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Probing the SARS-CoV-2 main protease active site as a target to inhibit viral replication
SARS-CoV-2 replication involves the synthesis of two large proteins, which are inactive and harmless until the viral main protease enzyme (3CL M pro ) uses them as substrates, cleaving them into many smaller, functional products. Dr Andrey Kovalevsky and his team from Oak Ridge National Laboratory propose a design of novel inhibitors and the repurposing of clinical drugs developed to treat other diseases for the treatment of COVID-19. Finally, the team uses room temperature crystallography with X-rays and neutrons to guide structure-based and computer-assisted drug design and to assess the ability of drugs to inhibit the SARS-CoV-2 main protease, causing virus replication to stop.
High Contagiousness and Rapid Spread of Severe Acute Respiratory Syndrome Coronavirus 2
Severe acute respiratory syndrome coronavirus 2 is the causative agent of the ongoing coronavirus disease pandemic. Initial estimates of the early dynamics of the outbreak in Wuhan, China, suggested a doubling time of the number of infected persons of 6–7 days and a basic reproductive number (R 0 ) of 2.2–2.7. We collected extensive individual case reports across China and estimated key epidemiologic parameters, including the incubation period (4.2 days). We then designed 2 mathematical modeling approaches to infer the outbreak dynamics in Wuhan by using high-resolution domestic travel and infection data. Results show that the doubling time early in the epidemic in Wuhan was 2.3–3.3 days. Assuming a serial interval of 6–9 days, we calculated a median R0 value of 5.7 (95% CI 3.8–8.9). Here, we further show that active surveillance, contact tracing, quarantine, and early strong social distancing efforts are needed to stop transmission of the virus.
Effect of in utero and lactational exposure to antiretroviral therapy on the gut microbial composition and metabolic function in aged rat offspring
Despite the highly effective impact of antiretroviral therapy (ART) in reducing mother-to-child transmission of human immunodeficiency virus (HIV), there are concerns of long-term impacts of ART on the health of the offspring. The implications of perinatal exposure to antiviral drugs on the gut bacterial population and metabolic function in the offspring is unclear but may influence health outcomes given the various reported effects of the microbiome in human health. This study aims to gain insight into the potential effect ofin uteroand lactational exposure to ART on gut microbiota populations and short‐chain fatty acids (SCFAs) production in aged rat offspring. Pregnant rats were administered a combination of antiretroviral drugs (abacavir/dolutegravir/lamivudine) at two different dose levels during gestation and throughout lactation, and the fecal bacterial abundance and SCFA levels of the offspring were analyzed when they reached 12 months of age. Our results showed dose-dependent and sex-based differences in fecal microbial abundance at various taxonomic levels. Specifically, we found a decline inFirmicutesin males, and an increase inActinobacteriaamong males and females. Furthermore, a sex-specific distribution reorganization ofLactobacillus,Bifidobacterium, andAkkermansiawas identified. No significant difference in the concentration of prominent SCFAs and IgA levels were identified. These findings provide preliminary information indicating the need to evaluate perinatal effects of ART more comprehensively on the gut bacterial and metabolic function in future studies, and their potential role in offspring health outcomes.
Improved Mobilome Delineation in Fragmented Genomes
The mobilome of a microbe, i.e., its set of mobile elements, has major effects on its ecology, and is important to delineate properly in each genome. This becomes more challenging for incomplete genomes, and even more so for metagenome-assembled genomes (MAGs), where misbinning of scaffolds and other losses can occur. Genomic islands (GIs), which integrate into the host chromosome, are a major component of the mobilome. Our GI-detection software TIGER, unique in its precise mapping of GI termini, was applied to 74,561 genomes from 2,473 microbial species, each species containing at least one MAG and one isolate genome. A species-normalized deficit of ∼1.6 GIs/genome was measured for MAGs relative to isolates. To test whether this undercount was due to the higher fragmentation of MAG genomes, TIGER was updated to enable detection of split GIs whose termini are on separate scaffolds or that wrap around the origin of a circular replicon. This doubled GI yields, and the new split GIs matched the quality of single-scaffold GIs, except that highly fragmented GIs may lack central portions. Cross-scaffold search is an important upgrade to GI detection as fragmented genomes increasingly dominate public databases. TIGER2 better captures MAG microdiversity, recovering niche-defining GIs and supporting microbiome research aims such as virus-host linking and ecological assessment.
