Nasal Mucosa Exploited by SARS-CoV-2 for Replicating and Shedding during Reinfection

Viruses

2022 Jul 23

Li, H;Zhao, X;Li, J;Zheng, H;Zhao, Y;Yang, J;Zhou, J;Yang, F;Chen, Y;Zuo, Y;Lai, Q;Long, H;Li, Y;Jin, W;Shi, H;Liu, L;
PMID: 35893674 | DOI: 10.3390/v14081608

Reinfection risk is a great concern with regard to the COVID-19 pandemic because a large proportion of the population has recovered from an initial infection, and previous reports found that primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques without viral presence and pathological injury; however, a high possibility for reinfection at the current stage of the pandemic has been proven. We found the reinfection of SARS-CoV-2 in Syrian hamsters with continuous viral shedding in the upper respiratory tracts and few injuries in the lung, and nasal mucosa was exploited by SARS-CoV-2 for replication and shedding during reinfection; meanwhile, no viral replication or enhanced damage was observed in the lower respiratory tracts. Consistent with the mild phenotype in the reinfection, increases in mRNA levels in cytokines and chemokines in the nasal mucosa but only slight increases in the lung were found. Notably, the high levels of neutralizing antibodies in serum could not prevent reinfection in hamsters but may play roles in benefitting the lung recovery and symptom relief of COVID-19. In summary, Syrian hamsters could be reinfected by SARS-CoV-2 with mild symptoms but with obvious viral shedding and replication, and both convalescent and vaccinated patients should be wary of the transmission and reinfection of SARS-CoV-2.

In vitro and in vivo differences in neurovirulence between D614G, Delta And Omicron BA.1 SARS-CoV-2 variants

Acta neuropathologica communications

2022 Sep 05

Bauer, L;Rissmann, M;Benavides, FFW;Leijten, L;van Run, P;Begeman, L;Veldhuis Kroeze, EJB;Lendemeijer, B;Smeenk, H;de Vrij, FMS;Kushner, SA;Koopmans, MPG;Rockx, B;van Riel, D;
PMID: 36058935 | DOI: 10.1186/s40478-022-01426-4

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with various neurological complications. Although the mechanism is not fully understood, several studies have shown that neuroinflammation occurs in the acute and post-acute phase. As these studies have predominantly been performed with isolates from 2020, it is unknown if there are differences among SARS-CoV-2 variants in their ability to cause neuroinflammation. Here, we compared the neuroinvasiveness, neurotropism and neurovirulence of the SARS-CoV-2 ancestral strain D614G, the Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529) variants using in vitro and in vivo models. The Omicron BA.1 variant showed reduced neurotropism and neurovirulence compared to Delta and D614G in human induced pluripotent stem cell (hiPSC)-derived cortical neurons co-cultured with astrocytes. Similar differences were obtained in Syrian hamsters inoculated with D614G, Delta and the Omicron BA.1 variant 5 days post infection. Replication in the olfactory mucosa was observed in all hamsters, but most prominently in D614G inoculated hamsters. Furthermore, neuroinvasion into the CNS via the olfactory nerve was observed in D614G, but not Delta or Omicron BA.1 inoculated hamsters. Furthermore, neuroinvasion was associated with neuroinflammation in the olfactory bulb of hamsters inoculated with D614G. Altogether, our findings suggest differences in the neuroinvasive, neurotropic and neurovirulent potential between SARS-CoV-2 variants using in vitro hiPSC-derived neural cultures and in vivo in hamsters during the acute phase of the infection.

The mast cell exosome-fibroblast connection: A novel pro-fibrotic pathway

Frontiers in medicine

2023 Feb 23

Savage, A;Risquez, C;Gomi, K;Schreiner, R;Borczuk, AC;Worgall, S;Silver, RB;
PMID: 36910476 | DOI: 10.3389/fmed.2023.1139397

In addition to the traditional activation of resident receptors by release of local mediators, new evidence favors the existence of exosomes in cell-to-cell communication that mediates delivery of specific cargo to modulate recipient cell function. We report that mast cell exosomes are an additional source of pro-fibrotic substances and constitute a unique pathway for the generation of excess collagen.We use primary human lung fibroblasts (HLFs) to demonstrate the uptake of labeled exosomes isolated from the human mast cell line HMC-1 (MC-EXOs), previously shown to contain protein cargo in common with human mast cell exosomes.The MC-EXO uptake by HLF is to the cytosol and increases both proline hydroxylation in HLF lysate and secreted collagen, within 24 h, which is sustained over 72 h, the same time required for transforming growth factor-β (TGF-β) to activate collagen synthesis in the HLFs. Unlike TGF-β, MC-EXO uptake does not induce fibrillar gene activation or invoke the Smad-nuclear transcription pathway. We show that MC-EXO uptake and TGF-β have an additive effect on collagen synthesis in HLF and postulate that MC-EXO uptake by HLFs is a contributing factor to excess collagen synthesis and represents a unique paradigm for understanding fibrosis.It is known that, in the lungs, mast cells are more activated and increase in number with inflammation, injury and viral infection associated with fibrosis. With the reported increased incidence of post-COVID-pulmonary fibrosis (PCPF), data from patients with severe COVID-19 are presented that show an increase in the mast cell number in lung parenchyma, the site of PCPF. Our findings provide a rationale for targeting multiple fibrogenic pathways in the management of lung fibrosis and the use of mast cell exosomes as a biomarker for the prognostic and diagnostic management of evolving fibrotic lung disease.

