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.

Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever.

There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

BACKGROUND: Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. METHODS: Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. RESULTS: The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 105-9.0 × 108 genomes/mL. CONCLUSIONS: The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015.

Treatment of Lassa virus infection in outbred guinea pigs with first-in-class human monoclonal antibodies.

Lassa fever is a significant health threat to West African human populations with hundreds of thousands of annual cases. There are no approved medical countermeasures currently available. Compassionate use of the antiviral drug ribavirin or transfusion of convalescent serum has resulted in mixed success depending on when administered or the donor source, respectively. We previously identified several recombinant human monoclonal antibodies targeting the glycoprotein of Lassa virus with strong neutralization profiles in vitro. Here, we demonstrate remarkable therapeutic efficacy using first-in-class human antibodies in a guinea pig model of Lassa infection thereby presenting a promising treatment alternative.

Field Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

BACKGROUND: The 2013-2016 West African Ebola virus disease (EVD) epidemic is the largest recorded. Triage on the basis of clinical signs had limited success, and the time to diagnosis by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) could exceed 5 days. Here we describe the development and field validation of the ReEBOV Antigen Rapid Test (ReEBOV RDT) to aid triage of individuals with suspected EVD. METHODS: Samples from patients with suspected EVD were submitted to Kenema Government Hospital, Sierra Leone, for Lassa fever and EVD screening throughout 2014. Banked residual clinical samples were tested in November 2014 and January 2015 in a blinded field trial to estimate the clinical effectiveness of the ReEBOV RDT, compared with EBOV-specific qRT-PCR. RESULTS: Preliminary ReEBOV RDT performance demonstrated a positive percentage agreement (PPA) of 91.1% (195 of 214 results; 95% confidence interval [CI], 86.5%-94.6%) and a negative percentage agreement (NPA) of 90.2% (175 of 194; 95% CI, 85.1%-94.0%). The final estimates used by the Food and Drug Administration to determine whether to grant emergency use authorization for the test, which excluded a qRT-PCR reference method threshold cutoff, were a PPA of 62.1% (72 of 116 results; 95% CI, 52.6%-70.9%) and a NPA of 96.7% (58 of 60; 95% CI, 88.5%-99.6%), with a diagnostic likelihood of 18.6. A subsequent, independent evaluation by the World Health Organization generated results consistent with the preliminary performance estimates. CONCLUSIONS: The ReEBOV RDT demonstrated the potential to provide clinically effective rapid and accurate point-of-care test results and, thus, to be a powerful tool for increasing triage efficiency.

Understanding the emergence of ebola virus disease in sierra leone: stalking the virus in the threatening wake of emergence.

Since Ebola Virus Disease (EVD) was first identified in 1976 in what is now the Democratic Republic of Congo, and despite the numerous outbreaks recorded to date, rarely has an epidemic origin been identified. Indeed, among the twenty-one most documented EVD outbreaks in Africa, an index case has been identified four times, and hypothesized in only two other instances. The initial steps of emergence and spread of a virus are critical in the development of a potential outbreak and need to be thoroughly dissected and understood in order to improve on preventative strategies. In the current West African outbreak of EVD, a unique index case has been identified, pinpointing the geographical origin of the epidemic in Guinea. Herein, we provide an accounting of events that serve as the footprint of EVD emergence in Sierra Leone and a road map for risk mitigation fueled by lessons learned.

Using modelling to disentangle the relative contributions of zoonotic and anthroponotic transmission: the case of lassa fever.

BACKGROUND: Zoonotic infections, which transmit from animals to humans, form the majority of new human pathogens. Following zoonotic transmission, the pathogen may already have, or may acquire, the ability to transmit from human to human. With infections such as Lassa fever (LF), an often fatal, rodent-borne, hemorrhagic fever common in areas of West Africa, rodent-to-rodent, rodent-to-human, human-to-human and even human-to-rodent transmission patterns are possible. Indeed, large hospital-related outbreaks have been reported. Estimating the proportion of transmission due to human-to-human routes and related patterns (e.g. existence of super-spreaders), in these scenarios is challenging, but essential for planned interventions. METHODOLOGY/PRINCIPAL FINDINGS: Here, we make use of an innovative modeling approach to analyze data from published outbreaks and the number of LF hospitalized patients to Kenema Government Hospital in Sierra Leone to estimate the likely contribution of human-to-human transmission. The analyses show that almost [Formula: see text] of the cases at KGH are secondary cases arising from human-to-human transmission. However, we found much of this transmission is associated with a disproportionally large impact of a few individuals ('super-spreaders'), as we found only [Formula: see text] of human cases result in an effective reproduction number (i.e. the average number of secondary cases per infectious case) [Formula: see text], with a maximum value up to [Formula: see text]. CONCLUSIONS/SIGNIFICANCE: This work explains the discrepancy between the sizes of reported LF outbreaks and a clinical perception that human-to-human transmission is low. Future assessment of risks of LF and infection control guidelines should take into account the potentially large impact of super-spreaders in human-to-human transmission. Our work highlights several neglected topics in LF research, the occurrence and nature of super-spreading events and aspects of social behavior in transmission and detection.

