The Importance of Lassa Fever and Its Disease Management in West Africa.

Lassa virus (LASV) is a zoonotic pathogen endemic throughout western Africa and is responsible for a human disease known as Lassa fever (LF). Historically, LASV has been emphasized as one of the greatest public health threats in West Africa, with up to 300,000 cases and 5000 associated deaths per year. This, and the fact that the disease has been reported in travelers, has driven a rapid production of various vaccine candidates. Several of these vaccines are currently in clinical development, despite limitations in understanding the immune response to infection. Alarmingly, the host immune response has been implicated in the induction of sensorineural hearing loss in LF survivors, legitimately raising safety questions about any future vaccines as well as efficacy in preventing potential hearing loss. The objective of this article is to revisit the importance and prevalence of LF in West Africa, with focus on Nigeria, and discuss current therapeutic approaches and ongoing vaccine development. In addition, we aim to emphasize the need for more scientific studies relating to LF-associated hearing loss, and to promote critical discussion about potential risks and benefits of vaccinating the population in endemic regions of West Africa.

Development of reverse genetics system for Guanarito virus: substitution of E1497K in the L protein of Guanarito virus S-26764 strain changes plaque phenotype and growth kinetics.

Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus Mammarenavirus, family Arenaviridae and has been classified as a Category A bioterrorism agent by the United States Centers for Disease Control and Prevention. Despite being a high-priority agent, vaccines and drugs against Venezuelan hemorrhagic fever are not available. GTOV S-26764, isolated from a non-fatal human case, produces an unclear cytopathic effect (CPE) in Vero cells, posing a significant obstacle to research and countermeasure development efforts. Vero cell-adapted GTOV S-26764 generated in this study produced clear CPE and demonstrated rapid growth and high yield in Vero cells compared to the original GTOV S-26764. We developed a reverse genetics system for GTOV to study amino acid changes acquired through Vero cell adaptation and leading to virus phenotype changes. The results demonstrated that E1497K in the L protein was responsible for the production of clear plaques as well as enhanced viral RNA replication and transcription efficiency. Vero cell-adapted GTOV S-26764, capable of generating CPE, will allow researchers to easily perform neutralization assays and anti-drug screening against GTOV. Moreover, the developed reverse genetics system will accelerate vaccine and antiviral drug development.IMPORTANCEGuanarito virus (GTOV) is a rodent-borne virus. GTOV causes fever, prostration, headache, arthralgia, cough, sore throat, nausea, vomiting, diarrhea, epistaxis, bleeding gums, menorrhagia, and melena in humans. The lethality rate is 23.1% or higher. Vero cell-adapted GTOV S-26764 shows a clear cytopathic effect (CPE), whereas the parental virus shows unclear CPE in Vero cells. We generated a reverse genetics system to rescue recombinant GTOVs and found that E1497K in the L protein was responsible for the formation of clear plaques as well as enhanced viral RNA replication and transcription efficiency. This reverse genetic system will accelerate vaccine and antiviral drug developments, and the findings of this study contribute to the understanding of the function of GTOV L as an RNA polymerase.

Inhibitors of cannabinoid receptor 1 suppress the cellular entry of Lujo virus.

Lujo virus (LUJV), which belongs to Mammarenavirus, family Arenaviridae, has emerged as a pathogen causing severe hemorrhagic fever with high mortality. Currently, there are no effective treatments for arenaviruses, including LUJV. Here, we screened chemical compound libraries of Food and Drug Administration (FDA)-approved drugs and G protein-coupled receptor-associated drugs to identify effective antivirals against LUJV targeting cell entry using a vesicular stomatitis virus-based pseudotyped virus bearing the LUJV envelope glycoprotein (GP). Cannabinoid receptor 1 (CB1) antagonists, such as rimonabant, AM251 and AM281, have been identified as robust inhibitors of LUJV entry. The IC50 of rimonabant was 0.26 and 0.53 µM in Vero and Huh7 cells, respectively. Analysis of the cell fusion activity of the LUJV GP in the presence of CB1 inhibitors revealed that these inhibitors suppressed the fusion activity of the LUJV GP. Moreover, rimonabant, AM251 and AM281 reduced the infectivity of authentic LUJV in vitro, suggesting that the antiviral activity of CB1 antagonists against LUJV is mediated, at least in part, by inhibition of the viral entry, especially, membrane fusion. These findings suggest promising candidates for developing new therapies against LUJV infections.

