Marburg virus pathogenesis - differences and similarities in humans and animal models.

Marburg virus (MARV) is a highly pathogenic virus associated with severe disease and mortality rates as high as 90%. Outbreaks of MARV are sporadic, deadly, and often characterized by a lack of resources and facilities to diagnose and treat patients. There are currently no approved vaccines or treatments, and the chaotic and infrequent nature of outbreaks, among other factors, makes testing new countermeasures during outbreaks ethically and logistically challenging. Without field efficacy studies, researchers must rely on animal models of MARV infection to assess the efficacy of vaccines and treatments, with the limitations being the accuracy of the animal model in recapitulating human pathogenesis. This review will compare various animal models to the available descriptions of human pathogenesis and aims to evaluate their effectiveness in modeling important aspects of Marburg virus disease.

A Comparison of the Pathogenesis of Marburg Virus Disease in Humans and Nonhuman Primates and Evaluation of the Suitability of These Animal Models for Predicting Clinical Efficacy under the 'Animal Rule'.

Marburg virus outbreaks are sporadic, infrequent, brief, and relatively small in terms of numbers of subjects affected. In addition, outbreaks most likely will occur in remote regions where clinical trials are not feasible; therefore, definitive, well-controlled human efficacy studies to test the effectiveness of a drug or biologic product are not feasible. Healthy human volunteers cannot ethically be deliberately exposed to a lethal agent such as Marburg virus in order to test the efficacy of a therapy or preventive prior to licensure. When human efficacy studies are neither ethical nor feasible, the US Food and Drug Administration may grant marketing approval of a drug or biologic product under the 'Animal Rule,' through which demonstration of the efficacy of a product can be 'based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is reasonably likely to produce clinical benefit in humans.' This process requires that the pathogenic determinants of the disease in the animal model are similar to those that have been identified in humans. After reviewing primarily English-language, peer-reviewed journal articles, we here summarize the clinical manifestations of Marburg virus disease and the results of studies in NHP showing the characteristics and progression of the disease. We also include a detailed comparison of the characteristics of the human disease relative to those for NHP. This review reveals that the disease characteristics of Marburg virus disease are generally similar for humans and 3 NHP species: cynomolgus macaques (Macaca fascicularis), rhesus macaques (Macaca mulatta), and African green monkeys (Chlorocebus aethiops).

Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques.

UNLABELLED: Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (>10(7) PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model. IMPORTANCE: In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure.

Transcriptional Profiling of the Immune Response to Marburg Virus Infection.

UNLABELLED: Marburg virus is a genetically simple RNA virus that causes a severe hemorrhagic fever in humans and nonhuman primates. The mechanism of pathogenesis of the infection is not well understood, but it is well accepted that pathogenesis is appreciably driven by a hyperactive immune response. To better understand the overall response to Marburg virus challenge, we undertook a transcriptomic analysis of immune cells circulating in the blood following aerosol exposure of rhesus macaques to a lethal dose of Marburg virus. Using two-color microarrays, we analyzed the transcriptomes of peripheral blood mononuclear cells that were collected throughout the course of infection from 1 to 9 days postexposure, representing the full course of the infection. The response followed a 3-stage induction (early infection, 1 to 3 days postexposure; midinfection, 5 days postexposure; late infection, 7 to 9 days postexposure) that was led by a robust innate immune response. The host response to aerosolized Marburg virus was evident at 1 day postexposure. Analysis of cytokine transcripts that were overexpressed during infection indicated that previously unanalyzed cytokines are likely induced in response to exposure to Marburg virus and further suggested that the early immune response is skewed toward a Th2 response that would hamper the development of an effective antiviral immune response early in disease. Late infection events included the upregulation of coagulation-associated factors. These findings demonstrate very early host responses to Marburg virus infection and provide a rich data set for identification of factors expressed throughout the course of infection that can be investigated as markers of infection and targets for therapy. IMPORTANCE: Marburg virus causes a severe infection that is associated with high mortality and hemorrhage. The disease is associated with an immune response that contributes to the lethality of the disease. In this study, we investigated how the immune cells circulating in the blood of infected primates respond following exposure to Marburg virus. Our results show that there are three discernible stages of response to infection that correlate with presymptomatic, early, and late symptomatic stages of infection, a response format similar to that seen following challenge with other hemorrhagic fever viruses. In contrast to the ability of the virus to block innate immune signaling in vitro, the earliest and most sustained response is an interferon-like response. Our analysis also identifies a number of cytokines that are transcriptionally upregulated during late stages of infection and suggest that there is a Th2-skewed response to infection. When correlated with companion data describing the animal model from which our samples were collected, our results suggest that the innate immune response may contribute to overall pathogenesis.

