Severe Acute Respiratory Syndrome Coronavirus 2 Vasculopathy in a Syrian Golden Hamster Model
2023; Elsevier BV; Volume: 193; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2023.02.013
ISSN1525-2191
AutoresErin E. Ball, Christopher M. Weiss, Hongwei Liu, Kenneth A. Jackson, M. Kevin Keel, Christopher J. Miller, Koen K. A. Van Rompay, Lark L. Coffey, Patricia A. Pesavento,
Tópico(s)Long-Term Effects of COVID-19
ResumoClinical evidence of vascular dysfunction and hypercoagulability as well as pulmonary vascular damage and microthrombosis are frequently reported in severe cases of human coronavirus disease 2019 (COVID-19). Syrian golden hamsters recapitulate histopathologic pulmonary vascular lesions reported in patients with COVID-19. Herein, special staining techniques and transmission electron microscopy further define vascular pathologies in a Syrian golden hamster model of human COVID-19. The results show that regions of active pulmonary inflammation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are characterized by ultrastructural evidence of endothelial damage with platelet marginalization and both perivascular and subendothelial macrophage infiltration. SARS-CoV-2 antigen/RNA was not detectable within affected blood vessels. Taken together, these findings suggest that the prominent microscopic vascular lesions in SARS-CoV-2–inoculated hamsters likely occur due to endothelial damage followed by platelet and macrophage infiltration. Clinical evidence of vascular dysfunction and hypercoagulability as well as pulmonary vascular damage and microthrombosis are frequently reported in severe cases of human coronavirus disease 2019 (COVID-19). Syrian golden hamsters recapitulate histopathologic pulmonary vascular lesions reported in patients with COVID-19. Herein, special staining techniques and transmission electron microscopy further define vascular pathologies in a Syrian golden hamster model of human COVID-19. The results show that regions of active pulmonary inflammation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are characterized by ultrastructural evidence of endothelial damage with platelet marginalization and both perivascular and subendothelial macrophage infiltration. SARS-CoV-2 antigen/RNA was not detectable within affected blood vessels. Taken together, these findings suggest that the prominent microscopic vascular lesions in SARS-CoV-2–inoculated hamsters likely occur due to endothelial damage followed by platelet and macrophage infiltration. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans varies from asymptomatic to severe respiratory disease, progressing to acute respiratory distress syndrome, multiorgan dysfunction, and death in a subset of patients. As the pandemic has progressed, there have been increasing reports documenting clinical evidence of coagulation abnormalities (coagulopathy) and microscopic indicators of pulmonary vascular damage (vasculopathy), including endotheliitis, defined as subendothelial leukocyte infiltration with lifting and/or damage to endothelial cells,1Shi Y. Dong K. Zhang Y.G. Michel R.P. Marcus V. Wang Y.Y. Chen Y. Gao Z.H. Sinusoid endotheliitis as a histological parameter for diagnosing acute liver allograft rejection.World J Gastroenterol. 2017; 23: 792-799Crossref PubMed Scopus (6) Google Scholar vasculitis, and microthrombosis associated with severe cases of coronavirus disease 2019 (COVID-19).2Ackermann M. Verleden S.E. Kuehnel M. Haverich A. Welte T. Laenger F. Vanstapel A. Werlein C. Stark H. Tzankov A. Li W.W. Li V.W. Mentzer S.J. Jonigk D. