The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters

Abstract

As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel variant of concern (VOC) designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and shown to enhance infectivity in vitro and decrease antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both strains exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most body weight loss among all 3 lineages. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three strains. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the upper respiratory tract (URT) but not in the lungs. In multi-virus in-vivo competition experiments, we found that epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the URT gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) variants in hamsters. These results demonstrate enhanced virulence and high relative fitness of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) strain.

Author summary: In the last 12 months new variants of SARS-CoV-2 have arisen in the UK, South Africa, Brazil, India, and California. New SARS-CoV-2 variants will continue to emerge for the foreseeable future in the human population and the potential for these new variants to produce severe disease and evade vaccines needs to be understood. In this study, we used the hamster model to determine the epsilon (B.1.427/429) SARS-CoV-2 strains that emerged in California in late 2020 cause more severe disease and infected hamsters have higher viral loads in the upper respiratory tract compared to the prior B.1 (614G) strain. These findings are consistent with human clinical data and help explain the emergence and rapid spread of this strain in early 2021.

Figure 1.. Change in body weight and lung histopathology scores in hamsters after intranasal inoculation with B.1 (614G), B.1.427 and B.1.429.

A) Change in body weight relative to the day of inoculation. A mixed effects model was used to compare the groups. B) Total lung histopathology score (see methods for explanation). C) Lung vascular histopathology score (see methods for explanation). In A, mean values from each group were compared by fitting a mixed model, and a post-hoc multiple comparison test was used to compare the mean values of the 427 or 429 groups individually to the 614G group. Top of bars indicate mean values in B and C..

Figure 2.. Extent of lung histopathology in hamsters after intranasal inoculation with B.1 (614G), B.1.427 and B.1.429.

A, F, K) histology of normal lungs from uninfected hamsters. Top row: Histology of B.1 (614G) infection after B) 2 days PI, C) 4 days PI, D) 6 days PI, E) 10 days PI. Middle row: Histology of B.1.427 infection after G) 2 days PI, H) 4 days PI, I) 6 days PI, J) 10 days PI. Bottom row: Histology of B.1.429 infection after L) 2 days PI, M) 4 days PI, N) 6 days PI, O) 10 days PI. Hematoxylin and eosin staining. Sub-gross magnification. Scale bars equal 1 mm.

Figure 3.. Nature of lung histopathology in hamsters after intranasal inoculation with B.1 (614G), B.1.427 and B.1.429.

A, F, K) histology of normal lungs from uninfected hamsters. Top row: Histology of B.1 (614G) infection after B) 2 days PI, C) 4 days PI, D) 6 days PI, E) 10 days PI. Middle row: Histology of B.1.427 infection after G) 2 days PI, H) 4 days PI, I) 6 days PI, J) 10 days PI. Bottom row: Histology of B.1.429 infection after L) 2 days PI, M) 4 days PI, N) 6 days PI, O) 10 days PI. Hematoxylin and eosin staining. 100x magnification. Scale bars equal 50 um, inset scale bars equal 20 um.

Figure 4.. Distribution of SARS-CoV-2 RNA+ cells in lungs of hamsters after intranasal inoculation with B.1 (614G), B.1.427 and B.1.429 by in-situ hybridization (ISH).

Sub-gross histology and magnified regions (inset) of ISH-labeled lung sections from hamsters infected with B.1 (614G) for A) 2 days PI, B) 4 days PI, C) 6 days PI and D) 10 days PI; or B.1.427 for E) 2 days PI, F) 4 days PI, G) 6 days PI and H) 10 days PI; or B.1.429 for I) 2 days PI, J) 4 days PI, K) 6 days PI and L) 10 days PI. Cells labeled by riboprobe in-situ hybridization stain red. Scale bars equal 1 mm.

Figure 5.. Viral loads in hamsters after intranasal inoculation with B.1 (614G), B.1.427 or B.1.429.

A) sg RNA copies in oral swabs collected daily until day 4 or necropsy. B) sgRNA copies in upper respiratory tract (URT) washes collected at necropsy on 2, 4, 6 or days 10 PI. D) sgRNA copies in lungs collected at necropsy on 2, 4, 6 or days 10 PI. D) Infectious virus titers in lungs collected at necropsy on 2, 4, 6 or 10 days PI. In A, mean values from each group were compared by fitting a mixed model, and a post-hoc multiple comparison test was used to compare the mean values of the B.1.427 or B.1.429 groups individually to the 614G group.

Figure 6.. sgRNA levels and the proportion of each virus in hamsters after intranasal inoculation with a 1:1 or 9:1 mixed inoculum of B.1 (614G) and B.1.427; and sgRNA levels in hamsters inoculated with a mixed inoculum of 7 SARS-CoV-2 variants: B.1 (614G), B.1.427, B.1.427, P.1, P.2, B.1.1.7 and B.1.351.

A) sgRNA copies in oral swabs collected daily for 4 days PI (copies/ug total RNA). B) sgRNA copies in lungs collected at necropsy on 2 and 4 days PI (copies/ug total RNA). C) proportion of the vRNA in lungs collected at necropsy on 2 and 4 days PI that was B.1.427. D) sgRNA copies in URT washes collected at necropsy on 2 and 4 days PI (copies/ug total RNA). E) proportion of the vRNA in URT washes collected at necropsy on 2, and 4 days PI that was B.1.427.

Figure 7.. Relative levels of SARS-CoV-2 variants in hamsters after intranasal inoculation with a mixed inoculum of 7 SARS-CoV-2 variants: B.1 (614G), B.1.427, B.1.429, P.1, P.2, B.1.1.7 and B.1.351.

A) Proportion of each variant RNA in the total vRNA of each virus stock and the mixed inoculum. B) Mean proportion of each variant RNA in the total vRNA at 2 and 4 days PI in the lungs (upper row) and URT (lower row) of hamsters infected with the mixed inoculum. C) Proportion of each variant RNA in the total vRNA at 2 and 4 days PI in the lungs of each hamster infected with the mixed inoculum. D) Proportion of each variant RNA in the total vRNA at 2 and 4 days PI in the URT washes of each hamster infected with the mixed inoculum.

Figure 8.. Prior infection with B.1 (614G) protects hamsters from subsequent challenge with B.1.427 or B.1.429.

A) sgRNA levels and infectious virus titer in hamster infected with B.1 (614G) and then challenged 21 days later with homologous B.1 (614G). B) sgRNA levels and infectious virus titer in hamster infected with B.1.427 and then challenged 21 days later with homologous B.1.427. C) sgRNA levels and infectious virus titer in hamster infected with B.1 (614G) and then challenged 21 days later with heterologous B.1.429. C) sgRNA levels and infectious virus titer in hamster infected with B.1 (614G) and then challenged 21 days later with heterologous B.1.427.

Figure 9.. Relative efficiency of B.1 (614G), B.1.427 and B.1.429 airborne hamster to hamster transmission.

A) sg RNA levels in oral swabs collected from donors 1 day after intranasal inoculation. B) percent of sentinel animals that are infected each day based on detection of sgRNA in oral swabs collected at least daily after co-housing.