Last data update: Jan 27, 2025. (Total: 48650 publications since 2009)
Records 1-15 (of 15 Records) |
Query Trace: Zivcec M[original query] |
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The use of mice lacking type I or both type I and type II interferon responses in research on hemorrhagic fever viruses. Part 2: Vaccine efficacy studies
Zivcec M , Spiropoulou CF , Spengler JR . Antiviral Res 2020 174 104702 For more than 20 years, researchers have used laboratory mice lacking type I or both type I and II interferon (IFN) responses to study high-containment viruses that cause hemorrhagic fevers (HF) in humans. With the exception of Rift Valley fever virus, agents that cause viral HF in humans, such as Ebola and Lassa virus, do not cause disease in mature immunocompetent mice. In contrast, IFN-deficient mice typically develop severe or fatal disease when inoculated with these agents. The sensitivity of IFN-deficient mice to disease has led to their widespread use in biocontainment laboratories to assess the efficacy of novel vaccines against HF viruses, often without considering whether adaptive immune responses in IFN-deficient mice accurately mirror those in immunocompetent humans. Failure to recognize these questions may lead to inappropriate expectations of the predictive value of mouse experiments. In two invited articles, we investigate these questions. The present article reviews the use of IFN-deficient mice for assessing novel vaccines against HF viruses, including Ebola, Lassa, Crimean-Congo hemorrhagic fever and Rift Valley fever viruses. A companion paper examines the general question of how the lack of IFN signaling may affect adaptive immune responses and the outcome of vaccine studies in mice. |
A cynomolgus macaque model for Crimean-Congo haemorrhagic fever
Haddock E , Feldmann F , Hawman DW , Zivcec M , Hanley PW , Saturday G , Scott DP , Thomas T , Korva M , Avsic-Zupanc T , Safronetz D , Feldmann H . Nat Microbiol 2018 3 (5) 556-562 Crimean-Congo haemorrhagic fever (CCHF) is the most medically significant tick-borne disease, being widespread in the Middle East, Asia, Africa and parts of Europe (1) . Increasing case numbers, westerly movement and broadly ranging case fatality rates substantiate the concern of CCHF as a public health threat. Ixodid ticks of the genus Hyalomma are the vector for CCHF virus (CCHFV), an arbovirus in the genus Orthonairovirus of the family Nairoviridae. CCHFV naturally infects numerous wild and domestic animals via tick bite without causing obvious disease(2,3). Severe disease occurs only in humans and transmission usually happens through tick bite or contact with infected animals or humans. The only CCHF disease model is a subset of immunocompromised mice(4-6). Here, we show that following CCHFV infection, cynomolgus macaques exhibited hallmark signs of human CCHF with remarkably similar viral dissemination, organ pathology and disease progression. Histopathology showed infection of hepatocytes, endothelial cells and monocytes and fatal outcome seemed associated with endothelial dysfunction manifesting in a clinical shock syndrome with coagulopathy. This non-human primate model will be an invaluable asset for CCHFV countermeasures development. |
Genome Sequences of Crimean-Congo Hemorrhagic Fever Virus Strains Isolated in South Africa, Namibia, and Turkey.
Zivcec M , Albarino CG , Guerrero LIW , Ksiazek TG , Nichol ST , Swanepoel R , Rollin PE , Spiropoulou CF . Genome Announc 2017 5 (42) ![]() We report here the full-length sequences of 16 historical isolates of Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) obtained in Turkey, Namibia, and South Africa. The strains may be useful for future work to develop molecular diagnostics or viral evolution studies. |
Structure-function assays for Crimean-Congo hemorrhagic fever virus polymerase
Zivcec M . Methods Mol Biol 2018 1604 229-235 The recently developed Crimean-Congo hemorrhagic fever virus (CCHFV) reverse genetics systems have paved the way for experiments looking to identify and characterize the roles played by viral and cellular proteins in the CCHFV life cycle. In particular, the development of the noninfectious minigenome and virus-like particle (VLP) systems is a tremendous technological advance, as these systems allow for precisely targeting proteins or nucleic acids and measuring the effects these mutations or treatments have on viral life cycle stages. Importantly, these systems can be used at low-containment levels. Presented are the materials and methods currently available to study CCHFV transcription, replication, and translation in the context of a minigenome or VLP. |
A DNA vaccine for Crimean-Congo hemorrhagic fever protects against disease and death in two lethal mouse models.
