key: cord-0261201-p21ph6ng
authors: Stone, Shannon; Rothan, Hussin A.; Natekar, Janhavi P.; Kumari, Pratima; Sharma, Shaligram; Pathak, Heather; Arora, Komal; Auroni, Tabassum T.; Kumar, Mukesh
title: SARS-CoV-2 variants of concern infect the respiratory tract and induce inflammatory response in wild-type laboratory mice
date: 2021-09-29
journal: bioRxiv
DOI: 10.1101/2021.09.29.462373
sha: 80878e009712187ac6e7ef888afce9bc3f5a903d
doc_id: 261201
cord_uid: p21ph6ng

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern poses a major threat to the public health due to possible enhanced virulence, transmissibility and immune escape. These variants may also adapt to new hosts in part through mutations in the spike protein. In this study, we evaluated the infectivity and pathogenicity of SARS-CoV-2 variants of concern in wild-type C57BL/6 mice. Six-week-old mice were inoculated intranasally with a representative virus from the original B.1 lineage or emerging B.1.1.7 and B.1.351 lineages. We also infected a group of mice with a mouse-adapted SARS-CoV-2 (MA10). Viral load and mRNA levels of multiple cytokines and chemokines were analyzed in the lung tissues on day 3 after infection. Our data show that unlike the B.1 virus, the B.1.1.7 and B.1.351 viruses are capable of infecting C57BL/6 mice and replicating at high concentrations in the lungs. The B.1.351 virus replicated to higher titers in the lungs compared to the B.1.1.7 and MA10 viruses. The levels of cytokines (IL-6, TNF-α, IL-1β) and chemokine (CCL2) were upregulated in response to the B.1.1.7 and B.1.351 infection in the lungs. Overall, these data indicate a greater potential for infectivity and adaptation to new hosts by emerging SARS-CoV-2 variants.

Coronaviruses are a family of positive-sense single strand RNA viruses whose large genomes and propensity for mutation have resulted in a diversity of strains that are capable of adaptation to new hosts. COVID-19, the disease caused by the new beta coronavirus, SARS-CoV-2, has caused significant human and economic burden [1] [2] [3] . Few therapies are available to treat COVID-19 disease in humans and the rapid evolution of SARS-CoV-2 variants threatens to diminish their efficacy [2, 4] . The lineage B.1.1.7, first identified in the United Kingdom, and lineage B.1.351, first described in South Africa, have been termed variants of concern because of the greater risk they pose due to possible enhanced transmissibility, disease severity and immune escape [4] [5] [6] [7] . These variants may also adapt to new hosts in part through mutations on the receptor binding domain (RBD) of the spike protein [6, 7] .

SARS-CoV-2 infection begins with the viral particles binding to the receptors on the host cell surface. The RBD of the spike protein binds to angiotensin-converting enzyme 2 (ACE-2) present on the host cellular surfaces [3, 8] . The RBD of the spike protein from the SARS-CoV-2 strain (Wuhan strain, lineage B.1) that started the pandemic does not efficiently bind mouse ACE-2, and therefore wild-type laboratory mice are not susceptible to infection with lineage B.1 virus [8] [9] [10] [11] [12] . Mouse-adapted SARS-CoV-2 variant (MA10) with binding affinity to mouse ACE-2 has been obtained after sequential passaging of virus in mouse lung tissues [11] . Infection of wild-type BALB/c mice with MA10 virus resulted in replication in both upper and lower airways [11, 13] . MA10 virus has several mutations, including multiple mutations in the spike protein compared to the Wuhan reference sequence. These mutations are also present in the B.1.1.7 and B.1.351 lineages [6, 7, 11, 14, 15] . Since the spike protein is necessary for direct interaction with the host receptor, mutations in the spike protein can affect SARS-CoV-2 infection efficiency depending on the host. However, the infectivity and pathogenicity of these emerging variants in mice have not yet been determined.

In this study, we evaluated the replication and pathogenicity of the original B.1 lineage and emerging SARS-CoV-2 lineages, B.1.1.7 and B.1.351, in wild-type C57BL/6 mice. We also used a mouse-adapted SARS-CoV-2 variant (MA10) that causes disease in the wild-type mice [11] . Our data show that the B. viruses trigger an inflammatory response in the lungs characterized by upregulation of inflammatory cytokines and chemokines.

C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All the animal experiments were conducted in a certified Animal Biosafety Level 3 (ABSL-3) laboratory at Georgia State University (GSU). The protocol was approved by the GSU Institutional Animal Care and Use Committee (Protocol number A20044). Six-week-old C57BL/6 mice were inoculated intranasally with PBS (mock) or 10 5 plaque-forming units (PFU) of SARS-CoV-2 as described previously [16] . We used B. 

The virus titers were analyzed in the lungs by plaque assay and quantitative real-time PCR (qRT-PCR). Briefly, frozen tissues were weighed and homogenized in a bullet blender (Next Advance, Averill Park, NY, USA) using stainless steel beads. Virus titers in tissue homogenates were measured by plaque assay using Vero cells [16, 17] . Quantitative RT-PCR was used to measure viral RNA levels with primers and probes specific for the SARS-CoV-2 N gene as described previously [18] . Viral genome copies were determined by comparison to a standard curve generated using a known amount of RNA extracted from previously titrated SARS-CoV-2 samples [18] . Frozen tissues harvested from mock and infected animals were weighed and lysed in RLT buffer (Qiagen), and RNA was extracted using a Qiagen RNeasy Mini kit (Qiagen, Germantown, MD, USA) [17, 19] . Total RNA extracted from the tissues was quantified and normalized, and viral RNA levels per !g of total RNA were calculated.

Total RNA was extracted from lungs using a Qiagen RNeasy Mini kit (Qiagen, Germantown, MD, USA). cDNA samples were prepared using an iScript™ cDNA Synthesis Kit (Bio-Rad). The expression levels of multiple host genes were determined using qRT-PCR, and the fold change in infected lungs compared to mock-infected controls was calculated after normalizing each sample to the level of the endogenous GAPDH gene mRNA [16, 17, 19] . The primer sequences used for qRT-PCR are listed in Table 1 . 

Mann-Whitney test and unpaired student t-test using GraphPad Prism 5.0 were used to calculate p values of the difference between viral titers and immune responses, respectively. Differences of p <0.05 were considered significant. [4, 6, 24] . More studies are needed to characterize the pathological consequences of infection with these variants in different mouse strains and mice with co-morbid conditions. It is possible that more severe condition could be observed in mice with co-morbid conditions such as old age, diabetes and hypertension. The ability of clinical SARS-CoV-2 isolates to replicate and induce inflammation in wild-type mice will facilitate studies to evaluate therapeutic interventions and pathogenesis studies. These data indicate the possibility of adaptation to new animal species by emerging SARS-CoV-2 variants.

Funding: This work was supported by a grant (R21OD024896) from the Office of the Director, National Institutes of Health, and Institutional funds. Acknowledgments: We thank members of the GSU High Containment Core and the Department for Animal Research for assistance with the experiments.

The authors declare no conflict of interest.

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