Kyle Rosenke

Mutation rate and genotype variation of Ebola virus from Mali case sequences

Evolution in the Ebola virus outbreak Has rapid mutation produced alarming new virus characteristics in the 2013–2015 Ebola virus outbreak in West Africa? Hoenen et al. sequenced isolates obtained 9 months into the epidemic from cases in Mali. The nucleotide substitution rate was consistent with rates estimated from past Central African outbreaks. In contrast, analysis of sequence data from early in the outbreak indicated rapid mutation. This more recent finding offers confidence that diagnostic methods, vaccines, and other treatment interventions will remain effective. Nevertheless, vigilance must be maintained: A few mutations can radically change the biological properties of other RNA viruses. Science, this issue p. 117 During the current outbreak in West Africa, Ebola virus has not mutated faster than historically observed. The occurrence of Ebola virus (EBOV) in West Africa during 2013–2015 is unprecedented. Early reports suggested that in this outbreak EBOV is mutating twice as fast as previously observed, which indicates the potential for changes in transmissibility and virulence and could render current molecular diagnostics and countermeasures ineffective. We have determined additional full-length sequences from two clusters of imported EBOV infections into Mali, and we show that the nucleotide substitution rate (1.3 × 10–3 substitutions per site per year) is consistent with rates observed in Central African outbreaks. In addition, overall variation among all genotypes observed remains low. Thus, our data indicate that EBOV is not undergoing rapid evolution in humans during the current outbreak. This finding has important implications for outbreak response and public health decisions and should alleviate several previously raised concerns.

Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool

Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions. We successfully used a novel, pocket-sized nanopore sequencer at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak.

The broad-spectrum antiviral favipiravir protects guinea pigs from lethal Lassa virus infection post-disease onset

With up to 500,000 infections annually, Lassa virus (LASV), the cause of Lassa fever, is one of the most prevalent etiological agents of viral hemorrhagic fever (VHF) in humans. LASV is endemic in several West African countries with sporadic cases and prolonged outbreaks observed most commonly in Sierra Leone, Liberia, Guinea and Nigeria. Additionally several cases of Lassa fever have been imported into North America, Europe and Asia making LASV a global threat to public health. Despite this, currently no approved therapeutic or vaccine exists to treat or prevent LASV infections. Here, using a passaged strain of LASV that is uniformly lethal in Hartley guinea pigs, we demonstrate that favipiravir, a broad-spectrum antiviral agent and leading treatment option for influenza, has potent activity against LASV infection. In this model, once daily treatment with favipiravir significantly reduced viral titers in tissue samples and reduced mortality rates when compared with animals receiving vehicle-only or ribavirin, the current standard of care for Lassa fever. Favipiravir remained highly effective against lethal LASV infection when treatments were initiated nine days post-infection, a time when animals were demonstrating advanced signs of disease. These results support the further preclinical evaluation of favipiravir for Lassa fever and other VHFs.

A Recombinant Vesicular Stomatitis Virus-Based Lassa Fever Vaccine Protects Guinea Pigs and Macaques against Challenge with Geographically and Genetically Distinct Lassa Viruses

Background Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a “universal” LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models. Methodologies/principle findings Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever. Conclusions/significance Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever.

Plasmodium Parasitemia Associated With Increased Survival in Ebola Virus–Infected Patients

