key: cord-0816493-hvlhfqsl
authors: Rizvi, Zaigham Abbas; Dalal, Rajdeep; Sadhu, Srikant; Kumar, Yashwant; Srivastava, Tripti; Gupta, Sonu Kumar; Agarwal, Suruchi; Tripathy, Manas Ranjan; Yadav, Amit Kumar; Medigeshi, Guruprasad R.; Pandey, Amit Kumar; Samal, Sweety; Asthana, Shailendra; Awasthi, Amit
title: Immunological and cardio-vascular pathologies associated with SARS-CoV-2 infection in golden syrian hamster
date: 2021-01-11
journal: bioRxiv
DOI: 10.1101/2021.01.11.426080
sha: dd6bbca94a907adbe0aa14a230a794608490e918
doc_id: 816493
cord_uid: hvlhfqsl

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection in golden Syrian hamster (GSH) causes lungs pathology and resembles to human corona virus disease (Covid-19). Extra-pulmonary pathologies and immunological parameters of SARS-CoV-2 infection remained undefined in GSH. Using in silico modelling, we identified the similarities between human and hamster angiotensin-converting enzyme-2 (ACE-2), neuropilin-1 (NRP-1) that bind to receptor-binding domain (RBD) and S1 fragment of spike protein of SARS-CoV-2. SARS-CoV-2 infection led to lung pathologies, and cardiovascular complications (CVC) marked by interstitial coronary fibrosis and acute inflammatory response. Serum lipidomic and metabolomic profile of SARS-CoV-2-infected GSH revealed changes in serum triglycerides (TG) and low-density lipoprotein (LDL), and alterations in metabolites that correlated with Covid19. Together, we propose GSH as an animal model to study SARS-CoV-2 infection and its therapy associated with pulmonary and extra-pulmonary pathologies.

First reported in Wuhan, China in December 2019, SARS-CoV-2 has infected nearly 0.9 % of 42 the total world population with around 2.17 % mortality rate as of January 2021 43 (https://covid19.who.int/). Symptomatic COVID-19 is typically characterized by symptoms 44 ranging from mild to acute respiratory distress which is associated with cytokine storm (Chen 45 and Li, 2020; Verity et al., 2020) . Other extra-pulmonary symptoms ranging from CVC, 46 inflammation, coagulopathies, multiple organ damage and neurological disorders have been involvement of other co-receptors, which may be crucial for the infectivity of SARS-CoV2. 59 Neuropilin-1 (NRP-1), which is abundantly expressed on the surface of endothelial and 60 epithelial cells and binds to furin cleaved substrates, has been shown to facilitate SARS-CoV2 61 infectivity (Cantuti-Castelvetri et al., 2020; Daly et al., 2020) . S1 fragment of the spike protein 62 cleaved by furin has been shown to directly bind to NRP1 expressed on the cell surface, and In the current study, we demonstrate using in-silico modelling, that host-virus interaction has 82 a striking similarity between humans and hamsters that involves TMPRSS2-mediated priming 83 5 of S protein, binding of S1 peptide with NRP-1 and subsequent binding of RBD to ACE2. 84 Intranasal infection of SARS-CoV2 in hamster resulted in high lung viral load with 85 significantly elevated lung injury on 2 and 4 dpi. Furthermore, our data shows aggressive 