Detection of SARS-CoV-2 infection in thyroid follicular cells from a COVID-19 autopsy series

European thyroid journal

2022 Aug 01

Macedo, S;Pestana, A;Santos, L;Neves, C;Guimarães, S;Duarte-Neto, A;Dolhnikoff, M;Saldiva, P;Alves, G;Oliveira, R;Cabanes, D;Carneiro, F;Sobrinho-Simões, M;Soares, P;
PMID: 35900859 | DOI: 10.1530/ETJ-22-0074

To understand whether thyroid cells can be directly infected by the SARS-CoV-2 virus and to establish a putative correlation with the expression of the host entry machinery: ACE-2, TMPRSS2, and furin.We assessed the presence of SARS-CoV-2 virus at the gene level by RT-PCR, viral RNA transcripts localization by in situ hybridization, and by detecting viral proteins by immunohistochemistry for the nucleocapsid and the spike proteins. Furthermore, we also described the immunoexpression of key host factors for virus entry in the COVID-19 thyroid samples.We performed RT-PCR for SARS-CoV-2 in all autopsy specimens and detected viral genome positivity in 13 of 15 thyroid tissues and in a lung specimen. In 9 of the 14 positive samples, we were also able to confirm SARS-CoV-2 signal by in situ hybridization. Immunohistochemistry for the viral nucleocapsid and spike protein was also positive for ten and nine of the RT-PCR-positive cases, respectively, but revealed a lower sensitivity. We also described, for the first time in a COVID-19 series, the immunohistochemical expression of ACE-2, TMPRSS2, and furin in the thyroid.Our results obtained in thyroid specimens from deceased COVID-19 patients indicate that thyrocytes can be directly infected by SARS-CoV-2 since we detected the presence of SARS-CoV-2 genome in follicular cells. Nevertheless, we did not find a clear correlation between the presence of viral genome and the expression of the host factors for virus entry, namely ACE-2, TMPRSS2, and furin.

The amplification of CNS damage in Alzheimer's disease due to SARS-CoV2 infection

Annals of Diagnostic Pathology

2022 Dec 01

Nuovo, GJ;Suster, D;Sawant, D;Mishra, A;Michaille, JJ;Tili, E;
| DOI: 10.1016/j.anndiagpath.2022.152057

Pre-existing Alzheimer's disease is a risk factor for severe/fatal COVID-19 and infection by SARS-CoV2 virus has been associated with an increased incidence of un-masked Alzheimer's disease. The molecular basis whereby SARS-CoV2 may amplify Alzheimer's disease is not well understood. This study analyzed the molecular changes in autopsy brain tissues from people with pre-existing dementia who died of COVID-19 (n = 5) which was compared to equivalent tissues of people who died of COVID-19 with no history of dementia (n = 8), Alzheimer's disease pre-COVID-19 (n = 10) and aged matched controls (n = 10) in a blinded fashion. Immunohistochemistry analyses for hyperphosphorylated tau protein, α-synuclein, and β-amyloid-42 confirmed the diagnoses of Alzheimer's disease (n = 4), and Lewy body dementia (n = 1) in the COVID-19 group. The brain tissues from patients who died of COVID-19 with no history of dementia showed a diffuse microangiopathy marked by endocytosis of spike subunit S1 and S2 in primarily CD31+ endothelia with strong co-localization with ACE2, Caspase-3, IL6, TNFα, and Complement component 6 that was not associated with SARS-CoV2 RNA. Microglial activation marked by increased TMEM119 and MCP1 protein expression closely paralleled the endocytosed spike protein. The COVID-19 tissues from people with no pre-existing dementia showed, compared to controls, 5-10× fold increases in expression of neuronal NOS and NMDAR2 as well as a marked decrease in the expression of proteins whose loss is associated with worsening Alzheimer's disease: MFSD2a, SHIP1, BCL6, BCL10, and BACH1. In COVID-19 tissues from people with dementia the widespread spike-induced microencephalitis with the concomitant microglial activation co-existed in the same areas where neurons had hyperphosphorylated tau protein suggesting that the already dysfunctional neurons were additionally stressed by the SARS-CoV2 induced microangiopathy. ACE2+ human brain endothelial cells treated with high dose (but not vaccine equivalent low dose) spike S1 protein demonstrated each of the molecular changes noted in the in vivo COVID-19 and COVID-19/Alzheimer's disease brain tissues. It is concluded that fatal COVID-19 induces a diffuse microencephalitis and microglial activation in the brain due to endocytosis of circulating viral spike protein that amplifies pre-existing dementia in at least two ways: 1) modulates the expression of proteins that may worsen Alzheimer's disease and 2) stresses the already dysfunctional neurons by causing an acute proinflammatory/hypercoagulable/hypoxic microenvironment in areas with abundant hyperphosphorylated tau protein and/or βA-42.