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19.N Engl J Med. 2020; 383: 120-128Crossref PubMed Scopus (3553) Google Scholar, 3Colling M.E. Kanthi Y. COVID–19-associated coagulopathy: an exploration of mechanisms.Vasc Med. 2020; 25: 471-478Crossref PubMed Scopus (174) Google Scholar, 4Panigada M. Bottino N. Tagliabue P. Grasselli G. Novembrino C. Chantarangkul V. Pesenti A. Peyvandi F. Tripodi A. Hypercoagulability of COVID-19 patients in intensive care unit: a report of thromboelastography findings and other parameters of hemostasis.J Thromb Haemost. 2020; 18: 1738-1742Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar, 5Gu S.X. Tyagi T. Jain K. Gu V.W. Lee S.H. Hwa J.M. Kwan JenniferM. Krause D.S. Lee A.I. Halene S. Martin K.A. Chun H.J. Hwa J. Thrombocytopathy and endotheliopathy: crucial contributors to COVID-19 thromboinflammation.Nat Rev Cardiol. 2021; 18: 194-209Crossref PubMed Scopus (237) Google Scholar Syrian golden hamsters (Mesocricetus auratus) are naturally susceptible to SARS-CoV-2 and closely recapitulate the clinical, virological, and histopathologic features of human COVID-19.6Imai M. Iwatsuki-Horimoto K. Hatta M. Loeber S. Halfmann P.J. Nakajima N. Watanabe T. Ujie M. Takahashi K. Ito M. Yamada S. Fan S. Chiba S. Kuroda M. Guan L. Takada K. Armbrust T. Balogh A. Furusawa Y. Okuda M. Ueki H. Yasuhara A. Sakai-Tagawa Y. Lopes Tiago J.S. Kiso M. Yamayoshi S. Kinoshita N. Ohmagari N. Hattori S.I. Takeda M. Mitsuya H. Krammer F. Suzuki T. Kawaoka Y. Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development.Proc Natl Acad Sci U S A. 2020; 117: 16587-16595Crossref PubMed Scopus (578) Google Scholar, 7Chan J.F.W. Zhang A.J. Yuan S. Poon V.K.M. Chan C.C.S. Lee A.C.Y. Chan Wan Mui Fan Z. Tsoi H.W. Wen L. Liang R. Cao J. Chen Y. Tang K. Luo C. Cai J.P. Kok K.H. Chu H. Chan K.H. Sridhar S. Chen Z. Chen H. To K.K.W. Yuen K.Y. Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility.Clin Infect Dis. 2020; 71: 2428-2446Crossref PubMed Scopus (210) Google Scholar, 8Roberts A. Vogel L. Guarner J. Hayes N. Murphy B. Zaki S. Subbarao K. Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters.J Virol. 2005; 79: 503-511Crossref PubMed Scopus (220) Google Scholar, 9Sia S.F. Yan L.M. Chin A.W.H. Fung K. Choy K.T. Wong A.Y.L. Kaewpreedee P. Perera R.A.P.M. Poon L.L.M. Nicholls J.M. Peiris M. Yen H.L. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.Nature. 2020; 583: 834-838Crossref PubMed Scopus (827) Google Scholar, 10Mulka K.R. Beck S.E. Solis C.V. Johanson A.L. Queen S.E. McCarron M.E. et al.Progression and resolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in golden Syrian hamsters.Am J Pathol. 2022; 192: 195-207Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 11Becker K. Beythien G. de Buhr N. Stanelle-Bertram S. Tuku B. Kouassi N.M. Beck S. Zickler M. Allnoch L. Gabriel G. von Köckritz-Blickwede M. Baumgärtner W. Vasculitis and neutrophil extracellular traps in lungs of golden Syrian hamsters with SARS-CoV-2.Front Immunol. 2021; 12: 1-11Crossref Scopus (38) Google Scholar Herein, a Syrian golden hamster model was used to define SARS-CoV-2–induced vasculopathies, which have not been fully characterized in this animal model. Hamsters show regions of active SARS-CoV-2–induced pulmonary inflammation that exhibit ultrastructural evidence of endothelial damage with detachment from the underlying basement membrane, platelet marginalization, and marked perivascular and subendothelial mononuclear inflammation composed primarily of macrophages. At 3 to 6 or 7 days post inoculation (dpi), SARS-CoV-2 antigen/RNA was not detectable within affected blood vessels. These findings suggest that the microscopic vascular lesions in