Human Monkeypox: A Comprehensive Overview of Epidemiology, Pathogenesis, Diagnosis, Treatment, and Prevention Strategies.

Monkeypox virus (MPXV) is an emerging zoonotic virus that belongs to the Orthopoxvirus genus and presents clinical symptoms similar to those of smallpox, such as fever and vesicular-pustular skin lesions. However, the differential diagnosis between smallpox and monkeypox is that smallpox does not cause lymphadenopathy but monkeypox generates swelling in the lymph nodes. Since the eradication of smallpox, MPXV has been identified as the most common Orthopoxvirus to cause human disease. Despite MPXV being endemic to certain regions of Africa, the current MPXV outbreak, which began in early 2022, has spread to numerous countries worldwide, raising global concern. As of the end of May 2023, over 87,545 cases and 141 deaths have been reported, with most cases identified in non-endemic countries, primarily due to human-to-human transmission. To better understand this emerging threat, this review presents an overview of key aspects of MPXV infection, including its animal reservoirs, modes of transmission, animal models, epidemiology, clinical and immunological features, diagnosis, treatments, vaccines, and prevention strategies. The material presented here provides a comprehensive understanding of MPXV as a disease, while emphasizing the significance and unique characteristics of the 2022 outbreak. This offers valuable information that can inform future research and aid in the development of effective interventions.

Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein.

High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. XL-MS of plasma isolated from patients with COVID-19 uncovered HDL protein interaction networks, dominated by acute-phase serum amyloid proteins, whereby serum amyloid A2 was shown to bind to apolipoprotein (Apo) D. XL-MS on isolated HDL confirmed ApoD to interact with SARS-CoV-2 spike but not SARS-CoV-1 spike. Other direct interactions of SARS-CoV-2 spike upon HDL included ApoA1 and ApoC3. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1. Mechanistically, XL-MS coupled with data-driven structural modeling determined that ApoD may interact within the receptor-binding domain of the spike. However, ApoD overexpression in multiple cell-based assays had no effect upon viral replication or infectivity. Thus, SARS-CoV-2 spike can bind to apolipoproteins on HDL, but these interactions do not appear to alter infectivity.

Drug repurposing based on a quantum-inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2.

The COVID-19 pandemic has accelerated the need to identify new antiviral therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir, the first antiviral against SARS-CoV-2 approved for human use, using a quantum-inspired device. We modelled Remdesivir and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of lead compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. We also demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Lastly, we employed an in vitro polymerization assay to demonstrate that these compounds directly inhibit the RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. Together, our data reveal that our QUBO model performs accurate comparisons (BMS-986094) that differed from those predicted by Tanimoto (different forms of vitamin B12); all compounds inhibited replication of SARS-CoV-2 via direct action on RdRP, with both models being useful. While Tanimoto may be employed when performing relatively small comparisons, QUBO is also accurate and may be well suited for very complex problems where computational resources may limit the number and/or complexity of possible combinations to evaluate. Our quantum-inspired screening method can therefore be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools.

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.

Neutralizing antibody activity in convalescent sera from infection in humans with SARS-CoV-2 and variants of concern.

COVID-19 vaccine design and vaccination rollout need to take into account a detailed understanding of antibody durability and cross-neutralizing potential against SARS-CoV-2 and emerging variants of concern (VOCs). Analyses of convalescent sera provide unique insights into antibody longevity and cross-neutralizing activity induced by variant spike proteins, which are putative vaccine candidates. Using sera from 38 individuals infected in wave 1, we show that cross-neutralizing activity can be detected up to 305 days pos onset of symptoms, although sera were less potent against B.1.1.7 (Alpha) and B1.351 (Beta). Over time, despite a reduction in overall neutralization activity, differences in sera neutralization potency against SARS-CoV-2 and the Alpha and Beta variants decreased, which suggests that continued antibody maturation improves tolerance to spike mutations. We also compared the cross-neutralizing activity of wave 1 sera with sera from individuals infected with the Alpha, the Beta or the B.1.617.2 (Delta) variants up to 79 days post onset of symptoms. While these sera neutralize the infecting VOC and parental virus to similar levels, cross-neutralization of different SARS-CoV-2 VOC lineages is reduced. These findings will inform the optimization of vaccines to protect against SARS-CoV-2 variants.

Drug repurposing based on a Quantum-Inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2 including vitamin B12.

The COVID-19 pandemic has accelerated the need to identify new therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir (RDV), the only antiviral against SARS-CoV-2 currently approved for human use, using a quantum-inspired device. We modelled RDV and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC 50 ) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. Lastly, we demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Our data reveal that BMS-986094 and different forms of vitamin B12 are effective at inhibiting replication of all these variants of SARS-CoV-2. While BMS-986094 can cause secondary effects in humans as established by phase II trials, these findings suggest that vitamin B12 deserves consideration as a SARS-CoV-2 antiviral, particularly given its extended use and lack of toxicity in humans, and its availability and affordability. Our screening method can be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

An Overview of Vaccines against SARS-CoV-2 in the COVID-19 Pandemic Era.

