Genes involved in the limited spread of SARS-CoV-2 in the lower respiratory airways of hamsters may be associated with adaptive evolution

Abstract

Novel respiratory viruses can cause a pandemic and then evolve to coexist with humans. The Omicron strain of severe acute respiratory syndrome coronavirus 2 has spread worldwide since its emergence in late 2021, and its sub-lineages are now established in human society. Compared to previous strains, Omicron is markedly less invasive in the lungs and causes less severe disease. One reason for this is that humans are acquiring immunity through previous infection and vaccination, but the nature of the virus itself is also changing. Using our newly established low-volume inoculation system, which reflects natural human infection, we show that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain. Furthermore, by characterizing chimeric viruses with the Omicron gene in the Wuhan strain genetic background and vice versa, we found that viral genes downstream of ORF3a, but not the S gene, were responsible for the limited spread of the Omicron strain in the lower airways of the virus-infected hamsters. Moreover, molecular evolutionary analysis of SARS-CoV-2 revealed a positive selection of genes downstream of ORF3a (M and E genes). Our findings provide insight into the adaptive evolution of the virus in humans during the pandemic convergence phase.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread worldwide since its emergence in late 2021, and its sub-lineages are established in human society. Compared to previous strains, the Omicron strain is less invasive in the lower respiratory tract, including the lungs, and causes less severe disease; however, the mechanistic basis for its restricted replication in the lower airways is poorly understood. In this study, using a newly established low-volume inoculation system that reflects natural human infection, we demonstrated that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain and found that viral genes downstream of ORF3a are responsible for replication restriction in the lower respiratory tract of Omicron-infected hamsters. Furthermore, we detected a positive selection of genes downstream of ORF3a (especially the M and E genes) in SARS-CoV-2, suggesting that these genes may undergo adaptive changes in humans.

Keywords: animal models; coronavirus; evolution.

Fig 1

Comparison of the low- and high-virus-volume hamster infection systems using Wuhan strains. Wild-type Syrian hamsters were intranasally inoculated with 10³ PFU in 30 µL (high virus volume) or 3 µL (low virus volume) of recombinant Wuhan strain. (a) Body weights of virus-infected (n = 12) animals were monitored daily for up to 7 days. Note that three animals were euthanized in each group at 1-, 3-, and 5-days post-infection. Data are presented as the mean percentages of the starting weight (±SD). (b) Virus titers in the organs of hamsters infected with each virus. Three hamsters per group were euthanized at 1-, 3-, 5-, and 7-days post-infection for virus titration. Virus titers in the nasal turbinate, trachea, and lung were determined by means of plaque assays in VeroE6/TMPRSS2 cells. Vertical bars show the virus titer, and the same row indicates the same individual hamsters. The detection limit was set at 0.5 (log₁₀ PFU/g). Mean values were compared by an analysis of variance, followed by Dunnett’s test (*P < 0.05 and **P < 0.01). (c) Immunohistochemical (IHC) detection of the SARS-CoV-2 nucleocapsid (N) antigen in the nasal turbinate, trachea, and lung of two individuals in each group. Viral antigen-positive regions are indicated in red. (d and e) Histopathological examination of the lungs, trachea, and nasal turbinate of an infected hamster at 5 days post-infection. (d and e) Representative results of IHC analysis for SARS-CoV-2 (N) antigen or hematoxylin and eosin (HE) staining images of infected nasal turbinate (d) and (e) lungs of hamsters infected with the Wuhan strain at 5 days post-infection using the high-volume (left panel) or low-volume infection system (right panel). Scale bars, 0.5 mm.

Fig 2

Low-virus-volume hamster infection system using Omicron strains. Wild-type Syrian hamsters were intranasally inoculated with 10³ PFU in 3 µL (low virus volume) of recombinant Omicron strain. Note, the Wuhan results are shown next to the Omicron results for comparison; these Wuhan data are the same as those shown in Fig. 1. (a) Body weights of virus-infected (n = 13) animals were monitored daily for up to 7 days. Note that three animals were euthanized in each group at 1-, 3-, and 5-days post-infection. Data are presented as the mean percentages of the starting weight (±SD). (b) Virus titers in the organs of hamsters infected with each virus. Three hamsters per group were euthanized at 1-, 3-, 5-, and 7-days post-infection for virus titration. Virus titers in the nasal turbinate, trachea, and lungs were determined by means of plaque assays in VeroE6/TMPRSS2 cells. Vertical bars show the virus titer, and the same row indicates the same individual hamsters. The detection limit was set at 0.5 (log₁₀ PFU/g). Mean values were compared by an analysis of variance, followed by Dunnett’s test (*P < 0.05 and **P < 0.01). (c) IHC analysis for SARS-CoV-2 (N) antigen in the nasal turbinate, trachea, and lungs of two individuals in each group. Viral antigen-positive regions are indicated in red. (d) SEM images of tracheal epithelia from mock-, Wuhan strain-, and Omicron strain-infected hamsters at 3 days post-infection.

Fig 3

Comparison of the spread of chimeric Wuhan and Omicron viruses in the lower airways of hamsters. (a) Schematic overview of the generated chimeric viruses derived from the Wuhan and Omicron strains. (b–d) Virus titers in the organs of hamsters infected with each chimeric virus and the parent strains [(b) S gene chimeric virus; (c), ORF1ab gene chimeric virus; and (d) ORF3-10 chimeric virus]. For virus titration, four hamsters per group were euthanized 3-days post-infection. Virus titers in the nasal turbinate, trachea, and lungs were determined by means of plaque assays in VeroE6/TMPRSS2 cells. Vertical bars show the average virus titers. Detection limits are indicated by dotted lines. Mean values were compared by an analysis of variance, followed by Tukey’s test (*P < 0.05 and **P < 0.01).