron VOCs led to robust SARS-CoV-2 infections in wild-type SD rats, with high replication titers in the lower and upper respiratory tracts and induction of adaptive and innate immunological responses. Nine IgG-positive rat samples (11%) and four IgM-positive rat samples (five percent) against Wuhan-Hu-1 S and S RBD were identified.
All samples subjected to microneutralization assays lacked SARS-CoV-2 neutralizing antibodies. Lung samples of only four rats were SARS-CoV-2-positive by qRT-PCR analysis. Of note, two of the four rats showed seropositivity and detectable SARS-CoV-2 RNA levels. However, viruses could not be retrieved from 293FT/human ACE2-positive (hACE2+) transmembrane serine protease (TMPRSS), Vero E6, rat lung tracheal epithelial cell line, or rat lung epithelial cells. A partial SARS-CoV-2 genome was detected among all rat samples with SARS-CoV-2 genome coverage ranging between two percent and 21%.
SARS-CoV-2 present in the rat samples was linked to the genetic B lineage. SARS-CoV-2 presence was detected in three qRT-PCR-positive rat samples, and one inconclusive sample and rat CoV was identified in an inconclusive rat sample. The detected rat CoV or SARS-CoV-2 reads aligned with several genes across the corresponding genomes. At two and four dpi, elevated SARS-CoV-2 RNA levels were observed in rat lungs and turbinates with infectious SARS-CoV-2 titers identified in lungs and/or turbinates with no loss of weight or any other clinical signs among infected rodents.
Delta-infected rat lungs demonstrated the greatest RNA levels and titers of infectious SARS-CoV-2 titers at two dpi. SARS-CoV-2 antigens were also expressed in lung tissues of any VOC-infected rats at two or four dpi, with the greatest antigen expression among Delta-infected rats. All VOC infections induced expression of interferon (IFN)-β, -γ, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, -1β, -6, -10, chemokine ligand 2 (CCL-2), and inducible protein 10 (IP-10). Delta-induced cytokine expression was greater than Omicron VOC and Alpha VOC.
At 21 dpi, neutralizing antibody titers and IgG titers were observed against all three VOCs; however, IgM titers were undetectable among infected rats. RBD IgG titers against Delta VOC were significantly greater than those against Omicron VOC, and Delta-neutralizing titers were significantly greater than Alpha- or Omicron-neutralizing titers. Lung samples sequenced from Alpha-, Delta-, or Omicron-infected rats showed no adapted mutations in their RBDs. However, N74K was observed in the S protein of Alpha-infected rats, and D950N and P681R were observed in S of Delta-infected rats.
Mutations were also detected in non-structural proteins (NSP)-6,13, and nucleocapsid (N) protein of Alpha- and Delta-challenged rats. Structural modeling analysis showed that the Alpha VOC, Delta VOC and Omicron VOC had enhanced rat ACE2 binding than the prototype.
Overall, the study findings showed that the Alpha VOC, Delta VOC, and Omicron VOC could infect SD rats and induce immunological responses with Delta VOC-infected rats replicating more efficiently than Alpha- and Omicron-challenged rats.
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