scholarly journals Generation and Characterization of a Mouse-Adapted Makona Variant of Ebola Virus

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 987 ◽  
Author(s):  
Mable Chan ◽  
Anders Leung ◽  
Bryan D. Griffin ◽  
Robert Vendramelli ◽  
Nikesh Tailor ◽  
...  
Keyword(s):  
Mouse Model ◽  
Reverse Genetics ◽  
Ebola Virus ◽  
Lethal Dose ◽  
West African ◽  
Zoonotic Pathogen ◽  
Immunocompetent Mice ◽  
Pro Inflammatory Cytokines ◽  
Prior Adaptation

Ebola virus (EBOV) is a zoonotic pathogen that poses a significant threat to public health, causing sporadic yet devastating outbreaks that have the potential to spread worldwide, as demonstrated during the 2013–2016 West African outbreak. Mouse models of infection are important tools for the development of therapeutics and vaccines. Exposure of immunocompetent mice to clinical isolates of EBOV is nonlethal; consequently, EBOV requires prior adaptation in mice to cause lethal disease. Until now, the only immunocompetent EBOV mouse model was based on the Mayinga variant, which was isolated in 1976. Here, we generated a novel mouse-adapted (MA)-EBOV based on the 2014 Makona isolate by inserting EBOV/Mayinga-MA mutations into the EBOV/Makona genome, followed by serial passaging of the rescued virus in suckling mice. The resulting EBOV/Makona-MA causes lethal disease in adult immunocompetent mice within 6 to 9 days and has a lethal dose (LD50) of 0.004 plaque forming units (PFU). Two additional mutations emerged after mouse-adaptation in the viral nucleoprotein (NP) and membrane-associated protein VP24. Using reverse genetics, we found the VP24 mutation to be critical for EBOV/Makona-MA virulence. EBOV/Makona-MA infected mice that presented with viremia, high viral burden in organs, increased release of pro-inflammatory cytokines/chemokines, and lymphopenia. Our mouse model will help advance pre-clinical development of countermeasures against contemporary EBOV variants.

scholarly journals Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection

2021 ◽  
Vol 102 (9) ◽  
Author(s):  
Constantina Borg ◽  
Aminu S. Jahun ◽  
Lucy Thorne ◽  
Frédéric Sorgeloos ◽  
Dalan Bailey ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Persistent Infection ◽  
Reverse Genetics ◽  
Viral Fitness ◽  
Murine Norovirus ◽  
Viral Loads ◽  
Immunocompetent Mice

Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and demonstrated its role as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Using reverse genetics, we generated MNV-3 viruses carrying a single or a triple termination codon inserted in the VF1 ORF. VF1-deleted MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. C57BL/6 mice infected with VF1-deleted MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a 3 month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonize the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilize multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

scholarly journals Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies

2016 ◽  
Vol 90 (8) ◽  
pp. 3890-3901 ◽  
Author(s):  
Philipp A. Ilinykh ◽  
Xiaoli Shen ◽  
Andrew I. Flyak ◽  
Natalia Kuzmina ◽  
Thomas G. Ksiazek ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Reverse Genetics ◽  
Ebola Virus ◽  
Fluorescent Protein ◽  
Marburg Virus ◽  
Antibody Neutralization ◽  
Surrogate Systems

ABSTRACTRecent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection.IMPORTANCEThe study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action.

scholarly journals Development and Characterization of a Mouse Model for Marburg Hemorrhagic Fever

2009 ◽  
Vol 83 (13) ◽  
pp. 6404-6415 ◽  
Author(s):  
Kelly L. Warfield ◽  
Steven B. Bradfute ◽  
Jay Wells ◽  
Loreen Lofts ◽  
Meagan T. Cooper ◽  
...  
Keyword(s):  
Mouse Model ◽  
Hemorrhagic Fever ◽  
Sampling Studies ◽  
Time To Death ◽  
Adult Mice ◽  
Serial Sampling ◽  
Liver Homogenates ◽  
Immunocompetent Mice ◽  
Spleen And Lymph Nodes

