key: cord-0953765-m5u6ryy9
authors: Boudewijns, Robbert; Thibaut, Hendrik Jan; Kaptein, Suzanne J. F.; Li, Rong; Vergote, Valentijn; Seldeslachts, Laura; De Keyzer, Carolien; Bervoets, Lindsey; Sharma, Sapna; Van Weyenbergh, Johan; Liesenborghs, Laurens; Ma, Ji; Jansen, Sander; Van Looveren, Dominique; Vercruysse, Thomas; Jochmans, Dirk; Wang, Xinyu; Martens, Erik; Roose, Kenny; De Vlieger, Dorien; Schepens, Bert; Van Buyten, Tina; Jacobs, Sofie; Liu, Yanan; Martí-Carreras, Joan; Vanmechelen, Bert; Wawina-Bokalanga, Tony; Delang, Leen; Rocha-Pereira, Joana; Coelmont, Lotte; Chiu, Winston; Leyssen, Pieter; Heylen, Elisabeth; Schols, Dominique; Wang, Lanjiao; Close, Lila; Matthijnssens, Jelle; Van Ranst, Marc; Compernolle, Veerle; Schramm, Georg; Van Laere, Koen; Saelens, Xavier; Callewaert, Nico; Opdenakker, Ghislain; Maes, Piet; Weynand, Birgit; Cawthorne, Christopher; Velde, Greetje Vande; Wang, Zhongde; Neyts, Johan; Dallmeier, Kai
title: STAT2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in SARS-CoV-2 infected hamsters
date: 2020-07-02
journal: bioRxiv
DOI: 10.1101/2020.04.23.056838
sha: 0eab90c85a55a8c8c0d73405a403f1a20bcbbb87
doc_id: 953765
cord_uid: m5u6ryy9

Since the emergence of SARS-CoV-2 causing COVID-19, the world is being shaken to its core with numerous hospitalizations and hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that productive SARS-CoV-2 infection in the lungs of mice is limited and restricted by early type I interferon responses. In contrast, we show that Syrian hamsters are highly permissive to SARS- CoV-2 and develop bronchopneumonia and a strong inflammatory response in the lungs with neutrophil infiltration and edema. Moreover, we identify an exuberant innate immune response as a key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Finally, we assess SARS-CoV- 2-induced lung pathology in hamsters by micro-CT alike used in clinical practice. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.

SARS-CoV-2 belongs to the family of Coronaviridae, which contains a large group of viruses that are constantly circulating in animals and humans. Illness in humans caused by coronaviruses is mostly mild and manifested by respiratory or digestive problems as leading symptoms 1 . However, some coronaviruses, such as SARS-CoV-1, MERS-CoV and the recent SARS-CoV-2, have been responsible for serious outbreaks of severe and lethal respiratory disease 2, 3 . Unlike the previous outbreaks with SARS-CoV-1 and MERS-CoV, the current SARS-CoV-2 outbreak has evolved to the largest global health threat to humanity in this century.

The unprecedented scale and rapidity of the current pandemic urges the development of efficient vaccines, antiviral and anti-inflammatory drugs. A key step in expediting this process is to have animal models that recapitulate and allow to understand viral pathogenesis, and that can in particular be used to identify new drug targets and preclinically assess preventive and therapeutic countermeasures.

Acute respiratory disease caused by SARS-CoV-1 and MERS infections is characterized by a dysregulated inflammatory response in which a delayed type I interferon (IFN) response promotes the accumulation of inflammatory monocyte-macrophages [4] [5] [6] . The severe lung disease in COVID-19 patients seems to result from a similar overshooting inflammatory response 7 . However, because even non-human primates do not fully replicate COVID-19, little information and no appropriate animal models are currently available to address this hypothesis 8 .

To address this knowledge gap, we compared the effect of SARS-CoV-2 infection in wild-type (WT) mice of different lineages (BALB/c and C57BL/6) and Syrian hamsters, as well as a panel of matched transgenic mouse and hamster strains with a knockout (KO) of key components of adaptive and innate immunity. We used an original patient isolate of SARS-CoV-2 (BetaCoV/Belgium/GHB-03021/2020) that was passaged on HuH7 and Vero E6 cells for these studies ( Fig. S1 and Fig. S2A ). For full characterization and to exclude possible contaminants, we performed deep sequencing on the inoculum that was used to infect the animals (Fig. S2A) . No adventitious agents could be detected (data not shown). However, two in-frame deletions in the N-terminal domain and the furin-cleavage site of Spike (S) glycoprotein (9aa and 5aa, respectively) had occurred between cell culture passage P4 (mixed population of 85% WT genomes and 15% (9+5aa del) mutant genomes) and P6 (100% (9+5aa del) mutant genomes) [9] [10] [11] , likely as adaptation to growth in Vero E6 cells in vitro (Fig. S2B ).

