Hemorrhagic Fever In Humans Research Articles

Targeting n-myristoyltransferases promotes a pan-Mammarenavirus inhibition through the degradation of the Z matrix protein.

Several Old World and New World Mammarenavirus are responsible for hemorrhagic fever in humans. These enveloped viruses have a bi-segmented ambisense RNA genome that encodes four proteins. All Mammarenavirus identified to date share a common dependency on myristoylation: the addition of the C14 myristic acid on the N-terminal G2 residue on two of their proteins. The myristoylation of the Z matrix protein is required for viral particle budding, while the myristoylation of the signal peptide to the envelope glycoproteins is important for the entry mechanism. Using Mopeia virus as a model, we characterized the interaction of the Z matrix protein with the N-Myristoyltransferases (NMT) 1 and 2, the two enzymes responsible for myristoylation in mammals. While both enzymes were capable to interact with Z, we showed that only NMT1 was important for the production of viral progeny, the endogenous expression of NMT2 being insufficient to make up for NMT1 in its absence. Using the high affinity inhibitors of NMTs, IMP1088 and DDD85646, we demonstrated a strong, dose dependent and specific inhibition at the nanomolar range for all Mammarenavirus tested, including the highly pathogenic Lassa, Machupo, Junin and Lujo viruses. Mechanistically, IMP1088 and DDD85646 blocked the interaction between Z and both NMTs, preventing myristoylation and further viral particle formation, egress and spread. Unexpectedly, we found that the matrix protein devoid of myristate, despite being fully translated, did not accumulate as the other viral proteins in infected cells but was instead degraded in a proteasome- and autophagy-independent manner. These molecules represent a new broad-spectrum class of inhibitors against Mammarenavirus.

Cryo-EM structure of single-layered nucleoprotein-RNA complex from Marburg virus

Marburg virus (MARV) causes lethal hemorrhagic fever in humans, posing a threat to global health. We determined by cryogenic electron microscopy (cryo-EM) the MARV helical ribonucleoprotein (RNP) complex structure in single-layered conformation, which differs from the previously reported structure of a double-layered helix. Our findings illuminate novel RNP interactions and expand knowledge on MARV genome packaging and nucleocapsid assembly, both processes representing attractive targets for the development of antiviral therapeutics against MARV disease.

Characterization of non-standard viral genomes during arenavirus infections identifies prominent S RNA intergenic region deletions.

Arenaviruses, a family of negative-sense RNA viruses spread by rodents, are a leading cause of severe hemorrhagic fever in humans. Due to a paucity of antivirals and vaccines for arenaviruses, there is a need to identify new mechanisms for interfering with arenavirus replication. In several negative-sense RNA viruses, natural viral interference results from the production of non-standard viral genomes (nsVGs) that activate the innate immune system and/or compete for essential viral products. Although it is well established that arenaviruses produce strong interfering activities, it is unknown if they produce interfering nsVGs. Here, we show that arenaviruses produce deletions within the intergenic region of their small (S) RNA genome, and these deletions inhibit viral glycoprotein production during minigenome replication. S RNA deletions are more abundant when arenaviruses are grown in high-interfering conditions and are associated with reduced viral replication. Overall, we found that arenaviruses produce internal deletions within the S RNA intergenic region that are capable of decreasing glycoprotein production. These natural arenavirus interfering molecules provide a new target for the generation of therapeutics against arenaviruses.IMPORTANCEArenaviruses are hemorrhagic fever-causing pathogens that infect millions of people a year. There are currently no approved antivirals that target arenaviruses, and understanding natural mechanisms that inhibit arenavirus replication is crucial for the development of effective therapeutics. Here, we identified multiple deletions within arenavirus genomes that remove major replicative elements of the viral genomes. We show that deletions that remove the intergenic region of the viral genome can prevent viral protein production. These deletions were found in all arenaviruses tested in this study representing a mechanism that could be harnessed for the development of antivirals that broadly target the arenavirus family.