The roles of XJ13 and XJ44-specific mutations within the Candid #1 GPC in Junin virus attenuation.

Junin virus (JUNV) is a member of the Arenaviridae family of viruses and is the pathogen responsible for causing Argentine hemorrhagic fever, a potentially lethal disease endemic to Argentina. A live attenuated vaccine for human use, called Candid#1, is approved only in Argentina. Candid#1 vaccine strain of Junin virus was obtained through serial passage in mouse brain tissues followed by passage in Fetal Rhesus macaque lung fibroblast (FRhL) cells. Previously, the mutations responsible for attenuation of this virus in Guinea pigs were mapped in the gene encoding for glycoprotein precursor (GPC) protein. The resulting Candid#1 glycoprotein complex has been shown to cause endoplasmic reticulum (ER) stress in vitro resulting in the degradation of the GPC. To evaluate the attenuating properties of specific mutations within GPC, we created recombinant viruses expressing GPC mutations specific to key Candid#1 passages and evaluated their pathogenicity in our outbred Hartley guinea pig model of Argentine hemorrhagic fever. Here, we provide evidence that early mutations in GPC obtained through serial passaging attenuate the visceral disease and increase immunogenicity in guinea pigs. Specific mutations acquired prior to the 13th mouse brain passage (XJ13) are responsible for attenuation of the visceral disease while having no impact on the neurovirulence of Junin virus. Additionally, our findings demonstrate that the mutation within an N-linked glycosylation motif, acquired prior to the 44th mouse brain passage (XJ44), is unstable but necessary for complete attenuation and enhanced immunogenicity of Candid#1 vaccine strain. The highly conserved N-linked glycosylation profiles of arenavirus glycoproteins could therefore be viable targets for designing attenuating viruses for vaccine development against other arenavirus-associated illnesses.

Vaccine Candidates against Arenavirus Infections.

The viral family Arenaviridae contains several members that cause severe, and often lethal, diseases in humans. Several highly pathogenic arenaviruses are classified as Risk Group 4 agents and must be handled in the highest biological containment facility, biosafety level-4 (BSL-4). Vaccines and treatments are very limited for these pathogens. The development of vaccines is crucial for the establishment of countermeasures against highly pathogenic arenavirus infections. While several vaccine candidates have been investigated, there are currently no approved vaccines for arenavirus infection except for Candid#1, a live-attenuated Junin virus vaccine only licensed in Argentina. Current platforms under investigation for use include live-attenuated vaccines, recombinant virus-based vaccines, and recombinant proteins. We summarize here the recent updates of vaccine candidates against arenavirus infections.

Depletion of CD4 and CD8 T Cells Reduces Acute Disease and Is Not Associated with Hearing Loss in ML29-Infected STAT1-/- Mice.

Lassa virus (LASV) is a zoonotic virus endemic to western Africa that can cause a potentially lethal and hemorrhagic disease, Lassa fever (LF). Survivors suffer a myriad of sequelae, most notably sudden onset sensorineural hearing loss (SNHL), the mechanism of which remains unclear. Unfortunately, studies aiming to identify the mechanism of these sequelae are limited due to the biosafety level 4 (BSL4) requirements of LASV itself. ML29, a reassortant virus proposed as an experimental vaccine candidate against LASV, is potentially an ideal surrogate model of LF in STAT1-/- mice due to similar phenotype in these animals. We intended to better characterize ML29 pathogenesis and potential sequelae in this animal model. Our results indicate that while both CD4 and CD8 T cells are responsible for acute disease in ML29 infection, ML29 induces significant hearing loss in a mechanism independent of either CD4 or CD8 T cells. We believe that this model could provide valuable information for viral-associated hearing loss in general.

Macrophage infection, activation, and histopathological findings in ebolavirus infection.

