key: cord-1056572-9dybtivg
authors: Abdelnabi, Rana; Boudewijns, Robbert; Foo, Caroline S.; Seldeslachts, Laura; Sanchez-Felipe, Lorena; Zhang, Xin; Delang, Leen; Maes, Piet; Kaptein, Suzanne J. F.; Weynand, Birgit; Velde, Greetje Vande; Neyts, Johan; Dallmeier, Kai
title: Comparative infectivity and pathogenesis of emerging SARS-CoV-2 variants in Syrian hamsters
date: 2021-02-26
journal: bioRxiv
DOI: 10.1101/2021.02.26.433062
sha: df2c9d98317fbec75625848234088c723c4453b1
doc_id: 1056572
cord_uid: 9dybtivg

Within one year after its emergence, more than 108 million people contracted SARS-CoV-2 and almost 2.4 million succumbed to COVID-19. New SARS-CoV-2 variants of concern (VoC) are emerging all over the world, with the threat of being more readily transmitted, being more virulent, or escaping naturally acquired and vaccine-induced immunity. At least three major prototypic VoC have been identified, i.e. the UK (B.1.1.7), South African (B.1.351) and Brazilian (B.1.1.28.1), variants. These are replacing formerly dominant strains and sparking new COVID-19 epidemics and new spikes in excess mortality. We studied the effect of infection with prototypic VoC from both B.1.1.7 and B.1.351 lineages in Syrian golden hamsters to assess their relative infectivity and pathogenicity in direct comparison to two basal SARS-CoV-2 strains isolated in early 2020. A very efficient infection of the lower respiratory tract of hamsters by these VoC is observed. In line with clinical evidence from patients infected with these VoC, no major differences in disease outcome were observed as compared to the original strains as was quantified by (i) histological scoring, (ii) micro-computed tomography, and (iii) analysis of the expression profiles of selected antiviral and pro-inflammatory cytokine genes. Noteworthy however, in hamsters infected with VoC B.1.1.7, a particularly strong elevation of proinflammatory cytokines was detected. Overall, we established relevant preclinical infection models that will be pivotal to assess the efficacy of current and future vaccine(s) (candidates) as well as therapeutics (small molecules and antibodies) against two important SARS-CoV-2 VoC.

Barely one year after surfacing and global spread of SARS-CoV-2, more than 113 Mio infected 50 people and 2.5 Mio fatal cases have been reported worldwide (as of February 26 th 2021). 51

Variants of SARS-CoV-2 variants are emerging in different parts of the world, posing a new 52 threat. Currently at least three major prototypic virus variants of concern (VoC) have been 53 detected respectively in the UK (lineage B.1.1.7; 1-3 earliest sample date 2020-02-03), South 54 Africa (B.1.351 or 501Y.V2; 1,4,5 earliest sample date 2020-10-08) and Brazil (B.1.1.28.1 or 55 P.1; 6,7 earliest sample date 2020-12-15). Even in highly endemic regions, these VoC started to 56 replace formerly dominant strains and appear to be at the root of new waves of infections 57 and new spikes in excess mortality, raising the concern of being more readily transmitted or 58 of being even more virulent. In general, numerous mutations have been identified in the VoC 59 genomes, which occur in different coronavirus genes. Though, the Spike (S) protein appears 60 to be particularly prone to accumulate amino acid changes. As such the VoC of UK, South 61

African and Brazil origin share the prominent mutation N501Y in the receptor-binding domain 62 (RBD) of S that may favor viral attachment to its cellular receptor hACE2. 8 Moreover, massive 63 outbreaks in previously heavily affected regions, such as in the Manaus area of Northern Brazil 64 with seropositivity rates of up to 76% prior to disease resurgence, raise the concern of VoC 65 escaping pre-existing immunity. 7 Likewise, several vaccine candidates either failed to show 66 efficacy, or at least displayed a marked drop in vaccine efficacy in Phase 3 clinical trials in 67 regions of South Africa where the VoC B.1.351 is circulating. 9,10 Escape from antibody 68 neutralization has been linked to amino acid changes in key residues of S, such as E484K, 69 found in both the South African and Brazilian variant and as well in some more recently 70 emerging UK (sub)lineages. [11] [12] [13] The evolution of such new SARS-CoV-2 VoC may be driven by 71 viral escape under host immune pressure during acute infection, 14 and resulting SARS-CoV-2 72 antigenic drift is feared to spark future COVID-19 epidemics. 73

Here, we investigate infection of Syrian hamsters with prototypic VoC, namely local Belgian 74 low-passage isolates from both B.1.1.7 and B.1.351 lineages (Fig 1A, B) . 15 Fig. 1A . 117

Housing and experimental infections of hamsters have been described [15] [16] [17] Virus loads were determined by titration and RT-qPCR from lung homogenates exactly as 127 described in detail before. 15-17 Analysis of differential expression of hamster interleukin (IL)-128 6, IL-10, interferon (IFN)-γ, INF-λ, IP-10, TNF-α, MX-2 and ACE by quantitative RT-qPCR has 129 been described. 17 130

