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1.  Host genetic diversity enables Ebola hemorrhagic fever pathogenesis and resistance 
Science (New York, N.Y.)  2014;346(6212):987-991.
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 non-human primates. Here we show that mice from the Collaborative Cross exhibit distinct disease phenotypes following 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, likely mediated by the susceptibility allele Tek. These data indicate that genetic background determines susceptibility to Ebola hemorrhagic fever.
doi:10.1126/science.1259595
PMCID: PMC4241145  PMID: 25359852
2.  Infection with MERS-CoV Causes Lethal Pneumonia in the Common Marmoset 
PLoS Pathogens  2014;10(8):e1004250.
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.
Author Summary
The development of vaccines and treatment strategies is aided by robust animal disease models that accurately depict the illness that is observed in humans. Here we describe a new, improved model for MERS-CoV using the common marmoset, whereby the severe, and even lethal, illness that has been observed in many human cases is recapitulated. Prior to the development of this model, the only available animal models for MERS-CoV infection were the rhesus macaque and a mouse model that requires adenovirus-transduced expression of the human version of the protein required for virus entry. The rhesus macaque model more closely mimics the mild to moderate disease observed in some patients—mainly those without significant comorbidities. The increased severity of illness in the common marmoset model is an important advance in the ability to evaluate potential therapeutic agents against MERS-CoV, as discrimination between successfully treated and control animals should be more apparent. In addition, the closer models recapitulate the disease observed in humans, the more likely findings can be eventually translated into use in humans.
doi:10.1371/journal.ppat.1004250
PMCID: PMC4140844  PMID: 25144235
3.  Influenza Virus A/Anhui/1/2013 (H7N9) Replicates Efficiently in the Upper and Lower Respiratory Tracts of Cynomolgus Macaques 
mBio  2014;5(4):e01331-14.
ABSTRACT
In March 2013, three fatal human cases of infection with influenza A virus (H7N9) were reported in China. Since then, human cases have been accumulating. Given the public health importance of this virus, we performed a pathogenicity study of the H7N9 virus in the cynomolgus macaque model, focusing on clinical aspects of disease, radiographic, histological, and gene expression profile changes in the upper and lower respiratory tracts, and changes in systemic cytokine and chemokine profiles during infection. Cynomolgus macaques developed transient, mild to severe disease with radiographic evidence of pulmonary infiltration. Virus replicated in the upper as well as lower respiratory tract, with sustained replication in the upper respiratory tract until the end of the experiment at 6 days after inoculation. Virus shedding occurred mainly via the throat. Histopathological changes in the lungs were similar to those observed in humans, albeit less severe, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, pneumocyte hyperplasia, and fibroproliferative changes. Analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage during H7N9 infection as well as leads for development of therapeutics targeting host responses rather than virus replication. Overall, H7N9 infection was not as severe in cynomolgus macaques as in humans, supporting the possible role of underlying medical complications in disease severity as discussed for human H7N9 infection (H. N. Gao et al., N. Engl. J. Med. 368:2277–2285, 2013, doi:10.1056/NEJMoa1305584).
IMPORTANCE
Influenza A virus H7N9 emerged early in 2013, and human cases have continued to emerge since then. Although H7N9 virus-induced disease in humans is often very severe and even lethal, the majority of reported H7N9 cases occurred in older people and people with underlying medical conditions. To better understand the pathogenicity of this virus, healthy cynomolgus macaques were inoculated with influenza A virus H7N9. Cynomolgus macaques were used as a model because the receptor distribution for H7N9 virus in macaques was recently shown to be more similar to that in humans than that of other frequently used animal models. From comparison with previous studies, we conclude that the emerging H7N9 influenza virus was more pathogenic in cynomolgus macaques than seasonal influenza A viruses and most isolates of the pandemic H1N1 virus but less pathogenic than the 1918 Spanish influenza virus or highly pathogenic avian influenza (HPAI) H5N1 virus.
doi:10.1128/mBio.01331-14
PMCID: PMC4145683  PMID: 25118237
4.  Interferon-α2b and ribavirin treatment improves outcome in MERS-CoV-infected rhesus macaques 
Nature medicine  2013;19(10):1313-1317.
