Distinct temporal regulation of intrinsic and innate intracellular immunity to Herpes Simplex Virus type 1 (HSV-1) infection

Intrinsic and innate immunity play pivotal roles in limiting the replication of invading viral pathogens. Intrinsic immunity is constitutive and mediated by pre-existing host cell restriction factors (e.g., promyelocytic leukemia-nuclear body (PML-NB) constituent proteins) which directly confer antiviral properties. On the other hand, innate immunity is inducible and upregulated in response to infection. Pattern recognition receptors (PRRs) (e.g., interferon gamma inducible protein 16 (IFI16)) sense pathogen-associated molecular patterns (PAMPs) and induce downstream signaling cascades leading to the induction of Interferon-stimulated gene (ISG) products that confer antiviral properties. These two arms of immunity represent the first line of intracellular defense to HSV-1 infection. Indeed, rapid recruitment of intrinsic and innate immune factors to viral DNA (vDNA) has a significant bearing on the outcome of infection. However, the spatial and temporal regulation of this recruitment remains poorly defined due to the technical challenges associated with vDNA detection at multiplicities of infection (MOI) that do not saturate intrinsic host factors. Utilizing 5-Ethynyl-2’-deoxyuridine (EdU) labeling of HSV-1 DNA in combination with click chemistry, we directly visualized input viral genomes under low MOI conditions (MOI of ≤ 3 PFU/cell) at 30-90 minutes post-addition of virus (mpi). This protocol is sensitive, specific, and compatible with indirect immunofluorescence (IF) staining protocols, providing a valuable assay to investigate the temporal recruitment of immune regulators to infecting vDNA. Upon entry of vDNA into the nucleus, PML-NB associated restriction factors (e.g., PML, SP100, and Daxx) were rapidly recruited to infecting viral genome foci. This process occurred in a PML-dependent manner and led to genome entrapment and silencing within PML-NBs. Interestingly, genome entrapment was observed during both wild-type (WT) and ICP0-null mutant (ΔICP0) HSV-1 infection. During WT HSV-1 infection, ICP0 induced PML degradation and the dispersal of PML-NB restriction factors, highlighting the importance of ICP0 to release viral genomes entrapped within PML-NBs to stimulate the onset of lytic HSV-1 replication. During ΔICP0 HSV-1 infection, vDNA remained stably entrapped within PML-NBs leading to a repression in viral gene expression and a restriction in plaque formation. Importantly, IFI16 was not stably recruited to vDNA entrapped within PML-NBs, and ISG expression was not induced under low MOI conditions that do not saturate PML-NB intrinsic host defenses. These data demonstrate that vDNA entry into the nucleus alone is not sufficient to stimulate the induction of innate immunity. Saturation of intrinsic host defenses under high MOI conditions stimulated the stable recruitment of IFI16 to infecting viral genomes, and induced ISG expression in a PML-, IFI16-, and Janus-associated kinase (JAK)-dependent manner. The induction of this innate immune response was dependent on the onset of vDNA replication, as treatment of the infected cell monolayers with phosphonoacetic acid (PAA), a vDNA polymerase inhibitor, inhibited ISG induction in a dose-dependent manner. Unlike PML depletion, inhibition of JAK signaling failed to relieve the plaque formation defect of ΔICP0 HSV-1, but instead significantly enhanced virus yields. Collectively, these data, for the first time, demonstrate a temporal and sequential induction of intrinsic and innate immunity during HSV-1 infection. Intrinsic immunity is induced within minutes of nuclear infection to restrict the initiation of viral gene transcription and the onset of lytic replication. Escape from this intrinsic repression and initiation of vDNA replication, which takes several hours, triggers the induction of innate immunity. ISG products establish an antiviral state within infected and neighboring uninfected cells to constrict viral propagation and limit the spread of infection. We identify dual roles for PML in the regulation of intrinsic and innate immunity to HSV-1 infection. However, these host defenses are counteracted by the viral ubiquitin ligase ICP0, which targets PML for degradation to promote vDNA release from PML-NBs in order to evade intrinsic viral genome silencing from the onset of nuclear infection

High Genetic Diversity of Human Rhinovirus among Pilgrims with Acute Respiratory Tract Infections during 2019 Hajj Pilgrimage Season

