Characterization of human tibrovirus envelope glycoproteins.

Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1 (EKV-1), Ekpoma virus 2, and Mundri virus. However, since none of them has been isolated, their biological properties are largely unknown. We aimed to characterize the human tibrovirus glycoprotein (G), which likely plays a pivotal role in viral tropism and pathogenicity. Human tibrovirus Gs were found to share some primary structures and display 14 conserved cysteine residues, although their overall amino acid homology was low (29%-48%). Multiple potential glycosylation sites were found on the G molecules, and endoglycosidase H- and peptide-N-glycosidase F-sensitive glycosylation was confirmed. AlphaFold-predicted three-dimensional (3D) structures of human tibrovirus Gs were overall similar. Membrane fusion mediated by these tibrovirus Gs was induced by acidic pH. The low pH-induced conformational change that triggers fusion was reversible. Virus-like particles (VLPs) were produced by transient expression of Gs in cultured cells and used to produce mouse antisera. Using vesicular stomatitis Indiana virus pseudotyped with Gs, we found that the antisera to the respective tibrovirus VLPs showed limited cross-neutralizing activity. It was also found that human C-type lectins and T-cell immunoglobulin mucin 1 acted as attachment factors for G-mediated entry into cells. Interestingly, BASV-G showed the highest ability to utilize these molecules. The viruses infected a wide range of cell lines with preferential tropism for human-derived cells whereas the preference of EKV-1 was unique compared with the other human tibroviruses. These findings provide fundamental information to understand the biological properties of the human tibroviruses. IMPORTANCE: Human tibroviruses are poorly characterized emerging rhabdoviruses associated with either asymptomatic infection or severe disease with a case fatality rate as high as 60% in humans. However, the extent and burden of human infection as well as factors behind differences in infection outcomes are largely unknown. In this study, we characterized human tibrovirus glycoproteins, which play a key role in virus-host interactions, mainly focusing on their structural and antigenic differences and cellular tropism. Our results provide critical information for understanding the biological properties of these novel viruses and for developing appropriate preparedness interventions such as diagnostic tools, vaccines, and effective therapies.

Immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccination in exposed and potentially exposed persons in the Democratic Republic of the Congo.

Despite more than 300,000 rVSVΔG-ZEBOV-glycoprotein (GP) vaccine doses having been administered during Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC) between 2018 and 2020, seroepidemiologic studies of vaccinated Congolese populations are lacking. This study examines the antibody response at 21 d and 6 mo postvaccination after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC. We conducted a longitudinal cohort study of 608 rVSVΔG-ZEBOV-GP-vaccinated individuals during an EVD outbreak in North Kivu Province, DRC. Participants provided questionnaires and blood samples at three study visits (day 0, visit 1; day 21, visit 2; and month 6, visit 3). Anti-GP immunoglobulin G (IgG) antibody titers were measured in serum by the Filovirus Animal Nonclinical Group anti-Ebola virus GP IgG enzyme-linked immunosorbent assay. Antibody response was defined as an antibody titer that had increased fourfold from visit 1 to visit 2 and was above four times the lower limit of quantification at visit 2; antibody persistence was defined as a similar increase from visit 1 to visit 3. We then examined demographics for associations with follow-up antibody titers using generalized linear mixed models. A majority of the sample, 87.2%, had an antibody response at visit 2, and 95.6% demonstrated antibody persistence at visit 3. Being female and of young age was predictive of a higher antibody titer postvaccination. Antibody response and persistence after Ebola vaccination was robust in this cohort, confirming findings from outside of the DRC.

The African Network for Improved Diagnostics, Epidemiology and Management of common infectious Agents.

