UNC-Chapel Hill Coronavirus Research // Carolina Digital Repository

Works (1029)

1001. Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines

Title Tesim:: Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines
Creator:: Dinnon III, Kenneth H., Baric, Ralph S., McAnarney, Eileen T., Gralinski, Lisa E., Menachery, Vineet D., Graham, Rachel L., Yount Jr., Boyd L., Mitchell, Hugh D., Leist, Sarah R., and Waters, Katrina M.
Date of publication:: 2018
Abstract Tesim:: With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2′O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2′O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2′O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines. IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2′O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2′O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2′O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/xghs-wp15
Identifier:: https://doi.org/10.1128/JVI.00710-18
ISSN:: 0022-538X
Journal Issue:: 17
Journal Title:: Journal of Virology
Journal Volume:: 92
Keyword:: vaccine, aged, coronavirus, live attenuated, SARS-CoV, and MERS-CoV
Language Label:: English
ORCID:
Other Affiliation:: , University of Texas Medical Branch, and Pacific Northwest National Laboratory
Person:: Dinnon III, Kenneth H., Baric, Ralph S., McAnarney, Eileen T., Gralinski, Lisa E., Menachery, Vineet D., Graham, Rachel L., Yount Jr., Boyd L., Mitchell, Hugh D., Leist, Sarah R., and Waters, Katrina M.
Rights Statement Label:: In Copyright
Source:: 1n79h885v

1002. The endonucleolytic RNA cleavage function of nsp1 of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines

Title Tesim:: The endonucleolytic RNA cleavage function of nsp1 of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines
Creator:: Wada, Masami, Makino, Shinji, Cajimat, Maria, Popov, Vsevolod, Narayanan, Krishna, Baric, Ralph S., and Nakagawa, Keisuke
Date of publication:: 2018
Abstract Tesim:: Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN-β and IFN-λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding. IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/4mqh-0k36
Identifier:: https://dx.doi.org/10.1128/JVI.01157-18
ISSN:: 0022-538X
Journal Issue:: 21
Journal Title:: Journal of Virology
Journal Volume:: 92
Keyword:: nsp1, virus assembly/budding, and MERS coronavirus
Language Label:: English
ORCID:
Other Affiliation:: The University of Texas Medical Branch and
Person:: Wada, Masami, Makino, Shinji, Cajimat, Maria, Popov, Vsevolod, Narayanan, Krishna, Baric, Ralph S., and Nakagawa, Keisuke
Rights Statement Label:: In Copyright
Source:: n583z0690

1003. Human coronaviruses and other respiratory infections in young adults on a university campus: Prevalence, symptoms, and shedding

Title Tesim:: Human coronaviruses and other respiratory infections in young adults on a university campus: Prevalence, symptoms, and shedding
Creator:: Martin, Emily T., Uzicanin, Amra, Monto, Arnold S., Foxman, Betsy, Baric, Ralph S., Rainey, Jeanette J., Aiello, Allison E., and Davis, Brian M.
Date of publication:: 2018
Abstract Tesim:: BACKGROUND: The prevalence, symptom course, and shedding in persons infected with the 4 most common human coronaviruses (HCoV)-229E, HKU1, NL63, and OC43 are poorly described. OBJECTIVES: We estimate their prevalence and associated symptoms among college students identified via a social network study design. PATIENTS/METHODS: We collected 1-3 samples (n = 250 specimens) from 176 participants between October 2012 and January 17, 2013: participants with acute respiratory infection (ARI; cough and body aches or chills or fever/feverishness) and their social contacts. Virus was detected using RT-PCR. RESULTS: 30.4% (76/250) of specimens tested positive for any virus tested, and 4.8% (12/250) were positive for 2 or more viruses. Human coronaviruses (HCoVs [22.0%; 55/250]), rhinovirus (7.6%; 19/250), and influenza A (6.4%; 16/250) were most prevalent. Symptoms changed significantly over time among ARI participants with HCoV: the prevalence of cough and chills decreased over 6 days (P = .04, and P = .01, respectively), while runny nose increased over the same period (P = .02). HCoV-NL63 was the most frequent virus detected 6 days following symptom onset (8.9%), followed by rhinovirus (6.7%). CONCLUSIONS: During a 3-month period covering a single season, HCoVs were common, even among social contacts without respiratory symptoms; specific symptoms may change over the course of HCoV-associated illness and were similar to symptoms from influenza and rhinovirus.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/8ydf-8k60
Identifier:: https://doi.org/10.1111/irv.12563
ISSN:: 1750-2640
Journal Issue:: 5
Journal Title:: Influenza and other Respiratory Viruses
Journal Volume:: 12
Keyword:: acute respiratory infection, human, university, influenza, coronavirus, and symptoms
Language Label:: English
ORCID:
Other Affiliation:: University of Michigan School of Public Health, Centers for Disease Control and Prevention, and
Page End:: 590
Page Start:: 582
Person:: Martin, Emily T., Uzicanin, Amra, Monto, Arnold S., Foxman, Betsy, Baric, Ralph S., Rainey, Jeanette J., Aiello, Allison E., and Davis, Brian M.
Rights Statement Label:: In Copyright
Source:: nz8064254