Immunogenicity of various variants of Ebola and Marburg virus glycoprotein genes in recombinant adenoviral vectors

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID50 assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

A Lipid Nanoparticle-Formulated Self-Amplifying RNA Rift Valley Fever Vaccine Induces a Robust Humoral Immune Response in Mice.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis that causes high fetal and neonatal mortality rates in ruminants and sometimes severe to fatal complications like encephalitis and hemorrhagic fever in humans. There is no licensed RVF vaccine for human use while approved livestock vaccines have suboptimal safety or efficacy. We designed self-amplifying RNA (saRNA) RVF vaccines and assessed their humoral immunogenicity in mice. Plasmid DNA encoding the Rift Valley fever virus (RVFV) medium (M) segment consensus sequence (WT consensus) and its derivatives mutated to enhance cell membrane expression of the viral surface glycoproteins n (Gn) and c (Gc) were assessed for in vitro expression. The WT consensus and best-expressing derivative (furin-T2A) were cloned into a Venezuelan equine encephalitis virus (VEEV) plasmid DNA replicon and in vitro transcribed into saRNA. The saRNA was formulated in lipid nanoparticles and its humoral immunogenicity in BALB/c mice was assessed. High quantities of dose-dependent RVFV Gn IgG antibodies were detected in the serum of all mice immunized with either WT consensus or furin-T2A saRNA RVF vaccines. Significant RVFV pseudovirus-neutralizing activity was induced in mice immunized with 1 µg or 10 µg of the WT consensus saRNA vaccine. The WT consensus saRNA RVF vaccine warrants further development.

A Longitudinal Analysis of Memory Immune Responses in Convalescent Crimean-Congo Hemorrhagic Fever Survivors in Uganda.

Evaluating the adaptive immune responses to natural infection with Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) in human survivors is critical to the development of medical countermeasures. However, the correlates of protection are unknown. As the most prevalent tick-borne human hemorrhagic fever virus with case fatality rates of 5%-30% and worldwide distribution, there is an urgent need to fill these knowledge gaps. Here, we describe adaptive immune responses in a cohort of Ugandan CCHF survivors via serial sampling over 6 years. We demonstrate persistent antibodies after infection and cross-neutralization against various clades of authentic CCHFV, as well as potent effector function. Moreover, we show for the first time persistent, polyfunctional antigen-specific memory T-cell responses to multiple CCHFV proteins up to 9 years after infection. Together, this data provides immunological benchmarks for evaluating CCHFV medical countermeasures and information that can be leveraged toward vaccine immunogen design and viral target identification for monoclonal antibody therapies.

Non-Ebola Filoviruses: Potential Threats to Global Health Security.

Filoviruses are negative-sense single-stranded RNA viruses often associated with severe and highly lethal hemorrhagic fever in humans and nonhuman primates, with case fatality rates as high as 90%. Of the known filoviruses, Ebola virus (EBOV), the prototype of the genus Orthoebolavirus, has been a major public health concern as it frequently causes outbreaks and was associated with an unprecedented outbreak in several Western African countries in 2013-2016, affecting 28,610 people, 11,308 of whom died. Thereafter, filovirus research mostly focused on EBOV, paying less attention to other equally deadly orthoebolaviruses (Sudan, Bundibugyo, and Taï Forest viruses) and orthomarburgviruses (Marburg and Ravn viruses). Some of these filoviruses have emerged in nonendemic areas, as exemplified by four Marburg disease outbreaks recorded in Guinea, Ghana, Tanzania, and Equatorial Guinea between 2021 and 2023. Similarly, the Sudan virus has reemerged in Uganda 10 years after the last recorded outbreak. Moreover, several novel bat-derived filoviruses have been discovered in the last 15 years (Lloviu virus, Bombali virus, Měnglà virus, and Dehong virus), most of which are poorly characterized but may display a wide host range. These novel viruses have the potential to cause outbreaks in humans. Several gaps are yet to be addressed regarding known and emerging filoviruses. These gaps include the virus ecology and pathogenicity, mechanisms of zoonotic transmission, host range and susceptibility, and the development of specific medical countermeasures. In this review, we summarize the current knowledge on non-Ebola filoviruses (Bombali virus, Bundibugyo virus, Reston virus, Sudan virus, Tai Forest virus, Marburg virus, Ravn virus, Lloviu virus, Měnglà virus, and Dehong virus) and suggest some strategies to accelerate specific countermeasure development.