OBJECTIVES: Acute Respiratory tract infections (ARI) due to Human Rhinoviruses (HRV) are common in pilgrims during the annual Hajj pilgrimage. The objective of this study was to investigate the genetic diversity of HRV among pilgrims with respiratory symptoms during Hajj 2019. METHODS: HRV infection was detected using multiplex real time RT-PCR. Cycle sequencing was performed on positive samples and the sequences were subjected to phylogenetic analysis. RESULTS: 19 HRV-positive respiratory samples were sequenced. All three serotypes of HRV were identified: HRV-A (13; [68.42%)) was more common than HRV-B (2; [10.53%]), and HRV-C (4; [21.05%]). HRV-A species were found to be of genotypes A101, A21, A30, A57, A23, A60 and A11. HRV-B species belonged to genotypes B4 and B84, and HRV-C species were of genotypes C15, C3 and C56. CONCLUSIONS: Sequencing studies of respiratory tract viruses in pilgrims are important. We provide preliminary evidence of high diversity of HRV genotypes circulating in pilgrims in a restricted area during Hajj. This requires further clinical and sequencing studies of viral pathogens in larger consorts of overseas and local pilgrims

Development of Serological Assays and Seroprevalence Studies of the New Coronavirus 2019 (COVID-19): Reports from Saudi Arabia

Serological assays are valuable tools for tracking COVID-19 spread, estimation of herd immunity, and evaluation of vaccine effectiveness. Several reports from Saudi Arabia describe optimized in-house protocols that enable detection of SARS-CoV-2 specific antibodies and measurement of their neutralizing activity. Notably, there were variations in the approaches utilized to develop and validate these immunoassays in term of sample size, validation methodologies, and statistical analyses. The developed enzyme-linked immunoassays (ELISAs) were based on the viral full-length spike (S), S1 subunit, and nucleocapsid (NP), and enabled detection of IgM and/or IgG. ELISAs were evaluated and validated against a microneutralization assay utilizing a local SARS-CoV-2 clinical isolate, FDA-approved commercially available immunoassays, and/or real-time polymerase chain reaction (RT-PCR). Overall, the performance of the described assays was high, reaching up to 100% sensitivity and 98.9% specificity with no cross-reactivity with other coronaviruses. In-house immunoassays, along with commercially available kits, were subsequently applied in a number of sero-epidemiological studies aiming to estimate sero-positivity status among local populations including healthcare workers, COVID-19 patients, non-COVID-19 patients, and healthy blood donors. The reported seroprevalence rates differed widely among these studies, ranging from 0.00% to 32.2%. These variations are probably due to study period, targeted population, sample size, and performance of the immunoassays utilized. Indeed, lack of sero-positive cases were reported among healthy blood donors during the lockdown, while the highest rates were reported when the number of COVID-19 cases peaked in the country, particularly among healthcare workers working in referral hospitals and quarantine sites. In this review, we aim to (1) provide a critical discussion about the developed in-house immunoassays, and (2) summarize key findings of the sero-epidemiological studies and highlight strengths and weaknesses of each study

Coronavirus disease of 2019 (COVID-19) in the Gulf Cooperation Council (GCC) countries: Current status and management practices

Coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global threat to human population. The numbers of cases and deaths due to COVID-19 are escalating daily, putting health care systems worldwide under tremendous pressure. Policymakers in the affected countries have adopted varying strategies to deal with this crisis. As a result, the current COVID-19 status in terms of number of cases and deaths hugely varies between countries. The Gulf Cooperation Council (GCC) countries have managed to limit the massive spread of the infection among their populations by implementing proactive plans and timely decisions in response to COVID-19 outbreak; measures taken included suspension of flights, closure of educational institutes, curfew and lockdown of major cities, and provision of free-of-charge healthcare to patients. This review summarizes the COVID-19 status as of 18 May 2020 and highlights prevention and control measures applied in the GCC countries

Exploration of Microbially Derived Natural Compounds against Monkeypox Virus as Viral Core Cysteine Proteinase Inhibitors

Monkeypox virus (MPXV) is a member of the Orthopoxvirus genus and the Poxviridae family, which instigated a rising epidemic called monkeypox disease. Proteinases are majorly engaged in viral propagation by catalyzing the cleavage of precursor polyproteins. Therefore, proteinase is essential for monkeypox and a critical drug target. In this study, high-throughput virtual screening (HTVS) and molecular dynamics simulation were applied to detect the potential natural compounds against the proteinase of the monkeypox virus. Here, 32,552 natural products were screened, and the top five compounds were selected after implementing the HTVS and molecular docking protocols in series. Gallicynoic Acid F showed the minimum binding score of −10.56 kcal/mole in the extra precision scoring method, which reflected the highest binding with the protein. The top five compounds showed binding scores ≤−8.98 kcal/mole. These compound complexes were tested under 100 ns molecular dynamics simulation, and Vaccinol M showed the most stable and consistent RMSD trend in the range of 2 Å to 3 Å. Later, MM/GBSA binding free energy and principal component analysis were performed on the top five compounds to validate the stability of selected compound complexes. Moreover, the ligands Gallicynoic Acid F and H2-Erythro-Neopterin showed the lowest binding free energies of −61.42 kcal/mol and −61.09 kcal/mol, respectively. Compared to the native ligand TTP-6171 (ΔGBind = −53.86 kcal/mol), these two compounds showed preferable binding free energy, suggesting inhibitory application against MPXV proteinase. This study proposed natural molecules as a therapeutic solution to control monkeypox disease

