An unconventional BST-2 function: down-regulation of transient protein expression.

While viral inhibition by tethering of budding virions to host cell membranes has been focused upon as one of the main functions of BST-2/tetherin, BST-2 is thought to possess other functions as well. Overexpression of BST-2 was found here to down-regulate transient protein expression. Removal of the N- and C-terminal regions of BST-2, previously described to be involved in signal transduction, reduced the impact of BST-2. These results suggest that BST-2-mediated signaling may play a role in regulating the levels of transiently expressed proteins, highlighting a new function for BST-2 that may also have implications for viral inhibition.

Pseudotyped zoonotic thogotoviruses exhibit broad entry range in mammalian cells.

Viruses in the thogotovirus genus of the family Orthomyxoviridae are much less well-understood than influenza viruses despite documented zoonotic transmission and association with human disease. This study therefore developed a cell-cell fusion assay and three pseudotyping tools and used them to assess envelope function and cell tropism. Envelope glycoproteins of Dhori (DHOV), Thogoto (THOV), Bourbon, and Sinu viruses were all revealed to exhibit pH-dependent triggering of membrane fusion. Lentivirus vectors were robustly pseudotyped with these glycoproteins while influenza virus vectors showed pseudotyping compatibility, albeit at lower efficiencies. Replication-competent vesicular stomatitis virus expressing DHOV or THOV glycoproteins were also successfully generated. These pseudotyped viruses mediated entry into a wide range of mammalian cell lines, including human primary cells. The promiscuousness of these viruses suggests the use of a relatively ubiquitous receptor and their entry into numerous mammalian cells emphasize their high potential as veterinary and zoonotic diseases.

Production and Immunogenicity Assessment of LTp50: An Escherichia coli-Made Chimeric Antigen Targeting S1- and S2-Epitopes from the SARS-CoV-2/BA.5 Spike Protein.

Subunit vaccines stand as a leading approach to expanding the current portfolio of vaccines to fight against COVID-19, seeking not only to lower costs but to achieve long-term immunity against variants of concern and have the main attributes that could overcome the limitations of the current vaccines. Herein a chimeric protein targeting S1 and S2 epitopes, called LTp50, was designed as a convenient approach to induce humoral responses against SARS-CoV-2. LTp50 was produced in recombinant Escherichia coli using a conventional pET vector, recovering the expected antigen in the insoluble fraction. LTp50 was purified by chromatography (purity > 90%). The solubilization and refolding stages helped to obtain a stable protein amenable for vaccine formulation. LTp50 was adsorbed onto alum, resulting in a stable formulation whose immunogenic properties were assessed in BALB/c mice. Significant humoral responses against the S protein (BA.5 variant) were detected in mice subjected to three subcutaneous doses (10 µg) of the LTp50/alum formulation. This study opens the path for the vaccine formulation optimization using additional adjuvants to advance in the development of a highly effective anti-COVID-19 vaccine directed against the antigenic regions of the S protein, which are less prone to mutations.

Unveiling the Potent Antiviral and Antioxidant Activities of an Aqueous Extract from Caesalpinia mimosoides Lamk: Cheminformatics and Molecular Docking Approaches.

Our group previously demonstrated that Caesalpinia mimosoides Lamk exhibits many profound biological properties, including anticancer, antibacterial, and antioxidant activities. However, its antiviral activity has not yet been investigated. Here, the aqueous extract of C. mimosoides was prepared from the aerial parts (leaves, stalks, and trunks) to see whether it exerts anti-influenza (H1N1) effects and to reduce the organic solvents consumed during extraction, making it a desirable approach for the large-scale production for medical uses. Our plant extract was quantified to contain 7 g of gallic acid (GA) per 100 g of a dry sample, as determined using HPLC analysis. It also exerts potent antioxidant activities comparable to those of authentic GA. According to untargeted metabolomics (UPLC-ESI(-)-QTOF-MS/MS) with the aid of cheminformatics tools (MetFrag (version 2.1), SIRIUS (version 5.8.3), CSI:FingerID (version 4.8), and CANOPUS), the major metabolite was best annotated as "gallic acid", phenolics (e.g., quinic acid, shikimic acid, and protocatechuic acid), sugar derivatives, and dicarboxylic acids were deduced from this plant species for the first time. The aqueous plant extract efficiently inhibited an influenza A (H1N1) virus infection of MDCK cells with an IC50 of 5.14 µg/mL. Of equal importance, hemolytic activity was absent for this plant extract, signifying its applicability as a safe antiviral agent. Molecular docking suggested that GA interacts with conserved residues (e.g., Arg152 and Asp151) located in the catalytic inner shell of the viral neuraminidase (NA), sharing the same pocket as those of anti-neuraminidase drugs, such as laninamivir and oseltamivir. Additionally, other metabolites were also found to potentially interact with the active site and the hydrophobic 430-cavity of the viral surface protein, suggesting a possibly synergistic effect of various phytochemicals. Therefore, the C. mimosoides aqueous extract may be a good candidate for coping with increasing influenza virus resistance to existing antivirals.