To first examine whether adaptive immunity contributed to the susceptibility to SARS-CoV-2 infection, we inoculated WT (immune-competent) and SCID mice (lacking functional T and B cells) from the same BALB/c background intranasally with a high 2 × 10 5 TCID50 viral dose (P4 virus) (Fig.   1A ). On day 3 p.i., a viral RNA peak in the lungs was observed ( Fig. 1B and Fig. S3 ) with no obvious differences in viral loads (Fig. 1B) nor lung pathology ( Fig. 1D and Fig. S4A and S4B ) between WT and SCID mice. These data indicate that mice that lack the human ACE2 receptor 12 , can in principle be infected with SARS-CoV-2, although inefficiently and likely transiently, as also observed for SARS-CoV-1 4,13 . However, adaptive immunity did not markedly contribute to this low susceptibility.

Interferons are the prototypic first-line innate immune defense against viral infections. To evaluate interferons, we compared viral RNA levels and lung pathology in WT C57BL/6 mice, and C57BL/6 mice with a genetic ablation of their type I (Ifnar1 -/-) and III interferon (IFN) receptors (Il28r -/-) (Fig.   1A ). Ifnar1 -/mice showed an enhanced replication of SARS-CoV-2 in the lung on day 3 p.i. compared to both WT and Il28r -/mice (Fig. 1C ). Similar to BALB/c mice, overall viral loads were low. Ifnar1 -/mice that were treated prior to infection with human convalescent SARS-CoV-2 patient serum or plasma that contains spike-specific antibodies (Fig. 1E, Fig. S4 ) had a 3-10-fold reduction in viral loads depending on the patient donor. This provides further evidence for active, although inefficient virus replication in Ifnar1 -/mice.

WT and knockout (Ifnar1 -/-, Il28r -/-) mouse strains, all on C57BL/6 background, presented consistently with only a mild lung pathology. However, Ifnar1 -/mice showed increased levels of intra-alveolar hemorrhage, sometimes accompanied by some peribronchiolar inflammation ( Fig. 1D and Fig. S5A and S5B ). Passive transfer of HCS did not result in an obvious improvement in histopathological scores ( [18] [19] [20] . Likewise, HCS treatment modulated, at least to some extent, the observed gene expression patterns ( Fig. 1F and cGAS (MB21D1, p=0.094) mRNA levels. In summary, our data are in line with restriction of SARS-CoV-2 infection by the interferon system in mice, and also suggest limited inflammatory responses in the lungs of mice, in contrast to COVID-19 in humans 21 . Taken together, mice were considered as a poor model to study COVID-19 pathogenesis, or to assess the efficacy of vaccines and treatments.

In contrast, Syrian hamsters have been reported to be highly susceptible to SARS-CoV-1 22 and SARS-CoV-2 23 and might thus provide a small animal model to study SARS-CoV-induced pathogenicity and the involvement of the immune response in aggravating lung disease. In contrast to mice, intranasal inoculation of SARS-CoV-2 in WT hamsters resulted in high viral RNA loads (Fig. 2B, Fig. S7 ) a proxy used for the quantification of viral loads (see Fig. S9C ), and in actual infectious titers (Fig. 2C) in the lungs, i.e. roughly 4 Log10 higher than in Ifnar -/mice (Fig. 2C) . Also, a marked lung pathology [median cumulative score (MCS) 9 out of maximal score of 18; IQR=8.5-10.5 (P4 virus)] characterized by a multifocal necrotizing bronchiolitis, massive leukocyte infiltration and edema was observed in infected hamsters but not in mice ( Fig. 2D and Fig. S8A -C). This resembles histopathological findings in humans suffering from severe bronchopneumonia 24 .