The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) is of global concern – causing severe respiratory illness with 97 confirmed cases and 46 deaths1. 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 disease model for MERS-CoV, developing an acute localized-to-widespread pneumonia with transient clinical disease2,3 that recapitulates mild-to-moderate human MERS-CoV cases4,5. The combination of interferon-α2b and ribavirin was effective in reducing MERS-CoV replication in vitro6; therefore, this strategy was initiated 8 h post-infection in the rhesus macaque model. Treated animals did not develop breathing abnormalities and showed no-to-very mild radiographic evidence of pneumonia. Moreover, treated animals showed reduced levels of systemic (serum) and local (lung) proinflammatory markers in addition to reduced viral genome copies, altered 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-α2b 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, IFN-α2b and ribavirin should be considered for management of MERS-CoV cases.
doi:10.1038/nm.3362
PMCID: PMC4093902  PMID: 24013700
5.  Speech disturbance at stroke onset is correlated with stroke early mortality 
BMC Neurology  2013;13:87.
Background
Speech disturbance is a common symptom of stroke and is important as a prompt identifier of the event. The frequency of the symptom among each stroke subtype, differences between patients with and without speech disturbance and its correlation to early mortality remain unclear.
Methods
The Kyoto prefecture of Japan has established a registry to enroll new stroke patients in cooperation with the Kyoto Medical Association and its affiliated hospitals. It is named the Kyoto Stroke Registry (KSR). We confirmed the existence or absence of speech disturbance in 1693 stroke patients registered to the KSR and investigated associations between speech disturbance and other characteristics.
Results
Speech disturbance was observed in 52.6% of cerebral infarction (CI), 47.5% of cerebral hemorrhage (CH), and 8.0% of subarachnoid hemorrhage (SAH) cases. Characteristics showing statistically significant differences between patients with and without speech disturbance and patients were age, blood pressure, history of hypertension, arrhythmia and diabetes mellitus, habit of tobacco and alcohol, and paresis. Mortality rates of patients with/without speech disturbance were 5.2%/1.2% for CI, 12.5% /4.1% for CH, and 62.5%/ 9.0% for SAH. Adjusted hazard ratios were 2.63 (1.14-6.13, p = 0.024) in CI, 4.15 (1.41-12.23, p = 0.010) in CH, and 20.46 (4.40-95.07, p < 0.001) in SAH).
Conclusion
Speech disturbance was frequently observed in stroke patients at the onset and therefore could be useful to identify the problem at the earliest stage. Hazard ratio for death was higher in stroke patients with speech disturbance than patients without. Speech disturbance is a prompt predictor of stroke early mortality.
Hiromi Nakano, Yoshiyuki Watanabe, Tatsuyuki Sekimoto, Kouichiro Shimizu, Akihiko Nishizawa, Atsushi Okumura and Masahiro Makino contributed equally to this work.
doi:10.1186/1471-2377-13-87
PMCID: PMC3720260  PMID: 23855651
6.  Characteristics, risk factors and mortality of stroke patients in Kyoto, Japan 
BMJ Open  2013;3(3):e002181.
Objectives
The aim of the study was to evaluate the characteristics, risk factors and outcome of recent stroke patients in Kyoto, Japan.
Design
We analysed stroke patients in the registry with regard to their characteristics, risk factors and mortality. Cox proportional hazards regressions were used to calculate adjusted HRs for death.
Settings
The Kyoto prefecture of Japan has established a registry to enrol new stroke patients in cooperation with the Kyoto Medical Association and its affiliated hospitals
Participants
The registry now has data on 14 268 patients enrolled from 1 January 1999 to 31 December 2009. Of these, 12 774(89.5%) underwent CT, 9232 (64.7%) MRI, 2504 (17.5%) angiography and 342 (2.4%) scintigraphy. Excluding 480 (3.3%) unclassified patients, 13 788 (96.6%) patients formed the basis of further analyses which were divided into three subtypes: cerebral infarction (CI), cerebral haemorrhage (CH) and subarachnoid haemorrhage (SAH).
Results
A total of 13 788 confirmed stroke patients in the study cohort comprised 9011 (86.3%) CI, 3549 (25.7%) CH and 1197 (8.7%) SAH cases. The mean age ±SD was 73.3±11.8, 69.1±13.6 and 62.7±13.5 in the CI, CH and SAH cases, respectively. Men were predominant in the CI and CH cases, whereas women were predominant in the SAH cases. The frequencies of risk factors were different among the subtypes. Mortality was worst in SAH, followed by CH, and least in CI. HRs for death adjusted for age, sex, histories of hypertension, arrhythmia, diabetes mellitus and hyperlipaemia and use of tobacco and/or alcohol showed a significant (p<0.001) difference among CI (as reference), CH (3.71; 3.11 to 4.43) and SAH (8.94; 7.21 to 11.11).