SARS-CoV-2–inoculated hamsters occur not primarily due to direct viral infection of the endothelium, but rather due to indirect endothelial damage with loss of endothelial attachment and vascular compromise followed by platelet and macrophage infiltration. All hamster experiments were conducted under protocol number 21868, approved by the University of California, Davis, Institutional Animal Care and Use Committee. Infectious virus was handled in certified animal biosafety level 3 laboratory spaces in compliance with approved institutional biological use authorization number R2813. University of California, Davis, is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and all work adhered to the NIH Guide for the Care and Use of Laboratory Animals.12Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar Two experiments, each using 8- to 10-week–old male and female Syrian golden hamsters, were performed [experiment 1, N = 15 (8 males and 7 females); experiment 2, N = 8 (4 males and 4 females)]; the animals were from Charles River Laboratories (Wilmington, MA) and were acclimated for up to 7 days at 22°C to 25°C and a 12:12-hours light/dark cycle. Rodent chow with 18% protein content and sterile bottled water were provided ad libitum for the duration of the experiment. SARS-CoV-2/human/USA/CA-CZB-59X002/2020 (GenBank, https://www.ncbi.nlm.nih.gov/nuccore/MT394528; accession number MT394528, data available to public) from a 2020 patient with COVID-19 in Northern California was passaged twice in Vero-E6 (ATCC, Manassas, VA) cells to achieve a titer of 2.2 × 107 plaque-forming units (PFUs)/mL and stored at −80°C. Randomly selected hamsters from experiment 1 and experiment 2 were anesthetized with isoflurane and administered 30 μL of phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA) [experiment 1, N = 4 (2 males and 2 females); experiment 2, N = 2 (1 male and 1 female)] or SARS-CoV-2 diluted in PBS at a dose of 104 PFUs [experiment 1, N = 11 (6 males and 5 females); experiment 2, N = 6 (3 males and 3 females)] intranasally by a hanging drop over both nares. Hamsters were monitored daily for clinical signs through the experimental end point and euthanized if weight loss exceeded 20% or if they appeared moribund. Hamsters were anesthetized daily with isoflurane, weighed, and throat swabbed (Puritan; Thermo Fisher Scientific). Swabs were vortexed in 400 μL of Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) and stored at −80°C. Before euthanasia, whole blood was collected via submandibular vein puncture, allowed to clot at room temperature for >10 minutes, and centrifuged for 5 minutes at 8000 × g. Serum was also stored at −80°C. Following humane euthanasia by isoflurane overdose and cervical dislocation, hamsters were perfused with cold sterile PBS, and a necropsy was performed. Tissues were divided and processed for virology assays and histopathology. Lung was homogenized in 1 to 10 μL/mg Dulbecco's modified Eagle's medium with a sterile glass bead using a TissueLyser (Qiagen, Germantown, MD) at 30 Hz for 4 minutes, centrifuged at 10,000 × g for 5 minutes, and stored at −80°C. Infectious SARS-CoV-2 was detected from thawed hamster samples and remaining inocula immediately after inoculation using Vero cell plaque assays. Briefly, samples were serially diluted 10-fold in Dulbecco's modified Eagle's medium with 1% bovine serum albumin (both from Thermo Fisher Scientific) starting at an initial dilution of 1:8. The 12-well plates of confluent Vero CCL-81 cells (ATCC) were inoculated with 125 μL of each dilution and incubated at 37°C and 5% CO2 for 1 hour. After incubation, each cell monolayer was