In order to investigate the roles of type I and III IFN in the pathogenesis of SARS-CoV-2 infection, we compared virus replication levels and lung pathology in WT hamsters and hamsters with ablated Signal Transducer and Activator of Transcription 2 (STAT2 -/lacking type I and III IFN signaling) 25, 26 and IL28R expression (IL28R-a -/lacking IFN type III signaling) ( Fig. 2A) . Of note, these receptor knockouts did not affect ACE2 expression in hamster lungs (Fig. S9A ), while interferon-stimulated genes (ISG) 27 such as MX-2 (strongly induced by IFNα/STAT2 signaling) and IP-10 (induced by both type I and type II IFNs) showed a differential expression pattern when comparing the different genotypes, triggered by SARS-CoV-2 infection (Fig. S9B) . Importantly, lower baseline expression of MX-2 and IP-10 and failure to respond to SARS-CoV-2 infection by MX-2 upregulation in STAT2 -/hamsters confirmed the functional knockout. As expected, IL28R-a -/hamsters showed an intermediate phenotype between that of WT and STAT2 -/concerning their antiviral response. For many respiratory viruses, including SARS-CoV-1, type I and III interferon signaling has been described to play an important role in restricting infection 28 . No marked differences were observed in viral RNA levels in the lung of WT, STAT2 -/or IL28R-a -/hamsters (Fig. 2B) . However, STAT2 -/hamsters had higher titers of infectious virus in the lung (Fig. 2C ), high titer viremia (measured by RT-qPCR and virus titration) 29 and Fig. S8C ). Matrix metalloprotease (MMP)-9 levels, which may serve as a sensitive marker for the infiltration and activation of neutrophils in inflamed tissues 31, 32 , were markedly elevated in the lungs of all infected hamsters (Fig. 2G) . However, higher MMP-9 levels were found in STAT2 -/animals, thereby inversely correlating with the histological findings (Fig. 2D ). In addition, biomarkers elevated in critically ill COVID-19 patients 2,7,33 such as the cytokines IL-6, IL-10 and IFN- were not found to be elevated in the serum of infected hamsters (Fig. S10B) , although mRNA levels of IP-10 (CXL- 10) were upregulated in the lungs of SARS-CoV-2 infected hamsters as reported for other cytokine/chemokines downstream of IFN- 23 (Fig. S9B) . Nonetheless, infected STAT2 -/and IL28R-a -/had clearly increased levels of IL-6 and IL-10 in their lungs (Fig. S10A ). Such an inverse correlation between biomarkers and pathology in WT versus STAT2 -/hamsters is in line with findings in mouse models of SARS-CoV-1 infection in which pathology correlated with the induction and dysregulation of alternatively activated "wound-healing" monocytes/macrophages 4,6 . To assess the utility of the hamsters for testing the effect of therapeutic interventions on SARS-CoV-2 replication, WT hamsters were treated with human convalescent plasma or a neutralizing SARS-CoV-1 and SARS-CoV-2specific single-domain antibody Fc fusion construct (VHH-72-Fc) 34 prior to infection (Fig. 2H) .

Unlike a single dose of convalescent plasma, which did not significantly reduce viral load in the lungs, pre-treatment with VHH-72-Fc reduced viral loads in the lung ~10 5 -fold compared to untreated control animals, validating hamsters as preclinical model for testing anti-SARS-CoV-2 therapies.

The lack of readily accessible serum markers or the absence of overt disease symptoms in hamsters prompted us to establish a non-invasive means to score for lung infection and SARS-CoV-2 induced lung disease by computed tomography (CT) as used in standard patient care to aid COVID-19 diagnosis with high sensitivity and monitor progression/recovery 7, 33, 35, 36 . Similar as in humans 37 , semiquantitative lung pathology scores were obtained from high-resolution chest micro-CT scans of freebreathing animals 38 The increase in replication of SARS-CoV-2 seen in IL28R-a -/hamsters, on one hand, combined with a tempered inflammatory response and lung injury as compared to WT hamsters, on the other hand, is in line with the role of type III IFN plays during respiratory virus infections, including SARS-CoV-1 53 . This observation also suggests that in humans pegylated IFN-lambda 54,55 (or similar modulators of innate immunity) may possibly be considered to protect medical staff and other frontline workers from SARS-CoV-2 infection or to dampen symptoms in critically ill patients 56 .

