Human Monkeypox in Sierra Leone after 44-Year Absence of Reported Cases.

We note the reemergence of human monkeypox in Sierra Leone following a 44-year absence of reported disease. The persons affected were an 11-month-old boy and, several years later, a 35-year-old man. The reappearance of monkeypox in this country suggests a need for renewed vigilance and awareness of the disease and its manifestations.

Animal Model of Sensorineural Hearing Loss Associated with Lassa Virus Infection.

UNLABELLED: Approximately one-third of Lassa virus (LASV)-infected patients develop sensorineural hearing loss (SNHL) in the late stages of acute disease or in early convalescence. With 500,000 annual cases of Lassa fever (LF), LASV is a major cause of hearing loss in regions of West Africa where LF is endemic. To date, no animal models exist that depict the human pathology of LF with associated hearing loss. Here, we aimed to develop an animal model to study LASV-induced hearing loss using human isolates from a 2012 Sierra Leone outbreak. We have recently established a murine model for LF that closely mimics many features of human disease. In this model, LASV isolated from a lethal human case was highly virulent, while the virus isolated from a nonlethal case elicited mostly mild disease with moderate mortality. More importantly, both viruses were able to induce SNHL in surviving animals. However, utilization of the nonlethal, human LASV isolate allowed us to consistently produce large numbers of survivors with hearing loss. Surviving mice developed permanent hearing loss associated with mild damage to the cochlear hair cells and, strikingly, significant degeneration of the spiral ganglion cells of the auditory nerve. Therefore, the pathological changes in the inner ear of the mice with SNHL supported the phenotypic loss of hearing and provided further insights into the mechanistic cause of LF-associated hearing loss. IMPORTANCE: Sensorineural hearing loss is a major complication for LF survivors. The development of a small-animal model of LASV infection that replicates hearing loss and the clinical and pathological features of LF will significantly increase knowledge of pathogenesis and vaccine studies. In addition, such a model will permit detailed characterization of the hearing loss mechanism and allow for the development of appropriate diagnostic approaches and medical care for LF patients with hearing impairment.

Serosurveillance of viral pathogens circulating in West Africa.

BACKGROUND: Sub-Saharan Africa is home to a variety of pathogens, but disease surveillance and the healthcare infrastructure necessary for proper management and control are severely limited. Lassa virus, the cause of Lassa fever, a severe hemorrhagic fever in humans is endemic in West Africa. In Sierra Leone at the Kenema Government Hospital Lassa Diagnostic Laboratory, up to 70 % of acute patient samples suspected of Lassa fever test negative for Lassa virus infection. This large amount of acute undiagnosed febrile illness can be attributed in part to an array of hemorrhagic fever and arthropod-borne viruses causing disease that goes undetected and untreated. METHODS: To better define the nature and extent of viral pathogens infecting the Sierra Leonean population, we developed a multiplexed MAGPIX® assay to detect IgG antibodies against Lassa, Ebola, Marburg, Rift Valley fever, and Crimean-Congo hemorrhagic fever viruses as well as pan-assays for flaviviruses and alphaviruses. This assay was used to survey 675 human serum samples submitted to the Lassa Diagnostic Laboratory between 2007 and 2014. RESULTS: In the study population, 50.2 % were positive for Lassa virus, 5.2 % for Ebola virus, 10.7 % for Marburg virus, 1.8 % for Rift Valley fever virus, 2.0 % for Crimean-Congo hemorrhagic fever virus, 52.9 % for flaviviruses and 55.8 % for alphaviruses. CONCLUSIONS: These data exemplify the importance of disease surveillance and differential diagnosis for viral diseases in Sierra Leone. We demonstrate the endemic nature of some of these viral pathogens in the region and suggest that unrecognized outbreaks of viral infection have occurred.

Control of immunopathology during chikungunya virus infection.