BACKGROUND: In sub-Saharan Africa, acute respiratory infections (ARI), acute gastrointestinal infections (GI) and acute febrile disease of unknown cause (AFDUC) have a large disease burden, especially among children, while respective aetiologies often remain unresolved. The need for robust infectious disease surveillance to detect emerging pathogens along with common human pathogens has been highlighted by the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. The African Network for Improved Diagnostics, Epidemiology and Management of Common Infectious Agents (ANDEMIA) is a sentinel surveillance study on the aetiology and clinical characteristics of ARI, GI and AFDUC in sub-Saharan Africa. METHODS: ANDEMIA includes 12 urban and rural health care facilities in four African countries (Côte d'Ivoire, Burkina Faso, Democratic Republic of the Congo and Republic of South Africa). It was piloted in 2018 in Côte d'Ivoire and the initial phase will run from 2019 to 2021. Case definitions for ARI, GI and AFDUC were established, as well as syndrome-specific sampling algorithms including the collection of blood, naso- and oropharyngeal swabs and stool. Samples are tested using comprehensive diagnostic protocols, ranging from classic bacteriology and antimicrobial resistance screening to multiplex real-time polymerase chain reaction (PCR) systems and High Throughput Sequencing. In March 2020, PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and analysis of full genomic information was included in the study. Standardised questionnaires collect relevant clinical, demographic, socio-economic and behavioural data for epidemiologic analyses. Controls are enrolled over a 12-month period for a nested case-control study. Data will be assessed descriptively and aetiologies will be evaluated using a latent class analysis among cases. Among cases and controls, an integrated analytic approach using logistic regression and Bayesian estimation will be employed to improve the assessment of aetiology and associated risk factors. DISCUSSION: ANDEMIA aims to expand our understanding of ARI, GI and AFDUC aetiologies in sub-Saharan Africa using a comprehensive laboratory diagnostics strategy. It will foster early detection of emerging threats and continued monitoring of important common pathogens. The network collaboration will be strengthened and site diagnostic capacities will be reinforced to improve quality management and patient care.

A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics.

BACKGROUND: Although several experimental therapeutics for Ebola virus disease (EVD) have been developed, the safety and efficacy of the most promising therapies need to be assessed in the context of a randomized, controlled trial. METHODS: We conducted a trial of four investigational therapies for EVD in the Democratic Republic of Congo, where an outbreak began in August 2018. Patients of any age who had a positive result for Ebola virus RNA on reverse-transcriptase-polymerase-chain-reaction assay were enrolled. All patients received standard care and were randomly assigned in a 1:1:1:1 ratio to intravenous administration of the triple monoclonal antibody ZMapp (the control group), the antiviral agent remdesivir, the single monoclonal antibody MAb114, or the triple monoclonal antibody REGN-EB3. The REGN-EB3 group was added in a later version of the protocol, so data from these patients were compared with those of patients in the ZMapp group who were enrolled at or after the time the REGN-EB3 group was added (the ZMapp subgroup). The primary end point was death at 28 days. RESULTS: A total of 681 patients were enrolled from November 20, 2018, to August 9, 2019, at which time the data and safety monitoring board recommended that patients be assigned only to the MAb114 and REGN-EB3 groups for the remainder of the trial; the recommendation was based on the results of an interim analysis that showed superiority of these groups to ZMapp and remdesivir with respect to mortality. At 28 days, death had occurred in 61 of 174 patients (35.1%) in the MAb114 group, as compared with 84 of 169 (49.7%) in the ZMapp group (P = 0.007), and in 52 of 155 (33.5%) in the REGN-EB3 group, as compared with 79 of 154 (51.3%) in the ZMapp subgroup (P = 0.002). A shorter duration of symptoms before admission and lower baseline values for viral load and for serum creatinine and aminotransferase levels each correlated with improved survival. Four serious adverse events were judged to be potentially related to the trial drugs. CONCLUSIONS: Both MAb114 and REGN-EB3 were superior to ZMapp in reducing mortality from EVD. Scientifically and ethically sound clinical research can be conducted during disease outbreaks and can help inform the outbreak response. (Funded by the National Institute of Allergy and Infectious Diseases and others; PALM ClinicalTrials.gov number, NCT03719586.).

Novel Polyomaviruses in Mammals from Multiple Orders and Reassessment of Polyomavirus Evolution and Taxonomy.