1004. Is regulation preventing the development of therapeutics that may prevent future coronavirus pandemics?

Title Tesim:: Is regulation preventing the development of therapeutics that may prevent future coronavirus pandemics?
Creator:: Baric, Ralph S. and Sheahan, Timothy P.
Date of publication:: 2018
Abstract Tesim:: In the last century, hundreds of new emerging infectious diseases (EIDs) have arisen in human populations most of which originate from wild animals as zoonoses [1]. The recent surge of zoonotic EIDs in human populations is driven by a constellation of socioeconomic factors including human population growth, eroding public health infrastructures, changes in land use and agriculture and ease of global travel. HIV, Ebola virus, avian influenza (H5N1, H7N9, etc.), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are but a few recent examples of highly virulent, zoonotic viral EIDs that have catastrophically affected global economies and public health [2]. Geopolitical flux since the 1990s and the potential for weaponizing EIDs provoked the creation of myriad policies aimed at protecting the USA from bioterrorist threats and the accidental release of potential pandemic pathogens from laboratories. Are these policies effective? Are they impacting countermeasure development for current and future EIDs? How are they shaping the direction of individual research programs, the recruitment of new investigators and the stability of impacted fields? Below, we discuss our experiences in developing therapeutics against SARS-, MERS- and zoonotic CoV in an ever changing regulatory environment.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/4mvw-jw13
Identifier:: https://doi.org/10.2217/fvl-2017-0143
ISSN:: 1746-0794
Journal Issue:: 3
Journal Title:: Future Virology
Journal Volume:: 13
Language Label:: English
ORCID:
Other Affiliation:
Page End:: 146
Page Start:: 143
Person:: Baric, Ralph S. and Sheahan, Timothy P.
Rights Statement Label:: In Copyright
Source:: cr56n5529

1005. Evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform

Title Tesim:: Evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform
Creator:: Deming, Damon J., Graham, Rachel L., Yount, Boyd, Deming, Meagan E., and Baric, Ralph S.
Date of publication:: 2018
Abstract Tesim:: Emerging and re-emerging zoonotic viral diseases are major threats to global health, economic stability, and national security. Vaccines are key for reducing coronaviral disease burden; however, the utility of live-attenuated vaccines is limited by risks of reversion or repair. Because of their history of emergence events due to their prevalence in zoonotic pools, designing live-attenuated coronavirus vaccines that can be rapidly and broadly implemented is essential for outbreak preparedness. Here, we show that coronaviruses with completely rewired transcription regulatory networks (TRNs) are effective vaccines against SARS-CoV. The TRN-rewired viruses are attenuated and protect against lethal SARS-CoV challenge. While a 3-nt rewired TRN reverts via second-site mutation upon serial passage, a 7-nt rewired TRN is more stable, suggesting that a more extensively rewired TRN might be essential for avoiding growth selection. In summary, rewiring the TRN is a feasible strategy for limiting reversion in an effective live-attenuated coronavirus vaccine candidate that is potentially portable across the Nidovirales order.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/7ees-5e49
Identifier:: https://dx.doi.org/10.1038/s42003-018-0175-7
ISSN:: 2399-3642
Journal Issue:: 1
Journal Title:: Communications Biology
Journal Volume:: 1
Language Label:: English
License Label:: Attribution 3.0 United States
ORCID:
Other Affiliation:
Person:: Deming, Damon J., Graham, Rachel L., Yount, Boyd, Deming, Meagan E., and Baric, Ralph S.
Rights Statement Label:: In Copyright
Source:: 9w032764v