overlaid with 0.5% agarose (Invitrogen, Carlsbad, CA) diluted in Dulbecco's modified Eagle's medium with 5% fetal bovine serum and 1× antibiotic-antimycotic (both from Thermo Fisher Scientific) and incubated for 3 days at 5% CO2 and 37°C. Cell monolayers were then fixed with 4% formalin for 30 minutes, agar plugs were gently removed, and viable cells were stained for 10 minutes with 0.05% crystal violet (Sigma, St. Louis, MO) in 20% ethanol, then rinsed with water. Plaques were counted and viral titers were recorded as the reciprocal of the highest dilution where plaques were noted (PFUs per swab, mg tissue, or mL inoculum). At necropsy, lung was inflated, and tissues were fixed for 48 hours at room temperature with a 10-fold volume of 10% neutral-buffered formalin (Thermo Fisher Scientific). A subset of tissues, including lung, brain, liver, spleen, heart, peripheral lymph nodes, eye, and nasal cavity, was embedded in paraffin, thin sectioned (4 μm thick), and stained routinely with hematoxylin and eosin. Hematoxylin and eosin–stained slides were scanned to ×40 magnification using an Aperio slide scanner (Leica Biosystems, Deer Park, IL) with a magnification doubler and a resolution of 0.25 μm/pixel. Image files were uploaded on a Leica hosted web-based site, and a board-certified veterinary anatomic pathologist (E.E.B.) blindly evaluated sections for SARS-CoV-2–induced histologic lesions. For immunofluorescence, fixed tissue was cryoprotected by rinsing overnight in PBS at 4°C, followed by transfer into 30% sucrose (Thermo Fisher Scientific) in 1× PBS for a second night, frozen over dry ice in Andwin Scientific Tissue-Tek cryomolds filled with Tissue-plus OCT compound, wrapped in parafilm (all from Thermo Fisher Scientific), and stored at −20°C. Colorimetric in situ hybridization was performed according to the manufacturer's instructions, using the RNAscope 2.5 HD Red Reagent Kit (Advanced Cell Diagnostics, Newark, CA) and RNAscope Probe - V-nCoV2019-S (Advanced Cell Diagnostics; catalog number 526 848,561). RNAscope Negative Control Probe - DapB (Advanced Cell Diagnostics; catalog number 310043) and lung tissue from a SARS-CoV-2–uninfected control animal hybridized with the SARS-CoV-2 probes served as negative controls. RNAscope Probe-Mau-Ppib (Advanced Cell Diagnostics; catalog number 890851) served as a positive control. Briefly, each deparaffinized section (5 μm thick) was pretreated with 1× Target Retrieval Buffer (Abcam, Eugene, OR) at 100°C for 15 minutes and Protease Plus (Advanced Cell Diagnostics) at 40°C for 30 minutes before hybridization at 40°C for 2 hours. This was followed by a cascade of signal amplification and signal detection using a Fast Red solution for 10 minutes at room temperature. Slides were counterstained with hematoxylin, dehydrated, coverslipped, and scanned to ×40 magnification, as described above. Positive cells (defined as those exhibiting red cytoplasmic staining) were identified on the basis of location and cell morphology. Slides were deparaffinized in xylene and 100% ethanol. Endogenous peroxidases were blocked by placing slides in 3% hydrogen peroxide in methanol (all from Thermo Fisher Scientific) for 30 minutes. After rehydration, ionized calcium-binding adaptor molecule 1 (Iba1), CD3, and CD79a slides underwent heat-induced antigen retrieval for 30 minutes using distilled water (Iba1) or citrate solution (CD3 and CD79a) composed of 20 mL 10× stock solution [12.9 g citric acid, trisodium salt, anhydrous, 500 mL distilled water, 10 mL 1N hydrogen chloride to adjust pH to 6.1, and 2.5 mL Tween 20 (all from Thermo Fisher Scientific)] and 180 mL deionized