In conclusion, hamsters may be preferred above mice as infection model for the preclinical assessment of antiviral therapies, of convalescent serum transfer and of approaches that aim at tempering the COVID-19 immune pathogenesis in critically ill patients 21, 57 . The latter may be achieved by repurposing anti-inflammatory drugs 58 such as IL-6 receptor antagonists (e.g. Tocilizumab) 59 , or small molecule Jak/STAT inhibitors (e.g. Ruxolitinib or Tofacitinib). Educated by our finding that STAT2 signaling plays a dual role in also limiting viral dissemination, targeting the virus-induced cytokine response and overshooting of macrophage activation may need to be complemented by (directly acting) antivirals 60 .

Wild-type Syrian hamsters (Mesocricetus auratus) were purchased from Janvier Laboratories. All other mouse (C57BL/6, Ifnar1 -/-, Il28r -/-, BALB/c and SCID) and hamster (STAT2 -/and IL28R-a -/-) strains were bred in-house. Six-to eight-weeks-old female mice and wild-type hamsters were used throughout the study. Knock-out hamsters were used upon availability; seven-to twelve-week old female STAT2 -/hamsters; five-to seven-week-old IL28R-a -/hamsters. 

Vero E6 (African green monkey kidney, kind gift from Peter Bredenbeek, LUMC, NL) and HuH7 (human hepatoma, JCRB0403) cells were maintained in minimal essential medium (Gibco) supplemented with 10% fetal bovine serum (Integro), 1% bicarbonate (Gibco), and 1% L-glutamine (Gibco). For maintenance of Calu-3 cells (human airway epithelium, kind gift from Lieve Naesens, KU Leuven, BE), the above medium was supplemented with 10mM HEPES (Gibco). All assays involving virus growth were performed using 2% (Vero E6 and HuH7) or 0.2% (Calu-3) fetal bovine serum instead of 10%.

SARS-CoV-2 strain BetaCov/Belgium/GHB-03021/2020 (EPI ISL 407976|2020-02-03) recovered from a nasopharyngeal swab taken from a RT-qPCR-confirmed asymptomatic patient returning from Wuhan, China beginning of February 2020 62 was directly sequenced on a MinION platform (Oxford Nanopore) as described previously 63 Antibody VHH-72-Fc was administered i.p. at a dose of 20mg/kg 1 day prior to infection. VHH-72-Fc was expressed in ExpiCHO cells (ThermoFisher Scientific) and purified from the culture medium as described 34 . Briefly, after transfection with pcDNA3.3-VHH-72-Fc plasmid DNA, followed by incubation at 32C and 5% CO2 for 6-7 days, the VHH-72-Fc protein in the cleared cell culture medium was captured on a 5 mL MabSelect SuRe column (GE Healthcare), eluted with a McIlvaine buffer pH 3, neutralized using a saturated Na3PO4 buffer, and buffer exchanged to storage buffer (25 mM L-Histidine, 125 mM NaCl). The antibody's identity was verified by protein-and peptide-level mass spectrometry.

Animals were euthanized at different time-points post-infection, organs were removed and lungs were homogenized manually using a pestle and a 12-fold excess of cell culture medium (DMEM/2%FCS).

RNA extraction was performed from homogenate of 4 mg of lung tissue with RNeasy Mini Kit (Qiagen), or 50µl of serum using the NucleoSpin kit (Macherey-Nagel), according to the manufacturer's instructions. Other organs were collected in RNALater (Qiagen) and homogenized in a bead mill (Precellys) prior to extraction. Of 100µl eluate, 4µl was used as template in RT-qPCR reactions. RT-qPCR was performed on a LightCycler96 platform (Roche) using the iTaq Universal Probes One-Step RT-qPCR kit (BioRad) with primers and probes (Table S1) infectious virus were used to express the amount of RNA as normalized viral genome equivalent (vge) copies per mg tissue, or as TCID50 equivalents per mL serum, respectively. The mean of housekeeping gene β-actin was used for normalization. The relative fold change was calculated using the 2 -ΔΔCt method 66 .

After extensive transcardial perfusion with PBS, lungs were collected, extensively homogenized using manual disruption (Precellys24) in minimal essential medium (5% w/v) and centrifuged (12,000 rpm, 10min, 4°C) to pellet the cell debris. Infectious SARS-CoV-2 particles were quantified by means of endpoint titrations on confluent Vero E6 cell cultures. Viral titers were calculated by the Spearman-Kärber method and expressed as the 50% tissue culture infectious dose (TCID50) per 100mg tissue.