In SARS-CoV-2 delta variants, Spike-P681R and D950N promote membrane fusion, Spike-P681R enhances spike cleavage, but neither substitution affects pathogenicity in hamsters

EBioMedicine

2023 Apr 10

Furusawa, Y;Kiso, M;Iida, S;Uraki, R;Hirata, Y;Imai, M;Suzuki, T;Yamayoshi, S;Kawaoka, Y;
PMID: 37043872 | DOI: 10.1016/j.ebiom.2023.104561

The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized.We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics in vitro and in vivo.S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3 cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected.Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity.A full list of funding bodies that contributed to this study can be found under Acknowledgments.

Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice

Nature communications

2023 Apr 21

Prompetchara, E;Ketloy, C;Alameh, MG;Tharakhet, K;Kaewpang, P;Yostrerat, N;Pitakpolrat, P;Buranapraditkun, S;Manopwisedjaroen, S;Thitithanyanont, A;Jongkaewwattana, A;Hunsawong, T;Im-Erbsin, R;Reed, M;Wijagkanalan, W;Patarakul, K;Techawiwattanaboon, T;Palaga, T;Lam, K;Heyes, J;Weissman, D;Ruxrungtham, K;
PMID: 37085495 | DOI: 10.1038/s41467-023-37795-0

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 μg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

Syrian hamster convalescence from prototype SARS-CoV-2 confers measurable protection against the attenuated disease caused by the Omicron variant

PLoS pathogens

2023 Apr 01

Ryan, KA;Bewley, KR;Watson, RJ;Burton, C;Carnell, O;Cavell, BE;Challis, A;Coombes, NS;Davies, ER;Edun-Huges, J;Emery, K;Fell, R;Fotheringham, SA;Gooch, KE;Gowan, K;Handley, A;Harris, DJ;Hesp, R;Hunter, L;Humphreys, R;Johnson, R;Kennard, C;Knott, D;Lister, S;Morley, D;Ngabo, D;Osman, KL;Paterson, J;Penn, EJ;Pullan, ST;Richards, KS;Summers, S;Thomas, SR;Weldon, T;Wiblin, NR;Rayner, EL;Vipond, RT;Hallis, B;Salguero, FJ;Funnell, SGP;Hall, Y;
PMID: 37014911 | DOI: 10.1371/journal.ppat.1011293

The mutation profile of the SARS-CoV-2 Omicron (lineage BA.1) variant posed a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2 ancestral isolate (Australia/VIC01/2020, VIC01) to protect against disease caused by BA.1. We established that BA.1 infection in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of the ancestral virus, with fewer clinical signs including less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of BA.1 50 days after an initial infection with ancestral virus. These data provide evidence that convalescent immunity against ancestral SARS-CoV-2 is protective against BA.1 in the Syrian hamster model of infection. Comparison with published pre-clinical and clinical data supports consistency of the model and its predictive value for the outcome in humans. Further, the ability to detect protection against the less severe disease caused by BA.1 demonstrates continued value of the Syrian hamster model for evaluation of BA.1-specific countermeasures.

SARS-CoV-2 Disease Severity in the Golden Syrian Hamster Model of Infection is Related to the Volume of Intranasal Inoculum.

preprints

2023 Feb 10

Handley, A;Ryan, K;Davies, E;Bewley, K;Carnell, O;Challis, A;Coombes, N;Fotheringham, S;Gooch, K;Charlton, M;Harris, D;Kennard, C;Ngabo, D;Weldon, T;Salguero, F;Funnell, S;Hall, Y;
| DOI: 10.20944/preprints202302.0171.v1

The Golden Syrian hamster (Mesocricetus auratus) is now commonly used in preclinical research for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the assessment of vaccines, drugs and therapeutics. Here we show that hamsters inoculated via the intranasal route with the same infectious virus dose of prototypical SARS-CoV-2 administered in a different volume present with different clinical signs, weight loss and viral shedding, with a reduced volume resulting in reduced severity of disease similar to that obtained by a 500-fold reduction in challenge dose. The tissue burden of virus and the severity of pulmonary pathology were also significantly affected by different challenge inoculum volumes. These findings suggest that direct comparison between the severity of SARS-CoV-2 variants or studies assessing the efficacy of treatments determined by hamster studies cannot be made unless both the challenge dose and inoculation volume are matched when using the intranasal route. Additionally, analysis of sub-genomic and total genomic RNA PCR data demonstrated no link between sub-genomic and live viral titres and that sub-genomic analyses do not provide any information beyond that provided by more sensitive total genomic PCR.