Design of a novel multi-epitope vaccine against Marburg virus using immunoinformatics studies

Marburg virus (MARV) is a highly contagious and virulent agent belonging to Filoviridae family. MARV causes severe hemorrhagic fever in humans and non-human primates. Owing to its highly virulent nature, preventive approaches are promising for its control. There is currently no approved drug or vaccine against MARV, and management mainly involves supportive care to treat symptoms and prevent complications. Our aim was to design a novel multi-epitope vaccine (MEV) against MARV using immunoinformatics studies. In this study, various proteins (VP35, VP40 and glycoprotein precursor) were used and potential epitopes were selected. CTL and HTL epitopes covered 79.44% and 70.55% of the global population, respectively. The designed MEV construct was stable and expressed in Escherichia coli (E. coli) host. The physicochemical properties were also acceptable. MARV MEV candidate could predict comprehensive immune responses such as those of humoral and cellular in silico. Additionally, efficient interaction to toll-like receptor 3 (TLR3) and its agonist (β-defensin) was predicted. There is a need for validation of these results using further in vitro and in vivo studies.

Development of epitope-based vaccine to prevent Marburg virus infection: an in silico approach

Marburg virus (MARV) is one of the deadliest zoonotic viruses, causing severe hemorrhagic fever in humans with high mortality rates. The development of an effective vaccine is crucial to prevent potential Marburg virus outbreaks. In this study, an in silico approach was employed to design an epitope-based vaccine to prevent MARV infections. The MARV proteins nominating NP, VP24, VP35, VP30, VP40, GP & Polymerase L was analyzed for antigenicity and non-allergenicity prediction, among these proteins VP30 protein has a 0.5636 (Probable Antigen) score and it was non-allergen. For that reason, VP30 was selected for further in silico analysis. After analysis it is found that the top ranked T–cell (MHC-I) epitopes LSKPPPPPK, ESSPTNHIPR, TQLPSKPHY, SPQDCGSPSL, FEAALWQGW, T-Cell (MHC-II) epitopes IHLDKGGQF, INTMTELHM, VTPTIYHET, YTNYHPRAR, YTGIHLDKG was epitopes & B-Cell epitopes SEIGKLDET, IHLDKGGQF, MNHENLPQDQNGV, PTCNRDHDLDNLTN was found non-toxic and non-allergen. The T-Cell (MHC-I)epitope TQLPSKPHY,T-Cell (MHC-II)epitope YTNYHPRAR & B-Cell epitope SEIGKLDET was found highly antigenic, non-toxic as well as non-allergen and it was selected for molecular docking analysis. The T-Cell (MHC-I) epitope TQLPSKPHY,T-Cell (MHC-II)epitope YTNYHPRAR shows strong structural similarity and potential binding affinity with antibody. The B-Cell epitope SEIGKLDET shows poor affinity towards antibody. In silico analysis indicate that both T-Cell epitopes becomes an effective peptide vaccine to prevent MARV infection. Our findings highlight the promise of in silico vaccine design in accelerating the development of vaccines against MARV, a highly pathogenic virus with no effective cure currently available

Filoviridae: insights into immune responses to Ebola virus

Ebola virus (EBOV) is a zoonotic virus comprising of six known different species, designated within the family Filoviridae and genus Ebolavirus. The first recorded outbreak of an EBOV disease (EVD) was in Yambuku, Zaire EBOV (ZEBOV) in 1976, followed by the Sudan EBOV (SUDV) later that year. Outbreaks have been increasing throughout the 21st century, and mortality rates can reach up to 90%. Such extraordinary virulence is evidenced by a few pathogens, similar to the Marburg virus (MARV) that originated in Uganda and was first detected in Germany in 1967. The virulent nature of filovirus disease has established these related viruses as a formidable global concern. There are currently four types of Ebolaviridae species known to infect humans, with two more recently identified in other animals that are genomically different concerning cellular pathogenesis or aetiology of disease. Recent advances in understanding the pathogenesis of filovirus disease infections have been remarkable, yet the immunological response to filovirus infection remains unknown. Scientific analysis of cellular mechanisms can provide insight into virulence factors utilised by other pathogenic viruses that also cause febrile illness with occasional haemorrhagic fever in humans. In this review, a brief summary of EBOV protein structure and functional cellular effects is covered. The role of innate and adaptive immune cells known since 1976 is considered with the relevance and implications of immunological proteins measured by cluster of differentiation (CD) molecule, alongside cytokine, chemokine, and other biologically relevant pathways, and through genetic research. A thorough understanding of immunological correlates affecting host responses to EBOV will facilitate clinical and applied research knowledge, contributing to protection against potential public health threats.