Chimeric Virus-like Particle-Based COVID-19 Vaccine Confers Strong Protection against SARS-CoV-2 Viremia in K18-hACE2 Mice.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.

A live measles-vectored COVID-19 vaccine induces strong immunity and protection from SARS-CoV-2 challenge in mice and hamsters.

Several COVID-19 vaccines have now been deployed to tackle the SARS-CoV-2 pandemic, most of them based on messenger RNA or adenovirus vectors.The duration of protection afforded by these vaccines is unknown, as well as their capacity to protect from emerging new variants. To provide sufficient coverage for the world population, additional strategies need to be tested. The live pediatric measles vaccine (MV) is an attractive approach, given its extensive safety and efficacy history, along with its established large-scale manufacturing capacity. We develop an MV-based SARS-CoV-2 vaccine expressing the prefusion-stabilized, membrane-anchored full-length S antigen, which proves to be efficient at eliciting strong Th1-dominant T-cell responses and high neutralizing antibody titers. In both mouse and golden Syrian hamster models, these responses protect the animals from intranasal infectious challenge. Additionally, the elicited antibodies efficiently neutralize in vitro the three currently circulating variants of SARS-CoV-2.

PEDV and PDCoV Pathogenesis: The Interplay Between Host Innate Immune Responses and Porcine Enteric Coronaviruses.

Enteropathogenic porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the coronavirus family, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. These two viruses pose significant economic and public health burdens, even as both continue to emerge and reemerge worldwide. The ability to evade, circumvent or subvert the host's first line of defense, namely the innate immune system, is the key determinant for pathogen virulence, survival, and the establishment of successful infection. Unfortunately, we have only started to unravel the underlying viral mechanisms used to manipulate host innate immune responses. In this review, we gather current knowledge concerning the interplay between these viruses and components of host innate immunity, focusing on type I interferon induction and signaling in particular, and the mechanisms by which virus-encoded gene products antagonize and subvert host innate immune responses. Finally, we provide some perspectives on the advantages gained from a better understanding of host-pathogen interactions. This includes their implications for the future development of PEDV and PDCoV vaccines and how we can further our knowledge of the molecular mechanisms underlying virus pathogenesis, virulence, and host coevolution.

The avian influenza virus PA segment mediates strain-specific antagonism of BST-2/tetherin.

BST-2 is an antiviral protein described as a powerful cross-species transmission barrier for simian immunodeficiency viruses. Influenza viruses appear to interact with BST-2, raising the possibility that BST-2 may be a barrier for cross-species transmission. An MDCK-based cell line expressing human BST-2 was generated to study human-derived A/Puerto Rico/8/36 (H1N1; PR8) as well as two low pathogenic avian influenza viruses (subtypes H4N6 and H6N1). The H4N6 and H6N1 viruses were less affected by BST-2 expression than PR8, due to their ability to decrease BST-2 levels, a function localized to the PA segment of both avian viruses. Experiments with PA-mutant and -chimeric viruses confirmed that the avian PA segment conferred BST-2 downregulation and antagonism. These results indicate a species-specific ability of PA from low pathogenic avian viruses to mitigate human BST-2 antiviral activity, suggesting that BST-2 is unlikely to be a general cross-species barrier to transmission of such viruses to humans.

Measles-derived vaccines to prevent emerging viral diseases.

Infectious disease epidemics match wars and natural disasters in their capacity to threaten lives and damage economies. Like SARS previously and Zika recently, the Ebola crisis in 2015 showed how vulnerable the world is to these epidemics, with over 11,000 people dying in the outbreak. In addition to causing immense human suffering, these epidemics particularly affect low- and middle-income countries. Many of these deadly infectious diseases that have epidemic potential can become global health emergencies in the absence of effective vaccines. But very few vaccines against these threats have been developed to create proven medical products. The measles vaccine is an efficient, live attenuated, replicating virus that has been safely administered to 2 billion children over the last 40 years, affording life-long protection after a single dose. Taking advantage of these characteristics, this attenuated virus was transformed into a versatile chimeric or recombinant vaccine vector with demonstrated proof-of-principle in humans and a preclinical track record of rapid adaptability and effectiveness for a variety of pathogens. Clinical trials have shown the safety and immunogenicity of this vaccine platform in individuals with preexisting immunity to measles. This review describes the potential of this platform to develop new vaccines against emerging viral diseases.