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.

Multiple circulating infections can mimic the early stages of viral hemorrhagic fevers and possible human exposure to filoviruses in Sierra Leone prior to the 2014 outbreak.

Lassa fever (LF) is a severe viral hemorrhagic fever caused by Lassa virus (LASV). The LF program at the Kenema Government Hospital (KGH) in Eastern Sierra Leone currently provides diagnostic services and clinical care for more than 500 suspected LF cases per year. Nearly two-thirds of suspected LF patients presenting to the LF Ward test negative for either LASV antigen or anti-LASV immunoglobulin M (IgM), and therefore are considered to have a non-Lassa febrile illness (NLFI). The NLFI patients in this study were generally severely ill, which accounts for their high case fatality rate of 36%. The current studies were aimed at determining possible causes of severe febrile illnesses in non-LF cases presenting to the KGH, including possible involvement of filoviruses. A seroprevalence survey employing commercial enzyme-linked immunosorbent assay tests revealed significant IgM and IgG reactivity against dengue virus, chikungunya virus, West Nile virus (WNV), Leptospira, and typhus. A polymerase chain reaction-based survey using sera from subjects with acute LF, evidence of prior LASV exposure, or NLFI revealed widespread infection with Plasmodium falciparum malaria in febrile patients. WNV RNA was detected in a subset of patients, and a 419 nt amplicon specific to filoviral L segment RNA was detected at low levels in a single patient. However, 22% of the patients presenting at the KGH between 2011 and 2014 who were included in this survey registered anti-Ebola virus (EBOV) IgG or IgM, suggesting prior exposure to this agent. The 2014 Ebola virus disease (EVD) outbreak is already the deadliest and most widely dispersed outbreak of its kind on record. Serological evidence reported here for possible human exposure to filoviruses in Sierra Leone prior to the current EVD outbreak supports genetic analysis that EBOV may have been present in West Africa for some time prior to the 2014 outbreak.

Undiagnosed acute viral febrile illnesses, Sierra Leone.

Sierra Leone in West Africa is in a Lassa fever-hyperendemic region that also includes Guinea and Liberia. Each year, suspected Lassa fever cases result in submission of ≈500-700 samples to the Kenema Government Hospital Lassa Diagnostic Laboratory in eastern Sierra Leone. Generally only 30%-40% of samples tested are positive for Lassa virus (LASV) antigen and/or LASV-specific IgM; thus, 60%-70% of these patients have acute diseases of unknown origin. To investigate what other arthropod-borne and hemorrhagic fever viral diseases might cause serious illness in this region and mimic Lassa fever, we tested patient serum samples that were negative for malaria parasites and LASV. Using IgM-capture ELISAs, we evaluated samples for antibodies to arthropod-borne and other hemorrhagic fever viruses. Approximately 25% of LASV-negative patients had IgM to dengue, West Nile, yellow fever, Rift Valley fever, chikungunya, Ebola, and Marburg viruses but not to Crimean-Congo hemorrhagic fever virus.

Review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for Lassa fever.

Lassa fever is an acute viral hemorrhagic illness; the virus is endemic in West Africa and also of concern with regard to bioterrorism. Transmission of Lassa virus between humans may occur through direct contact with infected blood or bodily secretions. Oral administration of the antiviral drug ribavirin is often considered for postexposure prophylaxis, but no systematically collected data or uniform guidelines exist for this indication. Furthermore, the relatively low secondary attack rates for Lassa fever, the restriction of the area of endemicity to West Africa, and the infrequency of high-risk exposures make it unlikely that controlled prospective efficacy trials will ever be possible. Recommendations for postexposure use of ribavirin can therefore be made only on the basis of a thorough understanding and logical extrapolation of existing data. Here, we review the pertinent issues and propose guidelines based on extensive review of the literature, as well as our experience in this field. We recommend oral ribavirin postexposure prophylaxis for Lassa fever exclusively for definitive high-risk exposures. These guidelines may also serve for exposure to other hemorrhagic fever viruses susceptible to ribavirin.

