Viral Replication in the Nasopharynx Is Associated with Diarrhea in Patients with Severe Acute Respiratory Syndrome

The global spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes COVID-19 has become a source of grave medical and socioeconomic concern to human society. Since its first appearance in the Wuhan region of China in December 2019, the most effective measures of managing the spread of SARS-CoV-2 infection have been social distancing and lockdown of human activity; the level of which has not been seen in our generations. Effective control of the viral infection and COVID-19 will ultimately depend on the development of either a vaccine or therapeutic agents. This article highlights the progresses made so far in these strategies by assessing key targets associated with the viral replication cycle. The key viral proteins and enzymes that could be targeted by new and repurposed drugs are discussed.

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Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22063163 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3163

Author(s):

Hirofumi Ohashi ◽

Feng Wang ◽

Frank Stappenbeck ◽

Kana Tsuchimoto ◽

Chisa Kobayashi ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Cultured Cells ◽

Peak Plasma ◽

Plasma Concentrations ◽

Membrane Vesicles ◽

Drug Candidates ◽

Increased Risk ◽

Derivatives Of

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

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Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines

Journal of General Virology ◽

10.1099/vir.0.2008/001891-0 ◽

2008 ◽

Vol 89 (9) ◽

pp. 2136-2146 ◽

Cited By ~ 40

Author(s):

Raymond H. See ◽

Martin Petric ◽

David J. Lawrence ◽

Catherine P. Y. Mok ◽

Thomas Rowe ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Respiratory Tract ◽

Viral Replication ◽

Lower Respiratory Tract ◽

Clinical Signs ◽

Neutralizing Antibody ◽

Lung Damage ◽

Upper Respiratory Tract ◽

Administration Routes ◽

Antibody Titres

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage.

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Possible Role of Accessory Proteins in the Viral Replication for the 20I/501Y.V1 (B.1.1.7) SARS CoV-2 Variant

Pathogens ◽

10.3390/pathogens10121586 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1586

Author(s):

Dimpal A. Nyayanit ◽

Prasad Sarkale ◽

Anita Shete-Aich ◽

Abhinendra Kumar ◽

Savita Patil ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Experimental Evidence ◽

Copy Number ◽

Transcriptional Response ◽

Accessory Proteins ◽

Maturation Time ◽

Transcriptional Pattern ◽

Global Concern

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) has been a global concern. The B.1.1.7 variant of SARS CoV-2 is reported to cause higher transmission. The study investigates the replication cycle and transcriptional pattern of the B.1.1.7 to hypothesis the possible role of different genes in viral replication. It was observed that the B.1.1.7 variant required a longer maturation time. The transcriptional response demonstrated higher expression of ORF6 and ORF8 compared to nucleocapsid transcript till the eclipse period which might influence higher viral replication. The number of infectious viruses titer is higher in the B.1.1.7, despite a lesser copy number than B.1, indicating higher transmissibility. The experimental evidence published linked ORF6 and ORF8 to play important role in replication and we also observed their higher expression. This leads us to hypothesis the possible role of ORF6 and ORF8 in B.1.1.7 higher replication which causes higher transmission.

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Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro

10.1101/2021.01.31.429001 ◽

2021 ◽

Author(s):

Hirofumi Ohashi ◽

Feng Wang ◽

Frank Stappenbeck ◽

Kana Tsuchimoto ◽

Chisa Kobayashi ◽

...

Keyword(s):

Cell Culture ◽

Severe Acute Respiratory Syndrome ◽

Antiviral Activity ◽

Viral Replication ◽

Cultured Cells ◽

Plasma Concentrations ◽

Hepatitis D ◽

Infection Assay ◽

Naturally Occurring ◽

Increased Risk

AbstractDevelopment of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

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The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication

Journal of Virology ◽

10.1128/jvi.79.21.13399-13411.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13399-13411 ◽

Cited By ~ 111

Author(s):

Rachel L. Graham ◽

Amy C. Sims ◽

Sarah M. Brockway ◽

Ralph S. Baric ◽

Mark R. Denison

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Cleavage Site ◽

Rna Synthesis ◽

Hepatitis Virus ◽

Nonstructural Proteins ◽

Murine Hepatitis Virus ◽

Retroviral Transduction ◽

Coding Sequence ◽

And Function

ABSTRACT The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Δnsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVΔnsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVΔnsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.

