Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection.

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

Pathogenicity of Lloviu and Bombali Viruses in Type I Interferon Receptor Knockout Mice.

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV.

Fluorescent and Bioluminescent Reporter Mouse-Adapted Ebola Viruses Maintain Pathogenicity and Can Be Visualized in Vivo.

Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA) EBOVs, including 1 based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 73% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using handheld blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models.

Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

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.

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.

Equine Encephalosis Virus in India, 2008.

A virus isolated from a sick horse from India in 2008 was confirmed by next-generation sequencing analysis to be equine encephalosis virus (EEV). EEV in India is concerning because several species of Culicoides midge, which play a major role in EEV natural maintenance and transmission, are present in this country.

Identification and characterization of novel mosquito-borne (Kammavanpettai virus) and tick-borne (Wad Medani) reoviruses isolated in India.

In 1954, a virus named Wad Medani virus (WMV) was isolated from Hyalomma marginatum ticks from Maharashtra State, India. In 1963, another virus was isolated from Sturnia pagodarum birds in Tamil Nadu, India, and named Kammavanpettai virus (KVPTV) based on the site of its isolation. Originally these virus isolates could not be identified with conventional methods. Here we describe next-generation sequencing studies leading to the determination of their complete genome sequences, and identification of both virus isolates as orbiviruses (family Reoviridae). Sequencing data showed that KVPTV has an AT-rich genome, whereas the genome of WMV is GC-rich. The size of the KVPTV genome is 18 234 nucleotides encoding proteins ranging 238-1290 amino acids (aa) in length. Similarly, the size of the WMV genome is 16 941 nucleotides encoding proteins ranging 214-1305 amino acids in length. Phylogenetic analysis of the VP1 gene, along with the capsid genes VP5 and VP7, revealed that KVPTV is likely a novel mosquito-borne virus and WMV is a tick-borne orbivirus. This study focuses on the phylogenetic comparison of these newly identified orbiviruses with mosquito-, tick- and Culicoides-borne orbiviruses isolated in India and other countries.

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.

Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin.

Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV's high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor's maturation to GP38, and Gn precursor's maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat.

Preliminary Evaluation of the Effect of Investigational Ebola Virus Disease Treatments on Viral Genome Sequences.

BACKGROUND: Several patients with Ebola virus disease (EVD) managed in the United States have received ZMapp monoclonal antibodies, TKM-Ebola small interfering RNA, brincidofovir, and/or convalescent plasma as investigational therapeutics. METHODS: To investigate whether treatment selected for Ebola virus (EBOV) mutations conferring resistance, viral sequencing was performed on RNA extracted from clinical blood specimens from patients with EVD following treatment, and putative viral targets were analyzed. RESULTS: We observed no major or minor EBOV mutations within regions targeted by therapeutics. CONCLUSIONS: This small subset of patients and clinical specimens suggests that evolution of resistance is not a direct consequence of antiviral treatment. As EVD antiviral treatments are introduced into wider use, it is essential that continuous viral full-genome surveillance is performed, to monitor for the emergence of escape mutations.

Prognostic Indicators for Ebola Patient Survival.

To determine whether 2 readily available indicators predicted survival among patients with Ebola virus disease in Sierra Leone, we evaluated information for 216 of the 227 patients in Bo District during a 4-month period. The indicators were time from symptom onset to healthcare facility admission and quantitative real-time reverse transcription PCR cycle threshold (Ct), a surrogate for viral load, in first Ebola virus-positive blood sample tested. Of these patients, 151 were alive when detected and had reported healthcare facility admission dates and Ct values available. Time from symptom onset to healthcare facility admission was not associated with survival, but viral load in the first Ebola virus-positive blood sample was inversely associated with survival: 52 (87%) of 60 patients with a Ct of >24 survived and 20 (22%) of 91 with a Ct of <24 survived. Ct values may be useful for clinicians making treatment decisions or managing patient or family expectations.

Characteristics and Clinical Management of a Cluster of 3 Patients With Ebola Virus Disease, Including the First Domestically Acquired Cases in the United States.

