Correction: Correction: A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever in Central Africa.

[This corrects the article DOI: 10.1371/journal.ppat.1002924.].

DNA indicative of human bocaviruses detected in non-human primates in the Democratic Republic of the Congo.

Bocaparvoviruses are members of the family Parvovirinae and human bocaviruses have been found to be associated with respiratory and gastrointestinal disease. There are four known human bocaviruses, as well as several distinct ones in great apes. The goal of the presented study was to detect other non-human primate (NHP) bocaviruses in NHP species in the Democratic Republic of the Congo using conventional broad-range PCR. We found bocavirus DNA in blood and tissues samples in 6 out of 620 NHPs, and all isolates showed very high identity (>97 %) with human bocaviruses 2 or 3. These findings suggest cross-species transmission of bocaviruses between humans and NHPs.

A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples.

Unbiased next-generation sequencing (NGS) approaches enable comprehensive pathogen detection in the clinical microbiology laboratory and have numerous applications for public health surveillance, outbreak investigation, and the diagnosis of infectious diseases. However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe. Here we describe SURPI ("sequence-based ultrarapid pathogen identification"), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. Deployable on both cloud-based and standalone servers, SURPI leverages two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. In fast mode, SURPI detects viruses and bacteria by scanning data sets of 7-500 million reads in 11 min to 5 h, while in comprehensive mode, all known microorganisms are identified, followed by de novo assembly and protein homology searches for divergent viruses in 50 min to 16 h. SURPI has also directly contributed to real-time microbial diagnosis in acutely ill patients, underscoring its potential key role in the development of unbiased NGS-based clinical assays in infectious diseases that demand rapid turnaround times.

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.

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.

Genomic variability of monkeypox virus among humans, Democratic Republic of the Congo.

Monkeypox virus is a zoonotic virus endemic to Central Africa. Although active disease surveillance has assessed monkeypox disease prevalence and geographic range, information about virus diversity is lacking. We therefore assessed genome diversity of viruses in 60 samples obtained from humans with primary and secondary cases of infection from 2005 through 2007. We detected 4 distinct lineages and a deletion that resulted in gene loss in 10 (16.7%) samples and that seemed to correlate with human-to-human transmission (p = 0.0544). The data suggest a high frequency of spillover events from the pool of viruses in nonhuman animals, active selection through genomic destabilization and gene loss, and increased disease transmissibility and severity. The potential for accelerated adaptation to humans should be monitored through improved surveillance.

Characterization of the Bas-Congo virus glycoprotein and its function in pseudotyped viruses.

Bas-Congo virus (BASV) is a novel rhabdovirus recently identified from a patient with acute hemorrhagic fever in the Bas-Congo province of the Democratic Republic of Congo (DRC). Here we show that the BASV glycoprotein (BASV-G) can be successfully used to pseudotype glycoprotein-deficient vesicular stomatitis virus (VSV), allowing studies of BASV-G-driven membrane fusion and viral entry into target cells without replication-competent virus. BASV-G displayed broad tissue and species tropism in vitro, and BASV-G-mediated membrane fusion was pH dependent. The conformational changes induced in BASV-G by acidification were fully reversible and did not lead to inactivation of the viral fusion protein. Our data combined with comparative sequence similarity analyses suggest that BASV-G shares structural and functional features with other rhabdovirus glycoproteins and falls into the group of class III viral fusion proteins. However, activation of BASV-G-driven fusion required a lower pH and higher temperatures than did VSV-G-mediated fusion. Moreover, in contrast to VSV-G, mature BASV-G in VSV pseudotypes consists of a mixture of high-mannose and complex glycans that enables it to bind to certain C-type lectins, thereby enhancing its attachment to target cells. Taken together, the results presented in this study will facilitate future investigations of BASV-G-mediated cell entry and its inhibition in the absence of an infectious cell culture assay for BASV and at lower biosafety levels. Moreover, serology testing based on BASV-G pseudotype neutralization can be used to uncover the prevalence and importance of BASV as a potential novel human pathogen in the DRC and throughout Central Africa.

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.

A novel rhabdovirus associated with acute hemorrhagic fever in central Africa.