Macrophages contribute to Ebola virus disease through their susceptibility to direct infection, their multi-faceted response to ebolaviruses, and their association with pathological findings in tissues throughout the body. Viral attachment and entry factors, as well as the more recently described influence of cell polarization, shape macrophage susceptibility to direct infection. Moreover, the study of Toll-like receptor 4 and the RIG-I-like receptor pathway in the macrophage response to ebolaviruses highlight important immune signaling pathways contributing to the breadth of macrophage responses. Lastly, the deep histopathological catalogue of macrophage involvement across numerous tissues during infection has been enriched by descriptions of tissues involved in sequelae following acute infection, including: the eye, joints, and the nervous system. Building upon this knowledge base, future opportunities include characterization of macrophage phenotypes beneficial or deleterious to survival, delineation of the specific roles macrophages play in pathological lesion development in affected tissues, and the creation of macrophage-specific therapeutics enhancing the beneficial activities and reducing the deleterious contributions of macrophages to the outcome of Ebola virus disease.

Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies.

Coronavirus disease 2019 (COVID-19) continues to significantly impact the global population, thus countermeasure platforms that enable rapid development of therapeutics against variants of SARS-CoV-2 are essential. We report use of a phage display human antibody library approach to rapidly identify neutralizing antibodies (nAbs) against SARS-CoV-2. We demonstrate the binding and neutralization capability of two nAbs, STI-2020 and STI-5041, against the SARS-CoV-2 WA-1 strain as well as the Alpha and Beta variants. STI-2020 and STI-5041 were protective when administered intravenously or intranasally in the golden (Syrian) hamster model of COVID-19 challenged with the WA-1 strain or Beta variant. The ability to administer nAbs intravenously and intranasally may have important therapeutic implications and Phase 1 healthy subjects clinical trials are ongoing.

Identification of cap-dependent endonuclease inhibitors with broad-spectrum activity against bunyaviruses.

Viral hemorrhagic fevers caused by members of the order Bunyavirales comprise endemic and emerging human infections that are significant public health concerns. Despite the disease severity, there are few therapeutic options available, and therefore effective antiviral drugs are urgently needed to reduce disease burdens. Bunyaviruses, like influenza viruses (IFVs), possess a cap-dependent endonuclease (CEN) that mediates the critical cap-snatching step of viral RNA transcription. We screened compounds from our CEN inhibitor (CENi) library and identified specific structural compounds that are 100 to 1,000 times more active in vitro than ribavirin against bunyaviruses, including Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Junin virus. To investigate their inhibitory mechanism of action, drug-resistant viruses were selected in culture. Whole-genome sequencing revealed that amino acid substitutions in the CEN region of drug-resistant viruses were located in similar positions as those of the CEN α3-helix loop of IFVs derived under drug selection. Thus, our studies suggest that CENi compounds inhibit both bunyavirus and IFV replication in a mechanistically similar manner. Structural analysis revealed that the side chain of the carboxyl group at the seventh position of the main structure of the compound was essential for the high antiviral activity against bunyaviruses. In LCMV-infected mice, the compounds significantly decreased blood viral load, suppressed symptoms such as thrombocytopenia and hepatic dysfunction, and improved survival rates. These data suggest a potential broad-spectrum clinical utility of CENis for the treatment of both severe influenza and hemorrhagic diseases caused by bunyaviruses.

The DNA glycosylase NEIL2 plays a vital role in combating SARS-CoV-2 infection.

Compromised DNA repair capacity of individuals could play a critical role in the severity of SARS-CoV-2 infection-induced COVID-19. We therefore analyzed the expression of DNA repair genes in publicly available transcriptomic datasets of COVID-19 patients and found that the level of NEIL2, an oxidized base specific mammalian DNA glycosylase, is particularly low in the lungs of COVID-19 patients displaying severe symptoms. Downregulation of pulmonary NEIL2 in CoV-2-permissive animals and postmortem COVID-19 patients validated these results. To investigate the potential roles of NEIL2 in CoV-2 pathogenesis, we infected Neil2-null (Neil2-/-) mice with a mouse-adapted CoV-2 strain and found that Neil2-/- mice suffered more severe viral infection concomitant with increased expression of proinflammatory genes, which resulted in an enhanced mortality rate of 80%, up from 20% for the age matched Neil2+/+ cohorts. We also found that infected animals accumulated a significant amount of damage in their lung DNA. Surprisingly, recombinant NEIL2 delivered into permissive A549-ACE2 cells significantly decreased viral replication. Toward better understanding the mechanistic basis of how NEIL2 plays such a protective role against CoV-2 infection, we determined that NEIL2 specifically binds to the 5'-UTR of SARS-CoV-2 genomic RNA and blocks protein synthesis. Together, our data suggest that NEIL2 plays a previously unidentified role in regulating CoV-2-induced pathogenesis, via inhibiting viral replication and preventing exacerbated proinflammatory responses, and also via its well-established role of repairing host genome damage.

