Vascular dysfunction in hemorrhagic viral fevers: opportunities for organotypic modeling.

The hemorrhagic fever viruses (HFVs) cause severe or fatal infections in humans. Named after their common symptom hemorrhage, these viruses induce significant vascular dysfunction by affecting endothelial cells, altering immunity, and disrupting the clotting system. Despite advances in treatments, such as cytokine blocking therapies, disease modifying treatment for this class of pathogen remains elusive. Improved understanding of the pathogenesis of these infections could provide new avenues to treatment. While animal models and traditional 2D cell cultures have contributed insight into the mechanisms by which these pathogens affect the vasculature, these models fall short in replicatingin vivohuman vascular dynamics. The emergence of microphysiological systems (MPSs) offers promising avenues for modeling these complex interactions. These MPS or 'organ-on-chip' models present opportunities to better mimic human vascular responses and thus aid in treatment development. In this review, we explore the impact of HFV on the vasculature by causing endothelial dysfunction, blood clotting irregularities, and immune dysregulation. We highlight how existing MPS have elucidated features of HFV pathogenesis as well as discuss existing knowledge gaps and the challenges in modeling these interactions using MPS. Understanding the intricate mechanisms of vascular dysfunction caused by HFV is crucial in developing therapies not only for these infections, but also for other vasculotropic conditions like sepsis.

Development of a non-infectious control for viral hemorrhagic fever PCR assays.

Assay validation is an essential component of disease surveillance testing, but can be problematic in settings where access to positive control material is limited and a safety risk for handlers. Here we describe a single non-infectious synthetic control that can help develop and validate the PCR based detection of the viral causes of Crimean-Congo hemorrhagic fever, Ebola virus disease, Lassa fever, Marburg virus disease and Rift Valley fever. We designed non-infectious synthetic DNA oligonucleotide sequences incorporating primer binding sites suitable for five assays, and a T7 promotor site which was used to transcribe the sequence. Transcribed RNA was used as template in a dilution series, extracted and amplified with RT-PCR and RT-qPCR to demonstrate successful recovery and determine limits of detection in a range of laboratory settings. Our results show this approach is adaptable to any diagnostic assay requiring validation of nucleic acid extraction and/or amplification, particularly where sourcing reliable, safe material for positive controls is infeasible.

Knowledge, perceptions, and exposure to bats in communities living around bat roosts in Bundibugyo district, Uganda: implications for viral haemorrhagic fever prevention and control.

BACKGROUND: Bats are a reservoir for many viruses causing haemorrhagic fevers. Proximity to bats is a risk factor for virus spillover to animals and humans. We conducted this study to assess knowledge, perceptions, and exposure to bats in communities living near bat roosts in Bundibugyo District, Uganda. METHODS: A cross-sectional study using mixed methods with both quantitative and qualitative data was conducted between September and December 2022. Participants for the quantitative data (survey) (n = 384) resided near bat caves and/or roost sites and were selected using multistage random sampling. The survey investigated participants' prior exposure to bats, as well as knowledge and perceptions of bat exposure. Logistic regression was used to determine factors associated with bat exposure. Participants for the qualitative data (focus group discussions) (n = 10, 6-8 participants each) were purposely selected based on engagement in guano mining, hunting, and farming activities. Perceived risk associated with bat-related activities were identified and ranked in the focus group discussions using participatory epidemiology tools. RESULTS: In total, (214/384, 55.7%) had a history of bat exposure and (208/384, 54.2%) had poor knowledge of risk factors associated with bat exposure. Increased exposure to bats was associated with being male (OR = 1.6; 95% CI: 1.0, 2.4 p-value = 0.038), staying in urban areas (OR = 1.9; p-value = 0.010), hunting (OR = 10.9; p-value = 0.024), and positive perception to bat guano being safe as fertiliser (OR = 2.5; p-value = 0.045). During the proportional piling process, a total of 7 risk factors were identified by 10 groups with hunting during an outbreak and consumption of bats being the most frequently identified. Overall, there was a strong statistical agreement in the ranking across the 10 focus groups (W = 0.52; p < 0.01; n = 10). Based on the provided data, the adjusted odds ratio of 0.7 for the good measures (p-value = 0.112), suggests a potential protective effect on the risk of bat exposure. CONCLUSION: Communities living around bat roosts frequently come into contact with bats, yet there is inadequate awareness regarding the behaviors that can lead to the transmission of bat- borne diseases to humans. It is essential to undertake educational initiatives and preventive measures to minimise the risks of bat-related infections. The need for targeted health communication and education efforts to address these knowledge gaps and promote an accurate understanding of bats and disease transmission. Understanding of diseases associated with bats will minimize bat-related health risks especially in communities engaged in wildlife hunting.

Characterization of antibodies targeting severe fever with thrombocytopenia syndrome virus glycoprotein Gc.