The voluntary HIV counselling and testing service in Kenema District, Sierra Leone, 2004-2006: a descriptive study.

BACKGROUND: Voluntary counselling and testing (VCT) is an important component of national HIV programs, which are necessary to realize the right to health. VCT data also provide valuable information on regional HIV epidemiology. METHODS: The study examines data on the population that obtained HIV VCT in Kenema District, Sierra Leone, from 2004 to 2006, using descriptive statistics and exploring potential HIV risk factors using bivariate and multivariable logistic regression. Analysis was performed separately for two subpopulations: those accessing VCT routinely as part of antenatal care and those specifically seeking VCT. RESULTS: During this period, 2230 people accessed VCT: 1213 through antenatal testing and 1017 specifically seeking VCT. The HIV prevalence was 0.6% in women presenting for antenatal care, 12.6% in women specifically accessing VCT, and 6.7% in men specifically accessing VCT. In both bivariate and multivariable analyses, being female was statistically significantly associated with testing positive in people specifically seeking VCT. CONCLUSIONS: These data from the VCT service in Kenema will be used to improve the accessibility of HIV testing. Questions raised by the analysis will be used to enhance data collection and to inform further research on risk factors.

Stress-induced senescence exaggerates postinjury neointimal formation in the old vasculature.

This study aims to demonstrate the role of stress-induced senescence in aged-related neointimal formation. We demonstrated that aging increases senescence-associated beta-galactosidase activity (SA-beta-Gal) after vascular injury and the subsequent neointimal formation (neointima-to-media ratio: 0.8 +/- 0.2 vs. 0.54 +/- 0.15) in rats. We found that senescent cells (SA-beta-Gal(+) p21(+)) were scattered throughout the media and adventitia of the vascular wall at day 7 after injury and reached their maximum number at day 14. However, senescent cells only persisted in the injured arteries of aged animals until day 30. No senescent cells were observed in the noninjured, contralateral artery. Interestingly, vascular senescent cells accumulated genomic 8-oxo-7,8-dihydrodeoxyguanine, indicating that these cells were under intense oxidative stress. To demonstrate whether senescence worsens intimal hyperplasia after injury, we seeded matrigel-embedded senescent and nonsenescent vascular smooth muscle cells around injured vessels. The neointima was thicker in arteries treated with senescent cells with respect to those that received normal cells (neointima-to-media ratio: 0.41 +/- 0.105 vs. 0.26 +/- 0.04). In conclusion, these results demonstrate that vascular senescence is not only a consequence of postinjury oxidative stress but is also a worsening factor for neointimal development in the aging vasculature.

The origin of post-injury neointimal cells in the rat balloon injury model.

AIMS: The origin of post-injury neointimal cells is still a matter of debate. This study aims to determine the anatomic source of neointimal cells in one of the most important animal models for the study of vascular stenosis in response to injury, the rat balloon injury model. METHODS AND RESULTS: Chimeric rats were generated by rescuing lethally irradiated animals with green fluorescent protein (GFP)(+) bone marrow (BM) cells from transgenic rats. Neointimal formation was induced in the right iliac artery of these animals using a balloon angioplasty catheter. Injured and non-injured contra-lateral arteries were harvested at 7, 14, and 30 days post-surgery. BM-derived monocytes/macrophages (CD68(+) GFP(+)) were abundant in the media and adventitia of injured vessels harvested at 7 days as determined by immunofluorescence and confocal microscopy. The number of GFP(+) cells declined in the vascular wall with time. Post-injury neointimal cells were mostly GFP(-)/smooth muscle actin (SMA)(+), which indicated that those cells originated in the recipient. Only a few neointimal cells seemed to come from circulating progenitors (GFP(+) SMA(+), 2.34% +/- 1.61). The vascular origin of cells in the neointima was further confirmed by transplanting injured GFP arteries into wild-type recipients. In these grafts, 94.23 +/- 0.44% of medial and 92.95 +/- 19.34% of neointimal cells were GFP(+) SMA(+). Finally, we tested the capacity of vascular smooth muscle cells (VSMC) to migrate through the vascular wall using a novel in vivo assay. As expected, VSMC migrated and populated the neointima only in response to injury. CONCLUSION: Our results suggest that neointimal cells in the rat balloon injury model mostly derive from pre-existing vascular cells and that only a small population of those cells come from BM-derived progenitors.