1006. Increasing the translation of mouse models of MERS coronavirus pathogenesis through kinetic hematological analysis

Title Tesim:: Increasing the translation of mouse models of MERS coronavirus pathogenesis through kinetic hematological analysis
Creator:: Baric, Ralph S., Sheahan, Timothy P., Jensen, Kara L., and Leist, Sarah R.
Date of publication:: 2019
Abstract Tesim:: Newly emerging viral pathogens pose a constant and unpredictable threat to human and animal health. Coronaviruses (CoVs) have a penchant for sudden emergence, as evidenced by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) and most recently, swine acute diarrhea syndrome coronavirus (SADS-CoV). Small animal models of emerging viral pathogenesis are crucial to better understand the virus and host factors driving disease progression. However, rodent models are often criticized for their limited translatability to humans. The complete blood count is the most ordered clinical test in the United States serving as the cornerstone of clinical medicine and differential diagnosis. We recently generated a mouse model for MERS-CoV pathogenesis through the humanization of the orthologous entry receptor dipeptidyl peptidase 4 (DPP4). To increase the translatability of this model, we validated and established the use of an automated veterinary hematology analyzer (VetScan HM5) at biosafety level 3 for analysis of peripheral blood. MERS-CoV lung titer peaked 2 days post infection concurrent with lymphopenia and neutrophilia in peripheral blood, two phenomena also observed in MERS-CoV infection of humans. The fluctuations in leukocyte populations measured by Vetscan HM5 were corroborated by standard flow cytometry, thus confirming the utility of this approach. Comparing a sublethal and lethal dose of MERS-CoV in mice, analysis of daily blood draws demonstrates a dose dependent modulation of leukocytes. Major leukocyte populations were modulated before weight loss was observed. Importantly, neutrophil counts on 1dpi were predictive of disease severity with a lethal dose of MERS-CoV highlighting the predictive value of hematology in this model. Taken together, the inclusion of hematological measures in mouse models of emerging viral pathogenesis increases their translatability and should elevate the preclinical evaluation of MERS-CoV therapeutics and vaccines to better mirror the complexity of the human condition.
Resource type:: Article
Admin Note:: deposited with permission from Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/0w7a-vt47
Identifier:: https://doi.org/10.1371/journal.pone.0220126
ISSN:: 1932-6203
Journal Issue:: 7
Journal Title:: PLoS ONE
Journal Volume:: 14
Language Label:: English
ORCID:
Other Affiliation:
Person:: Baric, Ralph S., Sheahan, Timothy P., Jensen, Kara L., and Leist, Sarah R.
Rights Statement Label:: In Copyright
Source:: 0r967843x

1007. Precision mouse models with expanded tropism for human pathogens

Title Tesim:: Precision mouse models with expanded tropism for human pathogens
Creator:: Newsome, Isabel G., Aguilera-Sandoval, Christian R., Boone, Allison, Goonetilleke, Nilu, Dayton, Paul A., De, Chandrav, Schramm, Nathaniel J., Vincent, Heather A., Johnson, Claire E., Sanders, Wes, Garcia, J. Victor, Xu, Yinyan, Rank, Laura M., Baric, Ralph S., Cockrell, Adam S., Lenarcic, Erik M., Moorman, Nathaniel J., Hildebrand, William H., Braunstein, Miriam, Fernandez, Maria Abad, Cleary, Rachel A., Pickles, Raymond J., and Wahl, Angela
Date of publication:: 2019
Abstract Tesim:: A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Joint Department of Biomedical Engineering, Center for AIDS Research, Department of Microbiology and Immunology, and Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/e4bv-1405
Identifier:: https://www.nature.com/articles/s41587-019-0225-9
ISSN:: 1087-0156
Journal Issue:: 10
Journal Title:: Nature Biotechnology
Journal Volume:: 37
Language Label:: English
ORCID:
Other Affiliation:: and University of Oklahoma Health Sciences Institute
Page End:: 1173
Page Start:: 1163
Person:: Newsome, Isabel G., Aguilera-Sandoval, Christian R., Boone, Allison, Goonetilleke, Nilu, Dayton, Paul A., De, Chandrav, Schramm, Nathaniel J., Vincent, Heather A., Johnson, Claire E., Sanders, Wes, Garcia, J. Victor, Xu, Yinyan, Rank, Laura M., Baric, Ralph S., Cockrell, Adam S., Lenarcic, Erik M., Moorman, Nathaniel J., Hildebrand, William H., Braunstein, Miriam, Fernandez, Maria Abad, Cleary, Rachel A., Pickles, Raymond J., and Wahl, Angela
Rights Statement Label:: In Copyright
Source:: s4655n668