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Methyltransferase-like 3 modulates severe acute respiratory syndrome coronavirus-2 RNA N6-methyladenosine modification and replication

10.1101/2020.10.14.338558 ◽

2020 ◽

Author(s):

Xueyan Zhang ◽

Haojie Hao ◽

Li Ma ◽

Yecheng Zhang ◽

Xiao Hu ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Viral Proteins ◽

Rna Modification ◽

Hairpin Rna ◽

Unknown Mechanism ◽

Public Crisis ◽

Rna Immunoprecipitation ◽

Short Hairpin ◽

Plasmid Transfection

ABSTRACTThe coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an ongoing global public crisis. Although viral RNA modification has been reported based on the transcriptome architecture, the types and functions of RNA modification are still unknown. In this study, we evaluated the roles of RNA N6-methyladenosine (m6A) modification in SARS-CoV-2. Our methylated RNA immunoprecipitation sequencing (MeRIP-Seq) analysis showed that SARS-CoV-2 RNA contained m6A modification. Moreover, SARS-CoV-2 infection not only increased the expression of methyltransferase-like 3 (METTL3) but also altered its distribution. Modification of METTL3 expression by short hairpin RNA or plasmid transfection for knockdown or overexpression, respectively, affected viral replication. Furthermore, the viral key protein RdRp interacted with METTL3, and METTL3 was distributed in both the nucleus and cytoplasm in the presence of RdRp. RdRp appeared to modulate the sumoylation and ubiquitination of METTL3 via an unknown mechanism. Taken together, our findings demonstrated that the host m6A modification complex interacted with viral proteins to modulate SARS-CoV-2 replication.

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Glycogen Synthase Kinase-3 Regulates the Phosphorylation of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein and Viral Replication

Journal of Biological Chemistry ◽

10.1074/jbc.m805747200 ◽

2008 ◽

Vol 284 (8) ◽

pp. 5229-5239 ◽

Cited By ~ 72

Author(s):

Chia-Hsin Wu ◽

Shiou-Hwei Yeh ◽

Yeou-Guang Tsay ◽

Ya-Hsiung Shieh ◽

Chuan-Liang Kao ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Nucleocapsid Protein ◽

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3

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Genomic Analysis Reveals Age-Dependent Innate Immune Responses to Severe Acute Respiratory Syndrome Coronavirus

Journal of Virology ◽

10.1128/jvi.00489-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9465-9476 ◽

Cited By ~ 38

Author(s):

Tracey Baas ◽

Anjeanette Roberts ◽

Thomas H. Teal ◽

Leatrice Vogel ◽

Jun Chen ◽

...

Keyword(s):

Gene Expression ◽

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Host Response ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Genomic Analysis ◽

Host Responses ◽

Aged Mice ◽

Young Mice

ABSTRACT The relationship between immunosenescence and the host response to virus infection is poorly understood at the molecular level. Two different patterns of pulmonary host responses to virus were observed when gene expression profiles from severe acute respiratory syndrome coronavirus (SARS-CoV)-infected young mice that show minimal disease were compared to those from SARS-CoV-infected aged mice that develop pneumonitis. In young mice, genes related to cellular development, cell growth, and cell cycle were downregulated during peak viral replication, and these transcripts returned to basal levels as virus was cleared. In contrast, aged mice had a greater number of upregulated immune response and cell-to-cell signaling genes, and the expression of many genes was sustained even after viral clearance, suggesting an exacerbated host response to virus. Interestingly, in SARS-CoV-infected aged mice, a subset of genes, including Tnfa, Il6, Ccl2, Ccl3, Cxcl10, and Ifng, was induced in a biphasic pattern that correlated with peak viral replication and a subsequent influx of lymphocytes and severe histopathologic changes in the lungs. We provide insight into gene expression profiles and molecular signatures underlying immunosenescence in the context of the host response to viral infection.

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Elevation in sphingolipid upon SARS-CoV-2 infection: possible implications for COVID-19 pathology

Life Science Alliance ◽

10.26508/lsa.202101168 ◽

2021 ◽

Vol 5 (1) ◽

pp. e202101168

Author(s):

Einat B Vitner ◽

Roy Avraham ◽

Boaz Politi ◽

Sharon Melamed ◽

Tomer Israely

Keyword(s):

Disease Management ◽

Severe Acute Respiratory Syndrome ◽

Viral Replication ◽

Murine Model ◽

Glucosylceramide Synthase ◽

Disease Outcomes ◽

Therapeutic Development

Understanding pathways that might impact coronavirus disease 2019 (COVID-19) manifestations and disease outcomes is necessary for better disease management and for therapeutic development. Here, we analyzed alterations in sphingolipid (SL) levels upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection induced elevation of SL levels in both cells and sera of infected mice. A significant increase in glycosphingolipid levels was induced early post SARS-CoV-2 infection, which was essential for viral replication. This elevation could be reversed by treatment with glucosylceramide synthase inhibitors. Levels of sphinganine, sphingosine, GA1, and GM3 were significantly increased in both cells and the murine model upon SARS-CoV-2 infection. The potential involvement of SLs in COVID-19 pathology is discussed.

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