Single nucleoprotein residue determines influenza A virus sensitivity to an intertypic suppression mechanism.

Several mechanisms underlying intertypic interference between co-infecting influenza types A and B viruses (IAV and IBV) have been proposed. We have recently described one in which IBV's nucleoprotein (BNP) sequestered IAV's nucleoprotein (ANP) and suppressed IAV polymerase and growth. However, its anti-IAV capacity and limitations have not been fully explored. Here, we showed that BNP's inhibitory effect was more potent toward a wide array of avian IAVs, whereas human IAVs revealed moderate resistance. BNP sensitivity was largely determined by ANP's residue 343 at the NP oligomerization interface. An avian IAV polymerase carrying an NP-V343L mutation switched from being highly BNP-sensitive to moderately BNP-resistant, and vice versa for a human IAV polymerase carrying a reverse mutation. To highlight its capacity, we demonstrated that the polymerases of highly-pathogenic H5N1 and the pandemic 2009 (H1N1) strains are strongly inhibited by BNP. Our work provides insights into lineage-specific sensitivity to BNP-mediated intertypic interference.

Deciphering the biology of porcine epidemic diarrhea virus in the era of reverse genetics.

Emergence of the porcine epidemic diarrhea virus (PEDV) as a global threat to the swine industry underlies the urgent need for deeper understanding of this virus. To date, we have yet to identify functions for all the major gene products, much less grasp their implications for the viral life cycle and pathogenic mechanisms. A major reason is the lack of genetic tools for studying PEDV. In this review, we discuss the reverse genetics approaches that have been successfully used to engineer infectious clones of PEDV as well as other potential and complementary methods that have yet to be applied to PEDV. The importance of proper cell culture for successful PEDV propagation and maintenance of disease phenotype are addressed in our survey of permissive cell lines. We also highlight areas of particular relevance to PEDV pathogenesis and disease that have benefited from reverse genetics studies and pressing questions that await resolution by such studies. In particular, we examine the spike protein as a determinant of viral tropism, entry and virulence, ORF3 and its association with cell culture adaptation, and the nucleocapsid protein and its potential role in modulating PEDV pathogenicity. Finally, we conclude with an exploration of how reverse genetics can help mitigate the global impact of PEDV by addressing the challenges of vaccine development.

In Vitro and In Vivo Attenuation of Vesicular Stomatitis Virus (VSV) by Phosphoprotein Deletion.

Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication.

Conserved determinants of enhanced CCR5 binding in the human immunodeficiency virus subtype D envelope third variable loop.

Human immunodeficiency virus 1 subtype D (HIV-1D) contributes to a significant portion of the HIV-1 disease burden in eastern and central Africa, and is associated with more rapid disease progression. Its viral envelope sequences, particularly in the third variable region (V3), are highly divergent from other major subtypes yet have rarely been studied to date. We evaluated the V3 and select bridging sheet residues of the HIV-1D 94UG114 envelope by alanine-scanning mutagenesis to determine the residues involved in CCR5 usage conservation in the face of sequence variability. We found most single alanine mutations capable of abolishing CCR5 binding, suggesting binding contacts that are highly sensitive to mutation. Despite drastic binding defects across the board, most mutants mediated fusion at or near wild-type levels, demonstrating an ability to accommodate changes in CCR5 affinity while maintaining the ability to complete entry. Three of the alanine mutations did not abolish CCR5 binding but rather resulted in enhanced CCR5 binding. The positions of these residues were found to be conserved between strains of two subtypes, revealing similar V3 elements that suggest a conservation of constraints in V3 loop conformation.

Relative concordance of human immunodeficiency virus oligomeric and monomeric envelope in CCR5 coreceptor usage.

A major difference between binding and fusion assays commonly used to study the human immunodeficiency virus (HIV) envelope is the use of monomeric envelope for the former assay and oligomeric envelope for the latter. Due to discrepancies in their readouts for some mutants, envelope regions involved in CCR5 coreceptor usage were systematically studied to determine whether the discordance is due to inherent differences between the two assays or whether it genuinely reflects functional differences at each entry step. By adding the binding inhibitor TAK-779 to delay coreceptor binding kinetics in the fusion assay, the readouts were found comparable between the assays for the mutants analysed in this study. Our finding indicates that monomeric binding reflects oligomeric envelope-CCR5 interaction, thus discordant results between binding and fusion assays do not necessarily indicate differences in coreceptor usage by oligomeric envelope and monomeric gp120.