1008. Antagonism of dsRNA-induced innate immune pathways by NS4a and NS4b accessory proteins during MERS coronavirus infection

Title Tesim:: Antagonism of dsRNA-induced innate immune pathways by NS4a and NS4b accessory proteins during MERS coronavirus infection
Creator:: Comar, Courtney E., Li, Yize, Goldstein, Stephen A., Weiss, Susan R., Baric, Ralph S., and Yount, Boyd
Date of publication:: 2019
Abstract Tesim:: Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in 2012 as a novel etiological agent of severe respiratory disease in humans. As during infection by other viruses, host sensing of viral double-stranded RNA (dsRNA) induces several antiviral pathways. These include interferon (IFN), oligoadenylate synthetase (OAS)-RNase L, and protein kinase R (PKR). Coronaviruses, including MERS-CoV, potently suppress the activation of these pathways, inducing only modest host responses. Our study describes the functions of two accessory proteins unique to MERS-CoV and related viruses, NS4a and NS4b, during infection in human airway epithelium-derived A549 cells. NS4a has been previously characterized as a dsRNA binding protein, while NS4b is a 2′,5′-phosphodiesterase with structural and enzymatic similarity to NS2 encoded by mouse hepatitis virus (MHV). We found that deletion of NS4a results in increased interferon lambda (IFNL1) expression, as does mutation of either the catalytic site or nuclear localization sequence of NS4b. All of the mutant viruses we tested exhibited slight decreases in replication. We previously reported that, like MHV NS2, NS4b antagonizes OAS-RNase L, but suppression of IFN is a previously unidentified function for viral phosphodiesterases. Unexpectedly, deletion of NS4a does not result in robust activation of the PKR or OAS-RNase L pathways. Therefore, MERS-CoV likely encodes other proteins that contribute to suppression or evasion of these antiviral innate immune pathways that should be an important focus of future work. This study provides additional insight into the complex interactions between MERS-CoV and the host immune response. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is the second novel zoonotic coronavirus to emerge in the 21st century and cause outbreaks of severe respiratory disease. More than 2,200 cases and 800 deaths have been reported to date, yet there are no licensed vaccines or treatments. Coronaviruses encode unique accessory proteins that are not required for replication but most likely play roles in immune antagonism and/or pathogenesis. Our study describes the functions of MERS-CoV accessory proteins NS4a and NS4b during infection of a human airway-derived cell line. Loss of these accessory proteins during MERS-CoV infection leads to host antiviral activation and modestly attenuates replication. In the case of both NS4a and NS4b, we have identified roles during infection not previously described, yet the lack of robust activation suggests much remains to be learned about the interactions between MERS-CoV and the infected host.
Resource type:: Article
Admin Note:: deposited with permission from Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/zh4n-4a64
Identifier:: https://dx.doi.org/10.1128/mBio.00319-19
ISSN:: 2161-2129
Journal Issue:: 2
Journal Title:: mBio
Journal Volume:: 10
Keyword:: MERS-CoV, coronavirus, interferon antagonist, and viral accessory proteins
Language Label:: English
ORCID:
Other Affiliation:: University of Pennsylvania and
Person:: Comar, Courtney E., Li, Yize, Goldstein, Stephen A., Weiss, Susan R., Baric, Ralph S., and Yount, Boyd
Rights Statement Label:: In Copyright
Source:: 6q182q928

