scholarly journals Small-Molecule Antiviral β-D-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Maria L. Agostini ◽  
Andrea J. Pruijssers ◽  
James D. Chappell ◽  
Jennifer Gribble ◽  
Xiaotao Lu ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Severe Disease ◽  
Nucleoside Analogues ◽  
Single Infection ◽  
Human Populations ◽  
Genetic Barrier ◽  
Low Level ◽  
Data Support ◽  
Animal Reservoirs ◽  
Mutagenic Mechanism

ABSTRACT Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3′-5′ exoribonuclease (ExoN). β-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 μM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication. IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

scholarly journals Psoriasiform Dermatitis Susceptibility in Itgb2tm1Bay PL/J Mice Requires Low-Level CD18 Expression and at Least Two Additional Loci for Progression to Severe Disease

2003 ◽  
Vol 163 (1) ◽  
pp. 197-202 ◽  
Author(s):  
Shayne C. Barlow ◽  
Robert G. Collins ◽  
Nigel J. Ball ◽  
Casey T. Weaver ◽  
Trenton R. Schoeb ◽  
...  
Keyword(s):  
Severe Disease ◽  
Low Level

scholarly journals Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Kevin W. Graepel ◽  
Xiaotao Lu ◽  
James Brett Case ◽  
Nicole R. Sexton ◽  
Everett Clinton Smith ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Hepatitis Virus ◽  
Nucleoside Analogues ◽  
High Fidelity ◽  
Genome Replication ◽  
Murine Hepatitis Virus ◽  
Replicase Proteins ◽  
Rna Genome ◽  
Inactivating Mutations

ABSTRACT The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

scholarly journals The coronavirus proofreading exoribonuclease mediates extensive viral recombination

2021 ◽  
Vol 17 (1) ◽  
pp. e1009226
Author(s):  
Jennifer Gribble ◽  
Laura J. Stevens ◽  
Maria L. Agostini ◽  
Jordan Anderson-Daniels ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Rna Synthesis ◽  
Recombination Frequency ◽  
Hepatitis Virus ◽  
Nucleoside Analogues ◽  
High Fidelity ◽  
Rna Recombination ◽  
Murine Hepatitis Virus ◽  
Critical Function ◽  
Viral Recombination

Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.

scholarly journals Pulmonary insults exacerbate susceptibility to oral Listeria monocytogenes infection through the production of IL-10 by NK cells

2021 ◽  
Vol 17 (4) ◽  
pp. e1009531
Author(s):  
Nikki Bortell ◽  
Elizabeth R. Aguilera ◽  
Laurel L. Lenz
Keyword(s):  
Listeria Monocytogenes ◽  
Nk Cells ◽  
Severe Disease ◽  
Bacterial Pathogen ◽  
Myeloid Cell ◽  
The Elderly ◽  
Interleukin 10 ◽  
Human Populations ◽  
Phagocytic Capacity ◽  
Increase Disease Susceptibility

Most individuals who consume foods contaminated with the bacterial pathogen Listeria monocytogenes (Lm) develop mild symptoms, while others are susceptible to life-threatening systemic infections (listeriosis). Although it is known that the risk of severe disease is increased in certain human populations, including the elderly, it remains unclear why others who consume contaminated food develop listeriosis. Here, we used a murine model to discover that pulmonary coinfections can impair the host’s ability to adequately control and eradicate systemic Lm that cross from the intestines to the bloodstream. We found that the resistance of mice to oral Lm infection was dramatically reduced by coinfection with Streptococcus pneumoniae (Spn), a bacterium that colonizes the respiratory tract and can also cause severe infections in the elderly. Exposure to Spn or microbial products, including a recombinant Lm protein (L1S) and lipopolysaccharide (LPS), rendered otherwise resistant hosts susceptible to severe systemic Lm infection. In addition, we show that this increase in susceptibility was dependent on an increase in the production of interleukin-10 (IL-10) from Ncr1+ cells, including Natural Killer (NK) cells. Lastly, the ability of Ncr1+ derived IL-10 to increase disease susceptibility correlated with a dampening of both myeloid cell accumulation and myeloid cell phagocytic capacity in infected tissues. These data suggest that efforts to minimize inflammation in response to an insult at the respiratory mucosa render the host more susceptible to infections by Lm and possibly other pathogens that access the oral mucosa.

scholarly journals Prevalence of Co-Infections With Respiratory Viruses in Individuals Investigated for Sars-Cov-2 in Ontario, Canada

2020 ◽  
Author(s):  
Adriana Peci ◽  
Vanessa Tran ◽  
Jennifer L Gurtherie ◽  
Ye Li ◽  
Paul Nelson ◽  
...  
Keyword(s):  
Respiratory Symptoms ◽  
Patient Management ◽  
Respiratory Virus ◽  
Severe Disease ◽  
Respiratory Viruses ◽  
Single Infection ◽  
Clinical Testing ◽  
Routine Testing ◽  
Wide Range ◽  
Bacteria And Fungi