The development and use of decision support framework for informing selection of select agent toxins with modelling studies to inform permissible toxin amounts
Many countries have worked diligently to establish and implement policies and processes to regulate high consequence pathogens and toxins that could have a significant public health impact if misused. In the United States, the Antiterrorism and Effective Death Penalty Act of 1996 (Public Law 104-132, 1996), as amended by the Bioterrorism Preparedness and Response Act of 2002 (Public Law 107-188, 2002) requires that the Department of Health and Human Services (HHS) [through the Centers for Disease Control and Prevention (CDC)] establish a list of bacteria, viruses, and toxins that have the potential to pose a severe threat to public health and safety. Currently, this list is reviewed and updated on a biennial basis using input from subject matter experts (SMEs). We have developed decision support framework (DSF) approaches to facilitate selection of select toxins and, where toxicity data are known, conducted modelling studies to inform selection of toxin amounts that should be excluded from select agent regulations. Exclusion limits allow laboratories to possess toxins under an established limit to support their research or teaching activities without the requirement to register with the Federal Select Agent Program. Fact sheets capturing data from a previously vetted SME workshop convened by CDC, literature review and SME input were developed to assist in evaluating toxins using the DSF approach. The output of the DSF analysis agrees with the current select toxin designations, and no other toxins evaluated in this study were recommended for inclusion on the select agent and toxin list. To inform the selection of exclusion limits, attack scenarios were developed to estimate the amount of toxin needed to impact public health. Scenarios consisted of simulated aerosol releases of a toxin in high-population-density public facilities and the introduction of a toxin into a daily consumable product supply chain. Using published inhalation and ingestion median toxic dose (TD 50 ) and median lethal dose (LD 50 ) values, where available, a range of toxin amounts was examined to estimate the number of people exposed to these amounts in these scenarios. Based on data generated by these models, we proposed toxin exclusion values corresponding to levels below those that would trigger a significant public health response (i.e., amounts estimated to expose up to ten people by inhalation or one hundred people by ingestion to LD 50 or TD 50 levels of toxin in the modeled scenarios).
Structure and Fc-Effector Function of Rhesusized Variants of Human Anti-HIV-1 IgG1s
Passive transfer of monoclonal antibodies (mAbs) of human origin into Non-Human Primates (NHPs), especially those which function predominantly by a Fc-effector mechanism, requires an a priori preparation step, in which the human mAb is reengineered to an equivalent NHP IgG subclass. This can be achieved by changing both the Fc and Fab sequence while simultaneously maintaining the epitope specificity of the parent antibody. This Ab reengineering process, referred to as rhesusization, can be challenging because the simple grafting of the complementarity determining regions (CDRs) into an NHP IgG subclass may impact the functionality of the mAb. Here we describe the successful rhesusization of a set of human mAbs targeting HIV-1 envelope (Env) epitopes involved in potent Fc-effector function against the virus. This set includes a mAb targeting a linear gp120 V1V2 epitope isolated from a RV144 vaccinee, a gp120 conformational epitope within the Cluster A region isolated from a RV305 vaccinated individual, and a linear gp41 epitope within the immunodominant Cys-loop region commonly targeted by most HIV-1 infected individuals. Structural analyses confirm that the rhesusized variants bind their respective Env antigens with almost identical specificity preserving epitope footprints and most antigen-Fab atomic contacts with constant regions folded as in control RM IgG1s. In addition, functional analyses confirm preservation of the Fc effector function of the rhesusized mAbs including the ability to mediate Antibody Dependent Cell-mediated Cytotoxicity (ADCC) and antibody dependent cellular phagocytosis by monocytes (ADCP) and neutrophils (ADNP) with potencies comparable to native macaque antibodies of similar specificity. While the antibodies chosen here are relevant for the examination of the correlates of protection in HIV-1 vaccine trials, the methods used are generally applicable to antibodies for other purposes.