Entry inhibitors as arenavirus antivirals.

Arenaviruses belonging to the Arenaviridae family, genus mammarenavirus, are enveloped, single-stranded RNA viruses primarily found in rodent species, that cause severe hemorrhagic fever in humans. With high mortality rates and limited treatment options, the search for effective antivirals is imperative. Current treatments, notably ribavirin and other nucleoside inhibitors, are only partially effective and have significant side effects. The high lethality and lack of treatment, coupled with the absence of vaccines for all but Junín virus, has led to the classification of these viruses as Category A pathogens by the Centers for Disease Control (CDC). This review focuses on entry inhibitors as potential therapeutics against mammarenaviruses, which include both New World and Old World arenaviruses. Various entry inhibition strategies, including small molecule inhibitors and neutralizing antibodies, have been explored through high throughput screening, genome-wide studies, and drug repurposing. Notable progress has been made in identifying molecules that target receptor binding, internalization, or fusion steps. Despite promising preclinical results, the translation of entry inhibitors to approved human therapeutics has faced challenges. Many have only been tested in in vitro or animal models, and a number of candidates showed efficacy only against specific arenaviruses, limiting their broader applicability. The widespread existence of arenaviruses in various rodent species and their potential for their zoonotic transmission also underscores the need for rapid development and deployment of successful pan-arenavirus therapeutics. The diverse pool of candidate molecules in the pipeline provides hope for the eventual discovery of a broadly effective arenavirus antiviral.

Mapping knowledge landscapes and emerging trends of Marburg virus: A text-mining study

BackgroundMarburg virus (MARV), a close relative of Ebola virus, could induce hemorrhagic fevers in humans with high mortality rate. In recent years, increasing attention has been paid to this highly lethal virus due to sporadic outbreaks observed in various African nations. This bibliometric analysis endeavors to elucidate the trends, dynamics, and focal points of knowledge that have delineated the landscape of research concerning MARV. MethodsRelevant literature on MARV from 1968 to 2023 was extracted from the Web of Science Core Collection database. Following this, the data underwent bibliometric analysis and visualization procedures utilizing online analysis platform, CiteSpace 6.2R6, and VOSviewer 1.6.20. Three different types of bibliometric indicators including quantitative indicator, qualitative indicators, and structural indicators were used to gauge a researcher's productivity, assess the quality of their work, and analyze publication relationships, respectively. ResultsMARV is mainly prevalent in Africa. And approximately 643 confirmed cases have been described in the literature to date, and mortality observed was 81.2 % in overall patients. A total of 1014 papers comprising 869 articles and 145 reviews were included. The annual publications showed an increasing growth pattern from 1968 to 2023 (R2 = 0.8838). The United States stands at the forefront of this discipline, having dedicated substantial financial and human resources to scientific inquiry. However, co-authorship analysis showed the international research collaboration needs to be further strengthened. Based on reference and keywords analysis, contemporary MARV research encompasses pivotal areas: primarily, prioritizing the creation of prophylactic vaccines to impede viral spread, and secondarily, exploring targeted antiviral strategies, including small-molecule antivirals or MARV-specific monoclonal antibodies. Additionally, a comprehensive grasp of viral transmission, transcription, and replication mechanisms remains a central focus in ongoing investigations. And future MARV studies are expected to focus on evaluating clinical trial safety and efficacy, developing inhibitors to contain viral spread, exploring vaccine immunogenicity, virus-host association studies, and elucidating the role of neutralizing antibodies in MARV treatment. ConclusionThe present study offered comprehensive insights into the contemporary status and trajectories of MARV over the past decades. This enables researchers to discern novel collaborative prospects, institutional partnerships, emerging topics, and research forefronts within this domain.

A novel MARV glycoprotein-specific antibody with potentials of broad-spectrum neutralization to filovirus

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

A novel MARV glycoprotein-specific antibody with potentials of broad-spectrum neutralization to filovirus.