In silico interaction predicts similarities in the host-virus interaction between hamster 101 and human 102 Interaction of SARS-CoV-2 RBD with host ACE2 receptor, TMPRSS protease and NRP-1 is 103 critical for virus entry into epithelial cells. However, the similarities of these interactions 104 between human (hu) and GSH (ha) remained unexplored. To compare residues of hu and ha 105 ACE2 that interact with RBD of SARS-CoV2, structure-guided sequence alignment was 106 performed with their respective sequences (Figures S1A and 1A) . The overlay of crystal of 107 6 108 huACE2 with modelled haACE2 was 1.2 Å, indicating the high structural resemblance. At 109 interface a total of 17 and 16 residues of the RBD are in contact with 20 residues of hu and ha 110 7 ACE2, respectively ( Figures 1B-1E) . The notable features that was common for both hu/ha 111 ACE2 interfaces with RBD was the networks of hydrophilic interactions like hydrogen bonds 112 (H-bonds) and salt bridges between Lys31 and Lys353 of ACE2 with Glu35 and Asp38 of 113 RBD (Figures 1E and 1F) . Aromatic residues of RBD, Tyr449, Tyr453, Phe456, Phe486, 114 Tyr489, Tyr495 and Tyr505, contributing to binding affinity remained common for both hu/ha 115 ACE2 ( Figure 1F ). Together, SARS-CoV2 RBD with hu/ha ACE2 interfaces shared a 116 substantial similarities in the buried surface area, the number of interacting residues and 117 hydrophilic interaction. hu/ha TMPRSS2 structurally looks very similar ( Figure S1B ). NRP-118 1, which bind to S1 fragment of spike protein, facilitates SARS-CoV2 entry within the cells 119 (Cantuti-Castelvetri et al., 2020; Daly et al., 2020) . Hu and ha NRP-1, the C-terminal R685 of 120 the CendR peptide, interacts identically with S1 fragment of spike protein residues, Y297, 121 W301, T316, D320, S346, T349 and Y353 (Figures 1G, 1H and 1I) . The R682 and R685 122 sidechains S1 fragment of spike protein engages with NRP-1 sidechains of Y297 and Y353 via 123 stacked cation-π interactions ( Fig. 1I and J) . In huNRP-1, the residues interacting from C-124 endR are R685 making H-bonds with residues Asp320, Ile415, Tyr353, Thr349 and Ser346 of 125 S1 fragment while in haNRP-1, the R685 remains stable but with less number of H-bonds with 126 residues Trp297, Gly318, Asp320, Tyr353 and Thr349. The other common interactions in 127 terms of hydrophobic contacts are with residues Thr316, Gly414, and Trp301. Glu348 and 128 Lys351 contributed only in huNRP-1 while Ser346 in haNRP-1. Among mutations at RBD, 129 the cause of stable Y501 over N501 was also compared in hu/ha, as Y501 gain additional 130 bonding with tyrosine residues (41@ACE2, 355@RBD) and H-bond with Arg353 ( Figure   131 1K). (Figure 2A ) (Chan et al., 2020; Sia et al., 2020) . This was accompanied by gross lungs 141 morphological changes characterized by pneumonitis regions with high viral load (~10.5 log10 142 copy number/g (lung mass) and TCID50 at 2 dpi that gradually decreased at later time points 143 marking the recovery of the animals (Figures 2B and 2C) . Covid-19 is marked by severity of respectively, the titre of these antibodies was further enhanced to 1:8900, 1:550 and 1:660 at 175 14 dpi (Figure 3B ), indicating for a steady recovery of infection. In addition, antigen-specific 176 frequency of CD4 + IFNγ + cells was elevated (~2.5 fold) without affecting the frequency of 177 CD4 + T cells at 2 dpi as compared to uninfected GSH (Figures 3C and S2B) . To define cellular dpi hamsters indicative of onset of inflammation and gastro-intestinal injury ( Figure S4A ). 261 However, there were no gross changes observed in brain, liver and kidney of infected as 262 compared to uninfected hamsters at 2 dpi ( Figure S4B ). correlation with severe covid19 metabolomic profile with sharp peak in abundance at 2dpi.

Both allantoin and N-acetylneuraminate increased abundance have been shown to be 374 biomarkers for increased heart risk. Together, our data provides first insight into the key 375 metabolites which could be used as the biomarkers for the efficient and rapid evaluation of 376 SARS-CoV2 disease severity.

As of now hamster model for SARS-CoV2 has been described as a suitable model to study IFNy  TGTTGCTCTGCCTCACTCAGG  AAGACGAGGTCCCCTCCATTC  TNFa  AGAATCCGGGCAGGTCTACT  TATCCCGGCAGCTTGTGTTT  IL13  AAATGGCGGGTTCTGTGC  AATATCCTCTGGGTCTTGTAGATGG  IL17A  ATGTCCAAACACTGAGGCCAA  GCGAAGTGGATCTGTTGAGGT  IL9  CTCTGCCCTGCTCTTTGGTT  CGAGGGTGGGTCATTCTTCA  TGFb  ACGGAGAAGAACTGCTGTG  GGTTGTGTTGGTTGTAGAGG  IL10  GGTTGCCAAACCTTATCAGAA ATG TTCACCTGTTCCACAGCCTTG  T-bet  ACAAGGGGGCTTCCAACAAT  CAGCTGAGTGATCTCGGCAT  GATA3 GAAGGCAGGGAGTGTGTGAA  GTC TGA CAG TTC GCA CAG GA  FOXP3  GGTCTTCGAGGAGCCAGAAGA  GCCTTGCCC TTC TCA TCC A  CCR5  TGT GAC ATC CGT TCC CCC T  GGC AGG GTG CTG ACA TAC TA  CCL5  CTACGCTCCTTCATC TGC CTC  CCT TCG GGT GAC AAA AAC GAC  CCL22  CGT GGC TCT CAT CCT TCT TGC  CAG ATG CTG TCT TCC ACG TTG G  CXCL9 TGG GTA TCA TCC TCC TGG AC  AAT GAG GAC CTG GAG CAA AC  CXCL10 TGGAAATTATTCCTGCAAGTCA  GTG ATC GGC TTC TCT CTG GT  PD1  CTGAAAAGGGTTAAG CCA GC  GCC TCC AGG ATT CTC TCT GTT  PDL1  TGATCATCCCAGACCCGCTC  CTC CTC GAA CTG CGT ATC GT  IL6 GGACAATGACTATGTGTTGTTAGAA AGGCAAATTTCCCAATTGTATCCAG iNOS TGAGCCACTGAGTTCTCCTAAGG TCCTATTTCAACTCCAAGATGTTCTG Lipidmatch flow was utilized with default settings for peak picking (using mzmine), lipid 623 annotation, blank filtration and combining positive and negative data (Koelmel et al., 2017) .

