RESUMEN
Since the largest and most fatal Ebola virus epidemic during 2014-2016, there have been several consecutive filoviral outbreaks in recent years, including those in 2021, 2022, and 2023. Ongoing outbreak prevalence and limited FDA-approved filoviral therapeutics emphasize the need for novel small molecule treatments. Here, we showcase the structure-activity relationship development of N-substituted pyrrole-based heterocycles and their potent, submicromolar entry inhibition against diverse filoviruses in a target-based pseudovirus assay. Inhibitor antiviral activity was validated using replication-competent Ebola, Sudan, and Marburg viruses. Mutational analysis was used to map the targeted region within the Ebola virus glycoprotein. Antiviral counter-screen and phospholipidosis assays were performed to demonstrate the reduced off-target activity of these filoviral entry inhibitors. Favorable antiviral potency, selectivity, and drug-like properties of the N-substituted pyrrole-based heterocycles support their potential as broad-spectrum antifiloviral treatments.
Asunto(s)
Antivirales , Ebolavirus , Pirroles , Internalización del Virus , Pirroles/farmacología , Pirroles/química , Pirroles/síntesis química , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Humanos , Relación Estructura-Actividad , Ebolavirus/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/síntesis química , Filoviridae/efectos de los fármacos , Marburgvirus/efectos de los fármacosRESUMEN
BACKGROUND: Recent outbreaks of Ebola virus disease (EVD) and Marburg virus disease (MVD) in sub-Saharan Africa illustrate the need to better understand animal reservoirs, burden of disease, and human transmission of filoviruses. This protocol outlines a systematic literature review to assess the prevalence of filoviruses that infect humans in sub-Saharan Africa. A secondary aim is to qualitatively describe and evaluate the assays used to assess prevalence. METHODS: The data sources for this systematic review include PubMed, Embase, and Web of Science. Titles, abstracts, and full texts will be reviewed for inclusion by a primary reviewer and then by a team of secondary reviewers, and data will be extracted using a pre-specified and piloted data extraction form. The review will include human cross-sectional studies, cohort studies, and randomized controlled trials conducted in sub-Saharan Africa up until March 13, 2024 that have been published in peer-reviewed scientific journals, with no language restrictions. Prevalence will be stratified by pathogen, population, assay, and sampling methodology and presented in forest plots with estimated prevalence and 95% confidence intervals. If there are enough studies within a stratum, I2 statistics will be calculated (using R statistical software), and data will be pooled if heterogeneity is low. In addition, assays used to detect infection will be evaluated. All studies included in the review will be assessed for quality and risk of bias using the JBI Prevalence Critical Appraisal Tool and for certainty using the GRADE certainty ratings. DISCUSSION: Accurately measuring the rate of exposure to filoviruses infecting humans in sub-Saharan Africa using prevalence provides an essential understanding of natural history, transmission, and the role of subclinical infection. This systematic review will identify research gaps and provide directions for future research seeking to improve our understanding of filovirus infections. Understanding the natural history, transmission, and the role of subclinical infection is critical for predicting the impact of an intervention on disease burden. SYSTEMATIC REVIEW REGISTRATION: In accordance with the guidelines outlined in the PRISMA-P methodology, this protocol was registered with PROSPERO on April 7, 2023 (ID: CRD42023415358).
Asunto(s)
Infecciones por Filoviridae , Revisiones Sistemáticas como Asunto , Humanos , África del Sur del Sahara/epidemiología , Prevalencia , Infecciones por Filoviridae/epidemiología , Metaanálisis como Asunto , Fiebre Hemorrágica Ebola/epidemiología , Animales , FiloviridaeRESUMEN
Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5' and 3' ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5' end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5' end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses.