As the phylogenetic organization of mammalian polyomaviruses is complex and currently incompletely resolved, we aimed at a deeper insight into their evolution by identifying polyomaviruses in host orders and families that have either rarely or not been studied. Sixteen unknown and two known polyomaviruses were identified in animals that belong to 5 orders, 16 genera, and 16 species. From 11 novel polyomaviruses, full genomes could be determined. Splice sites were predicted for large and small T antigen (LTAg, STAg) coding sequences (CDS) and examined experimentally in transfected cell culture. In addition, splice sites of seven published polyomaviruses were analyzed. Based on these data, LTAg and STAg annotations were corrected for 10/86 and 74/86 published polyomaviruses, respectively. For 25 polyomaviruses, a spliced middle T CDS was observed or predicted. Splice sites that likely indicate expression of additional, alternative T antigens, were experimentally detected for six polyomaviruses. In contrast to all other mammalian polyomaviruses, three closely related cetartiodactyl polyomaviruses display two introns within their LTAg CDS. In addition, the VP2 of Glis glis (edible dormouse) polyomavirus 1 was observed to be encoded by a spliced transcript, a unique experimental finding within the Polyomaviridae family. Co-phylogenetic analyses based on LTAg CDS revealed a measurable signal of codivergence when considering all mammalian polyomaviruses, most likely driven by relatively recent codivergence events. Lineage duplication was the only other process whose influence on polyomavirus evolution was unambiguous. Finally, our analyses suggest that an update of the taxonomy of the family is required, including the creation of novel genera of mammalian and non-mammalian polyomaviruses.

Varicella in Tshuapa Province, Democratic Republic of Congo, 2009-2014.

OBJECTIVE: To describe varicella cases in Tshuapa Province of the Democratic Republic of the Congo identified during monkeypox surveillance. METHODS: Demographic, clinical and epidemiological data were collected from each suspected monkeypox case 2009-2014. Samples were tested by PCR for both Orthopoxviruses and varicella-zoster virus (VZV); a subset of VZV-positive samples was genotyped. We defined a varicella case as a rash illness with laboratory-confirmed VZV. RESULTS: There were 366 varicella cases were identified; 66% were ≤19 years old. Most patients had non-typical varicella rash with lesions reported as the same size and stage of evolution (86%), deep and profound (91%), on palms of hands and/or soles of feet (86%) and not itchy (49%). Many had non-typical signs and symptoms, such as lymphadenopathy (70%) and sensitivity to light (23%). A higher proportion of persons aged ≥20 years than persons aged ≤19 years had ≥50 lesions (79% vs. 65%, P = 0.007) and were bedridden (15% vs. 9%, P = 0.056). All VZV isolates genotyped from 79 varicella cases were clade 5. During the surveillance period, one possible VZV-related death occurred in a 7-year-old child. CONCLUSIONS: A large proportion of patients presented with non-typical varicella rash and clinical signs and symptoms, highlighting challenges identifying varicella in an area with endemic monkeypox. Continued surveillance and laboratory diagnosis will help in rapid identification and control of both monkeypox and varicella and improve our understanding of varicella epidemiology in Africa.

OBJECTIF: Décrire les cas de varicelle identifiés dans la province de Tshuapa en République Démocratique du Congo (RDC) au cours de la surveillance de la variole du singe (monkeypox). MÉTHODES: Des données démographiques, cliniques et épidémiologiques ont été recueillies pour chaque cas présumé de monkeypox entre 2009 et 2014. Les échantillons ont été testés par PCR pour les orthopoxvirus et le virus varicelle-zona (VZV); un sous-ensemble d'échantillons positifs au VZV a été génotypé. Nous avons défini un cas de varicelle comme une éruption cutanée avec confirmation du VZV en laboratoire. RÉSULTATS: 366 cas de varicelle ont été identifiés; 66% avaient 19 ans ou moins. La plupart des patients présentaient une éruption non typique de varicelle avec des lésions rapportées de la même taille et le même stade d'évolution (86%), profonds (91%), sur la paume des mains et/ou la plante des pieds (86%), sans démangeaisons (49%). Nombre d'entre eux présentaient des signes et des symptômes inhabituels, tels qu'une adénopathie lymphatique (70%) et une sensibilité à la lumière (23%). Une proportion plus élevée de personnes âgées de 20 ans et plus que de personnes âgées de 19 ans et moins avaient 50 lésions ou plus (79% contre 65%, p = 0,007) et étaient alitées (15% contre 9%; p = 0,056). Tous les isolats de VZV génotypés chez 79 cas de varicelle appartenaient au clade 5. Au cours de la période de surveillance, un décès possible lié au VZV est survenu chez un enfant de 7 ans. CONCLUSIONS: Une forte proportion de patients ont présenté une éruption de varicelle ainsi que des signes et symptômes cliniques non typiques, soulignant les difficultés rencontrées pour identifier la varicelle dans une zone endémique pour le monkeypox. Une surveillance continue et des diagnostics de laboratoire aideront à identifier et à contrôler rapidement le monkeypox et la varicelle et à améliorer notre compréhension sur l'épidémiologie de la varicelle en Afrique.