Lethality and pathogenesis of airborne infection with filoviruses in A129 α/ß -/- interferon receptor-deficient mice.

Normal immunocompetent mice are not susceptible to non-adapted filoviruses. There are therefore two strategies available to establish a murine model of filovirus infection: adaptation of the virus to the host or the use of genetically modified mice that are susceptible to the virus. A number of knockout (KO) strains of mice with defects in either their adaptive or innate immunity are susceptible to non-adapted filoviruses. In this study, A129 α/ß -/- interferon receptor-deficient KO mice, strain A129 IFN-α/ß -/-, were used to determine the lethality of a range of filoviruses, including Lake Victoria marburgvirus (MARV), Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV) and Côte d'Ivoire ebolavirus (CIEBOV), administered by using intraperitoneal (IP) or aerosol routes of infection. One hundred percent mortality was observed in all groups of KO mice that were administered with a range of challenge doses of MARV and ZEBOV by either IP or aerosol routes. Mean time to death for both routes was dose-dependent and ranged from 5.4 to 7.4 days in the IP injection challenge, and from 10.2 to 13 days in the aerosol challenge. The lethal dose (50 % tissue culture infective dose, TCID(50)) of ZEBOV for KO mice was <1 TCID(50) ml(-1) when administered by either the IP or aerosol route of infection; for MARV the lethal dose was <1 TCID(50) ml(-1) by the IP route of infection and <10 TCID(50) ml(-1) by the aerosol route. In contrast, there was no mortality after infection with SEBOV or REBOV by either IP or aerosol routes of infection; all the mice lost weight (~15 % loss of group mean body weight with SEBOV and ~7 % with REBOV) but recovered to their original weights by day 14 post-challenge. There was no mortality in mice administered with CIEBOV via the IP route of infection and no clinical signs of infection were observed. The progression of disease was faster following infection with ZEBOV than with MARV but ultimately both viruses caused widespread infection with high titres of the infectious viruses in multiple organs. Histopathological observations were consistent with other animal models and showed widespread organ damage. This study suggests that MARV and ZEBOV are more virulent when administered via the IP route rather than by aerosol infection, although both are highly virulent by either route. The KO mouse may provide a useful model to test potential antiviral therapeutics against wild-type filoviruses.

Pathogenesis of Marburg hemorrhagic fever in cynomolgus macaques.

BACKGROUND: Marburg virus (MARV) infection causes a severe and often fatal hemorrhagic disease in primates; however, little is known about the development of MARV hemorrhagic fever. In this study we evaluated the progression of MARV infection in nonhuman primates. METHODS: Eighteen cynomolgus monkeys were infected with MARV; blood and tissues were examined sequentially over an 8-day period to investigate disease pathogenesis. RESULTS: Disease caused by MARV in cynomolgus macaques was very similar to disease previously described for Ebola virus-infected macaques. Monocytes, macrophages, Kupffer cells, and dendritic cells (DCs) were identified as the initial targets of MARV infection. Bystander lymphocyte apoptosis occurred at early stages in the disease course in intravascular and extravascular locations. The loss of splenic and lymph node DCs or downregulation of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on DCs as early as day 2 and continuing through day 8 after MARV infection was a prominent finding. Evidence of disseminated intravascular coagulation was noted; however, the degree of fibrin deposition in tissues was less prominent than was reported in Ebola-infected macaques. CONCLUSIONS: The sequence of pathogenic events identified in this study provides an understanding of the development of disease processes and also may provide new targets for rational prophylactic and chemotherapeutic interventions.

Plasma exchange for acute attacks of CNS demyelination: Predictors of improvement at 6 months.

BACKGROUND: Plasma exchange (PE) is used to treat severe episodes of CNS demyelination unresponsive to corticosteroids. Predictors of long-term response are not well known. METHODS: We retrospectively reviewed the medical records of 41 patients consecutively treated by PE between January 1995 and July 2007. The primary outcome was improvement at 6 months after PE defined as decrease of >or=1 point in the Expanded Disability Status Scale (EDSS) score for patients with EDSS or=8.0 or improvement of more than 2 lines in the visual acuity chart for patients with optic neuritis (ON). RESULTS: Twenty-five patients (61%) were women, and the median age was 33 years (range 14-57 years). Twenty-three (56%) had multiple sclerosis, 2 (5%) had clinically isolated syndrome, 2 (5%) had Marburg disease, 7 (17%) had acute disseminated encephalomyelitis, 4 (10%) had neuromyelitis optica, 2 (5%) had idiopathic ON, and 1 (2%) had idiopathic transverse myelitis. The median EDSS score before the attack was 1.0 (range 0-6.5). At PE onset, the median EDSS score was 7.0 (range 3.0-9.5). Sixteen patients (39%) improved at discharge, and 26 (63%) improved at 6 months. In the multivariate analysis, early initiation of PE (odds ratio [OR] 6.29, 95% confidence interval [CI] 1.18-52.96) and improvement at discharge (OR 7.32, 95% CI 1.21-44.38) were significantly associated with response at 6 months. CONCLUSIONS: Plasma exchange (PE) was associated with clinical improvement in 63% of patients at 6 months. Early initiation of PE and improvement at discharge were predictors of this response. Twelve patients (48%) who did not improve early did so during follow-up.