A Coarse-Grained Model of Affinity Maturation Indicates the Importance of B-Cell Receptor Avidity in Epitope Subdominance
The elicitation of broadly neutralizing antibodies (bnAbs) is a major goal in the design of vaccines against rapidly-mutating viruses. In the case of influenza, many bnAbs that target conserved epitopes on the stem of the hemagglutinin protein (HA) have been discovered. However, these antibodies are rare, are not boosted well upon reinfection, and often have low neutralization potency, compared to strain-specific antibodies directed to the HA head. Different hypotheses have been proposed to explain this phenomenon. We use a coarse-grained computational model of the germinal center reaction to investigate how B-cell receptor binding valency affects the growth and affinity maturation of competing B-cells. We find that receptors that are unable to bind antigen bivalently, and also those that do not bind antigen cooperatively, have significantly slower rates of growth, memory B-cell production, and, under certain conditions, rates of affinity maturation. The corresponding B-cells are predicted to be outcompeted by B-cells that bind bivalently and cooperatively. We use the model to explore strategies for a universal influenza vaccine, e.g., how to boost the concentrations of the slower growing cross-reactive antibodies directed to the stem. The results suggest that, upon natural reinfections subsequent to vaccination, the protectiveness of such vaccines would erode, possibly requiring regular boosts. Collectively, our results strongly support the importance of bivalent antibody binding in immunodominance, and suggest guidelines for developing a universal influenza vaccine.
A Path-Based Analysis of Infected Cell Line and COVID-19 Patient Transcriptome Reveals Novel Potential Targets and Drugs Against SARS-CoV-2
Most transcriptomic studies of SARS-CoV-2 infection have focused on differentially expressed genes, which do not necessarily reveal the genes mediating the transcriptomic changes. In contrast, exploiting curated biological network, our PathExt tool identifies central genes from the differentially active paths mediating global transcriptomic response. Here we apply PathExt to multiple cell line infection models of SARS-CoV-2 and other viruses, as well as to COVID-19 patient-derived PBMCs. The central genes mediating SARS-CoV-2 response in cell lines were uniquely enriched for ATP metabolic process, G1/S transition, leukocyte activation and migration. In contrast, PBMC response reveals dysregulated cell-cycle processes. In PBMC, the most frequently central genes are associated with COVID-19 severity. Importantly, relative to differential genes, PathExt-identified genes show greater concordance with several benchmark anti-COVID-19 target gene sets. We propose six novel anti-SARS-CoV-2 targets ADCY2, ADSL, OCRL, TIAM1, PBK, and BUB1, and potential drugs targeting these genes, such as Bemcentinib, Phthalocyanine, and Conivaptan.
The HHV-6B U20 glycoprotein binds ULBP1, masking it from recognition by NKG2D and interfering with natural killer cell activation
Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 – ULBP1 complex indicates some similarities to the m152-RAE1γ complex.
Complete Inactivation of Blood Borne Pathogen Trypanosoma cruzi in Stored Human Platelet Concentrates and Plasma Treated With 405 nm Violet-Blue Light
The introduction of pathogen reduction technologies (PRTs) to inactivate bacteria, viruses and parasites in donated blood components stored for transfusion adds to the existing arsenal toward reducing the risk of transfusion-transmitted infectious diseases (TTIDs). We have previously demonstrated that 405 nm violet-blue light effectively reduces blood-borne bacteria in stored human plasma and platelet concentrates. In this report, we investigated the microbicidal effect of 405 nm light on one important bloodborne parasite Trypanosoma cruzi that causes Chagas disease in humans. Our results demonstrated that a light irradiance at 15 mWcm -2 for 5 h, equivalent to 270 Jcm -2 , effectively inactivated T. cruzi by over 9.0 Log 10 , in plasma and platelets that were evaluated by a MK2 cell infectivity assay. Giemsa stained T. cruzi infected MK2 cells showed that the light-treated parasites in plasma and platelets were deficient in infecting MK2 cells and did not differentiate further into intracellular amastigotes unlike the untreated parasites. The light-treated and untreated parasite samples were then evaluated for any residual infectivity by injecting the treated parasites into Swiss Webster mice, which did not develop infection even after the animals were immunosuppressed, further demonstrating that the light treatment was completely effective for inactivation of the parasite; the light-treated platelets had similar in vitro metabolic and biochemical indices to that of untreated platelets. Overall, these results provide a proof of concept toward developing 405 nm light treatment as a pathogen reduction technology (PRT) to enhance the safety of stored human plasma and platelet concentrates from bloodborne T. cruzi, which causes Chagas disease.