Serologic Prevalence of Ebola Virus in Equatorial Africa.

We conducted a serologic survey of 2,430 serum samples collected during 1997-2012 for various studies to determine the prevalence of the hemorrhagic fever virus Ebola virus (EBOV) in equatorial Africa. We screened serum samples for neutralizing antibodies by using a pseudotype microneutralization assay and a newly developed luciferase immunoprecipitation system assay. Specimens seroreactive for EBOV were confirmed by using an ELISA. Our results suggest a serologic prevalence of 2%-3.5% in the Republic of the Congo and the Democratic Republic of the Congo, which have reported outbreaks of infection with EBOV. In addition we detected a seroprevalence of 1.3% in southern Cameroon, which indicated a low risk for exposure in this region.

Bushmeat Hunting and Zoonotic Transmission of Simian T-Lymphotropic Virus 1 in Tropical West and Central Africa.

Simian T-lymphotropic virus 1 (STLV-1) enters human populations through contact with nonhuman primate (NHP) bushmeat. We tested whether differences in the extent of contact with STLV-1-infected NHP bushmeat foster regional differences in prevalence of human T-lymphotropic virus 1 (HTLV-1). Using serological and PCR assays, we screened humans and NHPs at two Sub-Saharan African sites where subsistence hunting was expected to be less (Taï region, Côte d'Ivoire [CIV]) or more (Bandundu region, Democratic Republic of the Congo [DRC]) developed. Only 0.7% of human participants were infected with HTLV-1 in CIV (n = 574), and 1.3% of humans were infected in DRC (n = 302). Two of the Ivorian human virus sequences were closely related to simian counterparts, indicating ongoing zoonotic transmission. Multivariate analysis of human demographic parameters and behavior confirmed that participants from CIV were less often exposed to NHPs than participants from DRC through direct contact, e.g., butchering. At the same time, numbers of STLV-1-infected NHPs were higher in CIV (39%; n = 111) than in DRC (23%; n = 39). We conclude that similar ultimate risks of zoonotic STLV-1 transmission-defined as the product of prevalence in local NHP and human rates of contact to fresh NHP carcasses-contribute to the observed comparable rates of HTLV-1 infection in humans in CIV and DRC. We found that young adult men and mature women are most likely exposed to NHPs at both sites. In view of the continued difficulties in controlling zoonotic disease outbreaks, the identification of such groups at high risk of NHP exposure may guide future prevention efforts.IMPORTANCE Multiple studies report a high risk for zoonotic transmission of blood-borne pathogens like retroviruses through contact with NHPs, and this risk seems to be particularly high in tropical Africa. Here, we reveal high levels of exposure to NHP bushmeat in two regions of Western and Central tropical Africa. We provide evidence for continued zoonotic origin of HTLV-1 in humans at CIV, and we found that young men and mature women represent risk groups for zoonotic transmission of pathogens from NHPs. Identifying such risk groups can contribute to mitigation of not only zoonotic STLV-1 transmission but also transmission of any blood-borne pathogen onto humans in Sub-Saharan Africa.

Novel simian foamy virus infections from multiple monkey species in women from the Democratic Republic of Congo.