Deep sequencing was used to discover a novel rhabdovirus (Bas-Congo virus, or BASV) associated with a 2009 outbreak of 3 human cases of acute hemorrhagic fever in Mangala village, Democratic Republic of Congo (DRC), Africa. The cases, presenting over a 3-week period, were characterized by abrupt disease onset, high fever, mucosal hemorrhage, and, in two patients, death within 3 days. BASV was detected in an acute serum sample from the lone survivor at a concentration of 1.09 × 10(6) RNA copies/mL, and 98.2% of the genome was subsequently de novo assembled from ≈ 140 million sequence reads. Phylogenetic analysis revealed that BASV is highly divergent and shares less than 34% amino acid identity with any other rhabdovirus. High convalescent neutralizing antibody titers of >1:1000 were detected in the survivor and an asymptomatic nurse directly caring for him, both of whom were health care workers, suggesting the potential for human-to-human transmission of BASV. The natural animal reservoir host or arthropod vector and precise mode of transmission for the virus remain unclear. BASV is an emerging human pathogen associated with acute hemorrhagic fever in Africa.

Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.

Studies on the burden of human monkeypox in the Democratic Republic of the Congo (DRC) were last conducted from 1981 to 1986. Since then, the population that is immunologically naïve to orthopoxviruses has increased significantly due to cessation of mass smallpox vaccination campaigns. To assess the current risk of infection, we analyzed human monkeypox incidence trends in a monkeypox-enzootic region. Active, population-based surveillance was conducted in nine health zones in central DRC. Epidemiologic data and biological samples were obtained from suspected cases. Cumulative incidence (per 10,000 population) and major determinants of infection were compared with data from active surveillance in similar regions from 1981 to 1986. Between November 2005 and November 2007, 760 laboratory-confirmed human monkeypox cases were identified in participating health zones. The average annual cumulative incidence across zones was 5.53 per 10,000 (2.18-14.42). Factors associated with increased risk of infection included: living in forested areas, male gender, age < 15, and no prior smallpox vaccination. Vaccinated persons had a 5.2-fold lower risk of monkeypox than unvaccinated persons (0.78 vs. 4.05 per 10,000). Comparison of active surveillance data in the same health zone from the 1980s (0.72 per 10,000) and 2006-07 (14.42 per 10,000) suggests a 20-fold increase in human monkeypox incidence. Thirty years after mass smallpox vaccination campaigns ceased, human monkeypox incidence has dramatically increased in rural DRC. Improved surveillance and epidemiological analysis is needed to better assess the public health burden and develop strategies for reducing the risk of wider spread of infection.

High seroprevalence of enterovirus infections in apes and old world monkeys.

To estimate population exposure of apes and Old World monkeys in Africa to enteroviruses (EVs), we conducted a seroepidemiologic study of serotype-specific neutralizing antibodies against 3 EV types. Detection of species A, B, and D EVs infecting wild chimpanzees demonstrates their potential widespread circulation in primates.

Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome: case report.

Lassa fever is a neglected tropical disease with a significant impact on the health care system of endemic West African nations. To date, case reports of Lassa fever have focused on laboratory characterisation of serological, biochemical and molecular aspects of the disease imported by infected individuals from Western Africa to the United States, Canada, Europe, Japan and Israel. Our report presents the first comprehensive real time diagnosis and characterization of a severe, hemorrhagic Lassa fever case in a Sierra Leonean individual admitted to the Kenema Government Hospital Lassa Fever Ward. Fever, malaise, unresponsiveness to anti-malarial and antibiotic drugs, followed by worsening symptoms and onset of haemorrhaging prompted medical officials to suspect Lassa fever. A recombinant Lassa virus protein based diagnostic was employed in diagnosing Lassa fever upon admission. This patient experienced a severe case of Lassa hemorrhagic fever with dysregulation of overall homeostasis, significant liver and renal system involvement, the interplay of pro- and anti-inflammatory cytokines during the course of hospitalization and an eventual successful outcome. These studies provide new insights into the pathophysiology and management of this viral illness and outline the improved infrastructure, research and real-time diagnostic capabilities within LASV endemic areas.

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 origin and prevention of pandemics.