Asunto(s)
Ebolavirus , Filoviridae , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Viral , Análisis de Secuencia de ARN , ARN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ebolavirus/genética , Análisis de Secuencia de ARN/métodos , Filoviridae/genética , Marburgvirus/genética , Humanos , AnimalesRESUMEN
Ebola virus (EBOV) and Marburg virus (MARV), members of the Filoviridae family, are highly pathogenic and can cause hemorrhagic fevers, significantly impacting human society. Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats. However, due to the requirement for maximum containment laboratories when studying infectious viruses, the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems. In this study, we used RACE technology to sequence the 3'-leader and 5'-trailer of Menglà virus (MLAV) and constructed a minigenome. Similar to MARV, the transcription activities of the MLAV minigenome are independent of VP30. We further assessed the effects of polymorphisms at the 5' end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency, probably due to alterations in the RNA secondary structure. The reporter activity upon recombination of the 3'-leaders and 5'-trailers of MLAV, MARV, and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities. Additionally, we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system. Remdesivir efficiently inhibited MLAV as well as EBOV replication. In summary, this study provides new information on bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.
Asunto(s)
Ebolavirus , Genoma Viral , Marburgvirus , Animales , Genoma Viral/genética , Ebolavirus/genética , Humanos , Marburgvirus/genética , Mengovirus/genética , Replicación Viral , ARN Viral/genética , Alanina/análogos & derivados , Alanina/farmacología , Quirópteros/virología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/metabolismo , Filoviridae/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Antivirales , Ebolavirus , Fiebre Hemorrágica Ebola , Animales , Ratones , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/virología , Anticuerpos Antivirales/inmunología , Humanos , Filoviridae/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Monoclonales/inmunología , Femenino , Ratones Endogámicos BALB C , Infecciones por Filoviridae/inmunología , Infecciones por Filoviridae/terapia , Infecciones por Filoviridae/prevención & controlRESUMEN
Paramyxo- and filovirus genomes are equipped with bipartite promoters at their 3' ends to initiate RNA synthesis. The two elements, the primary promoter element 1 (PE1) and the secondary promoter element 2 (PE2), are separated by a spacer region that must be precisely a multiple of 6 nucleotides (nts), indicating these viruses adhere to the "rule of six." However, our knowledge of PE2 has been limited to a narrow spectrum of virus species. In this study, a comparative analysis of 1,647 paramyxoviral genomes from a public database revealed that the paramyxovirus PE2 can be clearly categorized into two distinct subcategories: one marked by C repeats at every six bases (exclusive to the subfamily Orthoparamyxovirinae) and another characterized by CG repeats every 6 nts (observed in the subfamilies Avulavirinae and Rubulavirinae). This unique pattern collectively mirrors the evolutionary lineage of these subfamilies. Furthermore, we showed that PE2 of the Rubulavirinae, with the exception of mumps virus, serves as part of the gene-coding region. This may be due to the fact that the Rubulavirinae are the only paramyxoviruses that cannot propagate without RNA editing. Filoviruses have three to eight consecutive uracil repeats every six bases (UN5) in PE2, which is located in the 3' end region of the genome. We obtained PE2 sequences from 2,195 filoviruses in a public database and analyzed the sequence conservation among virus species. Our results indicate that the continuity of UN5 hexamers is consistently maintained with a high degree of conservation across virus species. IMPORTANCE: The genomic intricacies of paramyxo- and filoviruses are highlighted by the bipartite promoters-promoter element 1 (PE1) and promoter element 2 (PE2)-at their 3' termini. The spacer region between these elements follows the "rule of six," crucial for genome replication. By a comprehensive analysis of paramyxoviral genome sequences, we identified distinct subcategories of PE2 based on C and CG repeats that were specific to Orthoparamyxovirinae and Avulavirinae/Rubulavirinae, respectively, mirroring their evolutionary lineages. Notably, the PE2 of Rubulavirinae is integrated into the gene-coding region, a unique trait potentially linked to its strict dependence on RNA editing for virus growth. This study also focused on the PE2 sequences in filovirus genomes. The strict conservation of the continuity of UN5 among virus species emphasizes its crucial role in viral genome replication.
