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1.
Sci Transl Med ; 14(632): eabi5735, 2022 Feb 16.
Article in English | MEDLINE | ID: covidwho-1691438

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 µg) SpFN vaccine, given twice 28 days apart, induced a Th1-biased CD4 T cell helper response and elicited neutralizing antibodies against SARS-CoV-2 wild-type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates following high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates supports the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.


Subject(s)
COVID-19 , Nanoparticles , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 Vaccines , Ferritins , Humans , Immunity , Macaca mulatta , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
2.
mBio ; : e0290621, 2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1649374

ABSTRACT

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health emergency. While most human disease is mild to moderate, some infections lead to a severe disease characterized by acute respiratory distress, hypoxia, anosmia, ageusia, and, in some instances, neurological involvement. Small-animal models reproducing severe disease, including neurological sequela, are needed to characterize the pathophysiological mechanism(s) of disease and to identify medical countermeasures. Transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the K18 promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge when high viral doses are used. Here, we report on SARS-CoV-2 infection of hamsters engineered to express the hACE2 receptor under the control of the K18 promoter. K18-hACE2 hamsters infected with a relatively low dose of 100 or 1,000 PFU of SARS-CoV-2 developed a severe and lethal disease, with most animals succumbing by day 5 postinfection. Hamsters developed severe lesions and inflammation within the upper and lower respiratory system, including infection of the nasal cavities causing marked destruction of the olfactory epithelium as well as severe bronchopneumonia that extended deep into the alveoli. Additionally, SARS-CoV-2 infection spread to the central nervous system (CNS), including the brain stem and spinal cord. Wild-type (WT) hamsters naturally support SARS-CoV-2 infection, with the primary lesions present in the respiratory tract and nasal cavity. Overall, infection in the K18-hACE2 hamsters is more extensive than that in WT hamsters, with more CNS involvement and a lethal outcome. These findings demonstrate the K18-hACE2 hamster model will be valuable for studying SARS-CoV-2. IMPORTANCE The rapid emergence of SARS-CoV-2 has created a global health emergency. While most human SARS-CoV-2 disease is mild, some people develop severe, life-threatening disease. Small-animal models mimicking the severe aspects of human disease are needed to more clearly understand the pathophysiological processes driving this progression. Here, we studied SARS-CoV-2 infection in hamsters engineered to express the human angiotensin-converting enzyme 2 viral receptor under the control of the K18 promoter. SARS-CoV-2 produces a severe and lethal infection in transgenic hamsters that mirrors the most severe aspects of COVID-19 in humans, including respiratory and neurological injury. In contrast to other animal systems, hamsters manifest disease with levels of input virus more consistent with natural human infection. This system will be useful for the study of SARS-CoV-2 disease and the development of drugs targeting this virus.

3.
NPJ Vaccines ; 6(1): 129, 2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1493109

ABSTRACT

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

4.
Sci Rep ; 11(1): 19458, 2021 09 30.
Article in English | MEDLINE | ID: covidwho-1447326

ABSTRACT

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Ebolavirus/drug effects , Hemorrhagic Fever, Ebola/drug therapy , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/pharmacology , Administration, Intravenous , Aerosols , Alanine/administration & dosage , Alanine/pharmacology , Animals , Antiviral Agents/administration & dosage , Disease Models, Animal , Female , Hemorrhagic Fever, Ebola/blood , Kaplan-Meier Estimate , Liver/drug effects , Liver/virology , Lung/pathology , Lung/virology , Lymph Nodes/drug effects , Lymph Nodes/pathology , Lymph Nodes/virology , Macaca mulatta , Male , Random Allocation , Systemic Inflammatory Response Syndrome/drug therapy , Systemic Inflammatory Response Syndrome/virology , Viral Load/drug effects , Viremia/drug therapy
5.
Respiration ; 101(2): 155-165, 2022.
Article in English | MEDLINE | ID: covidwho-1410800