After several decades of epidemiologic silence, chikungunya virus (CHIKV) has recently re-emerged, causing explosive outbreaks and reaching the 5 continents. Transmitted through the bite of Aedes species mosquitoes, CHIKV is responsible for an acute febrile illness accompanied by several characteristic symptoms, including cutaneous rash, myalgia, and arthralgia, with the latter sometimes persisting for months or years. Although CHIKV has previously been known as a relatively benign disease, more recent epidemic events have brought waves of increased morbidity and fatality, leading it to become a serious public health problem. The host's immune response plays a crucial role in controlling the infection, but it might also contribute to the promotion of viral spread and immunopathology. This review focuses on the immune responses to CHIKV in human subjects with an emphasis on early antiviral immune responses. We assess recent developments in the understanding of their possible Janus-faced effects in the control of viral infection and pathogenesis. Although preventive vaccination and specific therapies are yet to be developed, exploring this interesting model of virus-host interactions might have a strong effect on the design of novel therapeutic options to minimize immunopathology without impairing beneficial host defenses.

Mice lacking functional STAT1 are highly susceptible to lethal infection with Lassa virus.

Lassa fever (LF) is a potentially lethal human disease that is caused by the arenavirus Lassa virus (LASV). Annually, around 300,000 infections with up to 10,000 deaths occur in regions of Lassa fever endemicity in West Africa. Here we demonstrate that mice lacking a functional STAT1 pathway are highly susceptible to infection with LASV and develop lethal disease with pathology similar to that reported in humans.

Long-term persistence of Chikungunya virus neutralizing antibodies in human populations of North Eastern Thailand.

BACKGROUND: Chikungunya virus (CHIKV) outbreak recurrences in Thailand are unpredictable and separated by unexplained and often long silent epidemiological periods that can last for several years. These silent periods could be explained in part by the fact that infection with one CHIKV strain confers lasting natural immunity, even against other CHIKV strains. In this study we evaluated the persistence of CHIKV-specific neutralizing antibodies in the population of Chumpae District, Khon Kaen Province, nineteen years after a CHIKV outbreak occurred in the same area in 1991. FINDINGS: Overall 39% (44/111) of 111 former patients had neutralizing antibodies reacting against CHIKV ECSA strain. Consistently high titers of neutralizing antibodies were found in 75% (33/44) of all positively-reacting sera, 70% of which (23/33) were collected from individuals amongst the >60 years old age group. Although the prevalence found in Pong Haeng village (70%) was significantly higher than the prevalence detected in the Nong Thum village (14%), control study villages without known previous Chikungunya epidemics had a high Chikungunya neutralizing antibody prevalence (65%). CONCLUSIONS: More than one-third of the pre-exposed population had persisting natural immunity that was more likely boosted by recent and repetitive exposure to the emerging ECSA CHIKV in Thailand. Also, Chikungunya virus appears to largely circulate in the country with a great variability appears between villages or area probably associated with the vector abundance and efficiency. Altogether these results show a potential for a lifelong immunity against CHIKV. Given the rapid spread of the highly pathogenic ECSA strain in Southern Thailand, the development of CHIK vaccine is strongly recommended.

New member of Plasmodium (Vinckeia) and Plasmodium cyclopsi discovered in bats in Sierra Leone - nuclear sequence and complete mitochondrial genome analyses.

Malaria remains the most important arthropod-borne infectious disease globally. The causative agent, Plasmodium, is a unicellular eukaryote that develops inside red blood cells. Identifying new Plasmodium parasite species that infect mammalian hosts can shed light on the complex evolution and diversity of malaria parasites. Bats feature a high diversity of microorganisms including seven separate genera of malarial parasites. Three species of Plasmodium have been reported so far, for which scarce reports exist. Here we present data from an investigation of Plasmodium infections in bats in the western Guinean lowland forest in Sierra Leone. We discovered a new Plasmodium parasite in the horseshoe bat Rhinolophus landeri. Plasmodium cyclopsi infections in a member of leaf-nosed bats, Doryrhina cyclops, exhibited a high prevalence of 100%. Phylogenetic analysis of complete mitochondrial genomes and nine nuclear markers recovered a close relationship between P. cyclopsi and the new Plasmodium parasite with the rodent species Plasmodium berghei, a widely used in vivo model to study malaria in humans. The data suggests that the "rodent/bat" Plasmodium (Vinckeia) clade represents a diverse group of malarial parasites that would likely expand with a systematic sampling of small mammals in tropical Africa. Identifying the bat Plasmodium repertoire is central to our understanding of the evolution of Plasmodium parasites in mammals.

