Biology subjects

Menachery, V. D.

Publications and source records attributed to Menachery, V. D..

46 records · Page 3Linked to original sources

bioRxiv2020

Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures

The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. The innate immune response is critical for protection against Coronaviruses. However, little is known about the interplay between the innate immune system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNF, CXCL8. We also identified NF-{kappa}B and ATF4 transcription factors as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with type I and III IFNs dramatically reduced virus replication in pHAE cultures and this corresponded with an upregulation of antiviral effector genes. Our findings demonstrate that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients. IMPORTANCEThe current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines or antivirals approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human airway epithelial cultures induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients.

microbiology↗

bioRxiv2020

Topoisomerase III-beta is required for efficient replication of positive-sense RNA viruses

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-{beta} (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

microbiology↗

bioRxiv2020

SARS-CoV-2 sensitive to type I interferon pretreatment.

SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type-I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, while SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures (HAEC), we observe the absence of IFN-I stimulation by SARS-CoV-2 alone, but detect failure to counteract STAT1 phosphorylation upon IFN-I pretreatment resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment post infection and found SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame (ORF) 3b and changes to ORF6 suggest the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development. ImportanceWith the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection. Article SummarySARS-CoV-2 has similar replication kinetics to SARS-CoV, but demonstrates significant sensitivity to type I interferon treatment.

microbiology↗

bioRxiv2020

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures. Article SummaryScientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.

microbiology↗

bioRxiv2020

Mucin 4 Protects Female Mice from Coronavirus Pathogenesis

Using incipient lines of the Collaborative Cross (CC), a murine genetic reference population, we previously identified a quantitative trait loci (QTL) associated with low SARS-CoV titer. In this study, we integrated sequence information and RNA expression of genes within the QTL to identify mucin 4 (Muc4) as a high priority candidate for controlling SARS-CoV titer in the lung. To test this hypothesis, we infected Muc4-/- mice and found that female, but not male, Muc4-/- mice developed more weight loss and disease following infection with SARS-CoV. Female Muc4-/- mice also had more difficulty breathing despite reduced lung pathology; however, no change in viral titers was observed. Comparing across viral families, studies with chikungunya virus, a mosquito-borne arthralgic virus, suggests that Muc4s impact on viral pathogenesis may be widespread. Although not confirming the original titer QTL, our data identifies a role for Muc4 in the SARS-CoV disease and viral pathogenesis. ImportanceGiven the recent emergence of SARS-CoV-2, this work suggest that Muc4 expression plays a protective role in female mice not conserved in male mice following SARS-CoV infection. With the SARS-CoV-2 outbreak continuing, treatments that modulate or enhance Muc4 activity may provide an avenue for treatment and improved outcomes. In addition, the work highlights the importance of studying host factors including host genetics and biological sex as key parameters influencing infection and disease outcomes.

microbiology↗

bioRxiv2020

Novel ionophores active against La Crosse virus identified through rapid antiviral screening

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (Coxsackievirus, rhinovirus), flavirivuses (Zika), and coronaviruses (229E and MERS-CoV). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication. ImportanceNo antivirals are approved for the treatment of bunyavirus infection. The ability to rapidly screen compounds and identify novel antivirals is one means to accelerate drug discovery for viruses with no approved treatments. We used this approach to screen hundreds of compounds against La Crosse virus, an emerging bunyavirus that causes significant disease, including encephalitis. We identified several known and previously unidentified antivirals. We focused on a potassium ionophore, valinomycin, due to its promising in vitro antiviral activity. We demonstrate that valinomycin, as well as a selection of other ionophores, exhibits activity against La Crosse virus as well as several other distantly related bunyaviruses. We finally observe that valinomycin has activity against a wide array of human viral pathogens, suggesting that disrupting potassium ion homeostasis with valinomycin may be a potent host pathway to target to quell virus infection.

microbiology↗

bioRxiv2019

Trypsin treatment unlocks barrier for zoonotic coronaviruses infection

Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with SARS-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two MERS-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate infection of human gut cells, but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that addition of exogenous trypsin also rescues replication of HKU5-CoV, a second MERS-like group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate emergence potential of CoVs and offer a means to recover previously unrecoverable zoonotic CoV strains.\n\nImportanceOverall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans.

microbiology↗

bioRxiv2019

Peptidoglycan associated cyclic lipopeptide disrupts viral infectivity

Enteric viruses exploit bacterial components including lipopolysaccharides (LPS) and peptidoglycan (PG) to facilitate infection in humans. With origins in the bat enteric system, we wondered if severe acute respiratory syndrome-coronavirus (SARS-CoV) or Middle East respiratory syndrome-CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity finding no change following LPS treatment. However, PG from B. subtilis reduced infection >10,000-fold while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity and surfactin treatment of the virus inoculum ablated infection in vivo. Finally, similar cyclic lipopeptides had no effect on CoV infectivity and the inhibitory effect of surfactin extended broadly to enveloped viruses including influenza, Ebola, Zika, Nipah, Chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad virucidal activity and suggest bacteria byproducts may negatively modulate virus infection.\n\nImportanceIn this manuscript, we considered a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

microbiology↗

bioRxiv2018

Combination attenuation offers strategy for live-attenuated coronavirus vaccines

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 2O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the SARS-CoV mouse model, a 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 2O 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 2O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live-attenuated coronavirus vaccines.\n\nSignificanceEmergent 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 like the aged and immune-compromised. This study illustrates that despite success in young models, the NSP16 mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the NSP16 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 NSP16 attenuation as a major component of future live-attenuated coronavirus vaccines.

microbiology↗

bioRxiv2017

MERS-CoV NSP16 necessary for IFN resistance and viral pathogenesis

Coronaviruses encode a mix of highly conserved and novel genes as well as genetic elements necessary for infection and pathogenesis, raising the possibility for common targets for attenuation and therapeutic design. In this study, we focus on the highly conserved nonstructural protein (NSP) 16, a viral 2O methyl-transferase (MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of MERS NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2O MTase activity had only marginal impact on propagation and replication in Vero cells, the MERS dNSP16 mutant demonstrated significant attenuation relative to control both in primary human airway cultures and in vivo. Further examination indicated the MERS dNSP16 mutant had a type I IFN based attenuation and was partially restored in the absence of IFIT molecules. Importantly, the robust attenuation permitted use of MERS dNSP16 as a live attenuated vaccine platform protecting from challenge with a mouse adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2O MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.\n\nSignificanceCoronavirus emergence in both human and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of SARS-CoV, MERS-CoV, PEDV and swine delta coronavirus in the 21st century. These studies describe an approach that effectively targets the highly conserved 2O methyl-transferase activity of coronaviruses for attenuation. With clear understanding of the IFN/IFIT based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-coronavirus functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2O MTase function across coronaviruses.

microbiology↗