Single-Cell Genomics of Novel Actinobacteria With the Wood–Ljungdahl Pathway Discovered in a Serpentinizing System
Serpentinite-hosted systems represent modern-day analogs of early Earth environments. In these systems, water-rock interactions generate highly alkaline and reducing fluids that can contain hydrogen, methane, and low-molecular-weight hydrocarbons-potent reductants capable of fueling microbial metabolism. In this study, we investigated the microbiota of Hakuba Happo hot springs (~50°C; pH~10.5–11), located in Nagano (Japan), which are impacted by the serpentinization process. Analysis of the 16S rRNA gene amplicon sequences revealed that the bacterial community comprises Nitrospirae (47%), “Parcubacteria” (19%), Deinococcus-Thermus (16%), and Actinobacteria (9%), among others. Notably, only 57 amplicon sequence variants (ASV) were detected, and fifteen of these accounted for 90% of the amplicons. Among the abundant ASVs, an early-branching, uncultivated actinobacterial clade identified as RBG-16-55-12 in the SILVA database was detected. Ten single-cell genomes (average pairwise nucleotide identity: 0.98–1.00; estimated completeness: 33–93%; estimated genome size: ~2.3 Mb) that affiliated with this clade were obtained. Taxonomic classification using single copy genes indicates that the genomes belong to the actinobacterial class-level clade UBA1414 in the Genome Taxonomy Database. Based on metabolic pathway predictions, these actinobacteria are anaerobes, capable of glycolysis, dissimilatory nitrate reduction and CO 2 fixation via the Wood–Ljungdahl (WL) pathway. Several other genomes within UBA1414 and two related class-level clades also encode the WL pathway, which has not yet been reported for the Actinobacteria phylum. For the Hakuba actinobacterium, the energy metabolism related to the WL pathway is likely supported by a combination of the Rnf complex, group 3b and 3d [NiFe]-hydrogenases, [FeFe]-hydrogenases, and V-type (H + /Na + pump) ATPase. The genomes also harbor a form IV ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, also known as a RubisCO-like protein, and contain signatures of interactions with viruses, including clustered regularly interspaced short palindromic repeat (CRISPR) regions and several phage integrases. This is the first report and detailed genome analysis of a bacterium within the Actinobacteria phylum capable of utilizing the WL pathway. The Hakuba actinobacterium is a member of the clade UBA1414/RBG-16-55-12, formerly within the group “OPB41.” We propose to name this bacterium ‘Candidatus Hakubanella thermoalkaliphilus.'
Knowing the unknown: The underestimation of monkeypox cases. Insights and implications from an integrative review of the literature
Monkeypox is an emerging zoonotic disease caused by the monkeypox virus, which is an infectious agent belonging to the genus Orthopoxvirus. Currently, commencing from the end of April 2022, an outbreak of monkeypox is ongoing, with more than 43,000 cases reported as of 23 August 2022, involving 99 countries and territories across all the six World Health Organization (WHO) regions. On 23 July 2022, the Director-General of the WHO declared monkeypox a global public health emergency of international concern (PHEIC), since the outbreak represents an extraordinary, unusual, and unexpected event that poses a significant risk for international spread, requiring an immediate, coordinated international response. However, the real magnitude of the burden of disease could be masked by failures in ascertainment and under-detection. As such, underestimation affects the efficiency and reliability of surveillance and notification systems and compromises the possibility of making informed and evidence-based policy decisions in terms of the adoption and implementation of ad hoc adequate preventive measures. In this review, synthesizing 53 papers, we summarize the determinants of the underestimation of sexually transmitted diseases, in general, and, in particular, monkeypox, in terms of all their various components and dimensions (under-ascertainment, underreporting, under-detection, under-diagnosis, misdiagnosis/misclassification, and under-notification).