Garrison AR , Shoemaker CJ , Golden JW , Fitzpatrick CJ , Suschak JJ , Richards MJ , Badger CV , Six CM , Martin JD , Hannaman D , Zivcec M , Bergeron E , Koehler JW , Schmaljohn CS . PLoS Negl Trop Dis 2017 11 (9) e0005908 ![]() Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus capable of causing a severe hemorrhagic fever disease in humans. There are currently no licensed vaccines to prevent CCHFV-associated disease. We developed a DNA vaccine expressing the M-segment glycoprotein precursor gene of CCHFV and assessed its immunogenicity and protective efficacy in two lethal mouse models of disease: type I interferon receptor knockout (IFNAR-/-) mice; and a novel transiently immune suppressed (IS) mouse model. Vaccination of mice by muscle electroporation of the M-segment DNA vaccine elicited strong antigen-specific humoral immune responses with neutralizing titers after three vaccinations in both IFNAR-/- and IS mouse models. To compare the protective efficacy of the vaccine in the two models, groups of vaccinated mice (7-10 per group) were intraperitoneally (IP) challenged with a lethal dose of CCHFV strain IbAr 10200. Weight loss was markedly reduced in CCHFV DNA-vaccinated mice as compared to controls. Furthermore, whereas all vector-control vaccinated mice succumbed to disease by day 5, the DNA vaccine protected >60% of the animals from lethal disease. Mice from both models developed comparable levels of antibodies, but the IS mice had a more balanced Th1/Th2 response to vaccination. There were no statistical differences in the protective efficacies of the vaccine in the two models. Our results provide the first comparison of these two mouse models for assessing a vaccine against CCHFV and offer supportive data indicating that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV. |
Crimean-Congo hemorrhagic fever virus suppresses innate immune responses via a ubiquitin and ISG15 specific protease
Scholte FEM , Zivcec M , Dzimianski JV , Deaton MK , Spengler JR , Welch SR , Nichol ST , Pegan SD , Spiropoulou CF , Bergeron E . Cell Rep 2017 20 (10) 2396-2407 Antiviral responses are regulated by conjugation of ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU) superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae) is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights into viral OTU functions and support the development of therapeutics and vaccines. |
Identification of broadly neutralizing monoclonal antibodies against Crimean-Congo hemorrhagic fever virus
Zivcec M , Guerrero LIW , Albarino CG , Bergeron E , Nichol ST , Spiropoulou CF . Antiviral Res 2017 146 112-120 Despite the serious public health impact of Crimean-Congo hemorrhagic fever (CCHF), the efficacy of antivirals targeting the causative agent, CCHF virus (CCHFV), remains debatable. Neutralizing monoclonal antibodies (MAbs) targeting the CCHFV glycoprotein Gc have been reported to protect mice against challenge with the prototype CCHFV strain, IbAr10200. However, due to extensive sequence diversity of CCHFV glycoproteins, it is unknown whether these MAbs neutralize other CCHFV strains. We initially used a CCHF virus-like particle (VLP) system to generate 11 VLP moieties, each possessing a glycoprotein from a genetically diverse CCHFV strain isolated in either Africa, Asia, the Middle East, or southeastern Europe. We used these VLPs in biosafety level 2 conditions to efficiently screen MAb cross-neutralization potency. Of the 16 MAbs tested, 3 (8A1, 11E7, and 30F7) demonstrated cross-neutralization activity with most CCHF VLPs, with 8A1 neutralizing all VLPs tested. Although binding studies suggest that none of the MAbs compete for the same epitope, combining 11E7, 30F7, or both 11E7 and 30F7 with 8A1 had no additive effect on increasing neutralization in this system. To confirm our findings from the VLP system, the 3 MAbs capable of strain cross-neutralization were confirmed to effectively neutralize 5 diverse CCHFV strains in vitro. Passaging CCHFV strains in the presence of sub-neutralizing concentrations of MAbs did not generate escape mutants resistant to subsequent neutralization. This study demonstrates the utility of the VLP system for screening neutralizing MAbs against multiple CCHFV strains, and provides the first evidence that a single MAb can effectively neutralize a number of diverse CCHFV strains in vitro, which may lead to development of future CCHF therapeutics. |
Monitoring Innate Immune Gene Responses in the Hamster Model of Ebola Virus Disease by RT-PCR.