Discovery of Bispecific Lead Compounds from Azadirachta indica against ZIKA NS2B-NS3 Protease and NS5 RNA Dependent RNA Polymerase Using Molecular Simulations

Zika virus (ZIKV) has been characterized as one of many potential pathogens and placed under future epidemic outbreaks by the WHO. However, a lack of potential therapeutics can result in an uncontrolled pandemic as with other human pandemic viruses. Therefore, prioritized effective therapeutics development has been recommended against ZIKV. In this context, the present study adopted a strategy to explore the lead compounds from Azadirachta indica against ZIKV via concurrent inhibition of the NS2B-NS3 protease (ZIKVpro) and NS5 RNA dependent RNA polymerase (ZIKVRdRp) proteins using molecular simulations. Initially, structure-based virtual screening of 44 bioflavonoids reported in Azadirachta indica against the crystal structures of targeted ZIKV proteins resulted in the identification of the top four common bioflavonoids, viz. Rutin, Nicotiflorin, Isoquercitrin, and Hyperoside. These compounds showed substantial docking energy (−7.9 to −11.01 kcal/mol) and intermolecular interactions with essential residues of ZIKVpro (B:His51, B:Asp75, and B:Ser135) and ZIKVRdRp (Asp540, Ile799, and Asp665) by comparison to the reference compounds, O7N inhibitor (ZIKVpro) and Sofosbuvir inhibitor (ZIKVRdRp). Besides, long interval molecular dynamics simulation (500 ns) on the selected docked poses reveals stability of the respective docked poses contributed by intermolecular hydrogen bonds and hydrophobic interactions. The predicted complex stability was further supported by calculated end-point binding free energy using molecular mechanics generalized born surface area (MM/GBSA) method. Consequently, the identified common bioflavonoids are recommended as promising therapeutic inhibitors of ZIKVpro and ZIKVRdRp against ZIKV for further experimental assessment

Lack of Evidence on Association between Iron Deficiency and COVID-19 Vaccine-Induced Neutralizing Humoral Immunity

Iron is a crucial micronutrient for immunity induction in response to infections and vaccinations. This study aimed to investigate the effect of iron deficiency on COVID-19-vaccine-induced humoral immunity. We investigated the effectiveness of COVID-19 vaccines (BNT162b2, mRNA-1273, and ChAdOx nCov-2019) in iron-deficient individuals (n = 63) and provide a side-by-side comparison to healthy controls (n = 67). The presence of anti-SARS-CoV-2 spike (S) and anti-nucleocapsid (NP) IgG were assessed using in-house S- and NP-based ELISA followed by serum neutralization test (SNT). High concordance between S-based ELISA and SNT results was observed. The prevalence of neutralizing antibodies was 95.24% (60/63) in the study group and 95.52% (64/67) in the controls with no significant difference. The presence/absence of past infection, period since vaccination, vaccine type, and being iron-deficient or having iron-deficiency anemia did not exert any significant effect on the prevalence or titer of anti-SARS-CoV-2 neutralizing antibodies. NP-based ELISA identified individuals unaware of exposure to SARS-CoV-2. Moreover, absence of anti-NP IgG was noted in participants who were previously diagnosed with COVID-19 suggesting the unpredictability of after-infection immunity. To sum up, this study demonstrated an initial lack of evidence on the association between iron deficiency and the effectiveness of COVID-19-vaccine-induced neutralizing humoral immunity. Similar studies with larger sample size remain necessary to obtain comprehensive conclusions about the effect or lack of effect of iron on COVID-19-vaccine effectiveness

Cheminformatics Strategies Unlock Marburg Virus VP35 Inhibitors from Natural Compound Library