The emergence of SARS-CoV-2 in late 2019 led to the COVID-19 pandemic all over the world. When the virus was first isolated and its genome was sequenced in the early months of 2020, the efforts to develop a vaccine began. Based on prior well-known knowledge about coronavirus, the SARS-CoV-2 spike (S) protein was selected as the main target. Currently, more than one hundred vaccines are being investigated and several of them are already authorized by medical agencies. This review summarizes and compares the current knowledge about main approaches for vaccine development, focusing on those authorized and specifically their immunogenicity, efficacy preventing severe disease, adverse side effects, protection, and ability to cope with emergent SARS-CoV-2 variants.

Antibody longevity and cross-neutralizing activity following SARS-CoV-2 wave 1 and B.1.1.7 infections.

As SARS-CoV-2 variants continue to emerge globally, a major challenge for COVID-19 vaccination is the generation of a durable antibody response with cross-neutralizing activity against both current and newly emerging viral variants. Cross-neutralizing activity against major variants of concern (B.1.1.7, P.1 and B.1.351) has been observed following vaccination, albeit at a reduced potency, but whether vaccines based on the Spike glycoprotein of these viral variants will produce a superior cross-neutralizing antibody response has not been fully investigated. Here, we used sera from individuals infected in wave 1 in the UK to study the long-term cross-neutralization up to 10 months post onset of symptoms (POS), as well as sera from individuals infected with the B.1.1.7 variant to compare cross-neutralizing activity profiles. We show that neutralizing antibodies with cross-neutralizing activity can be detected from wave 1 up to 10 months POS. Although neutralization of B.1.1.7 and B.1.351 is lower, the difference in neutralization potency decreases at later timepoints suggesting continued antibody maturation and improved tolerance to Spike mutations. Interestingly, we found that B.1.1.7 infection also generates a cross-neutralizing antibody response, which, although still less potent against B.1.351, can neutralize parental wave 1 virus to a similar degree as B.1.1.7. These findings have implications for the optimization of vaccines that protect against newly emerging viral variants.

Development of a structural epitope mimic: an idiotypic approach to HCV vaccine design.

HCV vaccine development is stymied by the high genetic diversity of the virus and the variability of the envelope glycoproteins. One strategy to overcome this is to identify conserved, functionally important regions-such as the epitopes of broadly neutralizing antibodies (bNAbs)-and use these as a basis for structure-based vaccine design. Here, we report an anti-idiotype approach that has generated an antibody that mimics a highly conserved neutralizing epitope on HCV E2. Crucially, a mutagenesis screen was used to identify the antibody, designated B2.1 A, whose binding characteristics to the bNAb AP33 closely resemble those of the original antigen. Protein crystallography confirmed that B2.1 A is a structural mimic of the AP33 epitope. When used as an immunogen B2.1 A induced antibodies that recognized the same epitope and E2 residues as AP33 and most importantly protected against HCV challenge in a mouse model.

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation.

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.

Author Correction: Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models.

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

Human Organs-on-Chips for Virology.

While conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral diseases, models that can accurately recapitulate human responses to infection are still lacking. Human organ-on-a-chip (Organ Chip) microfluidic culture devices that recapitulate tissue-tissue interfaces, fluid flows, mechanical cues, and organ-level physiology have been developed to narrow the gap between in vitro experimental models and human pathophysiology. Here, we describe how recent developments in Organ Chips have enabled re-creation of complex pathophysiological features of human viral infections in vitro.

Phosphodiesterase-induced cAMP degradation restricts hepatitis B virus infection.

Hepatitis B virus (HBV) entry into hepatocytes is mediated via a high-affinity interaction between the preS1 glycoprotein and sodium/bile acid cotransporting polypeptide (NTCP). To date, in vitro model systems rely on high multiplicities of infection to achieve infection of cell lines overexpressing human NTCP. This study investigates a novel regulatory pathway for NTCP trafficking to the cell surface, induced by DMSO-mediated cellular differentiation. DMSO rapidly induces high cell surface expression of NTCP and results in increased susceptibility of cells to HBV infection. Additionally, DMSO treatment induces actin, as well as Tubulin reshaping within the cells. We show that direct disruption of the actin and Tubulin network directly enhances NTCP expression and the subsequent susceptibility of cells to HBV infection. DMSO induces these changes via alterations in the levels of cyclic (c)AMP, which participates in the observed actin rearrangements. Blocking of phosphodiesterases (PDEs), which degrade accumulated cAMP, had the same effect as DMSO differentiation and demonstrates that DMSO prevents phosphodiesterase-mediated cAMP degradation. This identifies adenylate cyclase as a novel target for blocking the entry of HBV via targeting the cell surface accumulation of NTCP. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