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

A Review on Crimean-Congo Hemorrhagic Fever Infections in Tunisia.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic tick-borne disease, caused by an arbovirus of the genus Orthonairovirus and the family Nairoviridae. Crimean-Congo hemorrhagic fever virus (CCHFV) is widespread in several regions of the world. While the virus is not pathogenic to all susceptible livestock and wild mammals, it can lead to severe hemorrhagic fever in humans. In this review, we compiled published data on CCHFV infections in humans, animals, and ticks in Tunisia. Based on that, we discussed the epidemiology and the distribution patterns of CCHFV infections highlighting the risk factors for this virus in the country. CCHF infection prevalence in humans was estimated to 2.76% (5/181) and 5% (2/38) in Tunisian febrile patients and Tunisian slaughterhouse workers, respectively. Concurrently, seroprevalence in domestic ungulates (sheep, goats, cattle, and dromedaries) ranged from zero to 89.7%, and only one Hyalomma impeltatum tick specimen collected from dromedaries in southern Tunisian was positive for CCHFV by reverse transcriptase-polymerase chain reaction (0.6%; 1/165). As Tunisian studies on CCHFV are geographically scattered and limited due to very small sample sizes, further studies are needed to improve the knowledge on the epidemiology of CCHF in Tunisia.

Interaction between the SFTSV envelope glycoprotein Gn and STING inhibits the formation of the STING-TBK1 complex and suppresses the NF-κB signaling pathway.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly discovered virus associated with severe hemorrhagic fever in humans. However, the role of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway during SFTSV infection is still unclear. We found that SFTSV infection inhibits downstream innate immune signaling pathways by activating the cytoplasmic DNA receptor cGAS. In addition, SFTSV Gn blocked the nuclear accumulation of interferon regulatory factor 3 and p65 to inhibit downstream innate immune signaling. Moreover, we determined that Gn of SFTSV inhibited K27-ubiquitin modification of STING to disrupt the assembly of the STING-TANK-binding kinase 1 complex and downstream signaling. We found that the SFTSV envelope glycoprotein Gn is a potent inhibitor of the cGAS-STING pathway. In conclusion, this study highlights the crucial function of the glycoprotein Gn in the antiviral innate immune response and reveals a new method of immune escape of SFTSV.

Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

An atlas of gross and histologic lesions and immunohistochemical immunoreactivity during the temporal progression of aerosolized Lassa virus induced hemorrhagic fever in cynomolgus macaques.

Lassa virus (LASV) causes an acute multisystemic hemorrhagic fever in humans known as Lassa fever, which is endemic in several African countries. This manuscript focuses on the progression of disease in cynomolgus macaques challenged with aerosolized LASV and serially sampled for the development and progression of gross and histopathologic lesions. Gross lesions were first noted in tissues on day 6 and persisted throughout day 12. Viremia and histologic lesions were first noted on day 6 commencing with the pulmonary system and hemolymphatic system and progressing at later time points to include all systems. Immunoreactivity to LASV antigen was first observed in the lungs of one macaque on day 3 and appeared localized to macrophages with an increase at later time points to include immunoreactivity in all organ systems. Additionally, this manuscript will serve as a detailed atlas of histopathologic lesions and disease progression for comparison to other animal models of aerosolized Arenaviral disease.

Design of universal Ebola virus vaccine candidates via immunofocusing

The Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV)-one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here, we describe a universal Ebola virus vaccine approach using a structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

Cellular Immune Responses Against γ-Inactivated Antigen in the Recovered Cases of Kyasanur Forest Disease.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne flavivirus that causes life-threatening hemorrhagic fever in humans with case fatality rates of 3-5%. Relatively little is known about the mechanism of its pathogenesis or host immune responses to KFDV infection. Here, we investigated KFDV-specific cellular immune responses in the recovered cases of Kyasanur Forest Disease (KFD). Peripheral blood mononuclear cells of the recovered KFD cases and healthy controls were exposed to γ-inactivated KFDV antigen ex vivo. The proliferation index was determined using an enzyme-linked immunosorbent assay-based lymphoproliferative assay. The frequencies of CD4+ and CD8+ T cells expressing intracellular interferon (IFN)-γ in response to stimulation with γ-inactivated KFDV antigen were determined using flow cytometry. A significant increase in lymphoproliferation and a high frequency of CD4+ and CD8+ T cells secreting IFN-γ against γ-inactivated KFDV antigen were found in the recovered KFD group compared to the healthy control group. In conclusion, the study indicated the generation of cellular immune responses in individuals who recovered from KFD and can be used as indicators of cellular immunity in KFD vaccine studies.