key: cord-0837861-vjtzdtk5
authors: Robertson, Shelly J; Bedard, Olivia; McNally, Kristin L.; Lewis, Matthew; Clancy, Chad; Shaia, Carl; Broeckel, Rebecca M.; Chiramel, Abhilash I.; Sturdevant, Gail L.; Forte, Elvira; Preuss, Christoph; Baker, Candice N.; Sturdevant, Daniel E.; Martens, Craig; Holland, Steven M.; Rosenthal, Nadia A.; Best, Sonja M.
title: Genetically diverse mouse models of SARS-CoV-2 infection model clinical variation and cytokine responses in COVID-19
date: 2021-09-18
journal: bioRxiv
DOI: 10.1101/2021.09.17.460664
sha: 4c5e8795cb4539b76cae089ed22b458cac86c789
doc_id: 837861
cord_uid: vjtzdtk5

Host genetics are a significant determinant of coronavirus disease 2019 (COVID-19)1. Animal models that reflect genetic diversity and a range of clinical outcomes observed in human populations are needed to understand mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection dynamics and disease2. Here, we report a mouse panel comprising the diverse genetic backgrounds of the Collaborative Cross (CC) founder strains crossed to C57BL/6J mice expressing the K18-hACE2 transgene3 that enables infection by SARS-CoV-2. Infection of CCxK18-hACE2 F1 progeny resulted in a spectrum of weight loss, survival, viral replication kinetics, histopathology, and cytokine profiles, some of which were sex-specific. Importantly, survival was closely associated with early type I interferon expression and a phased proinflammatory response distinct from mice with severe disease. Thus, dynamics of inflammatory responses observed in COVID-19 can be modeled in diverse mice that provide a genetically tractable platform for understanding antiviral immunity and evaluating countermeasures.

. Highly sensitive mice strains included C57BL/6J and A/JxK18-hACE2 that lost 70 approximately 20% of their starting weight between 4 and 7 dpi with no clear sex bias. In 71 contrast, CCxK18-hACE2 F1 progeny from PWK, NZO, 129S1/J (Fig. 1) , BALB/c and DBA 72 ( Fig. S1 ) were resistant to clinical disease, generally losing 5-10% starting weight with ~80% of 73 mice surviving infection. Finally, CCxK18-hACE2 F1 progeny of three strains (CAST, NOD, 74 WSB) had marked sexual dimorphism in response, with CAST and NOD F1 males susceptible to 75 lethal disease, although differences in weight loss between sexes were not apparent. Yet another 76 phenotype was obtained in SARS-CoV-2 infected WSB F1 progeny, where infection was 77 uniformly lethal in females. Weight loss in WSB F1 males began later than other groups at 6 dpi 78 and surviving males exhibited sustained weight loss until the end of the observation period in 79 contrast to the rapid weight gain associated with recovery of other CCxK18-hACE2 F1 cohorts. 80 81 In K18-hACE2 mice, SARS-CoV-2 titers peak in the lung at 2-3 dpi and high titers of virus can 82 be isolated from the CNS which may contribute to lethality in this model 24,25 . Thus, virus 83 replication kinetics were determined at 3 and 6 dpi in lung and brain of CCxK18-hACE2 F1 84 cohorts ( Fig. 2A, 2B ). F1 progeny of sensitive founder strains C57BL/6J and A/J showed high 85 levels of infectious SARS-CoV-2 at 3dpi in lung homogenates that was reduced by 1-2 log10 by 6 86 dpi with no differences between sexes. Infectious virus was generally not recovered from the 87 CNS at 3dpi, but 50% of C57BL/6J and A/J F1 progeny had high virus burden in the CNS by 88 6dpi (10 6 -10 8 PFU/g tissue). In contrast, peak lung virus titer in F1 progeny of the most resistant 89 founder strain, PWK, was 150-fold lower than the sensitive strains at 3 dpi and was below the 90 limit of detection at 6dpi. Control of virus replication in PWK F1s was also evident in the CNS 91