BACKGROUND: More than 26,000 cases of Ebola virus disease (EVD) have been reported in western Africa, with high mortality. Several patients have been medically evacuated to hospitals in the United States and Europe. Detailed clinical data are limited on the clinical course and management of patients with EVD outside western Africa. OBJECTIVE: To describe the clinical characteristics and management of a cluster of patients with EVD, including the first cases of Ebola virus (EBOV) infection acquired in the United States. DESIGN: Retrospective clinical case series. SETTING: Three U.S. hospitals in September and October 2014. PATIENTS: First imported EVD case identified in the United States and 2 secondary EVD cases acquired in the United States in critical care nurses who cared for the index case patient. MEASUREMENTS: Clinical recovery, EBOV RNA level, resolution of Ebola viremia, survival with discharge from hospital, or death. RESULTS: The index patient had high EBOV RNA levels, developed respiratory and renal failure requiring critical care support, and died. Both patients with secondary EBOV infection had nonspecific signs and symptoms and developed moderate illness; EBOV RNA levels were moderate, and both patients recovered. LIMITATION: Both surviving patients received uncontrolled treatment with multiple investigational agents, including convalescent plasma, which limits generalizability of the results. CONCLUSION: Early diagnosis, prompt initiation of supportive medical care, and moderate clinical illness likely contributed to successful outcomes in both survivors. The inability to determine the potential benefit of investigational therapies and the effect of patient-specific factors that may have contributed to less severe illness highlight the need for controlled clinical studies of these interventions, especially in the setting of a high level of supportive medical care. PRIMARY FUNDING SOURCE: None.

A new phlebovirus associated with severe febrile illness in Missouri.

Two men from northwestern Missouri independently presented to a medical facility with fever, fatigue, diarrhea, thrombocytopenia, and leukopenia, and both had been bitten by ticks 5 to 7 days before the onset of illness. Ehrlichia chaffeensis was suspected as the causal agent but was not found on serologic analysis, polymerase-chain-reaction (PCR) assay, or cell culture. Electron microscopy revealed viruses consistent with members of the Bunyaviridae family. Next-generation sequencing and phylogenetic analysis identified the viruses as novel members of the phlebovirus genus. Although Koch's postulates have not been completely fulfilled, we believe that this phlebovirus, which is novel in the Americas, is the cause of this clinical syndrome.

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens.

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 10(7) to 4 × 10(2) 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 10(2) TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD.

Hantavirus infections among overnight visitors to Yosemite National Park, California, USA, 2012.

In summer 2012, an outbreak of hantavirus infections occurred among overnight visitors to Yosemite National Park in California, USA. An investigation encompassing clinical, epidemiologic, laboratory, and environmental factors identified 10 cases among residents of 3 states. Eight case-patients experienced hantavirus pulmonary syndrome, of whom 5 required intensive care with ventilatory support and 3 died. Staying overnight in a signature tent cabin (9 case-patients) was significantly associated with becoming infected with hantavirus (p<0.001). Rodent nests and tunnels were observed in the foam insulation of the cabin walls. Rodent trapping in the implicated area resulted in high trap success rate (51%), and antibodies reactive to Sin Nombre virus were detected in 10 (14%) of 73 captured deer mice. All signature tent cabins were closed and subsequently dismantled. Continuous public awareness and rodent control and exclusion are key measures in minimizing the risk for hantavirus infection in areas inhabited by deer mice.

Novel paramyxovirus associated with severe acute febrile disease, South Sudan and Uganda, 2012.

In 2012, a female wildlife biologist experienced fever, malaise, headache, generalized myalgia and arthralgia, neck stiffness, and a sore throat shortly after returning to the United States from a 6-week field expedition to South Sudan and Uganda. She was hospitalized, after which a maculopapular rash developed and became confluent. When the patient was discharged from the hospital on day 14, arthralgia and myalgia had improved, oropharynx ulcerations had healed, the rash had resolved without desquamation, and blood counts and hepatic enzyme levels were returning to reference levels. After several known suspect pathogens were ruled out as the cause of her illness, deep sequencing and metagenomics analysis revealed a novel paramyxovirus related to rubula-like viruses isolated from fruit bats.

Lymphocytic choriomeningitis virus in employees and mice at multipremises feeder-rodent operation, United States, 2012.

We investigated the extent of lymphocytic choriomeningitis virus (LCMV) infection in employees and rodents at 3 commercial breeding facilities. Of 97 employees tested, 31 (32%) had IgM and/or IgG to LCMV, and aseptic meningitis was diagnosed in 4 employees. Of 1,820 rodents tested in 1 facility, 382 (21%) mice (Mus musculus) had detectable IgG, and 13 (0.7%) were positive by reverse transcription PCR; LCMV was isolated from 8. Rats (Rattus norvegicus) were not found to be infected. S-segment RNA sequence was similar to strains previously isolated in North America. Contact by wild mice with colony mice was the likely source for LCMV, and shipments of infected mice among facilities spread the infection. The breeding colonies were depopulated to prevent further human infections. Future outbreaks can be prevented with monitoring and management, and employees should be made aware of LCMV risks and prevention.