BACKGROUND The ongoing Ebola outbreak in West Africa has resulted in 28 646 suspected, probable, and confirmed Ebola virus infections. Nevertheless, malaria remains a large public health burden in the region affected by the outbreak. A joint Centers for Disease Control and Prevention/National Institutes of Health diagnostic laboratory was established in Monrovia, Liberia, in August 2014, to provide laboratory diagnostics for Ebola virus. METHODS All blood samples from suspected Ebola virus-infected patients admitted to the Médecins Sans Frontières ELWA3 Ebola treatment unit in Monrovia were tested by quantitative real-time polymerase chain reaction for the presence of Ebola virus and Plasmodium species RNA. Clinical outcome in laboratory-confirmed Ebola virus-infected patients was analyzed as a function of age, sex, Ebola viremia, and Plasmodium species parasitemia. RESULTS The case fatality rate of 1182 patients with laboratory-confirmed Ebola virus infections was 52%. The probability of surviving decreased with increasing age and decreased with increasing Ebola viral load. Ebola virus-infected patients were 20% more likely to survive when Plasmodium species parasitemia was detected, even after controlling for Ebola viral load and age; those with the highest levels of parasitemia had a survival rate of 83%. This effect was independent of treatment with antimalarials, as this was provided to all patients. Moreover, treatment with antimalarials did not affect survival in the Ebola virus mouse model. CONCLUSIONS Plasmodium species parasitemia is associated with an increase in the probability of surviving Ebola virus infection. More research is needed to understand the molecular mechanism underlying this remarkable phenomenon and translate it into treatment options for Ebola virus infection.

The Merits of Malaria Diagnostics during an Ebola Virus Disease Outbreak

Malaria is a major public health concern in the countries affected by the Ebola virus disease epidemic in West Africa. We determined the feasibility of using molecular malaria diagnostics during an Ebola virus disease outbreak and report the incidence of Plasmodium spp. parasitemia in persons with suspected Ebola virus infection.

Lassa Virus Seroprevalence in Sibirilia Commune, Bougouni District, Southern Mali.

The high rate documented in this study highlights the need for increased surveillance. Lassa Virus Seroprevalence, Mali

Vectorborne Infections, Mali.

