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An Improved Reverse Genetics System to Overcome Cell-Type-Dependent Ebola Virus Genome Plasticity.

ABSTRACT: Reverse genetics systems represent a key technique for studying replication and pathogenesis of viruses, including Ebola virus (EBOV). During the rescue of recombinant EBOV from Vero cells, a high frequency of mutations was observed throughout the genomes of rescued viruses, including at the RNA editing site of the glycoprotein gene. The influence that such genomic instability could have on downstream uses of rescued virus may be detrimental, and we therefore sought to improve the rescue system. Here we report an improved EBOV rescue system with higher efficiency and genome stability, using a modified full-length EBOV clone in Huh7 cells. Moreover, by evaluating a variety of cells lines, we revealed that EBOV genome instability is cell-type dependent, a fact that has significant implications for the preparation of standard virus stocks. Thus, our improved rescue system will have an impact on both basic and translational research in the filovirus field.

SUBMITTER: Tsuda Y

PROVIDER: S-EPMC4564527 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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An Improved Reverse Genetics System to Overcome Cell-Type-Dependent Ebola Virus Genome Plasticity.

Tsuda Yoshimi Y Hoenen Thomas T Banadyga Logan L Weisend Carla C Ricklefs Stacy M SM Porcella Stephen F SF Ebihara Hideki H

The Journal of infectious diseases 20150324

Reverse genetics systems represent a key technique for studying replication and pathogenesis of viruses, including Ebola virus (EBOV). During the rescue of recombinant EBOV from Vero cells, a high frequency of mutations was observed throughout the genomes of rescued viruses, including at the RNA editing site of the glycoprotein gene. The influence that such genomic instability could have on downstream uses of rescued virus may be detrimental, and we therefore sought to improve the rescue system. ...[more]

PMID: 25810440

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Project description:Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels.IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

Project description:Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures.IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.

Project description:UnlabelledSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that was first reported in China in 2009. Phylogenetic analysis of the viral genome showed that SFTS virus represents a new lineage within the Phlebovirus genus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the family Bunyaviridae. SFTS disease is characterized by gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia, and some hemorrhagic manifestations with a case fatality rate of about 2 to 15%. Here we report the development of reverse genetics systems to study STFSV replication and pathogenesis. We developed and optimized functional T7 polymerase-based M- and S-segment minigenome assays, which revealed errors in the published terminal sequences of the S segment of the Hubei 29 strain of SFTSV. We then generated recombinant viruses from cloned cDNAs prepared to the antigenomic RNAs both of the minimally passaged virus (HB29) and of a cell culture-adapted strain designated HB29pp. The growth properties, pattern of viral protein synthesis, and subcellular localization of viral N and NSs proteins of wild-type HB29pp (wtHB29pp) and recombinant HB29pp viruses were indistinguishable. We also show that the viruses fail to shut off host cell polypeptide production. The robust reverse genetics system described will be a valuable tool for the design of therapeutics and the development of killed and attenuated vaccines against this important emerging pathogen.ImportanceSFTSV and related tick-borne phleboviruses such as Heartland virus are emerging viruses shown to cause severe disease in humans in the Far East and the United States, respectively. Study of these novel pathogens would be facilitated by technology to manipulate these viruses in a laboratory setting using reverse genetics. Here, we report the generation of infectious SFTSV from cDNA clones and demonstrate that the behavior of recombinant viruses is similar to that of the wild type. This advance will allow for further dissection of the roles of each of the viral proteins in the context of virus infection, as well as help in the development of antiviral drugs and protective vaccines.

Dataset Information

An Improved Reverse Genetics System to Overcome Cell-Type-Dependent Ebola Virus Genome Plasticity.

Publications

An Improved Reverse Genetics System to Overcome Cell-Type-Dependent Ebola Virus Genome Plasticity.

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