Pathological Features and Neuroinflammatory Mechanisms of SARS-CoV-2 in the Brain and Potential Therapeutic Approaches

Biomolecules

2022 Jul 11

Sodagar, A;Javed, R;Tahir, H;Razak, SIA;Shakir, M;Naeem, M;Yusof, AHA;Sagadevan, S;Hazafa, A;Uddin, J;Khan, A;Al-Harrasi, A;
PMID: 35883527 | DOI: 10.3390/biom12070971

The number of deaths has been increased due to COVID-19 infections and uncertain neurological complications associated with the central nervous system. Post-infections and neurological manifestations in neuronal tissues caused by COVID-19 are still unknown and there is a need to explore how brainstorming promoted congenital impairment, dementia, and Alzheimer's disease. SARS-CoV-2 neuro-invasion studies in vivo are still rare, despite the fact that other beta-coronaviruses have shown similar properties. Neural (olfactory or vagal) and hematogenous (crossing the blood-brain barrier) pathways have been hypothesized in light of new evidence showing the existence of SARS-CoV-2 host cell entry receptors into the specific components of human nerve and vascular tissue. Spike proteins are the primary key and structural component of the COVID-19 that promotes the infection into brain cells. Neurological manifestations and serious neurodegeneration occur through the binding of spike proteins to ACE2 receptor. The emerging evidence reported that, due to the high rate in the immediate wake of viral infection, the olfactory bulb, thalamus, and brain stem are intensely infected through a trans-synaptic transfer of the virus. It also instructs the release of chemokines, cytokines, and inflammatory signals immensely to the blood-brain barrier and infects the astrocytes, which causes neuroinflammation and neuron death; and this induction of excessive inflammation and immune response developed in more neurodegeneration complications. The present review revealed the pathophysiological effects, molecular, and cellular mechanisms of possible entry routes into the brain, pathogenicity of autoantibodies and emerging immunotherapies against COVID-19.

OVX033, a nucleocapsid-based vaccine candidate, provides broad-spectrum protection against SARS-CoV-2 variants in a hamster challenge model

Frontiers in Immunology

2023 Jun 19

Primard, C;Monchâtre-Leroy, E;Del Campo, J;Valsesia, S;Nikly, E;Chevandier, M;Boué, F;Servat, A;Wasniewski, M;Picard-Meyer, E;Courant, T;Collin, N;Salguero, F;Le Vert, A;Guyon-Gellin, D;Nicolas, F;
| DOI: 10.3389/fimmu.2023.1188605

Spike-based COVID-19 vaccines induce potent neutralizing antibodies but their efficacy against SARS-CoV-2 variants decreases. OVX033 is a recombinant protein composed of the full-length nucleocapsid (N) protein of SARS-CoV-2 genetically fused to oligoDOM , a self-assembling domain which improves antigen immunogenicity. OVX033 including N as an antigenic target is proposed as new vaccine candidate providing broad-spectrum protection against sarbecoviruses. OVX033 demonstrated its ability to trigger cross-reactive T cell responses and cross-protection against three variants of SARS-CoV-2 (B.1 Europe, Delta B.1.617.2, and Omicron B.1.1.529) in a hamster challenge model, as evidenced by lower weight loss, lower lung viral loads, and reduced lung histopathological lesions.

Coronary Stent Abscess in the Setting of Arteriovenous Graft Infection following COVID-19: An Autopsy Case Report

Case reports in pathology

2023 May 03

Butler, JT;Chellappan, R;Litovsky, S;Leal, SM;Benson, PV;
PMID: 37180570 | DOI: 10.1155/2023/9998749

While rare, coronary stent infections present with significant mortality-with most infections and further complications occurring within months of percutaneous coronary intervention (PCI). Here, we discuss a post-COVID-19 patient who presented approximately one year after PCI for declotting of an arteriovenous graft (AVG). Upon admission, the patient was found to be bacteremic with multilobar pneumonia and an infection of the AVG. Empiric antibiotics were started, and blood cultures were subsequently positive for MRSA. Removal of the AVG was unsuccessful, and two days after admission, the patient passed. Autopsy revealed a perivascular abscess in the RCA near the origin of the stent with a ground section of the RCA with stent revealing abundant calcific atherosclerosis and marked necrosis of the artery wall. The cause of death was determined to be sepsis complicating coronary artery disease and chronic renal failure.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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