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Little is known about the temporal patterns of infection and transmission of Lassa virus (LASV) within its natural reservoir (Mastomys natalensis). Here, we characterize infection dynamics and transmissibility of a LASV isolate (Soromba-R) in adult lab-reared M. natalensis originating from Mali. The lab-reared M. natalenesis proved to be highly susceptible to LASV isolates from geographically distinct regions of West Africa via multiple routes of exposure, with 50% infectious doses of

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.

Antiviral therapeutics with existing clinical safety profiles would be highly desirable in an outbreak situation, such as the 2013–2016 emergence of Ebola virus (EBOV) in West Africa. Although, the World Health Organization declared the end of the outbreak early 2016, sporadic cases of EBOV infection have since been reported. Alisporivir is the most clinically advanced broad-spectrum antiviral that functions by targeting a host protein, cyclophilin A (CypA). A modest antiviral effect of alisporivir against contemporary (Makona) but not historical (Mayinga) EBOV strains was observed in tissue culture. However, this effect was not comparable to observations for an alisporivir-susceptible virus, the flavivirus tick-borne encephalitis virus. Thus, EBOV does not depend on (CypA) for replication, in contrast to many other viruses pathogenic to humans.

The Ebola virus (EBOV) epidemic in West Africa increased the focus on vaccine development against this hemorrhagic fever-causing pathogen, and as a consequence human clinical trials for a few selected platforms were accelerated. One of these vaccines is vesicular stomatitis virus (VSV)-EBOV, also known as rVSV-ZEBOV, a fast-acting vaccine against EBOV and so far the only vaccine with reported efficacy against EBOV infections in humans in phase III clinical trials. In this study, we analyzed the potential of VSV-EBOV for postexposure treatment of rhesus macaques infected with EBOV-Makona. We treated groups of animals with 1 dose of VSV-EBOV either in a single injection at 1 or 24 hours after EBOV exposure or with 2 injections, half the dose at each time point; 1 control group received the same dose of the VSV-based Marburg virus vaccine at both time points; another group remained untreated. Although all untreated animals succumbed to EBOV infection, 33%-67% of the animals in each treatment group survived the infection, including the group treated with the VSV-based Marburg virus vaccine. This result suggests that protection from postexposure vaccination may be antigen unspecific and due rather to an early activation of the innate immune system. In conclusion, VSV-EBOV remains a potent and fast-acting prophylactic vaccine but demonstrates only limited efficacy in postexposure treatment.

Ebola virus (EBOV) and Marburg virus (MARV) outbreaks are highly lethal, and infection results in a hemorrhagic fever with complex etiology. These zoonotic viruses dysregulate the immune system to cause disease, in part by replicating within myeloid cells that would normally innately control viral infection and shape the adaptive immune response. We used triple knockout (TKO)-bone marrow, liver, thymus (BLT) humanized mice to recapitulate the early in vivo human immune response to filovirus infection. Disease severity in TKO-BLT mice was dissimilar between EBOV and MARV with greater severity observed during EBOV infection. Disease severity was related to increased Kupffer cell infection in the liver, higher levels of myeloid dysfunction, and skewing of macrophage subtypes in EBOV compared with MARV-infected mice. Overall, the TKO-BLT model provided a practical in vivo platform to study the human immune response to filovirus infection and generated a better understanding of how these viruses modulate specific components of the immune system.

Summary: Ebola virus (EBOV), isolate Makona, the causative agent of the West African EBOV epidemic, has been the subject of numerous investigations to determine the genetic diversity and its potential implication for virus biology, pathogenicity, and transmissibility. Despite various mutations that have emerged over time through multiple human-to-human transmission chains, their biological relevance remains questionable. Recently, mutations in the glycoprotein GP and polymerase L, which emerged and stabilized early during the outbreak, have been associated with improved viral fitness in cell culture. Here, we infected mice and rhesus macaques with EBOV-Makona isolates carrying or lacking those mutations. Surprisingly, all isolates behaved very similarly independent of the genotype, causing severe or lethal disease in mice and macaques, respectively. Likewise, we could not detect any evidence for differences in virus shedding. Thus, no specific biological phenotype could be associated with these EBOV-Makona mutations in two animal models. : Marzi et al. demonstrate that recently identified mutations in the EBOV-Makona genome, which appeared during the West African epidemic, do not significantly alter pathogenicity in IFNAR−/− mice and rhesus macaques. Other factors may have been more important for increased case numbers, case fatalities, and human-to-human transmission during this unprecedented epidemic. Keywords: Ebola virus, Ebola Makona, glycoprotein GP, polymerase L, GP mutation A82V, L mutation D759G, West African epidemic, pathogenicity