water. von Willebrand factor slides were incubated for 10 minutes with proteinase K (Agilent Dako, Santa Clara, CA; catalog number S3020). Following antigen retrieval, all slides were blocked for 20 minutes in 20 mL normal horse serum (Vector Laboratories, Burlingame, CA) diluted in 180 mL PBS. Antibodies used for immunohistochemistry were as follows: rabbit polyclonal anti-Iba1 at 1:600 dilution (Wako Chemicals, Richmond, VA; catalog number 19-19741); anti-rat CD3 clone CD3-12 at 1:10 dilution (supplied by Dr. Peter Moore, University of California, Davis); mouse anti-human CD79a clone HM57 at 1:100 dilution (Bio Rad, Hercules, CA; catalog number MCA2538H); and rabbit polyclonal anti–von Willebrand factor at 1:2000 dilution (Agilent Dako; catalog number A0082). Slides were incubated with primary antibodies at room temperature for 1 hour with gentle agitation, then allowed to incubate at room temperature for 30 minutes with rat-on-canine (CD3), mouse-on-canine (CD79a), or rabbit-on-canine (Iba1 and von Willebrand factor) horseradish peroxidase–polymer (all from BioCare Medical, Pacheco, CA). Colorimetric detection was performed according to manufacturer's instructions using Vector NovaRED peroxidase substrate kit (Vector Laboratories), and slides were counterstained with hematoxylin and bluing reagent (both from Thermo Fisher Scientific) before mounting. Glass slides with sections (12 μm thick) of frozen, cryoprotected lung tissue cut with a Leica CM1860 cryotome (Leica Biosystems) were blocked in 2% bovine serum albumin, 0.3% Triton X-100 (Thermo Fisher Scientific), and 10% normal donkey serum (Jackson Laboratories, West Grove, PA) in 1× PBS for 1 hour at room temperature. Slides were incubated overnight with primary antibodies at 4°C: mouse anti-pan cytokeratin (Lu5) at 1:100 dilution (BioCare Medical; catalog number CM043C); mouse anti-CD31/platelet endothelial cell adhesion molecule 1 antibody (JC/70A) at 1:20 dilution (Novus Biologicals, Littleton, CO; catalog number NB600-562); rat anti-mouse CD41 (MWReg30) at 1:100 dilution (BD Pharmingen, Franklin Lakes, NJ; catalog number 553847); mouse anti-human CD61 (Y2/51) at 1:50 dilution (Bio Rad; catalog number MCA2588); and rabbit polyclonal anti-SARS2 nucleocapsid protein at 1:10,000 dilution (Sino Biologicals, Waye, PA; catalog number 40143-R019). Slides were incubated with secondary antibodies, including donkey anti-rabbit AF-594 (Invitrogen, Waltham, MA; catalog number A21207), donkey anti-mouse AF-488 (Invitrogen; catalog number A21202), and goat anti-rat AF-488 (Invitrogen; catalog number A48262), diluted 1:250 in PBS + 10% donkey serum for 1 hour, and mounted using prolong Gold antifade with DAPI (Thermo Fisher Scientific; catalog number P36931). Z-stacks in a 1.04-μm step size were acquired on a Leica SP8 STED 3× confocal microscope controlled by Leica LAS X software with a 20×/0.75-mm HC PL APO CS2 objective. Images were processed using ImageJ Fiji version 13.0.6 (NIH, Bethesda, MD; https://imagej.net/software/fiji, last accessed December 22, 2022). Lung tissues from two experiment 1 SARS-CoV-2–inoculated hamsters (one each at 3 dpi and 6 dpi) and two experiment 2 SARS-CoV-2–inoculated hamsters (one each at 3 dpi and 6 dpi) were evaluated by transmission electron microscopy (TEM). After fixation in 2.5% glutaraldehyde and 2% paraformaldehyde (both from Ted Pella, Redding, CA) in 0.1 mol/L sodium phosphate buffer (Thermo Fisher Scientific), tissues were post-fixed in 1% osmium tetroxide [Electron Microscopy Sciences (EMS), Hatfield, PA] in 0.1 mol/L sodium phosphate buffer for 1 hour, dehydrated for 30 minutes each in 50%, 75%, and 