Thereafter, data analysis was carried out as follows: the data was normalized by sum, perato 625 scaled, and log-transformed for analysis in metaboanalyst. for database search. We have also used online available spectral library for further confirmation 657 of identification. Cut-off for retention time match was 0.5 min and spectral similarity was more 658 than 30% fragmentation match in Progenesis QI. Peaks that had a coefcient of variation (CV) 659 less than 30% in pool QC samples were kept for the further analysis of data. Additionally, 660 manual verifcation of each detected feature has been done for the selection of right peaks. 

Covid-19 hyperinflammation and 732 post-Covid-19 illness may be rooted in mast cell activation syndrome

Kinetics of viral load, immunological 736 mediators and characterization of a SARS-CoV-2 isolate in mild COVID-19 patients during 737 acute phase of infection

CoV-2 cell entry and infectivity

Pathological Manifestations of Coronavirus Disease 2019 (COVID-19) in a Golden Syrian 744

Hamster Model: Implications for Disease Pathogenesis and Transmissibility. Clinical 745 Infectious Diseases

SARS-CoV-2: virus dynamics and host response

Neuropilin-1 is a 750 host factor for SARS-CoV-2 infection

Coronavirus and Cardiovascular Disease, Myocardial 753 Injury, and Arrhythmia

Targets of T Cell 756 Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed 757 Individuals

Identification 759 of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from 760 a naïve semisynthetic library

Isolation and characterization of SARS-CoV-2 from 763 the first US COVID-19 patient

Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Syrian hamsters as a small 770 animal model for SARS-CoV-2 infection and countermeasure development

C-mediated sodium glucose transporter 1 activation in precondition-induced cardioprotection

LipidMatch: an automated workflow 777 for rule-based lipid identification using untargeted high-resolution tandem mass spectrometry 778 data

The Pathogenesis of Cardiac Fibrosis

Metabolomic analysis of primary human skeletal muscle cells during myogenic progression

Medium-chain Fatty Acids as 785

Metabolic Therapy in Cardiac Disease

SARS-CoV-2 788 productively infects human gut enterocytes

Oral SARS-CoV-2 Inoculation Establishes Subclinical 791

Respiratory Infection with Virus Shedding in Golden Syrian Hamsters

Transcription factor Foxo1 is essential for IL-9 795 induction in T helper cells

Neurologic Manifestations of Hospitalized Patients With Coronavirus 798 Disease

Deep immune profiling of 801

COVID-19 patients reveals distinct immunotypes with therapeutic implications

Computational modeling suggests impaired interactions between NKX2.5 and GATA4 in 804 individuals carrying a novel pathogenic D16N NKX2.5 mutation

Two Detailed Plaque Assay 807 Protocols for the Quantification of Infectious SARS-CoV-2

ClustVis: a web tool for visualizing clustering of 810 multivariate data using Principal Component Analysis and heatmap

Cytokine release syndrome in severe COVID-19

Development of a Liquid Chromatography-High Resolution Mass

Using All Ion Fragmentation Acquisition

COVID-19 and 819 cardiovascular disease: from basic mechanisms to clinical perspectives

Evidence of CD1d pathway of lipid antigen 822 presentation in mouse primary lung epithelial cells and its up-regulation upon

Mycobacterium bovis BCG infection

Merging metabolomics and lipidomics into one analytical run

Proteomic and Metabolomic Characterization of COVID-19 Patient Sera

Envelope proteins of two HIV-1 clades induced different 832 epitope-specific antibody response

Pathogenesis and transmission 835 of SARS-CoV-2 in golden hamsters

Molecular dynamics 837 simulation reveals the possible druggable hot-spots of USP7

SARS-CoV-2 entry factors 840 are highly expressed in nasal epithelial cells together with innate immune genes

Cardiac 843 Fibrosis: The Fibroblast Awakens

Estimates of the 846 severity of coronavirus disease 2019: a model-based analysis

Comorbidities and 848 multi-organ injuries in the treatment of COVID-19

Cryo-EM structure of the 2019-nCoV spike in the prefusion 851 conformation

Nervous system involvement after infection with COVID-19 and other coronaviruses

Evidence for 856 Gastrointestinal Infection of SARS-CoV-2

Smell and 859 taste dysfunction in patients with COVID-19

Characterizes a Key Role for N-Acetylneuraminic Acid in Coronary Artery Diseases

All the results were analysed and plotted by using Graph pad prism 7.0 software. Body mass, 704 lung mass, gene expression, FACS, ELISA, TCID50 and qPCR studies were compared and 705 analyzed by using one-way ANOVA or student t-test for n = 6 hamsters samples per group.

Metabolomics and lipidomics analysis was carried out with n=9 serum samples per group. P-707 value of less than 0.05 and was considered as statistically significant.