where detection of infectious virus was sporadic. Interestingly, F1 progeny of additional CC 92 founder strains classed as resistant had equivalent peak viral titers in the lung to the sensitive 93 K18-hACE2 and A/J F1 progeny, but they tended to control replication by 6 dpi to levels 1log10 94 lower than sensitive mice and had less virus burden in the CNS. Finally, lung titers in (CAST, 95 NOD and WSB)xK18-hACE2 F1 progeny were not different between males and females despite 96 differences in clinical severity associated with sex. Thus, F1 progeny of CCxK18-hACE2 mouse 97 strains can be further stratified into a) sensitive F1 progeny with high sustained virus replication 98 in lung and CNS (C57BL/6J, A/J), b) resistant F1 progeny associated with lower peak virus titer 99 and earlier control of replication in the lung with no or low dissemination to other organs (PWK, 100 NZO, 129S1/J), and c) F1 progeny with sex bias where resistance is independent of virus titer in 101 the lung suggesting a sex-based difference in host response (CAST, NOD, WSB). Relative 102 expression of the K18-hACE2 transgene in lung suggested some variability, but expression was 103 not associated with clinical phenotypes or peak viral burden (Fig. S2 ). Together, these data 104 suggest that the range of clinical severity in mice is only partially associated with control of virus 105 replication (Summarized in Table 1) . 106 107 Pathological changes in SARS-CoV-2-infected K18-hACE2 mice were similar to those 108 previously described 7-9,26 . Inflammatory infiltrates evident by 3dpi included perivascular 109 lymphocytes with alveolar septa thickened by neutrophils, macrophages and edema. At 6 dpi, 110 pulmonary pathology was multifocal, and consistent with interstitial pneumonia including type II 111 pneumocyte hyperplasia, septal, alveolar and perivascular inflammation comprised of 112 lymphocytes, macrophages and neutrophils, with alveolar fibrin and edema evident. Bronchiolar 113 pathology was not observed in these mice. Pathology was classified as none, mild (rare scattered 114 inflammatory foci), moderate (coalescing inflammatory foci) or severe (widespread, large 115 inflammatory foci) as exemplified in Fig. 2c . The classification of 'severe' is a comparative term 116 for these mice, as we generally did not observe pathology equivalent to that of the more severe 117

Syrian hamster model of SARS-CoV-2 infection [27] [28] [29] . Surprisingly, in comparison to F1 progeny 118 of sensitive founder strains (K18-hACE2 and A/JxK18-hACE2), F1 progeny of resistant PWK, 119 female NZO and 129S/J mice, as well as those of NOD and WSB tended to have higher 120 pathology scores in lungs (Fig. 2d) . Lung distribution of viral RNA by RNAscope was limited to 121 type I and II pneumocytes in all strains (Fig. 2b) . Pathology in the CNS was scored as present or 122 absent, as it generally consisted of subtle inflammatory foci, including perivascular cuffing and 123 increased gliosis associated with necrotic cells observed in K18-hACE2 and DBAxK18-hACE2 124 F1 males ( Fig. S3A -C, G). However, pathology in the CNS of CASTxK18-hACE2 F1 progeny 125 was striking in that microthrombi were evident in capillaries with extensive hemorrhage in the 126 absence of encephalitis, and was associated with viral RNA distribution in the same area ( increased plasma levels of a core inflammatory signature of pro-inflammatory cytokines, 132 chemokines and growth factors (e.g., IL-1a, IL-1b, IFNa, IL-12 p70, and IL-17A) that are 133 effectively resolved 43 . In contrast, severe COVID-19 includes sustained expression of these 134 markers with additional signatures including IL-6, IL-10, IL-18, IL-23, TNF-a, and eotaxin 135 among others suggesting that specific timing and failure to resolve inflammatory responses are 136 important factors in disease progression 43 . In SARS-CoV-2-infected CCxK18-hACE2 F1 mice, 137 cytokines and chemokines were quantified in the serum and bronchoalveolar lavage fluid (BAL) 138 by multiplex analysis at 3 and 6 dpi. In general, cytokine levels were much higher in BAL fluid 139 than in serum (Fig. S4A and 129S1)xK18-hACE2 F1 progeny revealed that high IFNa expression at 3dpi was associated 142 with survival in both males and females (Fig. 3A, B) . Resistant mice also expressed 143 proinflammatory cytokine IL-6, Th1 cytokines (IL-12p70, IL-27) and chemokines (CXCL10, 144

Gro-a/KC, CCL2, CCL3, CCL4, CCL5, CCL7 and CXCL2) (designated as Group A) in BAL at 145 this early timepoint (Fig. 3B , Cluster 1). At 6 dpi, many Group A cytokines were resolving ( sexes, although responses in BAL were generally higher in males (Fig. S4C ). Together, these 156 genetically diverse mice model major dynamic phenotypes observed in human COVID-19. 157

Specifically, reduced disease severity in mice is associated with early type I IFN expression and 158 a phased, controlled inflammatory response. In contrast, a delayed and unorchestrated innate 159 response is associated with lethality. Infectious virus in lung and brain tissue was quantified by plaque assay. Tissues were collected in 0.5ml 213 of DMEM containing 2% FBS, 50U/ml penicillin and 50ug/ml streptomycin and immediately frozen.

Tissue samples were weighed, and then homogenized using 5mm steel beads and TissueLyzer II high-215 speed shaker (Qiagen). Ten-fold serial dilutions of homogenates were prepared in duplicate and used to 216 inoculate Vero cells grown in 48-well tissue culture plates. Following 1 hour incubation at 37ºC, the cells 217 were overlayed with 1.5% carboxymethyl cellulose (CMC) in MEM and incubated at 37ºC for 3-4 days.

Cells were then fixed in 10%formalin and plaques were visualized by staining with 1% crystal violet 219 diluted in 10% ethanol. concern by binding a highly conserved epitope. Absent Present 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 

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