Unconventional repertoire profile is imprinted during acute chikungunya infection for natural killer cells polarization toward cytotoxicity.

Chikungunya virus (CHIKV) is a worldwide emerging pathogen. In humans it causes a syndrome characterized by high fever, polyarthritis, and in some cases lethal encephalitis. Growing evidence indicates that the innate immune response plays a role in controlling CHIKV infection. We show here that CHIKV induces major but transient modifications in NK-cell phenotype and function soon after the onset of acute infection. We report a transient clonal expansion of NK cells that coexpress CD94/NKG2C and inhibitory receptors for HLA-C1 alleles and are correlated with the viral load. Functional tests reveal cytolytic capacity driven by NK cells in the absence of exogenous signals and severely impaired IFN-γ production. Collectively these data provide insight into the role of this unique subset of NK cells in controlling CHIKV infection by subset-specific expansion in response to acute infection, followed by a contraction phase after viral clearance.

Sequence optimized diagnostic assay for Ebola virus detection.

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013-2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

The acute phase of Chikungunya virus infection in humans is associated with strong innate immunity and T CD8 cell activation.

BACKGROUND: Rapidly spreading to new regions, including the islands of the Indian Ocean, Central Africa, and Europe, Chikungunya fever is becoming a major problem of public health. Unlike other members of the alphavirus genus, immune responses to Chikungunya virus (CHIKV) have been poorly investigated. METHODS: We conducted a large ex vivo multiplex study of 50 cytokine, chemokine, and growth factor plasma profiles in 69 acutely infected patients from the Gabonese outbreak of 2007. We also assessed a phenotypic study of T lymphocyte responses during human acute CHIKV infection. RESULTS: CHIKV infection in humans elicited strong innate immunity involving the production of numerous proinflammatory mediators. Interestingly, high levels of Interferon (IFN) α were consistently found. Production of interleukin (IL) 4, IL-10, and IFN-γ suggested the engagement of the adaptive immunity. This was confirmed by flow cytometry of circulating T lymphocytes that showed a CD8+ T lymphocyte response in the early stages of the disease, and a CD4+ T lymphocyte mediated response in the later stages. For the first time to our knowledge, we found evidence of CD95-mediated apoptosis of CD4+ T lymphocytes during the first 2 days after symptoms onset, ex vivo. CONCLUSIONS: Together, our findings suggest that strong innate immunity is required to control CHIKV infection.

Risk factors for Zaire ebolavirus--specific IgG in rural Gabonese populations.

BACKGROUND: In Gabon, several Ebolavirus outbreaks have occurred exclusively in the northeastern region. We conducted a large serosurvey to identify areas and populations at risk and potential demographic, clinical, and behavioral risk factors. METHODS: Blood samples and clinical and sociodemographic data were collected from 4349 adults and 362 children in a random sample of 220 villages in the 9 provinces of Gabon. An enzyme-linked immunosorbent assay was used to detect Zaire ebolavirus (ZEBOV)-specific IgG, and thin blood smears were used to detect parasites. Logistic regression was implemented using Stata software (Stata), and a probability level of <.05 was considered to be statistically significant. RESULTS: The prevalence of ZEBOV-specific IgG was 15.3% overall, increasing to 32.4% (P< .001) in forest areas. No sociodemographic risk factors were found, but the antibody prevalence increased linearly up to 20 years of age. Chronic arthralgia and amicrofilaremia were the only factors associated with ZEBOV seropositivity. CONCLUSIONS: These findings confirm the endemicity of ZEBOV in Gabon and its link to the ecosystem. Human antibody positivity would appear to be to the result of exposure to contaminated fruits.

Sustainable Laboratory Capacity Building After the 2014 Ebola Outbreak in the Republic of Guinea.

