Molecular characterization of the 2022 Sudan virus disease outbreak in Uganda.

IMPORTANCE: Ebola disease (EBOD) is a public health threat with a high case fatality rate. Most EBOD outbreaks have occurred in remote locations, but the 2013-2016 Western Africa outbreak demonstrated how devastating EBOD can be when it reaches an urban population. Here, the 2022 Sudan virus disease (SVD) outbreak in Mubende District, Uganda, is summarized, and the genetic relatedness of the new variant is evaluated. The Mubende variant exhibited 96% amino acid similarity with historic SUDV sequences from the 1970s and a high degree of conservation throughout the outbreak, which was important for ongoing diagnostics and highly promising for future therapy development. Genetic differences between viruses identified during the Mubende SVD outbreak were linked with epidemiological data to better interpret viral spread and contact tracing chains. This methodology should be used to better integrate discrete epidemiological and sequence data for future viral outbreaks.

A public, cross-reactive glycoprotein epitope confounds Ebola virus serology.

Ebola disease (EBOD) in humans is a severe disease caused by at least four related viruses in the genus Orthoebolavirus, most often by the eponymous Ebola virus. Due to human-to-human transmission and incomplete success in treating cases despite promising therapeutic development, EBOD is a high priority in public health research. Yet despite almost 50 years since EBOD was first described, the sources of these viruses remain undefined and much remains to be understood about the disease epidemiology and virus emergence and spread. One important approach to improve our understanding is detection of antibodies that can reveal past human infections. However, serosurveys routinely describe seroprevalences that imply infection rates much higher than those clinically observed. Proposed hypotheses to explain this difference include existence of common but less pathogenic strains or relatives of these viruses, misidentification of EBOD as something else, and a higher proportion of subclinical infections than currently appreciated. The work presented here maps B-cell epitopes in the spike protein of Ebola virus and describes a single epitope that is cross-reactive with an antigen seemingly unrelated to orthoebolaviruses. Antibodies against this epitope appear to explain most of the unexpected reactivity towards the spike, arguing against common but unidentified infections in the population. Importantly, antibodies of cross-reactive donors from within and outside the known EBOD geographic range bound the same epitope. In light of this finding, it is plausible that epitope mapping enables broadly applicable specificity improvements in the field of serology.

Lassa Virus Replicon Particle Vaccine Protects Strain 13/N Guinea Pigs Against Challenge With Geographically and Genetically Diverse Viral Strains.

Lassa virus (LASV) causes mild to severe hemorrhagic fever disease in humans. Strain 13/N guinea pigs are highly susceptible to infection with LASV strain Josiah (clade IV), providing a critical model system for therapeutics and vaccine development. To develop additional models of disease, we detail the clinical course in guinea pigs infected with 5 geographically and genetically diverse LASV strains. Two of the developed models (LASV clades II and III) were then used to evaluate efficacy of a virus replicon particle vaccine against heterologous LASV challenge, demonstrating complete protection against clinical disease after a single vaccination dose.

Antibody-Mediated Virus Neutralization Is Not a Universal Mechanism of Marburg, Ebola, or Sosuga Virus Clearance in Egyptian Rousette Bats.

Although bats are increasingly being recognized as natural reservoir hosts of emerging zoonotic viruses, little is known about how they control and clear virus infection in the absence of clinical disease. Here, we test >50 convalescent sera from Egyptian rousette bats (ERBs) experimentally primed or prime-boosted with Marburg virus, Ebola virus, or Sosuga virus for the presence of virus-specific neutralizing antibodies, using infectious reporter viruses. After serum neutralization testing, we conclude that antibody-mediated virus neutralization does not contribute significantly to the control and clearance of Marburg virus, Ebola virus, or Sosuga virus infection in ERBs.

Protection From Lethal Lassa Disease Can Be Achieved Both Before and After Virus Exposure by Administration of Single-Cycle Replicating Lassa Virus Replicon Particles.