BACKGROUND: Zoonotic transmission of simian retroviruses in Central Africa is ongoing and can result in pandemic human infection. While simian foamy virus (SFV) infection was reported in primate hunters in Cameroon and Gabon, little is known about the distribution of SFV in Africa and whether human-to-human transmission and disease occur. We screened 3,334 plasmas from persons living in rural villages in central Democratic Republic of Congo (DRC) using SFV-specific EIA and Western blot (WB) tests. PCR amplification of SFV polymerase sequences from DNA extracted from buffy coats was used to measure proviral loads. Phylogenetic analysis was used to define the NHP species origin of SFV. Participants completed questionnaires to capture NHP exposure information. RESULTS: Sixteen (0.5%) samples were WB-positive; 12 of 16 were from women (75%, 95% confidence limits 47.6%, 92.7%). Sequence analysis detected SFV in three women originating from Angolan colobus or red-tailed monkeys; both monkeys are hunted frequently in DRC. NHP exposure varied and infected women lived in distant villages suggesting a wide and potentially diverse distribution of SFV infections across DRC. Plasmas from 22 contacts of 8 WB-positive participants were all WB negative suggesting no secondary viral transmission. Proviral loads in the three women ranged from 14 - 1,755 copies/105 cells. CONCLUSIONS: Our study documents SFV infection in rural DRC for the first time and identifies infections with novel SFV variants from Colobus and red-tailed monkeys. Unlike previous studies, women were not at lower risk for SFV infection in our population, providing opportunities for spread of SFV both horizontally and vertically. However, limited testing of close contacts of WB-positive persons did not identify human-to-human transmission. Combined with the broad behavioral risk and distribution of NHPs across DRC, our results suggest that SFV infection may have a wider geographic distribution within DRC. These results also reinforce the potential for an increased SFV prevalence throughout the forested regions of Africa where humans and simians co-exist. Our finding of endemic foci of SFV infection in DRC will facilitate longitudinal studies to determine the potential for person-to-person transmissibility and pathogenicity of these zoonotic retroviral infections.

Identification and molecular characterization of new simian T cell lymphotropic viruses in nonhuman primates bushmeat from the Democratic Republic of Congo.

Four types of human T cell lymphotropic viruses (HTLV) have been described (HTLV-1 to HTLV-4) with three of them having closely related simian virus analogues named STLV-1, -2, and -3. To assess the risk of cross-species transmissions of STLVs from nonhuman primates to humans in the Democratic Republic of Congo, a total of 330 samples, derived from primate bushmeat, were collected at remote forest sites where people rely on bushmeat for subsistence. STLV prevalences and genetic diversity were estimated by PCR and sequence analysis of tax-rex and LTR fragments. Overall, 7.9% of nonhuman primate bushmeat is infected with STLVs. We documented new STLV-1 and STLV-3 variants in six out of the seven species tested and showed for the first time STLV infection in C. mona wolfi, C. ascanius whitesidei, L. aterrimus aterrimus, C. angolensis, and P. tholloni. Our results provide increasing evidence that the diversity and geographic distribution of PTLVs are much greater than previously thought.

Response to treatment in a prospective cohort of patients with large ulcerated lesions suspected to be Buruli Ulcer (Mycobacterium ulcerans disease).