ABSTRACT

BACKGROUND: Findings from autopsies have provided evidence on systemic microvascular damage as one of the underlying mechanisms of Coronavirus disease 2019 (CO-VID-19). The aim of this study was to correlate autopsy-based cause of death in SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive patients with chest imaging and severity grade of pulmonary and systemic morphological vascular pathology. METHODS: Fifteen SARS-CoV-2 positive autopsies with clinically distinct presentations (age 22-89 years) were retrospectively analyzed with focus on vascular, thromboembolic, and ischemic changes in pulmonary and in extrapulmonary sites. Eight patients died due to COVID-19 associated respiratory failure with diffuse alveolar damage in various stages and/or multi-organ failure, whereas other reasons such as cardiac decompensation, complication of malignant tumors, or septic shock were the cause of death in 7 further patients. The severity of gross and histopathological changes was semi-quantitatively scored as 0 (absent), 1 (mild), and 3 (severe). Severity scores between the 2 groups were correlated with selected clinical parameters, initial chest imaging, autopsy-based cause of death, and compared using Pearson χ2 and Mann-Whitney U tests. RESULTS: Severe pulmonary endotheliitis (p = 0.031, p = 0.029) and multi-organ involvement (p = 0.026, p = 0.006) correlated significantly with COVID-19 associated death. Pulmonary microthrombi showed limited statistical correlation, while tissue necrosis, gross pulmonary embolism, and bacterial superinfection did not differentiate the 2 study groups. Chest imaging at hospital admission did not differ either. CONCLUSIONS: Extensive pulmonary endotheliitis and multi-organ involvement are characteristic autopsy features in fatal CO-VID-19 associated deaths. Thromboembolic and ischemic events and bacterial superinfections occur frequently in SARS-CoV-2 infection independently of outcome.


Subject(s)
COVID-19/mortality , COVID-19/pathology , Endothelium, Vascular/pathology , Multiple Organ Failure/virology , Respiratory Distress Syndrome/virology , Vasculitis/virology , Adult , Aged , Aged, 80 and over , Autopsy , COVID-19/complications , Cause of Death , Cohort Studies , Female , Humans , Male , Middle Aged , Multiple Organ Failure/mortality , Multiple Organ Failure/pathology , Pulmonary Alveoli/pathology , Respiratory Distress Syndrome/mortality , Respiratory Distress Syndrome/pathology , Vasculitis/mortality , Vasculitis/pathology , Young Adult
6.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Article in English | MEDLINE | ID: covidwho-1392996

ABSTRACT

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/virology , Macaca mulatta/immunology , Nanoparticles/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/immunology , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , Ferritins/chemistry , SARS-CoV-2/metabolism , T-Lymphocytes/immunology
7.
JCI Insight ; 5(19)2020 10 02.
Article in English | MEDLINE | ID: covidwho-1388620

ABSTRACT

The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs.


Subject(s)
Cause of Death , Coronavirus Infections/pathology , Disease Progression , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/pathology , Severe Acute Respiratory Syndrome/pathology , Angiotensin-Converting Enzyme 2 , Animals , COVID-19 , Coronavirus Infections/physiopathology , Disease Models, Animal , Female , Humans , Lung/pathology , Male , Mice , Mice, Transgenic , Pandemics , Pneumonia, Viral/physiopathology , Severe Acute Respiratory Syndrome/physiopathology , Severity of Illness Index , Survival Rate , Virus Replication/genetics
8.
J Gen Virol ; 102(5)2021 05.
Article in English | MEDLINE | ID: covidwho-1219293

ABSTRACT

SARS-CoV-2 is the causative agent of COVID-19 and human infections have resulted in a global health emergency. Small animal models that reproduce key elements of SARS-CoV-2 human infections are needed to rigorously screen candidate drugs to mitigate severe disease and prevent the spread of SARS-CoV-2. We and others have reported that transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the Keratin 18 (K18) promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge. Here we report that some infected mice that survive challenge have residual pulmonary damages and persistent brain infection on day 28 post-infection despite the presence of anti-SARS-COV-2 neutralizing antibodies. Because of the hypersensitivity of K18-hACE2 mice to SARS-CoV-2 and the propensity of virus to infect the brain, we sought to determine if anti-infective biologics could protect against disease in this model system. We demonstrate that anti-SARS-CoV-2 human convalescent plasma protects K18-hACE2 against severe disease. All control mice succumbed to disease by day 7; however, all treated mice survived infection without observable signs of disease. In marked contrast to control mice, viral antigen and lesions were reduced or absent from lungs and absent in brains of antibody-treated mice. Our findings support the use of K18-hACE2 mice for protective efficacy studies of anti-SARS-CoV-2 medical countermeasures (MCMs). They also support the use of this system to study SARS-CoV-2 persistence and host recovery.


Subject(s)
COVID-19/therapy , Acute Lung Injury/prevention & control , Acute Lung Injury/virology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Brain/pathology , Brain/virology , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Disease Models, Animal , Female , Humans , Immunization, Passive , Lung/pathology , Lung/virology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Coronavirus/genetics , Receptors, Coronavirus/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Severity of Illness Index , Viral Load , Virus Replication
9.
PLoS One ; 16(2): e0246366, 2021.
Article in English | MEDLINE | ID: covidwho-1059447

ABSTRACT

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.