ABSTRACT The lack of a mouse model has hampered an understanding of the pathogenesis and immunity of Marburg hemorrhagic fever (MHF), the disease caused by marburgvirus (MARV), and has created a bottleneck in the development of antiviral therapeutics. Primary isolates of the filoviruses, i.e., ebolavirus (EBOV) and MARV, are not lethal to immunocompetent adult mice. Previously, pathological, virologic, and immunologic evaluation of a mouse-adapted EBOV, developed by sequential passages in suckling mice, identified many similarities between this model and EBOV infections in nonhuman primates. We recently demonstrated that serially passaging virus recovered from the liver homogenates of MARV-infected immunodeficient (SCID) mice was highly successful in reducing the time to death in these mice from 50 to 70 days to 7 to 10 days after challenge with the isolate MARV-Ci67, -Musoke, or -Ravn. In this study, we extended our findings to show that further sequential passages of MARV-Ravn in immunocompetent mice caused the MARV to kill BALB/c mice. Serial sampling studies to characterize the pathology of mouse-adapted MARV-Ravn revealed that this model is similar to the guinea pig and nonhuman primate MHF models. Infection of BALB/c mice with mouse-adapted MARV-Ravn caused uncontrolled viremia and high viral titers in the liver, spleen, lymph node, and other organs; profound lymphopenia; destruction of lymphocytes within the spleen and lymph nodes; and marked liver damage and thrombocytopenia. Sequencing the mouse-adapted MARV-Ravn strain revealed differences in 16 predicted amino acids from the progenitor virus, although the exact changes required for adaptation are unclear at this time. This mouse-adapted MARV strain can now be used to develop and evaluate novel vaccines and therapeutics and may also help to provide a better understanding of the virulence factors associated with MARV.

scholarly journals Characterization of Reverse Genetics-Derived Cold-Adapted Master Donor Virus A/Leningrad/134/17/57 (H2N2) and Reassortants with H5N1 Surface Genes in a Mouse Model

2014 ◽  
Vol 21 (5) ◽  
pp. 722-731 ◽  
Author(s):  
Irina Isakova-Sivak ◽  
Li-Mei Chen ◽  
Melissa Bourgeois ◽  
Yumiko Matsuoka ◽  
J. Theo M. Voeten ◽  
...  
Keyword(s):  
Mouse Model ◽  
Reverse Genetics ◽  
Protective Efficacy ◽  
Influenza Pandemic ◽  
Lethal Dose ◽  
Challenge Inoculation ◽  
Cold Adapted ◽  
Antibody Titers ◽  
Inactivated Vaccines ◽  
Clade 1

ABSTRACTLive attenuated influenza vaccines (LAIV) offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing processes and more cross-protective immune responses. Using an established reverse genetics (rg) system for wild-type (wt) A/Leningrad/134/1957 and cold-adapted (ca) A/Leningrad/134/17/1957 (Len17) master donor virus (MDV), we produced and characterized three rg H5N1 reassortant viruses carrying modified HA and intact NA genes from either A/Vietnam/1203/2004 (H5N1, VN1203, clade 1) or A/Egypt/321/2007 (H5N1, EG321, clade 2) virus. A mouse model of infection was used to determine the infectivity and tissue tropism of the parentalwtviruses compared to thecamaster donor viruses as well as the H5N1 reassortants. Allcaviruses showed reduced replication in lungs and enhanced replication in nasal epithelium. In addition, the H5N1 HA and NA enhanced replication in lungs unless it was restricted by the internal genes of thecaMDV. Mice inoculated twice 4 weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum hemagglutination inhibition HI and IgA antibody titers to the homologous and heterologous viruses consistent with protective immunity. These animals remained healthy after challenge inoculation with a lethal dose with homologous or heterologouswtH5N1 highly pathogenic avian influenza (HPAI) viruses. The profiles of viral replication in respiratory tissues and the immunogenicity and protective efficacy characteristics of the twocaH5N1 candidate LAIV viruses warrant further development into a vaccine for human use.

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