Micro-CT data for the lungs of free-breathing hamsters were acquired on a Skyscan 1278 132 system (Bruker Belgium) and analyzed as described before. 15-17 In brief, hamsters were 133 scanned in supine position under isoflurane anesthesia producing expiratory weighted three-134 dimensional data sets with 50-μm isotropic reconstructed voxel size 29 in an approximately 3 135 min scanning time. 136

Visualization and quantification of reconstructed micro-CT data were performed with as 137 primary outcome measure a semiquantitative scoring of micro-CT data. 29 Visual observations 138 were blindly scored on five different, predefined transversal tomographic sections for both 139 lung and airway disease by two independent observers, averaged, and scores for the five 140 sections summed up to obtain a score from 0 to 10, reflecting severity of lung and airway 141 abnormalities compared to healthy control hamsters. As secondary measure, imaging-142 derived biomarkers (nonaerated, aerated and total lung volume) were quantified as before. 15-143 17,30 144 Pathology assessment by histology 145 Assessment of lung pathology was performed as before. [15] [16] [17] In brief, for histological 146 examination, the lungs were fixed overnight in 4% formaldehyde, embedded in paraffin and 147 tissue sections (5 μm) after staining with H&E scored blindly for lung damage (cumulative 148 score of 1 to 3 each for congestion, intra-alveolar hemorrhage, apoptotic bodies in bronchial 149 epithelium, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular 150 inflammation, peribronchial inflammation, and vasculitis). 151

All statistical analyses were performed using GraphPad Prism 9 software (GraphPad, San 153 Diego, CA, USA). Results are presented as means ± SEM or medians ± IQR as indicated. 154

Statistical differences between groups were analyzed using Kruskal-Wallis with Dunn's 155 multiple comparisons test, or two-tailed Mann-Whitney U test for pairwise comparison, and 156 considered statistically significant at p-values ≤0.05 (* p≤0.05, ** p≤0.01, *** p≤0.001). 157

All of the data generated or analyzed during this study are included in this published article. 159 infection (4 dpi), SARS-CoV-2 replication (Fig. 1D, E) , pathology ( Fig. 2 and 3) , and cytokine 168 expression levels were determined in the lung tissue ( Fig. 4 and S2) . As documented before for the B.1-G strain in young female hamsters, 15,16 no overt signs of 179 disease or distress were observed throughout the study, and in general, none of the hamsters 180 showed a marked weight loss (i.e. more than 7%) in any of the infected groups (Fig. S1) . 181

Whether or not of biological relevance, some differences in weight evolution could be 182 calculated in a pairwise comparison of groups B.1-B (2% decrease between day 0 and 4 pi) 183 and B.1.1.7 (3% increase) (Fig. S1 ). Taken together, these results clearly show that VoC B.1.1.7 184 and B1.351 are able to replicate efficiently in the lower respiratory tract in Syrian hamsters 185 and this to a comparable extent as strains of basal lineages. 186

Basal lineages and VoC SARS-CoV-2-induced lung pathology was assessed by microcomputed 187 tomography (micro-CT) at 4 dpi immediately prior to euthanasia (Fig. 2) and by post-mortem 188 histopathological analysis (Fig.3) . Both semi-quantitative scores and quantitative biomarkers 189 in the surrounding alveoli (Fig. 3A) . The cumulative histopathological lung scores were 6-to 195 8-fold higher than the baseline score in untreated, non-infected hamsters (median score of 196 1·25), with no significant differences between the strains (Fig. 3B) . Taken were measured in the lung tissue of infected hamsters (Fig. 4) . Infection with either of the 202 four strains resulted in an up-regulation of IL-6, IL-10, IFN-λ, IFN-γ, IP-10, MX-2, and TNF-α 203 expression in the range of 10-to 1000-fold compared to non-infected hamsters (Fig. 4, Fig.  204 S2). Notably, IL-6, IL-10and IFN-γ, but not MX-2 expressions were most pronouncedly up-205 regulated in the B1.1.7 VoC-infected group as compared to the three other strains, with a 206 significant increase compared to B1.351 VoC (Fig. 4B ). Similar to lung viral loads (see above), 207 cytokine expression levels were hardly affected by lowering the inoculated virus dose (Fig. 4B  208 and Fig. S2 ; open circles), arguing for readily saturated conditions. ACE2 receptor expression 209 levels 35 remained rather unaffected and similar between all four strains (Fig. S2) SARS-CoV-2 genetic diversification was initially considered slow as the virus was spreading in 217 the first months around the globe. 36 However, recently more and more variants are emerging 218 and start dominating regional epidemics in widespread populations. 37 Notably, mutations 219 found in the SARS-CoV-2 spike protein that may be associated with (i) more efficient human-220 to-human transmission, (ii) increase virulence, 38 and/or (iii) escape from naturally acquired 7 221 or vaccine-induced 9,10 immunity raise concerns. The role of such mutations have been 222 experimentally addressed, mostly using synthetic viruses generated by targeted mutagenesis. 223