Severe fever with thrombocytopenia syndrome (SFTS) is a hemorrhagic fever caused by SFTS virus (SFTSV), which is primarily found in East Asian countries. Despite its high mortality rate and increasing incidence, no vaccines or therapeutics have yet been approved for use against SFTS. Antibody drugs have shown promise in treating lethal infectious diseases that currently have no established treatments. In the case of SFTS, however, only a limited amount of research has been done on SFTSV-neutralizing antibodies targeting the transmembrane proteins Gn and Gc, which play critical roles in viral infection. This study focuses on the production and characterization of antibodies targeting the SFTSV Gc protein. Monoclonal antibodies against Gc were generated through immunization of mice, and their antiviral activity was evaluated. Three out of four anti-Gc antibody clones from this study demonstrated dose-dependent SFTSV neutralization activity, two of which exhibited a synergistic effect on the neutralization activity of the anti-Gn antibody clone Mab4-5. Further studies are necessary to identify key sites on the SFTSV glycoprotein and to develop novel agents as well as antibodies with diverse mechanisms of action against SFTSV.

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.

Plasmid-Based Lassa Virus Reverse Genetics.

Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin whose efficacy remains controversial. The development of reverse genetics approaches has provided investigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of viral pathogenesis. Reverse genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise, reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without the need of high security biocontainment laboratories. In this chapter, we describe the experimental procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.

[Ebola and Marburg virus disease]. / Ebola-Virus- und Marburg-Viruserkrankung.

Viral hemorrhagic fevers (VHF) are serious, often fatal diseases that affect humans and non-human primates. The nomenclature of these diseases has changed in that they are now referred to as viral diseases because the previously named symptoms of fever or hemorrhages are not obligatory. In this article, the focus will be on the VHFs Ebola and Marburg viral disease with the potential for human-to-human transmission; these diseases are so-called high-consequence infectious diseases (HCID), some with considerable potential for epidemic spread and the risk of nosocomial transmission.

Universal primers for rift valley fever virus whole-genome sequencing.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease causing acute hemorrhagic fever. Accurate identification of mutations and phylogenetic characterization of RVF virus (RVFV) require whole-genome analysis. Universal primers to amplify the entire RVFV genome from clinical samples with low copy numbers are currently unavailable. Thus, we aimed to develop universal primers applicable for all known RVFV strains. Based on the genome sequences available from public databases, we designed eight pairs of universal PCR primers covering the entire RVFV genome. To evaluate primer universality, four RVFV strains (ZH548, Kenya 56 (IB8), BIME-01, and Lunyo), encompassing viral phylogenetic diversity, were chosen. The nucleic acids of the test strains were chemically synthesized or extracted via cell culture. These RNAs were evaluated using the PCR primers, resulting in successful amplification with expected sizes (0.8-1.7 kb). Sequencing confirmed that the products covered the entire genome of the RVFV strains tested. Primer specificity was confirmed via in silico comparison against all non-redundant nucleotide sequences using the BLASTn alignment tool in the NCBI database. To assess the clinical applicability of the primers, mock clinical specimens containing human and RVFV RNAs were prepared. The entire RVFV genome was successfully amplified and sequenced at a viral concentration of 108 copies/mL. Given the universality, specificity, and clinical applicability of the primers, we anticipate that the RVFV universal primer pairs and the developed method will aid in RVFV phylogenomics and mutation detection.

[Recent advances in the development of vaccines against hemorrhagic fevers caused by arenaviruses]. / Les fièvres hémorragiques causées par les arénavirus : de récentes avancées vaccinales.

Arenaviruses are a global threat, causing thousands of deaths each year in several countries around the world. Despite strong efforts in the development of vaccine candidates, vaccines against Lassa fever or Bolivian and Venezuelan hemorrhagic fevers are yet to be licensed for a use in humans. In this synthesis, we present the arenaviruses causing fatal diseases in humans and the main vaccine candidates that have been developed over the past decades with an emphasis on the measles-Lassa vaccine, the first Lassa vaccine ever tested in humans, and on the MOPEVAC platform that can potentially be used as a pan-arenavirus vaccine platform.

Title: Les fièvres hémorragiques causées par les arénavirus : de récentes avancées vaccinales. Abstract: Le développement de vaccins contre les arénavirus est un enjeu global. En effet, plusieurs milliers de personnes meurent chaque année de la fièvre de Lassa en Afrique occidentale et les virus Machupo, Guanarito ou Chapare continuent de ré-émerger en Amérique du Sud. Pourtant, il n'existe à ce jour aucun vaccin validé pour une utilisation dans l'espèce humaine pour lutter contre ces arénavirus. Dans cette synthèse, nous présentons les différents arénavirus causant des maladies mortelles chez l'espèce humaine et les principaux candidats vaccins développés au cours des dernières décennies contre ces virus. Nous décrivons plus particulièrement le vaccin rougeole-Lassa, premier vaccin contre la fièvre de Lassa à avoir été testé dans l'espèce humaine, et la plateforme MOPEVAC qui permet de générer avec succès des vaccins mono- ou multivalents contre potentiellement tous les arénavirus pathogènes connus.