Lassa fever is a frequently severe human disease that is endemic to several countries in West Africa. To date, no licensed vaccines are available to prevent Lassa virus (LASV) infection, even though Lassa fever is thought to be an important disease contributing to mortality and both acute and chronic morbidity. We have previously described a vaccine candidate composed of single-cycle LASV replicon particles (VRPs) and a stable cell line for their production. Here, we refine the genetic composition of the VRPs and demonstrate the ability to reproducibly purify them with high yields. Studies in the guinea pig model confirm efficacy of the vaccine candidate, demonstrate that single-cycle replication is necessary for complete protection by the VRP vaccine, and show that postexposure vaccination can confer protection from lethal outcome.

Lassa Virus Targeting of Anterior Uvea and Endothelium of Cornea and Conjunctiva in Eye of Guinea Pig Model.

Lassa virus (LASV), a hemorrhagic fever virus endemic to West Africa, causes conjunctivitis in patients with acute disease. To examine ocular manifestations of LASV, we histologically examined eyes from infected guinea pigs. In fatal disease, LASV immunostaining was most prominent in the anterior uvea, especially in the filtration angle, ciliary body, and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endothelial marker (platelet endothelial cell adhesion molecule). Antigen was primarily associated with infiltration of T-lymphocytes around vessels in the anterior uvea and with new vessel formation at the peripheral cornea. In animals that exhibited clinical signs but survived infection, eyes had little to no inflammation and no LASV immunostaining 6 weeks after infection. Overall, in this model, LASV antigen was restricted to the anterior uvea and was associated with mild chronic inflammation in animals with severe disease but was not detected in survivors.

Use of a Scalable Replicon-Particle Vaccine to Protect Against Lethal Lassa Virus Infection in the Guinea Pig Model.

Lassa fever is a viral zoonosis that can be transmitted from person to person, especially in the hospital setting. The disease is endemic to several countries in West Africa and can be a major contributor to morbidity and mortality in affected areas. There are no approved vaccines to prevent Lassa virus infection. In this work, we present a vaccine candidate that combines the scalability and efficacy benefits of a live vaccine with the safety benefits of single-cycle replication. The system consists of Lassa virus replicon particles devoid of the virus essential glycoprotein gene, and a cell line that expresses the glycoprotein products, enabling efficient vaccine propagation. Guinea pigs vaccinated with these particles showed no clinical reaction to the inoculum and were protected against fever, weight loss, and lethality after infection with Lassa virus.

Persistence of Ebola Virus in Ocular Fluid during Convalescence.

Among the survivors of Ebola virus disease (EVD), complications that include uveitis can develop during convalescence, although the incidence and pathogenesis of EVD-associated uveitis are unknown. We describe a patient who recovered from EVD and was subsequently found to have severe unilateral uveitis during convalescence. Viable Zaire ebolavirus (EBOV) was detected in aqueous humor 14 weeks after the onset of EVD and 9 weeks after the clearance of viremia.

Rapid Determination of Ebolavirus Infectivity in Clinical Samples Using a Novel Reporter Cell Line.

Modern ebolavirus diagnostics rely primarily on quantitative reverse transcription-polymerase chain reaction (qRT-PCR), a sensitive method to detect viral genetic material in the acute phase of the disease. However, qRT-PCR does not confirm presence of infectious virus, presenting limitations in patient and outbreak management. Attempts to isolate infectious virus rely on in vivo or basic cell culture approaches, which prohibit rapid results and screening. In this study, we present a novel reporter cell line capable of detecting live ebolaviruses. These cells permit sensitive, large-scale screening and titration of infectious virus in experimental and clinical samples, independent of ebolavirus species and variant.

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and ß-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

UNLABELLED: Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as ß-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and ß-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and ß-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE: Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and ß-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.

Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Ebola Virus Persistence in Semen of Male Survivors.