An internal ribosome entry site mediates the initiation of soluble guanylyl cyclase beta2 mRNA translation.

The soluble guanylyl cyclases (sGC), the receptor for nitric oxide, are heterodimers consisting of an alpha- and beta-subunit. This study aimed to investigate the translational mechanism of the sGC beta2-subunit. Two mRNA species for sGC beta2 were isolated from human kidney. These transcripts had dissimilar 5'-untranslated regions (5'-UTRs). The most abundant sGC beta2 mRNA showed numerous upstream open reading frames (ORFs) and stable secondary structures that inhibited in vivo and in vitro translation. To evaluate whether these 5'-UTRs harbored an internal ribosome entry site (IRES) that allows translation by an alternative mechanism, we inserted these regions between the two luciferase genes of a bicistronic vector. Transfection of those genetic constructs into HeLa cells demonstrated that both sGC beta2 leaders had IRES activity in a cell-type dependent manner. Finally, the secondary structural model of the sGC beta2 5'-UTR predicts a Y-type pseudoknot that characterizes the IRES of cellular mRNAs. In conclusion, our findings suggest that sGC beta2 5'-UTRs have IRES activity that may permit sGC beta2 expression under conditions that are not optimal for scanning-dependent translation.

New opportunities for field research on the pathogenesis and treatment of Lassa fever.

Unlike many viral hemorrhagic fevers (VHFs), Lassa fever (LF) is not a rare disease that emerges only as sporadic cases or in outbreak form. Although surveillance is inadequate to determine the true incidence, up to 300,000 infections and 5000 deaths from LF are estimated to occur yearly. The highest incidence is in the "Mano River Union (MRU) countries" of Sierra Leone, Liberia, and Guinea. Although civil unrest in this region over the past two decades has impeded capacity building and research, new-found peace in recent years presents new opportunities. In 2004, the Mano River Union Lassa Fever Network (MRU LFN) was established to assist MRU countries in the development of national and regional surveillance, diagnosis, treatment, control, and prevention of LF. Here, we review the present literature on treatment and pathogenesis of LF and outline priorities for future research in the field made possible by the improved research capacity of the MRU LFN.

Interactions of TRF2 with model telomeric ends.

Telomeres are DNA-protein complexes at the ends of eukaryotic chromosomes, the integrity of which is essential for chromosome stability. An important telomere binding protein, TTAGGG repeat factor 2 (TRF2), is thought to protect telomere ends by remodeling them into T-loops. We show that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the 3', guanine-strand (G-strand) overhang and double-stranded DNA sequence at the junction. Association of TRF2 with DNA junctions hinders cleavage by exonuclease T. TRF2 interactions with the G-strand overhang do not involve the TRF2 DNA binding domain or the linker region. However, mobility shifts and atomic force microscopy show that the previously uncharacterized linker region is involved in DNA-specific, TRF2 oligomerization. We suggest that T-loop formation at telomere ends involves TRF2 binding to the G-strand overhang and oligomerization through both the known TRFH domain and the linker region.

DNA structure-dependent recruitment of telomeric proteins to single-stranded/double-stranded DNA junctions.

Telomeres protect chromosome ends by assembling unique protein-DNA complexes. TRF2 is a telomere binding protein that is involved in protecting the G-strand overhang, a 3', guanine-rich, overhang at the telomere terminus. TRF2 may protect the G-strand overhang by recognizing some organizational aspect of the telomeric single-stranded/double-stranded (ss/ds) DNA junction. This work demonstrates that TRF2, purified or in crude extracts, recognizes telomeric ss/ds DNA junctions containing wild type telomeric sequence in the ds region and a G-strand overhang with at least one telomeric repeat. Telomeric complexes containing TRF2 and pot1 assemble less efficiently when the G-strand overhang is in the form of an intramolecular G-quadruplex. However, recruitment of the DNA repair proteins, WRN, Mre11, and Ku86, is not inhibited by a G-quadruplex. This suggests that an intramolecular G-quadruplex has the potential to disrupt certain telomeric assemblies, but efficient recruitment of appropriate DNA repair proteins provides the means to overcome this obstacle.