Hemostasis defects underlying the hemorrhagic syndrome caused by mammarenaviruses in a cynomolgus macaque model.

Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies it is found that arenaviruses, such as Lassa, Machupo, and Guanarito viruses cause HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito, and Lassa viruses is associated with cutaneomucosal, cerebral, digestive, and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation, and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic arenaviruses.

[Research progress on the role of monocytes in viral hemorrhagic fevers].

Monocytes are important target cells of various hemorrhagic fever viruses. In viral hemorrhagic fevers (VHFs), monocytes can be infected by viruses and produce different kinds of cytokines, which contribute to the antiviral immune response and participation in the immunopathogenesis of VHFs. During the pathogenesis of various VHFs (early stage), monocytes change in cell counting, subpopulation distribution and expression of surface molecules with an activated phenotype. Several hemorrhagic fever viruses can infect monocytes and induce immune response, which may play an important role in immunopathological injury. Monocytes and the cytokines they produce may interact with platelets and vascular endothelial cells, contributing to disease progression.

A narrative review of high-level isolation unit operational and infrastructure features.

High-level isolation units (HLIUs) are specially designed facilities for care and management of patients with suspected or confirmed high-consequence infectious diseases (HCIDs), equipped with unique infrastructure and operational features. While individual HLIUs have published on their experiences caring for patients with HCIDs and two previous HLIU consensus efforts have outlined key components of HLIUs, we aimed to summarise the existing literature that describes best practices, challenges and core features of these specialised facilities. A narrative review of the literature was conducted using keywords associated with HLIUs and HCIDs. A total of 100 articles were used throughout the manuscript from the literature search or from alternate methods like reference checks or snowballing. Articles were sorted into categories (eg, physical infrastructure, laboratory, internal transport); for each category, a synthesis of the relevant literature was conducted to describe best practices, experiences and operational features. The review and summary of HLIU experiences, best practices, challenges and components can serve as a resource for units continuing to improve readiness, or for hospitals in early stages of developing their HLIU teams and planning or constructing their units. The COVID-19 pandemic, a global outbreak of mpox, sporadic cases of viral haemorrhagic fevers in Europe and the USA, and recent outbreaks of Lassa fever, Sudan Ebolavirus, and Marburg emphasise the need for an extensive summary of HLIU practices to inform readiness and response.

Community knowledge, attitude and practices regarding zoonotic viral haemorrhagic fevers in five geo-ecological zones in Tanzania.

BACKGROUND: Viral haemorrhagic fevers (VHF) cause significant economic and public health impact in Sub-Saharan Africa. Community knowledge, awareness and practices regarding such outbreaks play a pivotal role in their management and prevention. This study was carried out to assess community knowledge, attitude and practices regarding VHF in five geo-ecological zones in Tanzania. METHODS: A cross-sectional study was conducted in Buhigwe, Kalambo, Kyela, Kinondoni, Kilindi, Mvomero, Kondoa and Ukerewe districts representing five geo-ecological zones in Tanzania. Study participants were selected by multistage cluster sampling design. A semi-structured questionnaire was used to collect socio-demographic and information related to knowledge, attitude and practices regarding VHFs. Descriptive statistics and logistic regression were used for the analysis. RESULTS: A total of 2,965 individuals were involved in the study. Their mean age was 35 (SD ± 18.9) years. Females accounted for 58.2% while males 41.8%. Most of the respondents (70.6%; n = 2093) had never heard of VHF, and those who heard, over three quarters (79%) mentioned the radio as their primary source of information. Slightly over a quarter (29.4%) of the respondents were knowledgeable, 25% had a positive attitude, and 17.9% had unfavourable practice habits. The level of knowledge varied between occupation and education levels (P < 0.005). Most participants were likely to interact with a VHF survivor or take care of a person suffering from VHF (75%) or visit areas with known VHF (73%). There were increased odds of having poor practice among participants aged 36-45 years (AOR: 3.566, 95% CI: 1.593-7.821) and those living in Western, North-Eastern and Lake Victoria zones (AOR: 2.529, 95% CI: 1.071-6.657; AOR: 2.639, 95% CI: 1.130-7.580 AOR: 2.248, 95% CI: 1.073-3.844, respectively). CONCLUSION: Overall, the knowledge on VHF among communities is low, while a large proportion of individuals in the community are involved in activities that expose them to the disease pathogens in Tanzania. These findings highlight the need for strengthening health educational and promotion efforts on VHF targeting specific populations.