Potential factors induced by filoviruses that lead to immune supression.

The filoviruses, Ebola (EBOV) and Marburg (MARV), are among the deadliest of human pathogens, causing acute diseases typified by rapidly fatal hemorrhagic fevers. Upon filoviral infection, innate immune cells become paralyzed and lose the capacity to properly co-stimulate and activate filovirus-specific, T-cell responses. Deleterious inflammation and upregulation of co-inhibitory molecules expressed by monocytic lineage cells (e.g., dendritic cells) and their co-inhibitory receptors on T- and B-cells may lead to incomplete humoral and T-cell immunity, anergy, exhaustion, apoptosis, and subsequent immune subversion. Hence, the dysregulation of inflammatory and co-inhibitory molecules may be exploited by filoviruses to further deteriorate host immune responses, ultimately leading to fulminant infections in susceptible species. Thus, in light of accumulating scientific observations, the challenge is now to characterize the molecular mechanisms that may result in rational strategies leading to new therapeutics and vaccines.

Disease modeling for Ebola and Marburg viruses.

The filoviruses Ebola and Marburg are zoonotic agents that are classified as both biosafety level 4 and category A list pathogens. These viruses are pathogenic in humans and cause isolated infections or epidemics of viral hemorrhagic fever, mainly in Central Africa. Their natural reservoir has not been definitely identified, but certain species of African bat have been associated with Ebola and Marburg infections. Currently, there are no licensed options available for either treatment or prophylaxis. Different animal models have been developed for filoviruses including mouse, guinea pig and nonhuman primates. The 'gold standard' animal models for pathogenesis, treatment and vaccine studies are rhesus and cynomolgus macaques. This article provides a brief overview of the clinical picture and the pathology/pathogenesis of human filovirus infections. The current animal model options are discussed and compared with regard to their value in different applications. In general, the small animal models, in particular the mouse, are the most feasible for high biocontainment facilities and they offer the most options for research owing to the greater availability of immunologic and genetic tools. However, their mimicry of the human diseases as well as their predictive value for therapeutic efficacy in primates is limited, thereby making them, at best, valuable initial screening tools for pathophysiology, treatment and vaccine studies.

Case definition for Ebola and Marburg haemorrhagic fevers: a complex challenge for epidemiologists and clinicians.

Viral haemorrhagic fevers (VHFs) represent a challenge for public health because of their epidemic potential, and their possible use as bioterrorism agents poses particular concern. In 1999 the World Health Organization (WHO) proposed a case definition for VHFs, subsequently adopted by other international institutions with the aim of early detection of initial cases/outbreaks in western countries. We applied this case definition to reports of Ebola and Marburg virus infections to estimate its sensitivity to detect cases of the disease. We analyzed clinical descriptions of 795 reported cases of Ebola haemorrhagic fever: only 58.5% of patients met the proposed case definition. A similar figure was obtained reviewing 169 cases of Marburg diseases, of which only 64.5% were in accordance with the case definition. In conclusion, the WHO case definition for hemorrhagic fevers is too specific and has poor sensitivity both for case finding during Ebola or Marburg outbreaks, and for early detection of suspected cases in western countries. It can lead to a hazardous number of false negatives and its use should be discouraged for early detection of cases.

Marburg hemorrhagic fever in Durba and Watsa, Democratic Republic of the Congo: clinical documentation, features of illness, and treatment.

The objective of the present study was to describe day of onset and duration of symptoms of Marburg hemorrhagic fever (MHF), to summarize the treatments applied, and to assess the quality of clinical documentation. Surveillance and clinical records of 77 patients with MHF cases were reviewed. Initial symptoms included fever, headache, general pain, nausea, vomiting, and anorexia (median day of onset, day 1-2), followed by hemorrhagic manifestations (day 5-8+), and terminal symptoms included confusion, agitation, coma, anuria, and shock. Treatment in isolation wards was acceptable, but the quality of clinical documentation was unsatisfactory. Improved clinical documentation is necessary for a basic evaluation of supportive treatment.