Discovery of a novel filamentous prophage in the genome of the Mimosa pudica microsymbiont Cupriavidus taiwanensis STM 6018
Integrated virus genomes (prophages) are commonly found in sequenced bacterial genomes but have rarely been described in detail for rhizobial genomes. Cupriavidus taiwanensis STM 6018 is a rhizobial Betaproteobacteria strain that was isolated in 2006 from a root nodule of a Mimosa pudica host in French Guiana, South America. Here we describe features of the genome of STM 6018, focusing on the characterization of two different types of prophages that have been identified in its genome. The draft genome of STM 6018 is 6,553,639 bp, and consists of 80 scaffolds, containing 5,864 protein-coding genes and 61 RNA genes. STM 6018 contains all the nodulation and nitrogen fixation gene clusters common to symbiotic Cupriavidus species; sharing >99.97% bp identity homology to the nod / nif / noeM gene clusters from C. taiwanensis LMG19424 T and “ Cupriavidus neocalidonicus” STM 6070. The STM 6018 genome contains the genomes of two prophages: one complete Mu-like capsular phage and one filamentous phage, which integrates into a putative dif site. This is the first characterization of a filamentous phage found within the genome of a rhizobial strain. Further examination of sequenced rhizobial genomes identified filamentous prophage sequences in several Beta-rhizobial strains but not in any Alphaproteobacterial rhizobia.
Host genetics and gut microbiota composition: Baseline gut microbiota composition as a possible prognostic factor for the severity of COVID-19 in patients with familial Mediterranean fever disease
It is known that the gut microbiome of a healthy person affects the process of COVID-19 after getting infected with SARS-CoV-2 virus. It is also believed that colchicine can alleviate the severity of COVID-19. Current investigations aimed to evaluate the associations between the baseline gut microbiota composition of healthy and Familial Mediterranean fever (FMF) - carrier Armenian men populations, and the severity of the COVID-19 disease after their infection with the SARS-CoV-2. The study has a purpose of answering three core questions: i. Do the characteristics of gut microbiome of Armenians affect the course of COVID-19 severity? ii. How does the COVID-19 disease course on go for FMF patients who have been taking colchicine as a medication over the years after getting infected with SARS-CoV-2? iii. Is there an initial gut micribiota structure pattern for non-FMF and FMF patients in the cases when COVID-19 appears in mild form? The gut microbiota composition in non-FMF and FMF patients before the first infection (mild and moderate course of COVID-19) was considered. COVID-19 was diagnosed by SARS-CoV-2 nucleic acid RT-PCR in nasopharyngeal swab and/or sputum. The number of patients with male FMF with mild COVID-19 was approximately two times higher than that of non-FMF male subjects with COVID-19. In addition, an association of COVID-19 disease severity with the baseline gut Prevotella, Clostridium hiranonis, Eubacterium biforme, Veillonellaceae, Coprococcus, and Blautia diversities in the non-FMF and FMF populations were revealed by us, which can be used as risk/prognostic factor for the severity of COVID-19.
Decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA in raw sewage from university dormitories
Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA has been frequently detected in sewage from many university dormitories to inform public health decisions during the COVID-19 pandemic, a clear understanding of SARS-CoV-2 RNA persistence in site-specific raw sewage is still lacking. To investigate the SARS-CoV-2 RNA persistence, a field trial was conducted in the University of Tennessee dormitories raw sewage, similar to municipal wastewater. The decay of enveloped SARS-CoV-2 RNA and non-enveloped Pepper mild mottle virus (PMMoV) RNA was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw sewage at 4°C and 20°C. Temperature, followed by the concentration level of SARS-CoV-2 RNA, was the most significant factors that influenced the first-order decay rate constants (k) of SARS-CoV-2 RNA. The mean k values of SARS-CoV-2 RNA were 0.094 day –1 at 4°C and 0.261 day –1 at 20°C. At high-, medium-, and low-concentration levels of SARS-CoV-2 RNA, the mean k values were 0.367, 0.169, and 0.091 day –1 , respectively. Furthermore, there was a statistical difference between the decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA at different temperature conditions. The first decay rates for both temperatures were statistically comparable for SARS-CoV-2 RNA, which showed sensitivity to elevated temperatures but not for PMMoV RNA. This study provides evidence for the persistence of viral RNA in site-specific raw sewage at different temperature conditions and concentration levels.