The Ebola virus and its close relative, the Marburg virus, both belong to the family Filoviridae and are highly hazardous and contagious viruses. With a mortality rate ranging from 23% to 90%, depending on the specific outbreak, the development of effective antiviral interventions is crucial for reducing fatalities and mitigating the impact of Marburg virus outbreaks. In this investigation, a virtual screening approach was employed to evaluate 2042 natural compounds for their potential interactions with the VP35 protein of the Marburg virus. Average and worst binding energies were calculated for all 20 poses, and compounds that exhibited binding energies <−6 kcal/mol in both criteria were selected for further analysis. Based on binding energies, only six compounds (Estradiol benzoate, INVEGA (paliperidone), Isosilybin, Protopanaxadiol, Permethrin, and Bufalin) were selected for subsequent investigations, focusing on interaction analysis. Among these selected compounds, Estradiol benzoate, INVEGA (paliperidone), and Isosilybin showed strong hydrogen bonds, while the others did not. In this study, the compounds Myricetin, Isosilybin, and Estradiol benzoate were subjected to a molecular dynamics (MD) simulation and free binding energy calculation using MM/GBSA analysis. The reference component Myricetin served as a control. Estradiol benzoate exhibited the most stable and consistent root-mean-square deviation (RMSD) values, whereas Isosilybin showed significant fluctuations in RMSD. The compound Estradiol benzoate exhibited the lowest ΔG binding free energy (−22.89 kcal/mol), surpassing the control compound’s binding energy (−9.29 kcal/mol). Overall, this investigation suggested that Estradiol benzoate possesses favorable binding free energies, indicating a potential inhibitory mechanism against the VP35 protein of the Marburg virus. The study proposes that these natural compounds could serve as a therapeutic option for preventing Marburg virus infection. However, experimental validation is required to further corroborate these findings

Virtual screening and molecular dynamics simulation analysis of Forsythoside A as a plant-derived inhibitor of SARS-CoV-2 3CLpro

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a more severe strain of coronavirus (CoV) that was first emerged in China in 2019. Available antiviral drugs could be repurposed and natural compounds with antiviral activity could be safer and cheaper source of medicine for SARS-CoV-2. 78 natural antiviral compounds database was identified from literature and virtual screening technique was applied to identify potential 3-chymotrypsin-like protease (3CLpro) inhibitors. Molecular docking studies were conducted to analyze the main protease (3CLpro) and inhibitors interactions with key residues of active site of target protein (PDB ID: 6LU7), active site constitute the part of active domain I and II of 3CLpro. 10 compounds with highest dock score were subjected to calculate ADMET parameters to figure out drug-likeness. Molecular dynamic (MD) simulation of the selected lead was performed by Amber simulation package to understand the conformational changes in docked complex. MD simulations analysis (RMSD, RMSF, Rg, BF, HBs, and SASA plots) of lead bounded with 3CLpro, hence revealed the important structural turns and twists during MD simulations from 0 to 100 ns. MM-PBSA/GBSA methods has also been applied for the estimation binding free energy (BFE) of the selected lead-complex. The present study has identified lead compound “Forsythoside A” an active extract of Forsythia suspense as SARS-CoV-2 3CLpro inhibitor that can block the viral replication and translation. Structural analysis of target protein and lead compound performed in this study could contribute to the development of potential drug against SARS-CoV-2 infection

A multi-targeted computational drug discovery approach for repurposing tetracyclines against monkeypox virus

Abstract Monkeypox viral infection is an emerging threat and a major concern for the human population. The lack of drug molecules to treat this disease may worsen the problem. Identifying potential drug targets can significantly improve the process of developing potent drug molecules for treating monkeypox. The proteins responsible for viral replication are attractive drug targets. Identifying potential inhibitors from known drug molecules that target these proteins can be key to finding a cure for monkeypox. In this work, two viral proteins, DNA-dependent RNA polymerase (DdRp) and viral core cysteine proteinase, were considered as potential drug targets. Sixteen antibiotic drugs from the tetracycline class were screened against both viral proteins through high-throughput virtual screening. These tetracycline class of antibiotic drugs have the ability to inhibit bacterial protein synthesis, which makes these antibiotics drugs a prominent candidate for drug repurposing. Based on the screening result obtained against DdRp, top two compounds, namely Tigecycline and Eravacycline with docking scores of − 8.88 and − 7.87 kcal/mol, respectively, were selected for further analysis. Omadacycline and minocycline, with docking scores of − 10.60 and − 7.51 kcal/mol, are the top two compounds obtained after screening proteinase with the drug library. These compounds, along with reference compounds GTP for DdRp and tecovirimat for proteinase, were used to form protein–ligand complexes, followed by their evaluation through a 300 ns molecular dynamic simulation. The MM/GBSA binding free energy calculation and principal components analysis of these selected complexes were also conducted for understanding the dynamic stability and binding affinity of these compounds with respective target proteins. Overall, this study demonstrates the repurposing of tetracycline-derived drugs as a therapeutic solution for monkeypox viral infection