Conclusions
The characteristics, risk factors and mortality were evaluated in a quantitative manner in a large Japanese study cohort to shed light on the present status of stroke medicine.
doi:10.1136/bmjopen-2012-002181
PMCID: PMC3612757  PMID: 23468468
7.  Evaluation of an Attenuated Vesicular Stomatitis Virus Vector Expressing Interferon-β for Use in Malignant Pleural Mesothelioma: Heterogeneity in Interferon Responsiveness Defines Potential Efficacy 
Human Gene Therapy  2009;21(1):51-64.
Abstract
Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-β (IFN-β) gene (VSV.IFN-β). We conducted this study to determine the ability of VSV.IFN-β to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-β did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-β. In the eight resistant lines, pretreatment with IFN-β prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-β protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2′,5′-oligo-adenylate-synthetase (2′5′-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-β protein and no IFN- or VSV-induced changes in PKR, MxA, and 2′5′-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-β by immunostaining for the presence of p48 protein.
doi:10.1089/hum.2009.088
PMCID: PMC2829454  PMID: 19715403
8.  Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles ▿  
Journal of Virology  2009;84(5):2294-2303.
The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.
doi:10.1128/JVI.02034-09
PMCID: PMC2820906  PMID: 20032189
9.  Antiviral Activity of Innate Immune Protein ISG15 
Journal of Innate Immunity  2009;1(5):397-404.
The host innate immune response, including the production of type-I IFN, represents the primary line of defense against invading viral pathogens. Of the hundreds of IFN-stimulated genes (ISGs) discovered to date, ISG15 was one of the first identified and shown to encode a ubiquitin-like protein that functions, in part, as a modifier of protein function. Evidence implicating ISG15 as an innate immune protein with broad-spectrum antiviral activity continues to accumulate rapidly. This review will summarize recent findings on the innate antiviral activity of ISG15, with a focus on the interplay between ubiquitination and ISGylation pathways resulting in modulation of RNA virus assembly/budding. Indeed, ubiquitination is known to be proviral for some RNA viruses, whereas the parallel ISGylation pathway is known to be antiviral. A better understanding of the antiviral activities of ISG15 will enhance our fundamental knowledge of host innate responses to viral pathogens and may provide insight useful for the development of novel therapeutic approaches designed to enhance the immune response against such pathogens.
doi:10.1159/000226245
PMCID: PMC2725329  PMID: 19680460
Antiviral; Budding; Innate immunity; Interferon-stimulated gene 15; ISGylation; Ubiquitin; Ubiquitination
10.  Interaction between Ebola Virus Glycoprotein and Host Toll-Like Receptor 4 Leads to Induction of Proinflammatory Cytokines and SOCS1 ▿ †  
Journal of Virology  2009;84(1):27-33.
Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection.
doi:10.1128/JVI.01462-09
PMCID: PMC2798428  PMID: 19846529
11.  Antiviral Activity of Innate Immune Protein ISG15 
Journal of innate immunity  2009;1(5):397-404.
The host innate immune response, including the production of type-I IFN, represents the primary line of defense against invading viral pathogens. Of the hundreds of IFN-stimulated genes (ISGs) discovered to date, ISG15 was one of the first identified and shown to encode a ubiquitin-like protein that functions, in part, as a modifier of protein function. Evidence implicating ISG15 as an innate immune protein with broad-spectrum antiviral activity continues to accumulate rapidly. This review will summarize recent findings on the innate antiviral activity of ISG15, with a focus on the interplay between ubiquitination and ISGylation pathways resulting in modulation of RNA virus assembly/budding. Indeed, ubiquitination is known to be proviral for some RNA viruses, whereas the parallel ISGylation pathway is known to be antiviral. A better understanding of the antiviral activities of ISG15 will enhance our fundamental knowledge of host innate responses to viral pathogens and may provide insight useful for the development of novel therapeutic approaches designed to enhance the immune response against such pathogens.
doi:10.1159/000226245
PMCID: PMC2725329  PMID: 19680460
Antiviral; Budding; Innate immunity; Interferon-stimulated gene 15; ISGylation; Ubiquitin; Ubiquitination
12.  Panencephalopathic type of Creutzfeldt-Jakob disease: primary involvement of the cerebral white matter 
Eight necropsy cases of a “panencephalopathic” type of Creutzfeldt-Jakob disease (CJD) in the Japanese are reported. The reasons why this type should be discussed separately from other types of CJD are that there is primary involvement of the cerebral white matter as well as the cerebral cortex, and that the white matter lesion of one Japanese human brain with CJD similar to the present group has been successfully transmitted to experimental animals.
Images
PMCID: PMC490840  PMID: 7012278

Results 1-12 (12)