Development of an imaging system for visualization of Ebola virus glycoprotein throughout the viral lifecycle

Ebola virus (EBOV) causes severe EBOV disease (EVD) in humans and non-human primates. Currently, limited countermeasures are available, and the virus must be studied in biosafety level-4 (BSL-4) laboratories. EBOV glycoprotein (GP) is a single transmembrane protein responsible for entry into host cells and is the target of multiple approved drugs. However, the molecular mechanisms underlying the intracellular dynamics of GP during EBOV lifecycle are poorly understood. In this study, we developed a novel GP monitoring system using transcription- and replication-competent virus-like particles (trVLPs) that enables the modeling of the EBOV lifecycle under BSL-2 conditions. We constructed plasmids to generate trVLPs containing the coding sequence of EBOV GP, in which the mucin-like domain (MLD) was replaced with fluorescent proteins. The generated trVLP efficiently replicated over multiple generations was similar to the wild type trVLP. Furthermore, we confirmed that the novel trVLP system enabled real-time visualization of GP throughout the trVLP replication cycle and exhibited intracellular localization similar to that of wild type GP. In summary, this novel monitoring system for GP will enable the characterization of the molecular mechanism of the EBOV lifecycle and can be applied for the development of therapeutics against EVD

Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability

Development of an Enzyme-Linked Immunosorbent Assay to Determine the Expression Dynamics of Ebola Virus Soluble Glycoprotein during Infection

Ebola virus (EBOV) is a highly pathogenic virus with human case fatality rates of up to 90%. EBOV uses transcriptional editing to express three different glycoproteins (GPs) from its GP gene: soluble GP (sGP), GP, and small sGP (ssGP). The molecular ratio of unedited to edited mRNA is about 70% (sGP): 25% (GP): 5% (ssGP), indicating that sGP is produced more abundantly than GP. While the presence of sGP has been confirmed in the blood during human EBOV infection, there is no report about its expression dynamics. In this study, we developed an EBOV-sGP-specific sandwich enzyme-linked immunosorbent assay (ELISA) using two different available antibodies and tested several animal serum samples to determine the concentration of sGP. EBOV-sGP was detected in nonhuman primate serum samples as early as 4 days after EBOV infection, correlating with RT-qPCR positivity. This ELISA might be further developed into a diagnostic tool for detection of EBOV in patients. Furthermore, this study provides insights into the expression dynamics of sGP during infection, which are important to decipher the function that sGP plays during infection

A complement component C1q-mediated mechanism of antibody-dependent enhancement of Ebola virus infection

Besides the common Fc receptor (FcR)-mediated mechanism of antibody-dependent enhancement (ADE), Ebola virus (EBOV) is known to utilize the complement component C1q for ADE of infection. This mechanism is FcR-independent and mediated by cross-linking of virus-antibody-C1q complexes to cell surface C1q receptors, leading to enhanced viral entry into cells. Using confocal microscopy, we found that virus-like particles (VLPs) consisting of EBOV glycoprotein, nucleoprotein, and matrix protein attached to the surface of human kidney 293 cells more efficiently in the presence of an ADE monoclonal antibody and C1q than with the antibody or C1q alone, and that there was no significant difference in the efficiency of VLP uptake into endosomes between the C1q-mediated ADE and non-ADE entry. Accordingly, both ADE and non-ADE infection were similarly decreased by inhibitors of the signaling pathways known to be required for endocytosis. These results suggest that C1q-mediated ADE of EBOV infection is simply caused by increased attachment of virus particles to the cell surface, which is distinct from the mechanism of FcR-mediated ADE requiring intracellular signaling to promote phagocytosis/macropinocytosis. Author summary Ebola virus (EBOV) utilizes the complement component C1q for antibody-dependent enhancement (ADE) of infection. We found that an ADE antibody increased viral attachment in the presence of C1q and that there was no significant difference in the efficiency of viral uptake into endosomes between the C1q-mediated ADE and non-ADE entry. Accordingly, both ADE and non-ADE infection were similarly decreased by inhibitors of the signaling pathways for endocytosis. These results suggest that C1q-mediated ADE of EBOV infection is simply caused by increased viral attachment to the cell surface, most likely via cross-linking of virus-antibody-C1q complexes to cellular C1q receptors

Ebola Virus Glycoprotein Domains Associated with Protective Efficacy

Ebola virus (EBOV) is the cause of sporadic outbreaks of human hemorrhagic disease in Africa, and the best-characterized virus in the filovirus family. The West African epidemic accelerated the clinical development of vaccines and therapeutics, leading to licensure of vaccines and antibody-based therapeutics for human use in recent years. The most widely used vaccine is based on vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) (VSV-EBOV). Due to its favorable immune cell targeting, this vaccine has also been used as a base vector for the development of second generation VSV-based vaccines against Influenza, Nipah, and Zika viruses. However, in these situations, it may be beneficial if the immunogenicity against EBOV GP is minimized to induce a better protective immune response against the other foreign immunogen. Here, we analyzed if EBOV GP can be truncated to be less immunogenic, yet still able to drive replication of the vaccine vector. We found that the EBOV GP glycan cap and the mucin-like domain are both dispensable for VSV-EBOV replication. The glycan cap, however, appears critical for mediating a protective immune response against lethal EBOV challenge in mice

The vesicular stomatitis virus-based Ebola virus vaccine: From concept to clinical trials

The devastating Ebola virus (EBOV) epidemic in West Africa in 2013–2016 accelerated the progress of several vaccines and antivirals through clinical trials, including the replication-competent vesicular stomatitis virus-based vaccine expressing the EBOV glycoprotein (VSV-EBOV). Extensive preclinical testing in animal models demonstrated the prophylactic and post-exposure efficacy of this vaccine, identified the mechanism of protection, and suggested it was safe for human use. Based on these data, VSV-EBOV was extensively tested in phase 1–3 clinical trials in North America, Europe and Africa. Although some side effects of vaccination were observed, these clinical trials showed that the VSV-EBOV was safe and immunogenic in humans. Moreover, the data supported the use of VSV-EBOV as an emergency vaccine in individuals at risk for Ebola virus disease. In this review, we summarize the results of the extensive preclinical and clinical testing of the VSV-EBOV vaccine

Discovery of an antibody for pan-ebolavirus therapy

During the latest outbreak of Ebola virus disease in West Africa, monoclonal antibody therapy (e.g., ZMapp) was utilized to treat patients. However, due to the antigenic differences among the five ebolavirus species, the current therapeutic monoclonal antibodies are only effective against viruses of the species Zaire ebolavirus. Although this particular species has indeed caused the majority of human infections in Central and, recently, West Africa, other ebolavirus species (e.g., Sudan ebolavirus and Bundibugyo ebolavirus) have also repeatedly caused outbreaks in Central Africa and thus should not be neglected in the development of countermeasures against ebolaviruses. Here we report the generation of an ebolavirus glycoprotein-specific monoclonal antibody that effectively inhibits cellular entry of representative isolates of all known ebolavirus species in vitro and show its protective efficacy in mouse models of ebolavirus infections. This novel neutralizing monoclonal antibody targets a highly conserved internal fusion loop in the glycoprotein molecule and prevents membrane fusion of the viral envelope with cellular membranes. The discovery of this highly cross-neutralizing antibody provides a promising option for broad-acting ebolavirus antibody therapy and will accelerate the design of improved vaccines that can selectively elicit cross-neutralizing antibodies against multiple species of ebolaviruses