BACKGROUND: The World Health Organization (WHO) advises treatment of Mycobacterium ulcerans disease, also called "Buruli ulcer" (BU), with a combination of the antibiotics rifampicin and streptomycin (R+S), whether followed by surgery or not. In endemic areas, a clinical case definition is recommended. We evaluated the effectiveness of this strategy in a series of patients with large ulcers of > or =10 cm in longest diameter in a rural health zone of the Democratic Republic of Congo (DRC). METHODS: A cohort of 92 patients with large ulcerated lesions suspected to be BU was enrolled between October 2006 and September 2007 and treated according to WHO recommendations. The following microbiologic data were obtained: Ziehl-Neelsen (ZN) stained smear, culture and PCR. Histopathology was performed on a sub-sample. Directly observed treatment with R+S was administered daily for 12 weeks and surgery was performed after 4 weeks. Patients were followed up for two years after treatment. FINDINGS: Out of 92 treated patients, 61 tested positive for M. ulcerans by PCR. PCR negative patients had better clinical improvement than PCR positive patients after 4 weeks of antibiotics (54.8% versus 14.8%). For PCR positive patients, the outcome after 4 weeks of antibiotic treatment was related to the ZN positivity at the start. Deterioration of the ulcers was observed in 87.8% (36/41) of the ZN positive and in 12.2% (5/41) of the ZN negative patients. Deterioration due to paradoxical reaction seemed unlikely. After surgery and an additional 8 weeks of antibiotics, 98.4% of PCR positive patients and 83.3% of PCR negative patients were considered cured. The overall recurrence rate was very low (1.1%). INTERPRETATION: Positive predictive value of the WHO clinical case definition was low. Low relapse rate confirms the efficacy of antibiotics. However, the need for and the best time for surgery for large Buruli ulcers requires clarification. We recommend confirmation by ZN stain at the rural health centers, since surgical intervention without delay may be necessary on the ZN positive cases to avoid progression of the disease. PCR negative patients were most likely not BU cases. Correct diagnosis and specific management of these non-BU ulcers cases are urgently needed.

Use of protective gear and the occurrence of occupational Marburg hemorrhagic fever in health workers from Watsa health zone, Democratic Republic of the Congo.

BACKGROUND: Occupational transmission to health workers (HWs) has been a typical feature of Marburg hemorrhagic fever (MHF) outbreaks. The goal of this study was to identify cases of occupational MHF in HWs from Durba and Watsa, Democratic Republic of the Congo; to assess levels of exposure and protection; and to explore reasons for inconsistent use of protective gear. METHODS: A serosurvey of 48 HWs who cared for patients with MHF was performed. In addition, HWs were given a questionnaire on types of exposure, use of protective gear, and symptoms after contact. Informal and in-depth interviews with HWs were also performed. RESULTS: We found 1 HW who was seropositive for MHF, in addition to 5 cases of occupational MHF known beforehand; 4 infections had occurred after the introduction of infection control. HWs protected themselves better during invasive procedures (injections, venipuncture, and surgery) than during noninvasive procedures, but the overall level of protection in the hospital remained insufficient, particularly outside of isolation wards. The reasons for inconsistent use of protective gear included insufficient availability of the gear, adherence to traditional explanatory models of the origin of disease, and peer bonding with sick colleagues. CONCLUSIONS: Infection control must not focus too exclusively on the establishment of isolation wards but should aim at improving overall hospital hygiene. Training of HWs should allow them to voice and discuss their doubts and prepare them for the peculiarities of caring for ill colleagues.

New enteroviruses, EV-93 and EV-94, associated with acute flaccid paralysis in the Democratic Republic of the Congo.

Surveillance of acute flaccid paralysis often identifies enteroviruses not typeable by virus neutralization in cell culture. During 2000 and 2001, 186 isolates from 138 children with acute flaccid paralysis in the Democratic Republic of the Congo were sent for typing to the National Reference Centre for Enteroviruses in Lyon, France. The 5' UTR of the viral genome could be amplified by PCR for 158 isolates from 114 patients. Isolates from 89 patients were neutralizable, and contained non-polio enterovirus types. Seventeen children were infected with more than one entero- or adenovirus; another three were co-infected with both these viruses. Serological typing failed with 19 isolates from 13 (9%) patients. The VP1 region of these strains could be amplified by PCR and sequenced, which revealed that five children were infected with CV-A17, EV-70, EV-76, EV-77, or CV-A13. Two patients were doubly infected, one with CV-A24 and E-9, and another with E-27 and EV-81. Isolates from six children contained strains with divergent VP1 region. The amino acid sequences of these complete VP1 regions diverged >or=28% from published types indicating that they represented two new enterovirus types, tentatively designated EV-93 belonging to HEV-B and EV-94 within HEV-D. The latter enterovirus has in parallel been isolated from sewage in Egypt. In conclusion, there was a high frequency of "untypable" enterovirus isolates from cases with acute flaccid paralysis in the Democratic Republic of the Congo. Six of these were shown to represent two enteroviruses not previously described.