Zivcec M . Methods Mol Biol 2017 1628 321-337 ![]() Ebola virus (EBOV) disease is a severe, acute human syndrome associated with high case fatality rates. Immune responses to EBOV are thought to be at least partially responsible for disease pathogenesis and must therefore be investigated to get a better understanding of underlying mechanisms of pathogenesis. Syrian hamsters are susceptible to EBOV infection and develop a disease more consistent with human EBOV disease than other rodent disease models. Quantitative RT-PCR (qRT-PCR) is ideal for monitoring immune responses during EBOV infection in low- to medium-throughput applications. A relatively straightforward protocol for monitoring immune responses, based on information gleaned from experimental EBOV infection of hamsters, is presented. |
Crimean-Congo hemorrhagic fever in humanized mice reveals glial cells as primary targets of neurological infection
Spengler JR , Keating MK , McElroy AK , Zivcec M , Coleman-McCray JD , Harmon JR , Bollweg BC , Goldsmith CS , Bergeron E , Keck JG , Zaki SR , Nichol ST , Spiropoulou CF . J Infect Dis 2017 216 (11) 1386-1397 Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral hemorrhagic disease seen exclusively in humans. Central nervous system (CNS) infection and neurological involvement have also been reported in CCHF. Here we inoculated NSGTM-SGM3 mice engrafted with human hematopoietic CD34+ stem cells with low passage CCHF virus strains isolated from human patients. Humanized mice develop lethal disease characterized by histopathological change in the liver and brain. To date, targets of neuroinfection and neuropathology have not been investigated in CCHF. CNS disease in humanized mice was characterized by gliosis, meningitis and meningoencephalitis, and glial cells were identified as principal targets of infection. Humanized mice represent a novel lethal model for studies of CCHF countermeasures, and CCHF-associated CNS disease. Our data suggests a role for astrocyte dysfunction in neurologic disease, and distinguish key regions of infection in the CNS for future investigations of CCHF. |
Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.
Erickson BR , Sealy TK , Flietstra T , Morgan L , Kargbo B , Matt-Lebby VE , Gibbons A , Chakrabarti AK , Graziano J , Presser L , Flint M , Bird BH , Brown S , Klena JD , Blau DM , Brault AC , Belser JA , Salzer JS , Schuh AJ , Lo M , Zivcec M , Priestley RA , Pyle M , Goodman C , Bearden S , Amman BR , Basile A , Bergeron E , Bowen MD , Dodd KA , Freeman MM , McMullan LK , Paddock CD , Russell BJ , Sanchez AJ , Towner JS , Wang D , Zemtsova GE , Stoddard RA , Turnsek M , Guerrero LW , Emery SL , Stovall J , Kainulainen MH , Perniciaro JL , Mijatovic-Rustempasic S , Shakirova G , Winter J , Sexton C , Liu F , Slater K , Anderson R , Andersen L , Chiang CF , Tzeng WP , Crowe SJ , Maenner MJ , Spiropoulou CF , Nichol ST , Stroher U . J Infect Dis 2016 214 S258-S262 ![]() During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses. |
Molecular Insights into Crimean-Congo Hemorrhagic Fever Virus.
Zivcec M , Scholte FE , Spiropoulou CF , Spengler JR , Bergeron E . Viruses 2016 8 (4) 106 ![]() Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen that causes high morbidity and mortality. Efficacy of vaccines and antivirals to treat human CCHFV infections remains limited and controversial. Research into pathology and underlying molecular mechanisms of CCHFV and other nairoviruses is limited. Significant progress has been made in our understanding of CCHFV replication and pathogenesis in the past decade. Here we review the most recent molecular advances in CCHFV-related research, and provide perspectives on future research. |
Assessment of Inhibitors of Pathogenic Crimean-Congo Hemorrhagic Fever Virus Strains Using Virus-Like Particles.