To study differential gene expression, RNA was extracted from lung tissues using Trizol, subjected to cDNA synthesis (High Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific), and qPCR using a custom Taqman qRT-PCR array (Thermo Fisher Scientific) of 30 genes known to be activated in response to virus infection 16 , as well as two housekeeping genes (Table S2) 

For histological examination, the lungs were fixed overnight in 4% formaldehyde and embedded in paraffin. Tissue sections (4 µm) were stained with hematoxylin and eosin to visualize and score for lung damage.

Calu-3 (human airway epithelial) cells were plated at 5×10 4 

Cytokine levels in lung homogenates and serum of hamsters were determined by ELISA for IFN- (EHA0005), IL-6 (EHA0008) and IL-10 (EHA0006) following the manufacturer's instructions (Wuhan Fine Biotech Co., Ltd).

The levels of gelatinase B/metalloproteinase (MMP)-9 present in lung homogenates were analyzed using gelatin zymography 68 , essentially as described previously 69 . For quantification of zymolytic bands internal control samples were spiked into each sample. Equivalent hamster enzyme concentrations were calculated with the use of known amounts of recombinant human pro-MMP-9

and recombinant human pro-MMP-9ΔOGHem as standards 70 .

Hamsters were anaesthetized using isoflurane (Iso-Vet) (2-3% in oxygen) and installed in prone position into the X-cube micro-CT scanner (Molecubes) using a dedicated imaging bed. Respiration was monitored throughout. A scout view was acquired and the lung was selected for a non-gated, helical CT acquisition using the High-Resolution CT protocol, with the following parameters: 50 kVp, 960 exposures, 32 ms/projection, 350 µA tube current, rotation time 120 s. Data were reconstructed using a regularized statistical (iterative) image reconstruction algorithm using nonnegative least squares 71 , using an isotropic 100 µm voxel size and scaled to Hounsfield Units (HUs) after calibration against a standard air/water phantom. The spatial resolution of the reconstruction was estimated at 200 µm by minimizing the mean squared error between the 3D reconstruction of the densest rod in a micro-CT multiple density rod phantom (Smart Scientific) summed in the axial direction and a digital phantom consisting of a 2D disk of 17.5 mm radius that was post-smoothed with Gaussian kernels using different full width half maxima (FWHM), after aligning the symmetry axis of the rod to the z-axis.

Visualization and quantification of reconstructed micro-CT data was performed with DataViewer and CTan software (Bruker micro-CT). As primary outcome parameter, a semi-quantitative scoring of micro-CT data was performed as previously described 38, 39, 72 with minor modifications towards optimization for COVID-19 lung disease in hamsters. In brief, visual observations were scored (from 0 -2 depending on severity, both for parenchymal and airway disease) on 5 different, predefined transversal tomographic sections throughout the entire lung image for both lung and airway disease by two independent observers (L.S. and G.V.V.) and averaged. Scores for the 5 sections were summed up to obtain a score from 0 to 10 reflecting severity of lung and airway abnormalities compared to scans of healthy, WT control hamsters. As secondary measures, image-derived biomarkers (nonaerated lung volume, aerated lung volume, total lung volume, the respective densities within these volumes and large airways volume) were quantified as in 38, 72 for a manually delineated VOI in the lung, avoiding the heart and main blood vessels. The threshold used to separate the airways and aerated (grey value 0-55) from non-aerated lung volume (grey value 56-255) was set manually on an 8-bit greyscale histogram and kept constant for all data sets. Il28r -/-(n=5) mice. At the indicated time intervals p.i., viral RNA levels were determined by RT-qPCR, normalized against β-actin mRNA levels and transformed to estimate viral genome equivalents (vge) content per weight of the lungs ( Figure S2 ). For heat-inactivation, SARS-CoV-2 was incubated for 30min at 56°C. Dotted line indicates lower limit of quantification (LLOQ). The data shown are means ± SEM. (D) Histopathological scoring of lungs for all different mouse strains. Mice were sacrificed on day 3 p.i. and lungs were stained with H&E and scored for signs of lung damage (inflammation and hemorrhage). Scores are calculated as percentage of the total maximal score. "No score" means not contributing to theoretical full cumulative score of 100%. Numbers (n) of animals analyzed per condition are given in the inner circle. (E) Viral RNA levels in Ifnar1 -/mice after treatment with anti-SARS-CoV-2 serum or plasma. Mice were either left untreated (IC, infection control), or treated intraperitoneally one day before infection with convalescent serum (patient #1), convalescent plasma (patient #2) or with negative control plasma (patient #3 NC, negative control) and sacrificed on day 3 p.i. Viral RNA levels were determined in the lungs, normalized against βactin and fold-changes were calculated using the 2 (-ΔΔCq) method compared to mean of IC. The data shown are means ± SEM. (F) Heatmap showing gene expression profiles of 30 selected marker genes in the lungs of uninfected and infected Ifnar1 -/mice that were either left untreated or treated with convalescent serum from patient #1 (n=3 per group). Analysis performed on day 3 p.i. The scale represents fold change compared to non-infected animals. Statistical significance between groups was calculated by the nonparametric two-tailed Mann-Whitney U-test (ns = not significant, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001).