These studies revealed the link between certain mutations and particular phenotypes such as 224 efficiently and consistently to high viral loads in hamster lungs (Fig. 1D, E) , causing a pathology 231 that resembles bronchopneumonia in COVID patients. However, despite uniformly high viral 232 replication, some variation in disease severity was observed as scored by micro-CT imaging 233 (Fig. 2) , histopathology scoring (Fig. 3) , and cytokine expression profiles ( Fig. 4 and Fig. S2 ) 234 both (i) between study groups as well as (ii) among individual animals within particular study 235 groups. At first glance, the hamster model may lack sensitivity to discriminate differences in 236 the replication fitness of SARS-CoV-2 from different lineages, in particular when using direct 237 intranasal infection with high-titred inocula. 31 hamster model (Fig. 2-4, Fig. S1 and S2) . In our study we focused on a single time point for 259 analysis (4 dpi), previously established to represent a peak of B.1-G virus replication. 15 variants, respectively. Statistical significance between variants was calculated by Kruskal-486

Wallis with two-sided Dunn's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. 487 

Tracking the international spread of SARS-CoV-2 306 lineages B.1.1.7 and B.1.351/501Y-V2

A dynamic nomenclature proposal for SARS-312

CoV-2 lineages to assist genomic epidemiology

Emergence and rapid spread of a new 316 severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with 317 multiple spike mutations in South Africa

Resurgence of COVID-19 in Manaus, Brazil, 321 despite high seroprevalence

Molecular Mechanism of the N501Y Mutation for 323 Enhanced Binding between SARS-CoV-2's Spike Protein and Human ACE2 Receptor

Covid-19: South Africa pauses use of Oxford vaccine after study casts 326 doubt on efficacy against variant

Covid-19: Novavax vaccine efficacy is 86% against UK variant and 60% 328 against South African variant

Complete Mapping of Mutations to the SARS-330

CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

mRNA vaccine-elicited antibodies to SARS-CoV-333 2 and circulating variants

Covid-19: The E484K mutation and the risks it poses

Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral 337 Immunity during Acute

STAT2 signaling restricts viral 339 dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters

Favipiravir at high doses has potent 342 antiviral activity in SARS-CoV-2−infected hamsters, whereas hydroxychloroquine lacks 343 activity

A single-dose live-attenuated YF17D-345 vectored SARS-CoV-2 vaccine candidate

Animal models for COVID-19. 347

Simulation of the clinical and pathological 349 manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster 350 model: implications for disease pathogenesis and transmissibility

Syrian hamsters as a small animal model 353 for SARS-CoV-2 infection and countermeasure development

SARS-CoV-2 vaccines in development

Ad26 vaccine protects against SARS-357

CoV-2 severe clinical disease in hamsters

EIDD-2801) inhibits SARS-CoV-2 359 replication in Syrian hamsters model

ALG-097111, a potent and selective SARS-361

CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo

Syrian Hamster model

Nelfinavir markedly improves lung pathology 364 in SARS-CoV-2-infected Syrian hamsters despite a lack of an antiviral effect

First cases of coronavirus disease 2019 (COVID-367 19) in the WHO European Region

Transmission of 2019-nCoV Infection from an 370

Asymptomatic Contact in Germany

Accounting for population 372 structure reveals ambiguity in the Zaire Ebolavirus reservoir dynamics

Radiosafe micro-computed tomography for 375 longitudinal evaluation of murine disease models

Longitudinal micro-CT provides 377 biomarkers of lung disease that can be used to assess the effect of therapy in 378 preclinical mouse models, and reveal compensatory changes in lung volume

SARS-CoV-2 D614G variant exhibits efficient 381 replication ex vivo and transmission in vivo

Evaluating the Effects of SARS-CoV-2 Spike Mutation 383 D614G on Transmissibility and Pathogenicity

Defining the Syrian hamster as a highly 385 susceptible preclinical model for SARS-CoV-2 infection

Transmission, Diagnosis, 388 and Treatment of Coronavirus Disease 2019 (COVID-19): A Review

Interferons and viruses induce a novel 391 truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor

A SARS-CoV-2 vaccine candidate would likely 394 match all currently circulating variants

Risk related to the spread of new SARS-CoV-2 variants of concern in the 396 EU/EEA-first update

Estimated transmissibility and severity of novel 400 SARS-CoV-2 Variant of Concern 202012/01 in England

The N501Y mutation in SARS-CoV-2 spike 403 leads to morbidity in obese and aged mice and is neutralized by convalescent and 404 post-vaccination human sera

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and 406 N501Y variants by BNT162b2 vaccine-elicited sera

Increased Resistance of SARS-CoV-2 Variants B.1.351 409 and B.1.1.7 to Antibody Neutralization

Loss of furin cleavage site attenuates SARS-CoV-2 411 pathogenesis

Attenuated SARS-CoV-2 variants with deletions at 413 the S1/S2 junction

From SARS and MERS to COVID-19: A brief summary and 415 comparison of severe acute respiratory infections caused by three highly pathogenic 416 human coronaviruses