The common small animal disease models for Zika virus (ZIKV) are mice lacking the interferon responses, but infection of interferon receptor α/β knock out (IFNAR−/−) mice is not uniformly lethal particularly in older animals. Here we sought to advance this model in regard to lethality for future countermeasure efficacy testing against more recent ZIKV strains from the Asian lineage, preferably the American sublineage. We first infected IFNAR−/− mice subcutaneously with the contemporary ZIKV-Paraiba strain resulting in predominantly neurological disease with ~50% lethality. Infection with ZIKV-Paraiba by different routes established a uniformly lethal model only in young mice (4-week old) upon intraperitoneal infection. However, intraperitoneal inoculation of ZIKV-French Polynesia resulted in uniform lethality in older IFNAR−/− mice (10–12-weeks old). In conclusion, we have established uniformly lethal mouse disease models for efficacy testing of antivirals and vaccines against recent ZIKV strains representing the Asian lineage.

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.

Nipah virus is a zoonotic paramyxovirus that causes severe respiratory and/or encephalitic disease in humans, often resulting in death. It is transmitted from pteropus fruit bats, which serve as the natural reservoir of the virus, and outbreaks occur on an almost annual basis in Bangladesh or India. Outbreaks are small and sporadic, and several cases of human-to-human transmission have been documented as an important feature of the epidemiology of Nipah virus disease. There are no approved countermeasures to combat infection and medical intervention is supportive. We recently generated a recombinant replication-competent vesicular stomatitis virus-based vaccine that encodes a Nipah virus glycoprotein as an antigen and is highly efficacious in the hamster model of Nipah virus disease. Herein, we show that this vaccine protects African green monkeys, a well-characterized model of Nipah virus disease, from disease one month after a single intramuscular administration of the vaccine. Vaccination resulted in a rapid and strong virus-specific immune response which inhibited virus shedding and replication. This vaccine platform provides a rapid means to afford protection from Nipah virus in an outbreak situation.

Both Ebola virus (EBOV) and Reston virus (RESTV) cause disease in nonhuman primates, yet only EBOV causes disease in humans. To investigate differences in viral pathogenicity, humanized mice (hu-NSG-SGM3) were inoculated with EBOV or RESTV. Consistent with differences in disease in human infection, pronounced weight loss and markers of hepatic damage and disease were observed exclusively in EBOV-infected mice. These abnormalities were associated with significantly higher EBOV replication in the liver but not in the spleen, suggesting that in this model, efficiency of viral replication in select tissues early in infection may contribute to differences in viral pathogenicity.

Highlights • In cell culture models, low-micromolar doses of alisporivir block SARS-CoV and MERS-CoV replication. • Combination treatment with alisporivir and ribavirin increases the anti-MERS-CoV activity in cell culture. • Combination treatment with alisporivir and ribavirin does not protect against SARS-CoV infection in a mouse model. • Cyclophilin-binding drugs should be explored further in the context of host-directed anti-coronaviral strategies., Currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like SARS- and MERS-coronavirus. We here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin A-analog, inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. Nevertheless, our data provide a basis to further explore the potential of Cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication.

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 Medecins Sans Frontieres 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.

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)

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.

International audience; In 2012, a novel betacoronavirus, designated Middle East respiratory syndrome coronavirus or MERS-CoV and associated with severe respiratory disease in humans, emerged in the Arabian Peninsula. To date, 108 human cases have been reported, including cases of human-to-human transmission. The availability of an animal disease model is essential for understanding pathogenesis and developing effective countermeasures. Upon a combination of intratracheal, ocular, oral, and intranasal inoculation with 7 x 10(6) 50% tissue culture infectious dose of the MERS-CoV isolate HCoV-EMC/2012, rhesus macaques developed a transient lower respiratory tract infection. Clinical signs, virus shedding, virus replication in respiratory tissues, gene expression, and cytokine and chemokine profiles peaked early in infection and decreased over time. MERS-CoV caused a multifocal, mild to marked interstitial pneumonia, with virus replication occurring mainly in alveolar pneumocytes. This tropism of MERS-CoV for the lower respiratory tract may explain the severity of the disease observed in humans and the, up to now, limited human-to-human transmission.

International audience; The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) is of global concern: the virus has caused severe respiratory illness, with 111 confirmed cases and 52 deaths1 at the time of this article's publication. Therapeutic interventions have not been evaluated in vivo; thus, patient management relies exclusively on supportive care, which, given the high case-fatality rate, is not highly effective. The rhesus macaque is the only known model organism for MERS-CoV infection, developing an acute localized to widespread pneumonia with transient clinical disease(2,3) that recapitulates mild to moderate human MERSCoV cases(4,5). The combination of interferon-alpha 2b and ribavirin was effective in reducing MERS-CoV replication in vitro(6); therefore, we initiated this treatment 8 h after inoculation of rhesus macaques. In contrast to untreated, infected macaques, treated animals did not develop breathing abnormalities and showed no or very mild radiographic evidence of pneumonia. Moreover, treated animals showed lower levels of systemic (serum) and local (lung) proinflammatory markers, in addition to fewer viral genome copies, distinct gene expression and less severe histopathological changes in the lungs. Taken together, these data suggest that treatment of MERS-CoV infected rhesus macaques with IFN-alpha 2b and ribavirin reduces virus replication, moderates the host response and improves clinical outcome. As these two drugs are already used in combination in the clinic for other infections, IFN-alpha 2b and ribavirin should be considered for the management of MERS-CoV cases.