The Medecins Sans Frontieres intervention in the Marburg hemorrhagic fever epidemic, Uige, Angola, 2005. I. Lessons learned in the hospital.

When the epidemic of Marburg hemorrhagic fever occurred in Uige, Angola, during 2005, the international response included systems of case detection and isolation, community education, the burial of the dead, and disinfection. However, despite large investments of staff and money by the organizations involved, only a fraction of the reported number of cases were isolated, and many cases were detected only after death. This article describes the response of Medecins Sans Frontieres Spain within the provincial hospital in Uige, as well as the lessons they learned during the epidemic. Diagnosis, management of patients, and infection control activities in the hospital are discussed. To improve the acceptability of the response to the host community, psychological and cultural factors need to be considered at all stages of planning and implementation in the isolation ward. More interventional medical care may not only improve survival but also improve acceptability.

Genomic differences between guinea pig lethal and nonlethal Marburg virus variants.

The complete genome sequences of 2 closely related plaque-derived variants of Marburg virus (MARV) species Lake Victoria marburgvirus, strain Musoke, indicate only a few regions of the RNA genome as underlying the differences between the 2 viruses. One variant is >90% lethal for guinea pigs and the other much less virulent, when guinea pigs are challenged with 1000 pfu of virus. Only 4 mutations that result in amino acid changes were identified, 1 in viral matrix protein VP40 and 3 in L, the RNA-dependent RNA polymerase. In addition, 6 differences were identified in noncoding regions of transcribed mRNA, and 1 silent codon change was identified in the L gene. Interestingly, the amino acid mutation identified in VP40 occurs in a nonconserved loop structure between 2 domains that are homologues only among MARV species. The L gene mutations were equally intriguing, clustering near a highly conserved motif in viral RNA-dependent RNA polymerases.

Breakdown of paraendothelial barrier function during Marburg virus infection is associated with early tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1.

Marburg virus (MARV) infection often causes fulminant shock due to pathologic immune responses and alterations of the vascular system. Cytokines released from virus-infected monocytes/macrophages provoke endothelial activation and vascular hyperpermeability and contribute to the development of shock. Tyrosine phosphorylation of cell-junction proteins is important for the regulation of paraendothelial barrier function. We showed that mediators released from MARV-infected monocytes/macrophages, as well as recombinant tumor necrosis factor (TNF)- alpha /H2O2 and interferon (IFN)- gamma , caused tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) but not of the vascular endothelial (VE) cadherin/catenin complex proteins. Tyrosine phosphorylation of PECAM-1 was associated with delayed opening of interendothelial junctions. Interestingly, we observed an early increase in water permeability in response to TNF- alpha /H2O2 that was not due to an opening of the interendothelial junctions. These data indicate 2 distinct mechanisms for the TNF- alpha /H2O2-mediated decrease in endothelial barrier function involving tyrosine phosphorylation of PECAM-1 but not requiring tyrosine phosphorylation of VE-cadherin or catenin proteins.

Short communication: a cluster of Marburg virus disease involving an infant.

A noteworthy cluster of six cases of Marburg haemorrhagic fever (MHF) was identified in the Democratic Republic of Congo. One of the cases is the first infant Marburg fever patient ever documented. Three of six cases presented surprisingly mild symptoms. The results of epidemiological and virological investigations are compatible with person-to-person transmission through body fluids and with mother-to-child transmission while nurturing. The findings show that mild cases of MHF have to be expected during an outbreak and point out the difficulty to base patient management decisions on clinical case definitions alone.

Respiratory Marburg virus infection in guinea pigs.

Marburg virus (MV) reproduction in organs, hematological and pathological changes were studied by virological and clinical methods, light and electron microscopy in guinea pigs respiratory challenged by the virus. Liver and spleen were most affected by MV, as in parenteral infection. The sequential involvement of cells in virus replication was also the same as in parenteral infection, with monocytoid-macrophagal cells infected first, followed by hepatocytes, spongiocytes, endotheliocytes and fibroblasts. Hemopoietic cells showed evidence of severe damage in respiratory infected guinea pigs. A distinguishing feature of the respiratory infection was close contact of leucocytes with MV infected cells. It is suggested that the entrapment and accumulation of MV in the lungs of respiratory infected guinea pigs makes possible the enfoldment leucocyte attack which does not, however, result in destruction of the infected cells.