Background: The 2014-2016 West Africa Ebola virus disease outbreak heavily impacted the Republics of Guinea, Sierra Leone, and Liberia. The outbreak uncovered the weaknesses of the public health systems, including inadequately trained and insufficient health personnel as well as limited and poorly equipped health infrastructures. These weaknesses represent significant threats to global health security. In the wake of the outbreak, affected countries made urgent requests for international engagement to help strengthening the public health systems. Methods: This work describes the successful multi-year implementation of a laboratory capacity building program in the Republic of Guinea. The program integrated biorisk and quality management systems training, infectious diseases diagnostic training, facility engineering and maintenance training, and mentorship to strengthen Guinea's bio-surveillance capacity. Results: The major outcome of these efforts was an established and local staff-operated public health laboratory that performs disease surveillance and reporting and diagnostic of priority diseases and pathogens of security concerns. Conclusions: This work has improved the Guinea country's capabilities to address country public health issues and preparedness to respond to future infectious disease threats.

Molecular analysis of the 2012 Bundibugyo virus disease outbreak.

Bundibugyo virus (BDBV) is one of four ebolaviruses known to cause disease in humans. Bundibugyo virus disease (BVD) outbreaks occurred in 2007-2008 in Bundibugyo District, Uganda, and in 2012 in Isiro, Province Orientale, Democratic Republic of the Congo. The 2012 BVD outbreak resulted in 38 laboratory-confirmed cases of human infection, 13 of whom died. However, only 4 BDBV specimens from the 2012 outbreak have been sequenced. Here, we provide BDBV sequences from seven additional patients. Analysis of the molecular epidemiology and evolutionary dynamics of the 2012 outbreak with these additional isolates challenges the current hypothesis that the outbreak was the result of a single spillover event. In addition, one patient record indicates that BDBV's initial emergence in Isiro occurred 50 days earlier than previously accepted. Collectively, this work demonstrates how retrospective sequencing can be used to elucidate outbreak origins and provide epidemiological contexts to a medically relevant pathogen.

The deployment of mobile diagnostic laboratories for Ebola virus disease diagnostics in Sierra Leone and Guinea.

BACKGROUND: Ebola virus emerged in West Africa in December 2013. The ease of mobility, porous borders, and lack of public health infrastructure led to the largest Ebola virus disease (EVD) outbreak to date. INTERVENTION: The 2013 EVD outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. As part of the United States' Department of Defense response, MRIGlobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (MDLs). The first laboratory analysed blood samples from patients in an adjacent Ebola Treatment Centre (ETC) and buccal swabs from the deceased in the community in Moyamba, Sierra Leone. The second laboratory was deployed to support an ETC in Conakry, Guinea. The Department of Defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site. LESSONS LEARNT: Prompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. Experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for MDL setup and operation. As the Ebola response slowed, the sustainment of the MDLs' operations was prioritised, including staff training and the transition of the MDLs to local governments. Training programmes for local staff were prepared in Sierra Leone and Guinea. RECOMMENDATIONS: The MRIGlobal MDL team significantly contributed to establishing increased laboratory capacity during the EVD outbreak in West Africa. Using the MDLs for molecular diagnosis is highly recommended until more sustainable solutions can be provided.

Association of KIR2DS1 and KIR2DS3 with fatal outcome in Ebola virus infection.

Zaïre ebolavirus (ZEBOV) infection rapidly outruns the host's immunity and leads to death within a week. Fatal cases have been associated with an aberrant innate, proinflammatory immune response followed by a suppressed adaptive response leading to the rapid depletion of peripheral NK cells and lymphocytes. A critical role for NK cells has been suggested but not elucidated. In this genetic study, we investigated the association of KIR genotype with disease outcome by comparing genotypes of a Gabonese control population, IgG+ contacts, survivors, and fatalities of ZEBOV infection. We showed that the activating KIR2DS1 and KIR2DS3 genes associate with fatal outcome in Ebola virus infection. In addition, this study brings supplemental evidence in favor of the specificity of the IgG+ contact population. The outcome of fulminating Ebola virus infection could depend in part on the host's inherited KIR gene repertoire. This supports a key role for KIRs in disease susceptibility to infections.

Acute dengue virus 2 infection in Gabonese patients is associated with an early innate immune response, including strong interferon alpha production.