95% ethanol, dehydrated twice for 20 minutes in 100% ethanol, and placed in propylene oxide (EMS) twice for 15 minutes. Samples were pre-infiltrated overnight in a 1:1 ratio of propylene oxide/resin [composed of 450 mL dodecenyl succinic anhydride, 250 mL araldite 6005 (both from EMS), 82.5 mL Epon 812 (Polysciences Inc., Warrington, PA), 12.5 mL dibutyl phthalate (Ted Pella), and 450 μL benzyldimethylamine (EMS)]. The following day, tissues were infiltrated in 100% resin for 5 hours, embedded with fresh resin, and polymerized at 60°C overnight. Embedded tissues were sectioned with a Leica EM UC6 ultramicrotome (Leica Biosystems) at a thickness of 90 nm, collected on copper mesh grids (EMS), and stained with 4% aqueous uranyl acetate for 20 minutes and 0.2% lead citrate for 2 minutes (both from Ted Pella) in 0.1N NaOH (Thermo Fisher Scientific). TEM imaging was done on FEI Talos L120C at 80 kV using a Ceta-M 16 MP camera (Thermo Fisher Scientific). For each hamster, two lung sections were examined. The images shown are from experiment 1 and 2 hamsters and are representative of changes in all SARS-CoV-2–inoculated hamsters evaluated. The experimental design is summarized in Supplemental Figure S1A. Briefly, two groups of hamsters (experiment 1, N = 11; experiment 2, N = 6) were inoculated intranasally with SARS-CoV-2 Alpha (B.1.1.7) or mock inoculated (experiment 1, N = 4; experiment 2, N = 2) with Dulbecco's PBS on day 0, monitored daily for weight loss and clinical disease and to swab the oropharyngeal cavity, and euthanized on 3, 6, or 7 dpi. A dose of 104 PFUs was chosen because previous studies modeling human COVID-19 in Syrian golden hamsters have shown that SARS-CoV-2 inoculum doses ranging from 103 to 105.6 PFUs reproducibly infect all animals and produce similar infectious lung and tracheal titers and lung and spleen inflammation scores.6Imai M. Iwatsuki-Horimoto K. Hatta M. Loeber S. Halfmann P.J. Nakajima N. Watanabe T. Ujie M. Takahashi K. Ito M. Yamada S. Fan S. Chiba S. Kuroda M. Guan L. Takada K. Armbrust T. Balogh A. Furusawa Y. Okuda M. Ueki H. Yasuhara A. Sakai-Tagawa Y. Lopes Tiago J.S. Kiso M. Yamayoshi S. Kinoshita N. Ohmagari N. Hattori S.I. Takeda M. Mitsuya H. Krammer F. Suzuki T. Kawaoka Y. Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development.Proc Natl Acad Sci U S A. 2020; 117: 16587-16595Crossref PubMed Scopus (578) Google Scholar, 7Chan J.F.W. Zhang A.J. Yuan S. Poon V.K.M. Chan C.C.S. Lee A.C.Y. Chan Wan Mui Fan Z. Tsoi H.W. Wen L. Liang R. Cao J. Chen Y. Tang K. Luo C. Cai J.P. Kok K.H. Chu H. Chan K.H. Sridhar S. Chen Z. Chen H. To K.K.W. Yuen K.Y. Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility.Clin Infect Dis. 2020; 71: 2428-2446Crossref PubMed Scopus (210) Google Scholar, 8Roberts A. Vogel L. Guarner J. Hayes N. Murphy B. Zaki S. Subbarao K. Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters.J Virol. 2005; 79: 503-511Crossref PubMed Scopus (220) Google Scholar, 9Sia S.F. Yan L.M. Chin A.W.H. Fung K. Choy K.T. Wong A.Y.L. Kaewpreedee P. Perera R.A.P.M. Poon L.L.M. Nicholls J.M. Peiris M. Yen H.L. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.Nature. 2020; 583: 834-838Crossref PubMed Scopus (827) Google Scholar,13Rosenke K. Meade-White K. Letko M. Clancy C. Hansen F. Liu Y. Okumura A. Tang-Huau T.S. Li R. Saturday G. Feldmann F. Scott D. Wang Z. Munster V. Jarvis M.A. Feldmann H. Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection.Emerg Microbes Infect. 2020; 9: 2673-2684Crossref PubMed Scopus (126) Google Scholar,14Song Z. Bao L. Yu P. Qi F. Gong S. Wang J. Zhao B. Liu M. Han Y. Deng W. Liu J. Wei Q. Xue J. Zhao W. Qin C. SARS-CoV-2 causes a systemically multiple organs damages and dissemination in hamsters.Front Microbiol. 