CD4 T-cell depletion prevents Lassa fever associated hearing loss in the mouse model.

Lassa virus (LASV) is the causative agent of Lassa fever (LF), which presents as a lethal hemorrhagic disease in severe cases. LASV-induced hearing loss in survivors is a huge socioeconomic burden, however, the mechanism(s) leading to hearing loss is unknown. In this study, we evaluate in a mouse LF model the auditory function using auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) to determine the mechanisms underlying LASV-induced hearing loss. In the process, we pioneered measures of ABR and DPOAE tests in rodents in biosafety level 4 (BSL-4) facilities. Our T cell depletion studies demonstrated that CD4 T-cells play an important role in LASV-induced hearing loss, while CD8 T-cells are critical for the pathogenicity in the acute phase of LASV infection. Results presented in this study may help to develop future countermeasures against acute disease and LASV-induced hearing loss.

Prolonged and extended impacts of SARS-CoV-2 on the olfactory neurocircuit.

The impact of SARS-CoV-2 on the olfactory pathway was studied over several time points using Syrian golden hamsters. We found an incomplete recovery of the olfactory sensory neurons, prolonged activation of glial cells in the olfactory bulb, and a decrease in the density of dendritic spines within the hippocampus. These data may be useful for elucidating the mechanism underlying long-lasting olfactory dysfunction and cognitive impairment as a post-acute COVID-19 syndrome.

Machupo Virus with Mutations in the Transmembrane Domain and Glycosylation Sites of the Glycoprotein Is Attenuated and Immunogenic in Animal Models of Bolivian Hemorrhagic Fever.

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuates MACV in an interferon alpha/beta and gamma (IFN-α/ß and -γ) receptor knockout (R-/-) mouse model. However, some capability to induce neurological pathology still remained. The highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. An F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in the GPC TMD. The MACV mutant is fully attenuated in IFN-α/ß and -γ R-/- mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV completely protected guinea pigs from wild-type MACV lethal challenge. Last, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which provide insight into the rational design of potential vaccine candidates for highly pathogenic arenaviruses. IMPORTANCE For arenaviruses, the only vaccine available is the live attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.

Recovery of anosmia in hamsters infected with SARS-CoV-2 is correlated with repair of the olfactory epithelium.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for a pandemic affecting billions of people worldwide. Apart from the extreme global economic impact, the pandemic will likely have a lasting impact through long-term sequelae not yet fully understood. Fully understanding the mechanisms driving the various symptoms and sequelae of SARS-CoV-2 infection will allow for the eventual development of therapeutics to prevent or treat such life-altering symptoms. In this study, we developed a behavioral test of anosmia in SARS-CoV-2-infected hamsters. We find a moderately strong correlation between the level of anosmia and the score of histological damage within the olfactory epithelium. We also find a moderately strong correlation between the level of anosmia and the thickness of the olfactory epithelium, previously demonstrated to be severely damaged upon infection. Thus, this food-searching behavioral test can act as a simple and effective screening method in a hamster model for various therapeutics for SARS-CoV-2-related anosmia.

Salicylanilides Reduce SARS-CoV-2 Replication and Suppress Induction of Inflammatory Cytokines in a Rodent Model.

SARS-CoV-2 virus has recently given rise to the current COVID-19 pandemic where infected individuals can range from being asymptomatic, yet highly contagious, to dying from acute respiratory distress syndrome. Although the world has mobilized to create antiviral vaccines and therapeutics to combat the scourge, their long-term efficacy remains in question especially with the emergence of new variants. In this work, we exploit a class of compounds that has previously shown success against various viruses. A salicylanilide library was first screened in a SARS-CoV-2 activity assay in Vero cells. The most efficacious derivative was further evaluated in a prophylactic mouse model of SARS-CoV-2 infection unveiling a salicylanilide that can reduce viral loads, modulate key cytokines, and mitigate severe weight loss involved in COVID-19 infections. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and a previously established favorable pharmacokinetic profile for the lead salicylanilide renders salicylanilides in general as promising therapeutics for COVID-19.

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity.

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

Regeneration Profiles of Olfactory Epithelium after SARS-CoV-2 Infection in Golden Syrian Hamsters.