Zivcec M , Metcalfe MG , Albarino CG , Guerrero LW , Pegan SD , Spiropoulou CF , Bergeron E . PLoS Negl Trop Dis 2015 9 (12) e0004259 ![]() Crimean-Congo hemorrhagic fever (CCHF) is an often lethal, acute inflammatory illness that affects a large geographic area. The disease is caused by infection with CCHF virus (CCHFV), a nairovirus from the Bunyaviridae family. Basic research on CCHFV has been severely hampered by biosafety requirements and lack of available strains and molecular tools. We report the development of a CCHF transcription- and entry-competent virus-like particle (tecVLP) system that can be used to study cell entry and viral transcription/replication over a broad dynamic range (~4 orders of magnitude). The tecVLPs are morphologically similar to authentic CCHFV. Incubation of immortalized and primary human cells with tecVLPs results in a strong reporter signal that is sensitive to treatment with neutralizing monoclonal antibodies and by small molecule inhibitors of CCHFV. We used glycoproteins and minigenomes from divergent CCHFV strains to generate tecVLPs, and in doing so, we identified a monoclonal antibody that can prevent cell entry of tecVLPs containing glycoproteins from 3 pathogenic CCHFV strains. In addition, our data suggest that different glycoprotein moieties confer different cellular entry efficiencies, and that glycoproteins from the commonly used strain IbAr10200 have up to 100-fold lower ability to enter primary human cells compared to glycoproteins from pathogenic CCHFV strains. |
RIG-I mediates an antiviral response to Crimean-Congo hemorrhagic fever virus.
Spengler JR , Patel JR , Chakrabarti AK , Zivcec M , Garcia-Sastre A , Spiropoulou CF , Bergeron E . J Virol 2015 89 (20) 10219-29 ![]() In the cytoplasm, the retinoic acid-inducible gene I (RIG-I) senses the RNA genomes of several RNA viruses. RIG-I binds to viral RNA, eliciting an antiviral response via the cellular adaptor MAVS. Crimean-Congo hemorrhagic fever virus (CCHFV), a negative sense RNA virus with a 5' -monophosphorylated genome, is a highly pathogenic zoonotic agent with significant public health and clinical implications. We found that, during CCHFV infection, RIG-I mediated a type-I interferon (IFN) response via MAVS. Interfering with RIG-I signaling reduced IFN production and IFN-stimulated gene expression, and increased viral replication. Immunostimulatory RNA was isolated from CCHFV-infected cells and from virion preparations, and RIG-I co-immunoprecipitation of infected cell lysates isolated immunostimulatory CCHFV RNA. This report serves as the first description of a pattern recognition receptor for CCHFV and highlights a critical signaling pathway in the antiviral response to CCHFV. IMPORTANCE: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus with significant public health and clinical impact. In order for cells to respond to virus infection, they must recognize the virus as foreign and initiate antiviral signaling. To date, the receptors involved in recognition of CCHFV are not known. Herein we investigate and identify retinoic acid-inducible gene I (RIG-I) as a receptor involved in initiating an antiviral response to CCHFV. This receptor was initially not expected to play a role in CCHFV recognition because of characteristics of the viral genome. These findings are important in understanding the antiviral response to CCHFV and support continued investigation into the spectrum of potential viruses recognized by RIG-I. |
Ebola virus diagnostics: the US Centers for Disease Control and Prevention laboratory in Sierra Leone, August 2014 to March 2015
Flint M , Goodman CH , Bearden S , Blau DM , Amman BR , Basile AJ , Belser JA , Bergeron E , Bowen MD , Brault AC , Campbell S , Chakrabarti AK , Dodd KA , Erickson BR , Freeman MM , Gibbons A , Guerrero LW , Klena JD , Lash RR , Lo MK , McMullan LK , Momoh G , Massally JL , Goba A , Paddock CD , Priestley RA , Pyle M , Rayfield M , Russell BJ , Salzer JS , Sanchez AJ , Schuh AJ , Sealy TK , Steinau M , Stoddard RA , Taboy C , Turnsek M , Wang D , Zemtsova GE , Zivcec M , Spiropoulou CF , Stroher U , Towner JS , Nichol ST , Bird BH . J Infect Dis 2015 212 Suppl 2 S350-8 In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone. |
Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin.
Bergeron E , Zivcec M , Chakrabarti AK , Nichol ST , Albarino CG , Spiropoulou CF . PLoS Pathog 2015 11 (5) e1004879 ![]() Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV's high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor's maturation to GP38, and Gn precursor's maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat. |
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