We investigated the duration of Ebola virus (EBOV) RNA and infectious EBOV in semen specimens of 5 Ebola virus disease (EVD) survivors. EBOV RNA and infectious EBOV was detected by real-time RT-PCR and virus culture out to 290 days and 70 days, respectively, after EVD onset.

Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.

UNLABELLED: The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. IMPORTANCE: Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

Knowledge, attitudes, and practices and long-term immune response after rVSVΔG-ZEBOV-GP Ebola vaccination in healthcare workers in high-risk districts in Uganda.

BACKGROUND: The rVSVΔG-ZEBOV-GP Ebola vaccine (rVSV-ZEBOV) has been used in response to Ebola disease outbreaks caused by Ebola virus (EBOV). Understanding Ebola knowledge, attitudes, and practices (KAP) and the long-term immune response following rVSV-ZEBOV are critical to inform recommendations on future use. METHODS: We administered surveys and collected blood samples from healthcare workers (HCWs) from seven Ugandan healthcare facilities. Questionnaires collected information on demographic characteristics and KAP related to Ebola and vaccination. IgG ELISA, virus neutralization, and interferon gamma ELISpot measured immunological responses against EBOV glycoprotein (GP). RESULTS: Overall, 37 % (210/565) of HCWs reported receiving any Ebola vaccination. Knowledge that rVSV-ZEBOV only protects against EBOV was low among vaccinated (32 %; 62/192) and unvaccinated (7 %; 14/200) HCWs. Most vaccinated (91 %; 192/210) and unvaccinated (92 %; 326/355) HCWs wanted to receive a booster or initial dose of rVSV-ZEBOV, respectively. Median time from rVSV-ZEBOV vaccination to sample collection was 37.7 months (IQR: 30.5, 38.3). IgG antibodies against EBOV GP were detected in 95 % (61/64) of HCWs with vaccination cards and in 84 % (162/194) of HCWs who reported receiving a vaccination. Geometric mean titer among seropositive vaccinees was 0.066 IU/mL (95 % CI: 0.058-0.076). CONCLUSION: As Uganda has experienced outbreaks of Sudan virus and Bundibugyo virus, for which rVSV-ZEBOV does not protect against, our findings underscore the importance of continued education and risk communication to HCWs on Ebola and other viral hemorrhagic fevers. IgG antibodies against EBOV GP were detected in most vaccinated HCWs in Uganda 2─4 years after vaccination; however, the duration and correlates of protection warrant further investigation.

Induction of DNA damage signaling upon Rift Valley fever virus infection results in cell cycle arrest and increased viral replication.

Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus infecting a wide range of vertebrate hosts. Of particular interest is the nonstructural NSs protein, which forms large filamentous fibril bundles in the nucleus. Past studies have shown NSs to be a multifaceted protein important for virulence through modulation of the interferon response as well acting as a general inhibitor of transcription. Here we investigated the regulation of the DNA damage signaling cascades by RVFV infection and found virally inducted phosphorylation of the classical DNA damage signaling proteins, ataxia-telangiectasia mutated (ATM) (Ser-1981), Chk.2 (Thr-68), H2A.X (Ser-139), and p53 (Ser-15). In contrast, ataxia-telangiectasia mutated and Rad3-related kinase (ATR) (Ser-428) phosphorylation was decreased following RVFV infection. Importantly, both the attenuated vaccine strain MP12 and the fully virulent strain ZH548 showed strong parallels in their up-regulation of the ATM arm of the DNA damage response and in the down-regulation of the ATR pathway. The increase in DNA damage signaling proteins did not result from gross DNA damage as no increase in DNA damage was observed following infection. Rather the DNA damage signaling was found to be dependent on the viral protein NSs, as an NSs mutant virus was not found to induce the equivalent signaling pathways. RVFV MP12-infected cells also displayed an S phase arrest that was found to be dependent on NSs expression. Use of ATM and Chk.2 inhibitors resulted in a marked decrease in S phase arrest as well as viral production. These results indicate that RVFV NSs induces DNA damage signaling pathways that are beneficial for viral replication.