Olfactory dysfunction is one of the most frequent and specific symptoms of coronavirus disease 2019 (COVID-19). Information on the damage and repair of the neuroepithelium and its impact on olfactory function after COVID-19 is still incomplete. While severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes the ongoing worldwide outbreak of COVID-19, little is known about the changes triggered by SARS-CoV-2 in the olfactory epithelium (OE) at the cellular level. Here, we report profiles of the OE after SARS-CoV-2 infection in golden Syrian hamsters, which is a reliable animal model of COVID-19. We observed severe damage in the OE as early as 3 days postinoculation and regionally specific damage and regeneration of the OE within the nasal cavity; the nasal septal region demonstrated the fastest recovery compared to other regions in the nasal turbinates. These findings suggest that anosmia related to SARS-CoV-2 infection may be fully reversible.

Auditory function analysis in immunodeficient STAT1 knock-out mice: Considerations for viral infection models.

The STAT1 knock-out (KO) mouse is a frequently used transgenic immunodeficient strain to model human viral and bacterial diseases. The Lassa fever model was established in the STAT1 KO mice mimicking phenotypes seen in human patients including deafness in survivors. This model develops hearing loss at high prevalence and is a valuable tool to investigate viral infection-induced hearing loss. However, Lassa virus is a highly contagious and regulated agent requiring the unique logistics of the biosafety level 4 posing limitations for experimental work. Therefore, we did a detailed auditory analysis of the STAT1 KO mice to assess baseline auditory function in preparation for further auditory behavioral studies. Auditory brainstem response and distortion product otoacoustic emission tests were performed on males and females of the STAT1 KO mice and was compared to 129S6/SvEv wild type (WT) mice. The male WT mice had the best auditory performance and the female WT mice had the worst hearing performance. The male and female STAT1 KO mice had similar auditory performance to each other, which was intermediate between WT males and females. We conclude that both male and female STAT1 KO mice are suitable for studying viral infection-induced hearing loss.

Receptor-Mediated Host Cell Preference of a Bat-Derived Filovirus, Lloviu Virus.

Lloviu virus (LLOV), a bat-derived filovirus that is phylogenetically distinct from human pathogenic filoviruses such as Ebola virus (EBOV) and Marburg virus (MARV), was discovered in Europe. However, since infectious LLOV has never been isolated, the biological properties of this virus remain poorly understood. We found that vesicular stomatitis virus (VSV) pseudotyped with the glycoprotein (GP) of LLOV (VSV-LLOV) showed higher infectivity in one bat (Miniopterus sp.)-derived cell line than in the other bat-derived cell lines tested, which was distinct from the tropism of VSV pseudotyped with EBOV (VSV-EBOV) and MARV GPs. We then focused on the interaction between GP and Niemann-Pick C1 (NPC1) protein, one of the cellular receptors of filoviruses. We introduced the Miniopterus bat and human NPC1 genes into NPC1-knockout Vero E6 cells and their susceptibilities to the viruses were compared. The cell line expressing the bat NPC1 showed higher susceptibility to VSV-LLOV than that expressing human NPC1, whereas the opposite preference was seen for VSV-EBOV. Using a site-directed mutagenesis approach, amino acid residues involved in the differential tropism were identified in the NPC1 and GP molecules. Our results suggest that the interaction between GP and NPC1 is an important factor in the tropism of LLOV to a particular bat species.

A single mutation (V64G) within the RING Domain of Z attenuates Junin virus.

Junin virus (JUNV) is a New World arenavirus that is the causative agent of Argentine hemorrhagic fever (AHF). Candid#1 (Can) is a live-attenuated vaccine strain of JUNV that since its introduction has resulted in a marked decrease in AHF incidence within the endemic regions of the Pampas in Argentina. Originally, the viral determinants and mechanisms of Can attenuation were not well understood. Recent work has identified the glycoprotein as the major attenuating factor for Can. The establishment of attenuating strategies based on any of the other viral proteins, however, has not been pursued. Here, we document the role of Can Z resulting in incompatibilities with wild type JUNV that results in decreased growth in vitro. In addition, this incompatibility results in attenuation of the virus in the guinea pig model. Further, we identify a single mutation (V64G) in the Z protein that is able to confer this demonstrated attenuation. By establishing and characterizing a novel attenuation strategy for New World mammarenaviruses, we hope to aid future vaccine development for related emerging pathogens including Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV).