Elaine Haddock

Host genetic diversity enables Ebola hemorrhagic fever pathogenesis and resistance

Existing mouse models of lethal Ebola virus infection do not reproduce hallmark symptoms of Ebola hemorrhagic fever, neither delayed blood coagulation and disseminated intravascular coagulation nor death from shock, thus restricting pathogenesis studies to nonhuman primates. Here we show that mice from the Collaborative Cross panel of recombinant inbred mice exhibit distinct disease phenotypes after mouse-adapted Ebola virus infection. Phenotypes range from complete resistance to lethal disease to severe hemorrhagic fever characterized by prolonged coagulation times and 100% mortality. Inflammatory signaling was associated with vascular permeability and endothelial activation, and resistance to lethal infection arose by induction of lymphocyte differentiation and cellular adhesion, probably mediated by the susceptibility allele Tek. These data indicate that genetic background determines susceptibility to Ebola hemorrhagic fever. Intercrossed mice infected with Ebola virus show a spectrum of pathology from prolonged coagulation to total resistance. Variety of Ebola symptoms in mice Apart from monkeys, there are no animal models available that show the same symptoms of Ebola virus infection as those of humans. Rasmussen et al. tested the effects of Ebola virus in mice with defined genetic backgrounds in a series of pains-taking experiments performed under stringent biosafety conditions. Resistance and susceptibility to Ebola virus was associated with distinct genetic profiles in inflammation, blood coagulation, and vascular function. This panel of mice could prove valuable for preliminary screens of candidate therapeutics and vaccines. Science, this issue p. 987

Infection with MERS-CoV causes lethal pneumonia in the common marmoset.

The availability of a robust disease model is essential for the development of countermeasures for Middle East respiratory syndrome coronavirus (MERS-CoV). While a rhesus macaque model of MERS-CoV has been established, the lack of uniform, severe disease in this model complicates the analysis of countermeasure studies. Modeling of the interaction between the MERS-CoV spike glycoprotein and its receptor dipeptidyl peptidase 4 predicted comparable interaction energies in common marmosets and humans. The suitability of the marmoset as a MERS-CoV model was tested by inoculation via combined intratracheal, intranasal, oral and ocular routes. Most of the marmosets developed a progressive severe pneumonia leading to euthanasia of some animals. Extensive lesions were evident in the lungs of all animals necropsied at different time points post inoculation. Some animals were also viremic; high viral loads were detected in the lungs of all infected animals, and total RNAseq demonstrated the induction of immune and inflammatory pathways. This is the first description of a severe, partially lethal, disease model of MERS-CoV, and as such will have a major impact on the ability to assess the efficacy of vaccines and treatment strategies as well as allowing more detailed pathogenesis studies.

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Does Not Replicate in Syrian Hamsters

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.

VSV-EBOV rapidly protects macaques against infection with the 2014/15 Ebola virus outbreak strain

Shortening the time to protection Although Ebola vaccine candidates have entered clinical trials in West Africa, there is little information available on the mechanism of protection. A single dose of the recombinant vesicular stomatitis virus–Ebola vaccine protects nonhuman primates, acting primarily through antibody responses. Marzi et al. found that this vaccine generates a robust immune response in macaques to a West African strain of Ebola virus within days of immunization (see the Perspective by Klenk and Becker). Innate immune responses developed in as little as 3 days and increased the chances of survival, with complete antibody protection acquired 7 days after immunization. Science, this issue p. 739; see also p. 693 A recombinant vaccine stimulates protective immunity against West African Ebola virus within days. [Also see Perspective by Klenk and Becker] The latest Ebola virus (EBOV) epidemic spread rapidly through Guinea, Sierra Leone, and Liberia, creating a global public health crisis and accelerating the assessment of experimental therapeutics and vaccines in clinical trials. One of those vaccines is based on recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (VSV-EBOV), a live-attenuated vector with marked preclinical efficacy. Here, we provide the preclinical proof that VSV-EBOV completely protects macaques against lethal challenge with the West African EBOV-Makona strain. Complete and partial protection was achieved with a single dose given as late as 7 and 3 days before challenge, respectively. This indicates that VSV-EBOV may protect humans against EBOV infections in West Africa with relatively short time to immunity, promoting its use for immediate public health responses.

A Syrian Golden Hamster Model Recapitulating Ebola Hemorrhagic Fever

Ebola hemorrhagic fever (EHF) is a severe viral infection for which no effective treatment or vaccine is currently available. While the nonhuman primate (NHP) model is used for final evaluation of experimental vaccines and therapeutic efficacy, rodent models have been widely used in ebolavirus research because of their convenience. However, the validity of rodent models has been questioned given their low predictive value for efficacy testing of vaccines and therapeutics, a result of the inconsistent manifestation of coagulopathy seen in EHF. Here, we describe a lethal Syrian hamster model of EHF using mouse-adapted Ebola virus. Infected hamsters displayed most clinical hallmarks of EHF, including severe coagulopathy and uncontrolled host immune responses. Thus, the hamster seems to be superior to the existing rodent models, offering a better tool for understanding the critical processes in pathogenesis and providing a new model for evaluating prophylactic and postexposure interventions prior to testing in NHPs.

Host Response Dynamics Following Lethal Infection of Rhesus Macaques With Zaire ebolavirus

To gain further insight into the interdependent pathogenic processes in Ebola hemorrhagic fever (EHF), we have examined the dynamics of host responses in individual rhesus macaques infected with Zaire ebolavirus over the entire disease course. Examination of coagulation parameters revealed that decreased coagulation inhibitor activity triggered severe coagulopathy as indicated by prolonged coagulation times and decreased fibrinogen levels. This has been proposed as one of the significant mechanisms underlying disseminated intravascular coagulation in EHF patients. Furthermore, monitoring of expression levels for cytokines/chemokines suggested a mixed anti-inflammatory response syndrome (MARS), which indicates that a catastrophic uncontrolled immunological status contributes to the development of fatal hemorrhagic fever. These results highlight the pathological analogies between EHF and severe sepsis and not only contribute to our understanding of the pathogenic process, but will also help to establish novel postexposure treatment modalities.

Validation of assays to monitor immune responses in the Syrian golden hamster (Mesocricetus auratus).

Abstract The Syrian golden hamster (Mesocricetus auratus) is a valuable but under-utilized animal model for studies of human viral pathogens such as bunyaviruses, arenaviruses, flaviviruses, henipaviruses, and SARS-coronavirus. A lack of suitable reagents and specific assays for monitoring host responses has limited the use of this animal model to clinical observations, pathology and humoral immune responses. The objective of this study was to establish and validate assays to monitor host immune responses in the hamster including important pro-inflammatory, anti-inflammatory and innate immune responses, as well as markers of apoptosis, cell proliferation, cell junction integrity and coagulation. Commercially available mouse and rat ELISA and luminex panels were screened for potential cross-reactivity, but were found to be of limited value for studying host responses in hamsters. Subsequently, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays for the detection of 51 immune-related and four internal reference genes were developed. To validate the immune-related assays, hamsters were infected with vesicular stomatitis virus (VSV), Indiana species, or treated with lipopolysaccharide (LPS) and host immune responses were monitored in selected organs. Ribosomal protein L18 was identified as the most stable internal reference gene. In conclusion, these new assays will greatly improve the use of the hamster as an important small animal model in infectious disease research.

Discovery of an antibody for pan-ebolavirus therapy

During the latest outbreak of Ebola virus disease in West Africa, monoclonal antibody therapy (e.g., ZMapp) was utilized to treat patients. However, due to the antigenic differences among the five ebolavirus species, the current therapeutic monoclonal antibodies are only effective against viruses of the species Zaire ebolavirus. Although this particular species has indeed caused the majority of human infections in Central and, recently, West Africa, other ebolavirus species (e.g., Sudan ebolavirus and Bundibugyo ebolavirus) have also repeatedly caused outbreaks in Central Africa and thus should not be neglected in the development of countermeasures against ebolaviruses. Here we report the generation of an ebolavirus glycoprotein-specific monoclonal antibody that effectively inhibits cellular entry of representative isolates of all known ebolavirus species in vitro and show its protective efficacy in mouse models of ebolavirus infections. This novel neutralizing monoclonal antibody targets a highly conserved internal fusion loop in the glycoprotein molecule and prevents membrane fusion of the viral envelope with cellular membranes. The discovery of this highly cross-neutralizing antibody provides a promising option for broad-acting ebolavirus antibody therapy and will accelerate the design of improved vaccines that can selectively elicit cross-neutralizing antibodies against multiple species of ebolaviruses.

Pathogenesis and Host Response in Syrian Hamsters following Intranasal Infection with Andes Virus

Hantavirus pulmonary syndrome (HPS), also referred to as hantavirus cardiopulmonary syndrome (HCPS), is a rare but frequently fatal disease caused by New World hantaviruses. In humans HPS is associated with severe pulmonary edema and cardiogenic shock; however, the pathogenesis of this disease remains unclear largely due to a lack of suitable animal models for the study of disease progression. In this study we monitored clinical, virological, pathophysiological parameters and host immunological responses to decipher pathological factors and events in the lethal Syrian hamster model of HPS following intranasal inoculation of Andes virus. Transcriptional profiling of the host gene responses demonstrated a suppression of innate immune responses in most organs analyzed during the early stage of infection, except for in the lung which had low level activation of several pro-inflammatory genes. During this phase Andes virus established a systemic infection in hamsters, with viral antigen readily detectable in the endothelium of the majority of tissues analyzed by 7–8 days post-inoculation. Despite wide-spread infection, histological analysis confirmed pathological abnormalities were almost exclusively found in the lungs. Immediately preceding clinical signs of disease, intense activation of pro-inflammatory and Th1/Th2 responses were observed in the lungs as well as the heart, but not in peripheral organs, suggesting that localized immune-modulations by infection is paramount to pathogenesis. Throughout the course of infection a strong suppression of regulatory T-cell responses was noted and is hypothesized to be the basis of the aberrant immune activations. The unique and comprehensive monitoring of host immune responses to hantavirus infection increases our understanding of the immuno-pathogenesis of HPS and will facilitate the development of treatment strategies targeting deleterious host immunological responses.

Pathophysiology of hantavirus pulmonary syndrome in rhesus macaques

Significance Hantavirus pulmonary syndrome (HPS) is a rare but often fatal disease caused by infection with New World hantaviruses. A limitation to understanding the pathogenesis of HPS and developing medical countermeasures against this disease is a lack of experimental disease models. In this study we describe the characterization of a novel nonhuman primate model of HPS. After infection with deer mouse-only–passaged Sin Nombre virus, macaques developed severe respiratory disease indicative of HPS. Viremia and hematological abnormalities were the earliest markers of ensuing disease, and the hyperpermeability associated with the onset of respiratory distress coincided with dysregulation of host responses exclusively in the pulmonary endothelium. This model will help advance our understanding of HPS and preclinical development of therapeutic strategies. The pathophysiology of hantavirus pulmonary syndrome (HPS) remains unclear because of a lack of surrogate disease models with which to perform pathogenesis studies. Nonhuman primates (NHP) are considered the gold standard model for studying the underlying immune activation/suppression associated with immunopathogenic viruses such as hantaviruses; however, to date an NHP model for HPS has not been described. Here we show that rhesus macaques infected with Sin Nombre virus (SNV), the primary etiological agent of HPS in North America, propagated in deer mice develop HPS, which is characterized by thrombocytopenia, leukocytosis, and rapid onset of respiratory distress caused by severe interstitial pneumonia. Despite establishing a systemic infection, SNV differentially activated host responses exclusively in the pulmonary endothelium, potentially the mechanism leading to acute severe respiratory distress. This study presents a unique chronological characterization of SNV infection and provides mechanistic data into the pathophysiology of HPS in a closely related surrogate animal model. We anticipate this model will advance our understanding of HPS pathogenesis and will greatly facilitate research toward the development of effective therapeutics and vaccines against hantaviral diseases.