1009. Small-Molecule Antiviral β-d-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance

Title Tesim:: Small-Molecule Antiviral β-d-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance
Creator:: Agostini, Maria L., Pruijssers, Andrea J., Sims, Amy C., Natchus, Michael G., Sheahan, Timothy, Saindane, Manohar, Chappell, James D., Denison, Mark R., Lu, Xiaotao, Kolykhalov, Alexander A., Andres, Erica L., Lockwood, Mark A., Baric, Ralph S., Painter, George, Gribble, Jennifer, and Bluemling, Gregory R.
Date of publication:: 2019
Abstract Tesim:: 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.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Department of Epidemiology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/83ze-tt28
Identifier:: https://dx.doi.org/10.1128/JVI.01348-19
ISSN:: 1098-5514
Journal Issue:: 24
Journal Title:: Journal of Virology
Journal Volume:: 93
Keyword:: RNA-dependent RNA polymerase, pandemic, nucleoside analogue, antiviral resistance, coronavirus, RdRp, MERS-CoV, and SARS-CoV
Language Label:: English
ORCID:
Other Affiliation:: Vanderbilt University School of Medicine, , and Emory University
Person:: Agostini, Maria L., Pruijssers, Andrea J., Sims, Amy C., Natchus, Michael G., Sheahan, Timothy, Saindane, Manohar, Chappell, James D., Denison, Mark R., Lu, Xiaotao, Kolykhalov, Alexander A., Andres, Erica L., Lockwood, Mark A., Baric, Ralph S., Painter, George, Gribble, Jennifer, and Bluemling, Gregory R.
Rights Statement Label:: In Copyright
Source:: z603r309p

1010. The role of EGFR in influenza pathogenicity: Multiple network-based approaches to identify a key regulator of non-lethal infections

Title Tesim:: The role of EGFR in influenza pathogenicity: Multiple network-based approaches to identify a key regulator of non-lethal infections
Creator:: Stratton, Kelly G., Le, Mai Q., Gralinski, Lisa E., Heller, Natalie C., Kawaoka, Yoshihiro, Sims, Amy C., Waters, Katrina, Eisfeld, Amie J., McDermott, Jason E., Baric, Ralph S., Bramer, Lisa M., Wen, Ji, and Mitchell, Hugh D.
Date of publication:: 2019
Abstract Tesim:: Despite high sequence similarity between pandemic and seasonal influenza viruses, there is extreme variation in host pathogenicity from one viral strain to the next. Identifying the underlying mechanisms of variability in pathogenicity is a critical task for understanding influenza virus infection and effective management of highly pathogenic influenza virus disease. We applied a network-based modeling approach to identify critical functions related to influenza virus pathogenicity using large transcriptomic and proteomic datasets from mice infected with six influenza virus strains or mutants. Our analysis revealed two pathogenicity-related gene expression clusters; these results were corroborated by matching proteomics data. We also identified parallel downstream processes that were altered during influenza pathogenesis. We found that network bottlenecks (nodes that bridge different network regions) were highly enriched in pathogenicity-related genes, while network hubs (highly connected network nodes) were significantly depleted in these genes. We confirmed that this trend persisted in a distinct virus: Severe Acute Respiratory Syndrome Coronavirus (SARS). The role of epidermal growth factor receptor (EGFR) in influenza pathogenesis, one of the bottleneck regulators with corroborating signals across transcript and protein expression data, was tested and validated in additional mouse infection experiments. We demonstrate that EGFR is important during influenza infection, but the role it plays changes for lethal versus non-lethal infections. Our results show that by using association networks, bottleneck genes that lack hub characteristics can be used to predict a gene’s involvement in influenza virus pathogenicity. We also demonstrate the utility of employing multiple network approaches for analyzing host response data from viral infections.
Resource type:: Article
Admin Note:: deposited with permission of Ralph Baric
Affiliation Label Tesim:: Department of Microbiology and Immunology
Type:: http://purl.org/dc/dcmitype/Text
DOI:: https://doi.org/10.17615/1736-x458
Identifier:: https://doi.org/10.3389/fcell.2019.00200
ISSN:: 2296-634X
Journal Title:: Frontiers in Cell and Developmental Biology
Journal Volume:: 7
Keyword:: systems biology, SARS-CoV, data integration, network topology, and influenza
Language Label:: English
License Label:: Attribution 3.0 United States
ORCID:
Other Affiliation:: Pacific Northwest National Laboratory, National Institute of Hygiene and Epidemiology, , and University of Wisconsin-Madison
Person:: Stratton, Kelly G., Le, Mai Q., Gralinski, Lisa E., Heller, Natalie C., Kawaoka, Yoshihiro, Sims, Amy C., Waters, Katrina, Eisfeld, Amie J., McDermott, Jason E., Baric, Ralph S., Bramer, Lisa M., Wen, Ji, and Mitchell, Hugh D.
Rights Statement Label:: In Copyright
Source:: sb397d70d

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