2021; 11: 1-15Crossref Scopus (34) Google Scholar Herein, SARS-CoV-2–inoculated hamsters from experiments 1 and 2 showed similar trends in body weight and recovery of infectious virus from the respiratory tract. A subset of experiment 1 and 2 hamsters inoculated with SARS-CoV-2 exhibited mild weight loss from 2 to 3 dpi (Supplemental Figure S1B), although none reached euthanasia criteria (loss of 20% of day 0 weight) or displayed clinical signs of illness. In both experiments, infectious virus was recovered from the upper respiratory tract between 1 and 3 dpi and from the lung at 3 dpi (Supplemental Figure S1, C and D). Most hamsters did not become viremic, although infectious virus was detected in the plasma of one female hamster from experiment 1, which also had a low infectious virus titer in the duodenum at 3 dpi. Otherwise, infectious virus was not detected in other hamster tissues. Overall, SARS-CoV-2–inoculated hamsters developed nonlethal infection with infectious virus recoverable until 3 dpi from the upper airway and lung. All hamsters inoculated with SARS-CoV-2 developed moderate to severe microscopic pulmonary lesions (Figure 1). No significant microscopic abnormalities were noted in the brain, heart, spleen, liver, gastrointestinal tract, kidneys, or male/female reproductive tissues of any examined hamster. Inoculation of additional hamsters with emerging SARS-CoV-2 variants, including Delta (B.1.617.2) and Epsilon (B.1.427 and B.1.429) variants, produced similar histopathologic lesions and is described elsewhere.15Carroll T. Fox D. van Doremalen N. Ball E. Morris M.K. Sotomayor-Gonzalez A. Servellita V. Rustagi A. Yinda C.K. Fritts L. Port J.R. Ma Z.M. Holbrook M.G. Schulz J. Blish C.A. Hanson C. Chiu CY Munster V. Stanley S. Miller C.J. The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters.PLoS Pathog. 2022; 18: 1-24Crossref Scopus (12) Google Scholar,16Saito A. Irie T. Suzuki R. Maemura T. Nasser H. Uriu K. et al.Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.Nature. 2021; 602: 300-306Crossref PubMed Scopus (235) Google Scholar SARS-CoV-2–induced airway disease and pneumonia in two independent experimental groups of hamsters were assessed using two methods. First, ImageJ was used to quantify the approximate lung surface area exhibiting microscopic lesions consistent with COVID-19 (defined as inflammation, necrosis, hemorrhage, and/or edema visible at subgross magnification) as a proxy for the overall extent and severity of pulmonary lesions. Next, pathologic lesions specifically affecting pulmonary vasculature were scored according to the criteria outlined in Supplemental Table S1. Both scores were significantly higher in SARS-CoV-2–inoculated hamsters when compared with uninfected controls (Figure 1A). Experiment 1 and 2 SARS-CoV-2–inoculated hamsters exhibited a progressive increase in the extent and severity of lung lesions from 3 to 7 dpi (Figure 1). At 3 dpi, there was patchy neutrophilic and histiocytic inflammation centered on large airways (bronchitis and bronchiolitis), as well as variable bronchiolar epithelial and alveolar septal necrosis with replacement by fibrin, hemorrhage, edema, necrotic debris, numerous neutrophils, and macrophages, and scattered multinucleated syncytial cells (Figure 1C) that was absent from uninfected control hamsters (Figure 1B). By 6 dpi, lesions progressed to widespread necrotizing broncho-interstitial pneumonia (Figure 1D). Reparative changes, consisting of bronchiolar epithelial hyperplasia (Figure 1D) and