BACKGROUND: Dengue is now a leading cause of morbidity and mortality throughout the tropics. We conducted the first ex vivo study of dengue fever (DF) in African patients infected during the first Gabonese dengue virus 2 (DENV-2) outbreak in 2007, in order to investigate cytokine production, including the antiviral cytokine IFN-α, reported to be a potent inhibitor of DENV replication in vitro. METHODS: Levels of 50 cytokines, chemokines and growth factors were measured in plasma from 36 patients with DENV-2 infection, and in uninfected controls, using Luminex multiplex technology. The results were interpreted according to the day of sampling after symptom onset. PBMC from six patients were also studied for T lymphocyte cell surface marker expression by flow cytometry. RESULTS: Acute DENV-2 infection elicited high levels of several pro-inflammatory cytokines (IL-6 and IL-17), chemokines (MIF, RANTES, IP-10 and MCP-1) and growth factors (G-CSF, GM-CSF and VEGF-A). We also observed high levels of IFN-α for the first time in adult DF patients, and CD4+ and CD8+ T cell activation at symptom onset. CONCLUSION: Acute DENV-2 infection in African patients elicits a strong innate response involving IFN-α production, as well as an adaptive immune response.

HLA-C-restricted viral epitopes are associated with an escape mechanism from KIR2DL2+ NK cells in Lassa virus infection.

BACKGROUND: Lassa virus (LASV) is the etiologic agent of an acute hemorrhagic fever endemic in West Africa. Natural killer (NK) cells control viral infections in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their ligands. LASV infection is associated with defective immune responses, including inhibition of NK cell activity in the presence of MHC-class 1+-infected target cells. METHODS: We compared individual KIR and HLA-class 1 genotypes of 68 healthy volunteers to 51 patients infected with LASV in Sierra Leone, including 37 survivors and 14 fatalities. Next, potential HLA-C1, HLA-C2, and HLA-Bw4 binding epitopes were in silico screened among LASV nucleoprotein (NP) and envelope glycoprotein (GP). Selected 10-mer peptides were then tested in peptide-HLA stabilization, KIR binding and polyfunction assays. FINDINGS: LASV-infected patients were similar to healthy controls, except for the inhibitory KIR2DL2 gene. We found a specific increase in the HLA-C1:KIR2DL2 interaction in fatalities (10/11) as compared to survivors (12/19) and controls (19/29). We also identified that strong of NP and GP viral epitopes was only observed with HLA-C molecules, and associated with strong inhibition of degranulation in the presence of KIR2DL+ NK cells. This inhibitory effect significantly increased in the presence of the vGP420 variant, detected in 28.1% of LASV sequences. INTERPRETATION: Our finding suggests that presentation of specific LASV epitopes by HLA-C alleles to the inhibitory KIR2DL2 receptor on NK cells could potentially prevent the killing of infected cells and provides insights into the mechanisms by which LASV can escape NK-cell-mediated immune pressure.

Virus-encoded miRNAs in Ebola virus disease.

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

Contact tracing performance during the Ebola virus disease outbreak in Kenema district, Sierra Leone.

Contact tracing in an Ebola virus disease (EVD) outbreak is the process of identifying individuals who may have been exposed to infected persons with the virus, followed by monitoring for 21 days (the maximum incubation period) from the date of the most recent exposure. The goal is to achieve early detection and isolation of any new cases in order to prevent further transmission. We performed a retrospective data analysis of 261 probable and confirmed EVD cases in the national EVD database and 2525 contacts in the Contact Line Lists in Kenema district, Sierra Leone between 27 April and 4 September 2014 to assess the performance of contact tracing during the initial stage of the outbreak. The completion rate of the 21-day monitoring period was 89% among the 2525 contacts. However, only 44% of the EVD cases had contacts registered in the Contact Line List and 6% of probable or confirmed cases had previously been identified as contacts. Touching the body fluids of the case and having direct physical contact with the body of the case conferred a 9- and 20-fold increased risk of EVD status, respectively. Our findings indicate that incompleteness of contact tracing led to considerable unmonitored transmission in the early months of the epidemic. To improve the performance of early outbreak contact tracing in resource poor settings, our results suggest the need for prioritized contact tracing after careful risk assessment and better alignment of Contact Line Listing with case ascertainment and investigation.This article is part of the themed issue 'The 2013-2016 West African Ebola epidemic: data, decision-making and disease control'.