Background: Co-infections of SARS-CoV-2 with respiratory viruses, bacteria and fungi have been reported to cause a wide range of illness. Objectives: We asses s the prevalence of co-infection of SARS-CoV-2 with seasonal respiratory viruses, document the respiratory viruses detected among individuals tested for SARS-CoV-2, and describe characteristics of individuals with respiratory virus co-infection detected. Methods: Specimens included in this study were submitted as part of routine clinical testing to Public Health Ontario Laboratory from individuals requiring testing for SARS-CoV-2 and/or seasonal respiratory viruses. Results: Co-infection was detected in a smaller proportion (2.5%) of individuals with laboratory confirmed SARS-CoV-2 than those with seasonal respiratory viruses (4.3%); this difference was not significant. Individuals with any respiratory virus co-infection were more likely to be younger than 65 years of age and male than those with single infection. Those with SARS-CoV-2 co-infection manifested mostly mild respiratory symptoms. Conclusions: Findings of this study may not support routine testing for seasonal respiratory viruses among all individuals tested for SARS-CoV-2, as they were rare during the study period nor associated with severe disease. However, testing for seasonal respiratory viruses should be performed in severely ill individuals, in which detection of other viruses may assist with patient management.

scholarly journals Surveillance of endemic human coronaviruses (HCoV-NL63, OC43 and 229E) associated with pneumonia in Kilifi, Kenya

2020 ◽  
Vol 5 ◽  
pp. 150
Author(s):  
Grieven P. Otieno ◽  
Nickson Murunga ◽  
Charles N. Agoti ◽  
Katherine E. Gallagher ◽  
Juliet O. Awori ◽  
...  
Keyword(s):  
Respiratory Virus ◽  
Seasonal Pattern ◽  
Severe Disease ◽  
Age Distribution ◽  
Coastal Region ◽  
Severe Pneumonia ◽  
Human Populations ◽  
Surveillance Period ◽  
Human Coronaviruses ◽  
Epidemiological Characteristics

Introduction: Human coronaviruses (HCoVs) circulate endemically in human populations, often with seasonal variation. We describe the long-term patterns of paediatric disease associated with three of these viruses, HCoV-NL63, OC43 and 229E, in coastal Kenya. Methods: Continuous surveillance of pneumonia admissions was conducted at the Kilifi county hospital (KCH) located in the northern coastal region of Kenya. Children aged <5 years admitted to KCH with clinically defined syndromic severe or very severe pneumonia were recruited. Respiratory samples were taken and tested for 15 virus targets, using real-time polymerase chain reaction. Unadjusted odds ratios were used to estimate the association between demographic and clinical characteristics and HCoV positivity. Results: From 2007 to 2019, we observed 11,445 pneumonia admissions, of which 314 (3.9%) tested positive for at least one HCoV type. There were 129 (41.1%) OC43, 99 (31.5%) 229E, 74 (23.6%) NL63 positive cases and 12 (3.8%) cases of HCoV to HCoV coinfection.  Among HCoV positive cases, 47% (n=147) were coinfected with other respiratory virus pathogens. The majority of HCoV cases were among children aged <1 year (66%, n=208), though there was no age-dependence in the proportion testing positive. HCoV-OC43 was predominant of the three HCoV types throughout the surveillance period. Evidence for seasonality was not identified. Conclusions: Overall, 4% of paediatric pneumonia admissions were associated with three endemic HCoVs, with a high proportion of cases co-occurring with another respiratory virus, with no clear seasonal pattern, and with the age-distribution of cases following that of pneumonia admissions (i.e. highest in infants). These observations suggest, at most, a small severe disease contribution of endemic HCoVs in this tropical setting and offer insight into the potential future burden and epidemiological characteristics of SARS-CoV-2.

scholarly journals Potential Health and Performance Effects of High-Level and Low-Level Blast: A Scoping Review of Two Decades of Research

2021 ◽  
Vol 12 ◽  
Author(s):  
Jennifer N. Belding ◽  
Robyn M. Englert ◽  
Shannon Fitzmaurice ◽  
Jourdan R. Jackson ◽  
Hannah G. Koenig ◽  
...  
Keyword(s):  
Scoping Review ◽  
Animal Studies ◽  
Human Subjects ◽  
Animal Health ◽  
Medical Specialty ◽  
Future Research ◽  
Human Populations ◽  
Potential Health ◽  
Low Level ◽  
Blast Exposure

Although blast exposure has been recognized as a significant source of morbidity and mortality in military populations, our understanding of the effects of blast exposure, particularly low-level blast (LLB) exposure, on health outcomes remains limited. This scoping review provides a comprehensive, accessible review of the peer-reviewed literature that has been published on blast exposure over the past two decades, with specific emphasis on LLB. We conducted a comprehensive scoping review of the scientific literature published between January 2000 and 2019 pertaining to the effects of blast injury and/or exposure on human and animal health. A three-level review process with specific inclusion and exclusion criteria was used. A full-text review of all articles pertaining to LLB exposure was conducted and relevant study characteristics were extracted. The research team identified 3,215 blast-relevant articles, approximately half of which (55.4%) studied live humans, 16% studied animals, and the remainder were non-subjects research (e.g., literature reviews). Nearly all (99.49%) of the included studies were conducted by experts in medicine or epidemiology; approximately half of these articles were categorized into more than one medical specialty. Among the 51 articles identified as pertaining to LLB specifically, 45.1% were conducted on animals and 39.2% focused on human subjects. Animal studies of LLB predominately used shock tubes to induce various blast exposures in rats, assessed a variety of outcomes, and clearly demonstrated that LLB exposure is associated with brain injury. In contrast, the majority of LLB studies on humans were conducted among military and law enforcement personnel in training environments and had remarkable variability in the exposures and outcomes assessed. While findings suggest that there is the potential for LLB to harm human populations, findings are mixed and more research is needed. Although it is clear that more research is needed on this rapidly growing topic, this review highlights the detrimental effects of LLB on the health of both animals and humans. Future research would benefit from multidisciplinary collaboration, larger sample sizes, and standardization of terminology, exposures, and outcomes.

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