Despite the fact that most emerging diseases stem from the transmission of pathogenic agents from animals to humans, the factors that mediate this process are still ill defined. What is known, however, is that the interface between humans and animals is of paramount importance in the process. This review will discuss the importance of the human-animal interface to the disease emergence process. We also provide an overview of factors that are believed to contribute to the origin and global spread of emerging infectious diseases and offer suggestions that may serve as future prevention strategies, such as social mobilization, public health education, behavioral change, and communication strategies. Because there exists no comprehensive global surveillance system to monitor zoonotic disease emergence, the intervention measures discussed herein may prove effective temporary alternatives.

Uncoupling GP1 and GP2 expression in the Lassa virus glycoprotein complex: implications for GP1 ectodomain shedding.

BACKGROUND: Sera from convalescent Lassa fever patients often contains antibodies to Lassa virus (LASV) glycoprotein 1 (GP1), and glycoprotein 2 (GP2); Immunization of non-human primates with viral vectors expressing the arenaviral glycoprotein complex (GPC) confers full protective immunity against a lethal challenge with LASV. Thus, the development of native or quasi native recombinant LASV GP1 and GP2 as soluble, uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever. To this end, mammalian expression systems were engineered for production and purification of secreted forms of soluble LASV GP1 and GP2 proteins. RESULTS: Determinants for mammalian cell expression of secreted uncoupled Lassa virus (LASV) glycoprotein 1 (GP1) and glycoprotein 2 (GP2) were established. Soluble GP1 was generated using either the native glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the intracellular domain (IC) was fused to the ectodomain, and the gene was co-expressed with a complete GP1 gene in cis; (2) the TM and IC domains were deleted and GP1 was co-expressed in cis; (3) expression of GP1 was driven by the native GPC SP. These data implicate GP1 as a chaperone for processing and shuttling GP2 to the cell surface. The soluble forms of GP1 and GP2 generated through these studies were secreted as homogeneously glycosylated proteins that contained high mannose glycans. Furthermore, observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC represents a novel aspect of arenaviral glycoprotein expression. CONCLUSION: These results implicate GP1 as a chaperone for the correct processing and shuttling of GP2 to the cell surface, and suggest that native GPC SP plays a role in this process. In the absence of GP1 and GPC SP the GP2 protein may be processed by an alternate pathway that produces heterogeneously glycosylated protein, or the polypeptide may not fully mature in the secretory cascade in mammalian cells. The expression constructs developed in these studies resulted in the generation and purification of soluble, uncoupled GP1 and GP2 proteins from mammalian cells with quasi-native properties. The observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC establishes new correlates of disease progression and highlights potential opportunities for development of diagnostics targeting the early stages of Lassa fever.

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance.

BACKGROUND: There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). RESULTS: Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). CONCLUSION: These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

First Detection of an Enterovirus C99 in a Captive Chimpanzee with Acute Flaccid Paralysis, from the Tchimpounga Chimpanzee Rehabilitation Center, Republic of Congo.

Enteroviruses, members of the Picornaviridae family, are ubiquitous viruses responsible for mild to severe infections in human populations around the world. In 2010 Pointe-Noire, Republic of Congo recorded an outbreak of acute flaccid paralysis (AFP) in the humans, caused by wild poliovirus type 1 (WPV1). One month later, in the Tchimpounga sanctuary near Pointe-Noire, a chimpanzee developed signs similar to AFP, with paralysis of the lower limbs. In the present work, we sought to identify the pathogen, including viral and bacterial agents, responsible for this illness. In order to identify the causative agent, we evaluated a fecal specimen by PCR and sequencing. A Human enterovirus C, specifically of the EV-C99 type was potentially responsible for the illness in this chimpanzee. To rule out other possible causative agents, we also investigated the bacteriome and the virome using next generation sequencing. The majority of bacterial reads obtained belonged to commensal bacteria (95%), and the mammalian virus reads matched mainly with viruses of the Picornaviridae family (99%), in which enteroviruses were the most abundant (99.6%). This study thus reports the first identification of a chimpanzee presenting AFP most likely caused by an enterovirus and demonstrates once again the cross-species transmission of a human pathogen to an ape.

Characterization of a genogroup I sapovirus isolated from chimpanzees in the republic of congo.

Sapoviruses, which are members of the Caliciviridae family, are small nonenveloped viruses known to infect a large spectrum of mammalian hosts. We report here the first complete genome sequences of two genogroup I sapoviruses isolated from fecal samples from chimpanzees living in the Tchimpounga sanctuary, Republic of Congo.