type II pneumocyte hyperplasia, were also common by 6 dpi. Vascular lesions, including marked perivascular cuffing and subendothelial mononuclear inflammatory cell infiltration (endotheliitis) with variable transmigration of the vessel wall, were noted frequently at both time points (Figure 1, C and D); however, necrotizing vasculitis and thrombosis were not prominent features. These histologic features are consistent with published data from hamster models,6Imai M. Iwatsuki-Horimoto K. Hatta M. Loeber S. Halfmann P.J. Nakajima N. Watanabe T. Ujie M. Takahashi K. Ito M. Yamada S. Fan S. Chiba S. Kuroda M. Guan L. Takada K. Armbrust T. Balogh A. Furusawa Y. Okuda M. Ueki H. Yasuhara A. Sakai-Tagawa Y. Lopes Tiago J.S. Kiso M. Yamayoshi S. Kinoshita N. Ohmagari N. Hattori S.I. Takeda M. Mitsuya H. Krammer F. Suzuki T. Kawaoka Y. Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development.Proc Natl Acad Sci U S A. 2020; 117: 16587-16595Crossref PubMed Scopus (578) Google Scholar, 7Chan J.F.W. Zhang A.J. Yuan S. Poon V.K.M. Chan C.C.S. Lee A.C.Y. Chan Wan Mui Fan Z. Tsoi H.W. Wen L. Liang R. Cao J. Chen Y. Tang K. Luo C. Cai J.P. Kok K.H. Chu H. Chan K.H. Sridhar S. Chen Z. Chen H. To K.K.W. Yuen K.Y. Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility.Clin Infect Dis. 2020; 71: 2428-2446Crossref PubMed Scopus (210) Google Scholar, 8Roberts A. Vogel L. Guarner J. Hayes N. Murphy B. Zaki S. Subbarao K. Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters.J Virol. 2005; 79: 503-511Crossref PubMed Scopus (220) Google Scholar, 9Sia S.F. Yan L.M. Chin A.W.H. Fung K. Choy K.T. Wong A.Y.L. Kaewpreedee P. Perera R.A.P.M. Poon L.L.M. Nicholls J.M. Peiris M. Yen H.L. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.Nature. 2020; 583: 834-838Crossref PubMed Scopus (827) Google Scholar, 10Mulka K.R. Beck S.E. Solis C.V. Johanson A.L. Queen S.E. McCarron M.E. et al.Progression and resolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in golden Syrian hamsters.Am J Pathol. 2022; 192: 195-207Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar including lesions reported in association with emerging SARS-CoV-2 variants.15Carroll T. Fox D. van Doremalen N. Ball E. Morris M.K. Sotomayor-Gonzalez A. Servellita V. Rustagi A. Yinda C.K. Fritts L. Port J.R. Ma Z.M. Holbrook M.G. Schulz J. Blish C.A. Hanson C. Chiu CY Munster V. Stanley S. Miller C.J. The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters.PLoS Pathog. 2022; 18: 1-24Crossref Scopus (12) Google Scholar,16Saito A. Irie T. Suzuki R. Maemura T. Nasser H. Uriu K. et al.Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.Nature. 2021; 602: 300-306Crossref PubMed Scopus (235) Google Scholar Immunohistochemistry, immunofluorescence, in situ hybridization, and TEM were used to further characterize foci of endotheliitis identified on hematoxylin and eosin–stained slides. Immunohistochemistry staining with antibodies against von Willebrand factor/factor 8 (endothelial cells) (Figure 2A), Iba1 (macrophages), CD3 (T lymphocytes), and CD79a (B lymphocytes) revealed that, at 3, 6, and 7 dpi, the mononuclear cells adhered to the endothelial surface or infiltrating subendothelial vascular layers were consistent primarily with macrophages (Figure 2B). There were also low numbers of T lymphocytes (Figure 2C) and few B lymphocytes (Figure 2D). Internal immunohistochemistry controls are shown in Supplemental Figure S2. Immunofluorescence antibodies against SARS-CoV-2 nucleocapsid protein demonstrated viral antigen in hamster terminal bronchiolar epithelium, type I pneumocytes, and, less c
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