Asunto(s)
Filoviridae , Genoma Viral , Filogenia , Regiones Promotoras Genéticas , Regiones Promotoras Genéticas/genética , Genoma Viral/genética , Filoviridae/genética , Filoviridae/clasificación , Paramyxoviridae/genética , Paramyxoviridae/clasificación , Humanos , ARN Viral/genética , Evolución Molecular , AnimalesRESUMEN
Bats are associated with the circulation of most mammalian filoviruses (FiVs), with pathogenic ones frequently causing deadly hemorrhagic fevers in Africa. Divergent FiVs have been uncovered in Chinese bats, raising concerns about their threat to public health. Here, we describe a long-term surveillance to track bat FiVs at orchards, eventually resulting in the identification and isolation of a FiV, Dehong virus (DEHV), from Rousettus leschenaultii bats. DEHV has a typical filovirus-like morphology with a wide spectrum of cell tropism. Its entry into cells depends on the engagement of Niemann-Pick C1, and its replication is inhibited by remdesivir. DEHV has the largest genome size of filoviruses, with phylogenetic analysis placing it between the genera Dianlovirus and Orthomarburgvirus, suggesting its classification as the prototype of a new genus within the family Filoviridae. The continuous detection of viral RNA in the serological survey, together with the wide host distribution, has revealed that the region covering southern Yunnan, China, and bordering areas is a natural circulation sphere for bat FiVs. These emphasize the need for a better understanding of the pathogenicity and potential risk of FiVs in the region.
Asunto(s)
Quirópteros , Filoviridae , Animales , Filogenia , China , MamíferosRESUMEN
Bats are increasingly recognized as reservoirs of emerging zoonotic pathogens. Egyptian rousette bats (ERBs) are the known reservoir of Marburg virus (MARV), a filovirus that causes deadly Marburg virus disease (MVD) in humans. However, ERBs harbor MARV asymptomatically, likely due to a coadapted and specific host immunity-pathogen relationship. Recently, we measured transcriptional responses in MARV-infected ERB whole tissues, showing that these bats possess a disease tolerant strategy that limits pro-inflammatory gene induction, presumably averting MVD-linked immunopathology. However, the host resistant strategy by which ERBs actively limit MARV burden remains elusive, which we hypothesize requires localized inflammatory responses unresolvable at bulk-tissue scale. Here, we use dexamethasone to attenuate ERB pro-inflammatory responses and assess MARV replication, shedding and disease. We show that MARV-infected ERBs naturally mount coordinated pro-inflammatory responses at liver foci of infection, comprised of recruited mononuclear phagocytes and T cells, the latter of which proliferate with likely MARV-specificity. When pro-inflammatory responses are diminished, ERBs display heightened MARV replication, oral/rectal shedding and severe MVD-like liver pathology, demonstrating that ERBs balance immunoprotective tolerance with discreet MARV-resistant pro-inflammatory responses. These data further suggest that natural ERB immunomodulatory stressors like food scarcity and habitat disruption may potentiate viral shedding, transmission and therefore outbreak risk.
Asunto(s)
Quirópteros , Filoviridae , Enfermedad del Virus de Marburg , Marburgvirus , Animales , Humanos , Marburgvirus/genética , InmunidadRESUMEN
Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1-3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness.
Asunto(s)
Filoviridae , Infecciones por Orthomyxoviridae , Animales , Humanos , Inmunoinformática , Macaca , AntiviralesRESUMEN
Filoviruses are causative agents of severe hemorrhagic fevers with high case fatality rates in humans. For studies of virus biology and the subsequent development of countermeasures, reverse genetic systems, and especially those facilitating the generation of recombinant filoviruses, are indispensable. Here, we describe the generation of recombinant filoviruses from cDNA.