Subject(s)
COVID-19/physiopathology , Disease Models, Animal , Macaca mulatta , SARS-CoV-2/physiology , Animals , COVID-19/pathology , COVID-19/transmission , Chlorocebus aethiops , Disease Transmission, Infectious , Female , Lung/pathology , Macaca fascicularis , Male , Virus Shedding
10.
NPJ Vaccines ; 6(1): 16, 2021 Jan 25.
Article in English | MEDLINE | ID: covidwho-1047957

ABSTRACT

A worldwide effort to counter the COVID-19 pandemic has resulted in hundreds of candidate vaccines moving through various stages of research and development, including several vaccines in phase 1, 2 and 3 clinical trials. A relatively small number of these vaccines have been evaluated in SARS-CoV-2 disease models, and fewer in a severe disease model. Here, a SARS-CoV-2 DNA targeting the spike protein and delivered by jet injection, nCoV-S(JET), elicited neutralizing antibodies in hamsters and was protective in both wild-type and transiently immunosuppressed hamster models. This study highlights the DNA vaccine, nCoV-S(JET), we developed has a great potential to move to next stage of preclinical studies, and it also demonstrates that the transiently-immunosuppressed Syrian hamsters, which recapitulate severe and prolonged COVID-19 disease, can be used for preclinical evaluation of the protective efficacy of spike-based COVID-19 vaccines.

11.
EBioMedicine ; 63: 103182, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1014448

ABSTRACT

BACKGROUND: SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. METHODS: Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. FINDINGS: COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. INTERPRETATION: ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. FUNDING: No external funding was necessary for this study.


Subject(s)
Capillaries/pathology , Coronary Vessels/pathology , SARS-CoV-2/metabolism , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Capillaries/metabolism , Coronary Vessels/metabolism , Female , Humans , Inflammation/pathology , Male , Microscopy, Fluorescence , Middle Aged , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism
12.
J Virol ; 94(22)2020 10 27.
Article in English | MEDLINE | ID: covidwho-982503

ABSTRACT

Animal models recapitulating human COVID-19 disease, especially severe disease, are urgently needed to understand pathogenesis and to evaluate candidate vaccines and therapeutics. Here, we develop novel severe-disease animal models for COVID-19 involving disruption of adaptive immunity in Syrian hamsters. Cyclophosphamide (CyP) immunosuppressed or RAG2 knockout (KO) hamsters were exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the respiratory route. Both the CyP-treated and RAG2 KO hamsters developed clinical signs of disease that were more severe than those in immunocompetent hamsters, notably weight loss, viral loads, and fatality (RAG2 KO only). Disease was prolonged in transiently immunosuppressed hamsters and was uniformly lethal in RAG2 KO hamsters. We evaluated the protective efficacy of a neutralizing monoclonal antibody and found that pretreatment, even in immunosuppressed animals, limited infection. Our results suggest that functional B and/or T cells are not only important for the clearance of SARS-CoV-2 but also play an early role in protection from acute disease.IMPORTANCE Syrian hamsters are in use as a model of disease caused by SARS-CoV-2. Pathology is pronounced in the upper and lower respiratory tract, and disease signs and endpoints include weight loss and viral RNA and/or infectious virus in swabs and organs (e.g., lungs). However, a high dose of virus is needed to produce disease, and the disease resolves rapidly. Here, we demonstrate that immunosuppressed hamsters are susceptible to low doses of virus and develop more severe and prolonged disease. We demonstrate the efficacy of a novel neutralizing monoclonal antibody using the cyclophosphamide transient suppression model. Furthermore, we demonstrate that RAG2 knockout hamsters develop severe/fatal disease when exposed to SARS-CoV-2. These immunosuppressed hamster models provide researchers with new tools for evaluating therapies and vaccines and understanding COVID-19 pathogenesis.


Subject(s)
Coronavirus Infections/immunology , Coronavirus Infections/pathology , Disease Models, Animal , Mesocricetus , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Adaptive Immunity , Animals , Animals, Genetically Modified , Betacoronavirus/physiology , COVID-19 , Cyclophosphamide , DNA-Binding Proteins/genetics , Gene Knockout Techniques , Immunosuppressive Agents , Pandemics , SARS-CoV-2 , Severity of Illness Index
13.
JCI Insight ; 5(12)2020 06 18.
Article in English | MEDLINE | ID: covidwho-215032

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Pneumonia, Viral/virology , Animals , Antibodies, Viral/immunology , Antigens, Viral/isolation & purification , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Formaldehyde , Humans , Immunohistochemistry , In Situ Hybridization , Mice , Nucleocapsid Proteins/immunology , Pandemics , Paraffin Embedding/methods , Pathology, Molecular/methods , Pneumonia, Viral/pathology , RNA, Viral/isolation & purification , Rabbits , SARS-CoV-2 , Tissue Fixation/methods
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