The identification of a novel β coronavirus, nCoV, as the causative agent of severe respiratory illness in humans originating in Saudi Arabia, Qatar and Jordan has raised concerns about the possibility of a coronavirus pandemic similar to that of SARS-CoV. As a definitive treatment regimen has never been thoroughly evaluated for coronavirus infections, there is an urgent need to rapidly identify potential therapeutics to address future cases of nCoV. To determine an intervention strategy, the effect of interferon-α2b and ribavirin on nCoV isolate hCoV-EMC/2012 replication in Vero and LLC-MK2 cells was evaluated. hCoV-EMC/2012 was sensitive to both interferon-α2b and ribavirin alone in Vero and LLC-MK2 cells, but only at relatively high concentrations; however, when combined, lower concentrations of interferon-α2b and ribavirin achieved comparable endpoints. Thus, a combination of interferon-α2b and ribavirin, which are already commonly used in the clinic, may be useful for patient management in the event of future nCoV infections.

In 2012 a novel coronavirus, MERS-CoV, associated with severe respiratory disease emerged in the Arabian Peninsula. To date, 55 human cases have been reported, including 31 fatal cases. Several of the cases were likely a result of human-to-human transmission. The emergence of this novel coronavirus prompts the need for a small animal model to study the pathogenesis of this virus and to test the efficacy of potential intervention strategies. In this study we explored the use of Syrian hamsters as a small animal disease model, using intratracheal inoculation and inoculation via aerosol. Clinical signs of disease, virus replication, histological lesions, cytokine upregulation nor seroconversion were observed in any of the inoculated animals, indicating that MERS-CoV does not replicate in Syrian hamsters.

A novel betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), emerged on the Arabian Peninsula in 2012. MERS-CoV was found to produce severe respiratory illness with case-fatality rates near 50%, and may be transmitted person-to-person in instances of close physical contact, such as among family members and to health care workers. As mice, hamsters, ferrets, and cynomolgus macaques are either not susceptible to MERS-CoV infection or do not develop observable clinical disease, we sought to determine whether rhesus macaques (Macaca mulatta) can be used as a model of MERS-CoV infection and disease. Animals challenged with MERS-CoV via multiple combined infection routes rapidly developed pneumonia, and we observed other clinical signs of illness, virus shedding, and virus replication in respiratory tissues. Using microarrays to compare transcripts associated with histopathologic lesions removed from the lungs of infected animals, we identified 173 significant differentially expressed genes (DEG) associated with lung lesions on day 3 post-infection concurrent with peak clinical signs. Functional analysis indicated that these genes were predominantly associated with proinflammatory processes, recruitment and chemotaxis of inflammatory cells, and antiviral immunity. Using singular value decomposition coupled multidimensional scaling (SVD-MDS), we also identified changes in similar inflammatory genes in peripheral blood mononuclear cells, suggesting a rapid activation of innate immune and inflammatory processes in leukocytes after infection. These data were consistent with cytokine and chemokine profiles in the blood of animals. Due to the urgent need for an effective therapeutic means of treating MERS-CoV patients, we used this rhesus macaque model to evaluate post-exposure treatment with combination interferon (IFN)-a2b and ribavirin (RBV), and identified key transcriptional signatures associated with disease severity and treatment efficacy. Animals treated with IFN-a2b/RBV beginning 8 h post-infection did not develop significant clinical or severe histopathologic signs of disease, breathing abnormalities, or radiographic evidence of pneumonia, and showed reduced viral loads and lower levels of serum proinflammatory cytokines. Microarray analysis of treated animals showed significant induction of interferon-stimulated genes (ISGs) and fewer upregulated transcripts associated with inflammatory processes. Additionally, we identified a subset of statistically significant DEGs associated with signaling pathways such as hedgehog, which has been linked to airway function and protection against pulmonary injury. Together, these data indicate that treatment of MERS-CoV-infected rhesus macaques with IFN-a2b and ribavirin reduces virus replication and improves clinical outcome, and suggests a transcriptomic basis for therapeutic efficacy. As these two drugs are already used together in the clinic, IFN-a2b and ribavirin may be useful for therapeutic intervention and patient management of new human MERS-CoV cases.