Asunto(s)
Ebolavirus , Filoviridae , Fiebre Hemorrágica Ebola , Humanos , Filoviridae/genética , Genética Inversa , ADN Complementario/genética , Ebolavirus/genéticaRESUMEN
IMPORTANCE: Filoviruses are the causative agents of severe and often fatal hemorrhagic disease in humans. Menglà virus (MLAV) is a recently reported filovirus, isolated from fruit bats that is capable to replicate in human cells, representing a potential risk for human health. An in-depth structural and functional knowledge of MLAV proteins is an essential step for antiviral research on this virus that can also be extended to other emerging filoviruses. In this study, we determined the first crystal structures of the C-terminal domain (CTD) of the MLAV nucleoprotein (NP), showing important similarities to the equivalent domain in MARV. The structural data also show that the NP CTD has the ability to form large helical oligomers that may participate in the control of cytoplasmic inclusion body formation during viral replication.
Asunto(s)
Ebolavirus , Filoviridae , Humanos , Nucleoproteínas/química , Filoviridae/química , Filoviridae/metabolismo , Proteínas Virales/metabolismoRESUMEN
Viruses in the family Filoviridae, including the commonly known Ebola (EBOV) and Marburg (MARV) viruses, can cause severe hemorrhagic fever in humans and nonhuman primates. Sporadic outbreaks of filovirus disease occur in sub-Saharan Africa with reported case fatality rates ranging from 25% to 90%. The high mortality and increasing frequency and magnitude of recent outbreaks along with the increased potential for spread from rural to urban areas highlight the importance of pandemic preparedness for these viruses. Despite their designation as high-priority pathogens, numerous scientific gaps exist in critical areas. In this review, these gaps and an assessment of potential prototype pathogen candidates are presented for this important virus family.
Asunto(s)
Ebolavirus , Filoviridae , Fiebre Hemorrágica Ebola , Marburgvirus , Animales , Humanos , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/prevención & control , Brotes de EnfermedadesRESUMEN
A new filovirus named Menglà virus was found in bats in southern China in 2015. This species has been assigned to the new genus Dianlovirus and has only been detected in China. In this article, we report the detection of filoviruses in bats captured in Vietnam. We studied 248 bats of 15 species caught in the provinces of Lai Chau and Son La in northern Vietnam and in the province of Dong Thap in the southern part of the country. Filovirus RNA was found in four Rousettus leschenaultii and one Rousettus amplexicaudatus from Lai Chau Province. Phylogenetic analysis of the polymerase gene fragment showed that three positive samples belong to Dianlovirus, and two samples form a separate clade closer to Orthomarburgvirus. An enzyme-linked immunosorbent assay showed that 9% of Rousettus, 13% of Eonycteris, and 10% of Cynopterus bats had antibodies to the glycoprotein of marburgviruses.
Asunto(s)
Quirópteros , Filoviridae , Marburgvirus , Animales , Vietnam/epidemiología , FilogeniaRESUMEN
Although there are now approved treatments and vaccines for Ebola virus disease, the case fatality rate remains unacceptably high even when patients are treated with the newly approved therapeutics. Furthermore, these countermeasures are not expected to be effective against disease caused by other filoviruses. A meeting of subject-matter experts was held during the 10th International Filovirus Symposium to discuss strategies to address these gaps. Several investigational therapeutics, vaccine candidates, and combination strategies were presented. The greatest challenge was identified to be the implementation of well-designed clinical trials of safety and efficacy during filovirus disease outbreaks. Preparing for this will require agreed-upon common protocols for trials intended to bridge multiple outbreaks across all at-risk countries. A multinational research consortium including at-risk countries would be an ideal mechanism to negotiate agreement on protocol design and coordinate preparation. Discussion participants recommended a follow-up meeting be held in Africa to establish such a consortium.