To the Editor: As in many West Africa nations, vectorborne diseases represent a substantial health burden in Mali; however, beyond malaria, the incidence and etiology of many of these diseases is poorly understood. Of the estimated 14.1 million persons living in sub-Saharan Mali, ≈70% live in remote rural settings with an ecologic landscape that puts inhabitants at an increased risk for contact with rodent and arthropodborne diseases. We retrospectively analyzed serum samples for evidence of recent (IgM+) and previous (IgG+) infection with chikungunya (CHIKV), dengue (DENV), West Nile (WNV), Lassa (LASV), Crimean-Congo hemorrhagic fever (CCHFV), and Ebola (EBOV) virus, as well as Old World hantaviruses (OW-HANV) and Leptospira spp., which is regularly misdiagnosed as an acute viral infection. We tested 376 deidentified serum samples collected from acutely ill patients who had a history of fever and hemorrhagic, diarrheal, or icteric syndromes (Technical Appendix Figure). Research on samples from humans was conducted in accordance with the policies and regulations of the National Institutes of Health and adhered to the principles of the Belmont Report (1979) (http://www.hhs.gov/ohrp/humansubjects/guidance/belmont.html). This research was conducted under an institutional review board–approved document. Samples had previously tested negative for acute Plasmodium falciparum malaria and yellow fever virus infections. Commercially available IgM capture and conventional IgG ELISAs were used for serologic testing for CHIKV (GenWay Biotech, San Diego, CA, USA); DENV (all four serotypes) and WNV (both from Focus Diagnostics, Cypress, CA, USA); OW-HANVs (Euroimmun, Luebeck, Germany); and Leptospira spp. (Abnova, Taipei City, Taiwan). Conventional IgM/IgG ELISAs were used for LASV (Corgenix, Broomfield, CO, USA) and CCHFV (Vector-Best, Novosibirsk, Russia), and reagents for the EBOV IgM/IgG ELISA (infected/uninfected cell lysates) were prepared at the Rocky Mountain Laboratories (Hamilton, MT, USA) and validated with serum from experimentally infected monkeys. With the exception of the CHIKV, Leptospira spp., and in-house EBOV assays, the tests conducted in this study are under preclinical development for human diagnostic assays. Samples were tested at a 1:100 dilution according to manufacturer specifications (CHIKV, CCHFV, WNV, DENV, OW-HANVs, LASV, and Leptospira spp.) or in-house quality-control assessments (EBOV), in a blinded fashion. Serologic reactivity was assessed according to manufacturer recommendations. For the EBOV ELISA, samples were deemed positive if optical density at 405 nm was >3 SD above that of the average of known negative samples. Serologic evidence suggestive of acute infection (IgM+) with 1 of the pathogens tested for was observed for 39.9% of samples (Table). At 14.4%, Leptospira spp. was the most prevalent probable etiologic agent of acute disease identified. Of mosquitoborne viruses tested, DENV had the highest prevalence at 7.7%, followed by CHIKV (5.3%) and WNV (0.27%). Of rodentborne pathogens, OW-HANVs had a seroprevalence of 7.2%, whereas LASV was considerably lower (0.27%). CCHFV IgM was documented in 4.8% of samples. Overall, little annual variation in the IgM seroprevalence was noted, except for Leptospira spp., for which 2 obvious peaks in seroprevalence were observed (Table). Table IgM and IgG seroprevalence rates of selected vectorborne pathogens in samples submitted from suspected yellow fever cases in Mali, 2009–2013* Most IgM+ samples demonstrated serologic reactivity in only 1 assay. The exception was 2 samples that were IgM+ for hantaviruses and Leptospira spp., an acute dual infection that might be underrecognized (1). With the exception of DENV, few samples were both IgM+ and IgG+, suggesting the results were not attributable to IgM persistence. The DENV IgM+/IgG+ results might represent IgM persistence. However, because the ELISA detected all 4 serotypes, it is plausible that some results represent recent infection with DENV in the presence of IgG reactive with a different serotype. The relatively high IgG seroprevalence for most of the pathogens tested supports the findings of the IgM assays and further suggest the circulation of and potential for human exposure to these agents in Mali (Table). Geographically, serologic evidence of infections with Leptospira spp., DENV, WNV, OW-HANVs, and CHIKV was observed throughout Mali (online Technical Appendix). No samples were reactive with EBOV, and the low incidence of LASV infection is not surprising because the samples analyzed here were collected outside of the 1 documented LASV-endemic region in Mali (2). We used commercially available diagnostic platforms, primarily IgM capture and conventional IgG ELISAs, many of which are validated for human diagnostics. Ideally, diagnostics for zoonotic diseases would not rely on IgM/IgG serologic analysis because of caveats including IgM persistence and cross-reactivity between closely related pathogens (3,4). In the industrialized world, as well as in several countries throughout Africa, molecular approaches are often used to genetically identify pathogens, or follow-up convalescent-phase serum samples are collected to determine seroconversion or increased antibody titers or to conduct plaque reduction neutralization assays. Unfortunately, because of the nature of the samples available, including time of collection, storage history, and remaining volume, many of these tests were not feasible for our study. Despite these limitations, these serologic findings indicate that flaviviruses, bunyaviruses, and togaviruses, as well as Leptospira spp., are contributing to human illness in Mali. These results add to those recently documented in studies conducted in Sierra Leone, implying that several of these zoonotic pathogens are widely distributed yet underreported throughout West Africa (5,6). Technical Appendix. Distribution of serum samples tested, by region and year. IgM and IgG seroprevalence rates of selected vectorborne pathogens, by region of sample collection. Click here to view.(192K, pdf)

Domestic Pig Unlikely Reservoir for MERS-CoV

We tested the suitability of the domestic pig as a model for Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Inoculation did not cause disease, but a low level of virus replication, shedding, and seroconversion were observed. Pigs do not recapitulate human MERS-CoV and are unlikely to constitute a reservoir in nature.

Annual Incidence of Lassa Virus Infection in Southern Mali

AbstractPreviously, we reported a high seroprevalence rate of Lassa virus antibodies in inhabitants of three villages in southern Mali where infected rodents have been demonstrated. Herein, we report a 1-year follow-up study in which we were able to collect a second blood samples from 88.7% of participants of the same cohort. We identified 23 seroconversions for IgG antibodies reactive against Lassa virus, representing an incidence of 6.3% (95% confidence interval = 3.8-8.8%). Seroconversion was frequently seen in preteenage children (12/23, 51.7%) and two household/familial clusters were identified. These results confirm active transmission of Lassa virus is occurring in southern Mali and appropriate diagnostic testing should be established for this etiological agent of severe viral hemorrhagic fever.