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Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate.

ABSTRACT: Recent investigations have shown the Egyptian fruit bat (Rousettus aegyptiacus) to be a natural reservoir for marburgviruses. To better understand the life cycle of these viruses in the natural host, a new reverse genetics system was developed for the reliable rescue of a Marburg virus (MARV) originally isolated directly from a R. aegyptiacus bat (371Bat). To develop this system, the exact terminal sequences were first determined by 5' and 3' RACE, followed by the cloning of viral proteins NP, VP35, VP30 and L into expression plasmids. Novel conditions were then developed to efficiently replicate virus mini-genomes followed by the construction of full-length genomic clones from which recombinant wild type and GFP-containing MARVs were rescued. Surprisingly, when these recombinant MARVs were propagated in primary human macrophages, a dramatic difference was found in their ability to grow and to elicit anti-viral cytokine responses.

SUBMITTER: Albarino CG

PROVIDER: S-EPMC5683708 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate.

Albariño César G CG Uebelhoer Luke S LS Vincent Joel P JP Khristova Marina L ML Chakrabarti Ayan K AK McElroy Anita A Nichol Stuart T ST Towner Jonathan S JS

Virology 20130905 1-2

Recent investigations have shown the Egyptian fruit bat (Rousettus aegyptiacus) to be a natural reservoir for marburgviruses. To better understand the life cycle of these viruses in the natural host, a new reverse genetics system was developed for the reliable rescue of a Marburg virus (MARV) originally isolated directly from a R. aegyptiacus bat (371Bat). To develop this system, the exact terminal sequences were first determined by 5' and 3' RACE, followed by the cloning of viral proteins NP, V ...[more]

PMID: 24074586

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Project description:BackgroundRespiratory Syncytial Virus (RSV) is an important human respiratory pathogen, particularly of infants and older adults, and despite several decades of research and development, no licensed vaccine is available. Studies have confirmed that enhancement of RSV disease does not occur after inoculation with RSV live-attenuated vaccine candidates, making such vaccines preferable. In this paper, reverse genetics was used to construct two recombinant viruses, a recombinant Long strain (rLong) and rLong-∆G-EGFP; rLong-∆G-EGFP is a recombinant mutant in which G was replaced with the EGFP gene, based on the Long strain of RSV.ResultsBoth rLong and rLong-∆G-EGFP were constructed successfully and recovered in Hep-2 cells, and autofluorescence was observed in rLong-∆G-EGFP-infected cells during consecutive passages. Titers of rLong and rLong-∆G-EGFP were ~100-fold lower than the parental strain. Although virulence was attenuated, high titers of neutralizing antibodies were induced in BALB/c mice after being inoculated with recombinant viruses in a three-dose schedule. Unexpectedly, the neutralizing antibody titer in rLong-∆G-EGFP-immunized recipients did not decline significantly compared with the rLong strain. Protective efficacy of recombinant viruses in lung tissue was up to 100%, and the serum neutralizing antibody levels could stabilize at 21 days with no significant fall post-challenge. Enzyme-linked immunospot (ELISPOT) assays showed that both recombinant viruses were capable of inducing CD8+ T cell immune responses, which are crucial for virus clearance, and that rLong stimulated a higher level of IFN-γ production by comparison. In terms of inducing a balanced immune response, rLong-∆G-EGFP elicited slightly higher levels of IgG2a antibodies and lower levels of IgG1/IgG2a than the rLong virus.ConclusionsThis study suggested that immunization with rLong and rLong-∆G-EGFP were immunogenic and protected against RSV infection in the lower respiratory tract of BALB/c mice better than in the nose. Because of a relative low IgG1/IgG2a ratio, rLong-∆G-EGFP was more inclined to make CD4+ T cells, shifting toward a Th1-type response, indicating that the generation of a more balanced Th1/Th2 response was desirable. This explorative study on the recombinant Long viruses also contributed to obtaining more RSV attenuated candidates by a reverse genetics approach.

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: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.

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Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate.

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Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate.

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