Kinetics of Early Innate Immune Activation during HIV-1 Infection of Humanized Mice.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with aberrant immune activation; however, most model systems for HIV-1 have been used during established infection. Here, we utilize ultrasensitive HIV-1 quantification to delineate early events during the eclipse, burst, and chronic phases of HIV-1 infection in humanized mice. We show that very early in infection, HIV-1 suppresses peripheral type I interferon (IFN) and interferon-stimulated gene (ISG) responses, including the HIV-1 restriction factor IFI44. At the peak of innate immune activation, prior to CD4 T cell loss, HIV-1 infection differentially affects peripheral and lymphoid Toll-like receptor (TLR) expression profiles in T cells and macrophages. This results in a trend toward an altered activation of nuclear factor κB (NF-κB), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3). The subsequent type I and III IFN responses result in preferential induction of peripheral ISG responses. Following this initial innate immune activation, peripheral expression of the HIV-1 restriction factor SAM domain- and HD domain-containing protein 1 (SAMHD1) returns to levels below those observed in uninfected mice, suggesting that HIV-1 interferes with their basal expression. However, peripheral cells still retain their responsiveness to exogenous type I IFN, whereas splenic cells show a reduction in select ISGs in response to IFN. This demonstrates the highly dynamic nature of very early HIV-1 infection and suggests that blocks to the induction of HIV-1 restriction factors contribute to the establishment of viral persistence.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) infection is restricted to humans and some nonhuman primates (e.g., chimpanzee and gorilla). Alternative model systems based on simian immunodeficiency virus (SIV) infection of macaques are available but do not recapitulate all aspects of HIV-1 infection and disease. Humanized mice, which contain a human immune system, can be used to study HIV-1, but only limited information on early events and immune responses is available to date. Here, we describe very early immune responses to HIV-1 and demonstrate a suppression of cell-intrinsic innate immunity. Furthermore, we show that HIV-1 infection interacts differently with innate immune responses in blood and lymphoid organs.

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry.

Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.

In Vivo Model Systems for Hepatitis B Virus Research.

Hepatitis B virus (HBV) affects more than 257 million people globally, resulting in progressively worsening liver disease, manifesting as fibrosis, cirrhosis, and hepatocellular carcinoma. The exceptionally narrow species tropism of HBV restricts its natural hosts to humans and non-human primates, including chimpanzees, gorillas, gibbons, and orangutans. The unavailability of completely immunocompetent small-animal models has contributed to the lack of curative therapeutic interventions. Even though surrogates allow the study of closely related viruses, their host genetic backgrounds, immune responses, and molecular virology differ from those of HBV. Various different models, based on either pure murine or xenotransplantation systems, have been introduced over the past years, often making the choice of the optimal model for any given question challenging. Here, we offer a concise review of in vivo model systems employed to study HBV infection and steps in the HBV life cycle or pathogenesis.

A Hitchhiker's guide to humanized mice: new pathways to studying viral infections.

Humanized mice are increasingly appreciated as an incredibly powerful platform for infectious disease research. The often very narrow species tropism of many viral infections, coupled with the sometimes misleading results from preclinical studies in animal models further emphasize the need for more predictive model systems based on human cells rather than surrogates. Humanized mice represent such a model and have been greatly enhanced with regards to their immune system reconstitution as well as immune functionality in the past years, resulting in their recommendation as a preclinical model by the US Food and Drug Administration. This review aims to give a detailed summary of the generation of human peripheral blood lymphocyte-, CD34+ haematopoietic stem cell- and bone marrow/liver/thymus-reconstituted mice and available improved models (e.g. myeloid- or T-cell-only mice, MISTRG, NSG-SGM3). Additionally, we summarize human-tropic viral infections, for which humanized mice offer a novel approach for the study of disease pathogenesis as well as future perspectives for their use in biomedical, drug and vaccine research.

Immune Evasion Strategies during Chronic Hepatitis B and C Virus Infection.

Both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are a major global healthcare problem with more than 240 million and 70 million infected, respectively. Both viruses persist within the liver and result in progressive liver disease, resulting in liver fibrosis, cirrhosis and hepatocellular carcinoma. Strikingly, this pathogenesis is largely driven by immune responses, unable to clear an established infection, rather than by the viral pathogens themselves. Even though disease progression is very similar in both infections, HBV and HCV have evolved distinct mechanisms, by which they ensure persistence within the host. Whereas HCV utilizes a cloak-and-dagger approach, disguising itself as a lipid-like particle and immediately crippling essential pattern-recognition pathways, HBV has long been considered a "stealth" virus, due to the complete absence of innate immune responses during infection. Recent developments and access to improved model systems, however, revealed that even though it is among the smallest human-tropic viruses, HBV may, in addition to evading host responses, employ subtle immune evasion mechanisms directed at ensuring viral persistence in the absence of host responses. In this review, we compare the different strategies of both viruses to ensure viral persistence by actively interfering with viral recognition and innate immune responses.