Asunto(s)
Ebolavirus , Infecciones por Filoviridae , Filoviridae , Fiebre Hemorrágica Ebola , Humanos , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/epidemiología , Brotes de Enfermedades/prevención & control , ÁfricaRESUMEN
The International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group continues to prospectively refine the established nomenclature for taxa included in family Filoviridae in an effort to decrease confusion of genus, species, and virus names and to adhere to amended stipulations of the International Code of Virus Classification and Nomenclature (ICVCN). Recently, the genus names Ebolavirus and Marburgvirus were changed to Orthoebolavirus and Orthomarburgvirus, respectively. Additionally, all established species names in family Filoviridae now adhere to the ICTV-mandated binomial format. Virus names remain unchanged and valid. Here, we outline the revised taxonomy of family Filoviridae as approved by the ICTV in April 2023.
Asunto(s)
Ebolavirus , Filoviridae , Marburgvirus , VirusRESUMEN
Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses.
Asunto(s)
Quirópteros , Filoviridae , Marburgvirus , Animales , Filoviridae/genética , Línea Celular , Italia , FilogeniaRESUMEN
Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no pan-filovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3-31.7 copies/reaction and was evaluated against the field collected samples. The assay's performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples.
Asunto(s)
Biovigilancia , Quirópteros , Ebolavirus , Filoviridae , Fiebre Hemorrágica Ebola , Animales , Filoviridae/genética , Ebolavirus/genética , MamíferosRESUMEN
A One Health cross-sectoral surveillance approach was implemented to screen biological samples from bats, pigs, and humans at high-risk interfaces for zoonotic viral spillover for five viral families with zoonotic potential in Viet Nam. Over 1600 animal and human samples from bat guano harvesting sites, natural bat roosts, and pig farming operations were tested for coronaviruses (CoVs), paramyxoviruses, influenza viruses, filoviruses and flaviviruses using consensus PCR assays. Human samples were also tested using immunoassays to detect antibodies against eight virus groups. Significant viral diversity, including CoVs closely related to ancestors of pig pathogens, was detected in bats roosting at the human-animal interfaces, illustrating the high risk for CoV spillover from bats to pigs in Viet Nam, where pig density is very high. Season and reproductive period were significantly associated with the detection of bat CoVs, with site-specific effects. Phylogeographic analysis indicated localized viral transmission among pig farms. Our limited human sampling did not detect any known zoonotic bat viruses in human communities living close to the bat cave and harvesting bat guano, but our serological assays showed possible previous exposure to Marburg virus-like (Filoviridae), Crimean-Congo hemorrhagic fever virus-like (Bunyaviridae) viruses and flaviviruses. Targeted and coordinated One Health surveillance helped uncover this viral pathogen emergence hotspot.
Asunto(s)
Quirópteros , Infecciones por Coronavirus , Coronavirus , Filoviridae , Salud Única , Humanos , Animales , Porcinos , Vietnam/epidemiología , Filogenia , ZoonosisRESUMEN
AIMS: Filoviruses encompass highly pathogenic viruses placing significant public health burden on countries affected. Efforts for improved diagnostics and surveillance are needed. The requirement for high-containment can be circumvented by using pseudotype viruses (PV), which can be handled safely, in tropism, drug screening, vaccine evaluation, and serosurveillance studies. We assessed the stability and functionality after long-term storage of lyophilised filovirus pseudotypes for use in neutralisation assays. METHODS AND RESULTS: We generated a panel of filovirus lentiviral pseudotypes followed by lyophilisation and storage in different conditions. Next, we reconstituted and tested PVs in infection experiments and pseudotype neutralisation assays where possible. Lyophilised Ebola and Marburg PVs retained production titres for at least two years when stored at +4ËC or less. Lyophilised Ebola PVs performed similarly to non-lyophilised PVs in neutralisation assays after reconstitution. When stored at high temperatures (+37ËC), lyophilised PVs did not retain titres after 1-month storage, however, when lyophilised using pilot-scale facilities EBOV PVs retained titres and performed as standard in neutralisation assays after on 1-month storage at 37ËC. CONCLUSIONS: Filovirus PVs are amenable to lyophilisation and can be stored for at least 2 years in a household fridge to be used in antibody assays. Lyophilisation performed in the right conditions would allow transportation at room temperature, even in warmer climates.