Multiparametric homogeneous method for identification of ligand binding to G protein-coupled receptors: receptor-ligand binding and ß-arrestin assay.

Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to ß(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of ß(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.

Recombinant Sudan virus and evaluation of humoral cross-reactivity between Ebola and Sudan virus glycoproteins after infection or rVSV-ΔG-ZEBOV-GP vaccination.

Ebola disease outbreaks are major public health events because of human-to-human transmission and high mortality. These outbreaks are most often caused by Ebola virus, but at least three related viruses can also cause the disease. In 2022, Sudan virus re-emerged causing more than 160 confirmed and probable cases. This report describes generation of a recombinant Sudan virus and demonstrates its utility by quantifying antibody cross-reactivity between Ebola and Sudan virus glycoproteins after human infection or vaccination with a licensed Ebola virus vaccine.

Development of a neutralization assay using a vesicular stomatitis virus expressing Nipah virus glycoprotein and a fluorescent protein.

Nipah virus (NiV) is a highly pathogenic paramyxovirus with a high case fatality rate. Due to its high pathogenicity, pandemic potential, and lack of therapeutics or approved vaccines, its study requires biosafety level 4 (BSL4) containment. In this report, we developed a novel neutralization assay for use in biosafety level 2 laboratories. The assay uses a recombinant vesicular stomatitis virus expressing NiV glycoprotein and a fluorescent protein. The recombinant virus propagates as a replication-competent virus in a cell line constitutively expressing NiV fusion protein, but it is restricted to a single round of replication in wild-type cells. We used this system to evaluate the neutralization activity of monoclonal and polyclonal antibodies, plasma from NiV-infected hamsters, and serum from human patients. Therefore, this recombinant virus could be used as a surrogate for using pathogenic NiV and may constitute a powerful tool to develop therapeutics in low containment laboratories.

Single-dose mucosal replicon-particle vaccine protects against lethal Nipah virus infection up to 3 days after vaccination.

Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.

Epidemiologic and Genomic Evidence for Zoonotic Transmission of SARS-CoV-2 among People and Animals on a Michigan Mink Farm, United States, 2020.

Farmed mink are one of few animals in which infection with SARS-CoV-2 has resulted in sustained transmission among a population and spillback from mink to people. In September 2020, mink on a Michigan farm exhibited increased morbidity and mortality rates due to confirmed SARS-CoV-2 infection. We conducted an epidemiologic investigation to identify the source of initial mink exposure, assess the degree of spread within the facility's overall mink population, and evaluate the risk of further viral spread on the farm and in surrounding wildlife habitats. Three farm employees reported symptoms consistent with COVID-19 the same day that increased mortality rates were observed among the mink herd. One of these individuals, and another asymptomatic employee, tested positive for SARS-CoV-2 by real-time reverse transcription PCR (RT-qPCR) 9 days later. All but one mink sampled on the farm were positive for SARS-CoV-2 based on nucleic acid detection from at least one oral, nasal, or rectal swab tested by RT-qPCR (99%). Sequence analysis showed high degrees of similarity between sequences from mink and the two positive farm employees. Epidemiologic and genomic data, including the presence of F486L and N501T mutations believed to arise through mink adaptation, support the hypothesis that the two employees with SARS-CoV-2 nucleic acid detection contracted COVID-19 from mink. However, the specific source of virus introduction onto the farm was not identified. Three companion animals living with mink farm employees and 31 wild animals of six species sampled in the surrounding area were negative for SARS-CoV-2 by RT-qPCR. Results from this investigation support the necessity of a One Health approach to manage the zoonotic spread of SARS-CoV-2 and underscores the critical need for multifaceted public health approaches to prevent the introduction and spread of respiratory viruses on mink farms.