WT, STAT2 -/and IL28R-a -/hamster strains were intranasally inoculated with 2 × 10 5 TCID50 of passage 4 or 2 × 10 6 of passage 6 SARS-CoV-2. Outcomes derived from inoculation with passage 4 or passage 6 SARS-CoV-2 is designated by circles (P4) or squares (P6). On the indicated days post inoculation (d.p.i.), organs and blood were collected to determine viral RNA levels, infectious viral load and score for lung damage. Viral loads in the indicated organs were quantified by RT-qPCR (B, E and F) or virus titration (C). (B,F) Viral RNA levels in the indicated organs were normalized against β-actin mRNA levels and transformed to estimate viral genome equivalents (vge) content per weight of the lungs ( Figure S5 ). (C) Infectious viral loads in the lung are expressed as the number of infectious virus particles per 100 mg of lung tissue. (E) Viral RNA levels in the blood were calculated from a standard of infectious virus and expressed as TCID50 equivalents per ml blood. Dotted lines indicate lower limit of quantification (LLOQ) or lower limit of detection (LLOD) (D) Histopathological scoring of lungs. Hamsters were sacrificed on day 4 p.i. with passage 4 SARS-CoV-2 and lungs were stained with H&E and scored for signs of lung damage (apoptotic bodies, necrotizing bronchiolitis, edema, pneumonia and inflammation). Scores are calculated as percentage of the total maximal score. (G) Levels of matrix metalloproteinase (MMP)-9 levels in lung homogenates of SARS-CoV-2 infected hamsters, relative to non-infected controls of the same strain. Statistical significance was calculated between infected and non-infected animals within each group. Values for infected animals (n=7 each) compiled from two independent experiments using either P4 (n=3, circles) and P6 (n=4, squares) SARS-CoV-2. (H) Viral RNA levels in hamsters after treatment with convalescent SARS-CoV-2 plasma or with a previously described antibody. Hamsters were either left untreated (IC, infection control, n=5) or treated with a single-domain antibody (VHH-72-Fc, n=4), convalescent plasma (patient #2, n=4) or negative control plasma (patient #3 NC, negative control, n=4) and sacrificed on day 4 p.i. Viral RNA levels were determined in the lungs, normalized against β-actin and fold-changes were calculated using the 2 (-ΔΔCq) method compared to the mean of IC. The data shown are means ± SEM. Statistical significance between groups was calculated by the nonparametric two-tailed Mann-Whitney U-test (ns P > 0.05, * P < 0.05, ** P < 0.01, **** P < 0.0001). . 2C) . Lines indicate matched samples. The data shown are means ± SEM. Statistical significance between groups was calculated by the nonparametric two-tailed Mann Whitney U-test (ns P > 0.05, * P < 0.05).

D wrote the original draft with input from co-authors

developed the STAT2 -/-and IL28R-a -/-hamster strains

are named as inventors on US patent application no. 62/988,610, entitled ''Coronavirus Binders

are named as inventors on US patent application no. 62/991,408, entitled ''SARS-CoV-2 Virus Binders

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Modular approach to customize sample preparation procedures for viral metagenomics

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We thank Kathleen Van