Bunyavirus Reverse Genetics and Applications to Studying Interactions with Host Cells

Elliott, Richard M.

doi:10.1002/9781118405338.ch7

Cited by 5 publications

(5 citation statements)

References 95 publications

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“…Bunyavirus minigenomes comprise a reporter gene, such as luciferase, cloned in the negative sense between the 3= and 5= untranslated regions of a viral segment, under the control of T7 promoter and hepatitis ribozyme sequences (41). When transfected into cells expressing T7 RNA polymerase, a negative-sense genome analogue is transcribed (the minigenome), and in the presence of coexpressed viral N and L proteins, a functional ribonucleoprotein that allows viral transcription and replication to be assessed by reporter gene activity is produced.…”

Section: Resultsmentioning

confidence: 99%

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

Brennan

Zhang

et al. 2015

J Virol

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Severe 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.T he family Bunyaviridae is one of the largest taxonomic groupings of RNA viruses, containing over 350 named isolates that are classified into five genera: Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, and Tospovirus (1). All viruses share a genome structure that comprises three segments of negative-sense or ambisense RNA, named small (S), medium (M), and large (L). The S segment encodes the nucleocapsid (N) protein; the M segment encodes the virion glycoproteins Gn and Gc; and the L segment encodes the L protein, the viral RNA-dependent RNA polymerase. Some viruses also encode nonstructural proteins on the S and M segments. Viruses that impinge on human health, either directly by causing human disease or indirectly by causing economic losses of domestic animals or crop plants, are found in each of the five gener...

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Section: Resultsmentioning

confidence: 99%

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

Brennan

Zhang

et al. 2015

J Virol

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“…The complete nucleotide sequence of rSBV was determined and matched that of the parental plasmids. Thus, SBV, like BUNV, LACV and RVFV, can be recovered from cDNAs expressing full-length antigenome-sense RNAs without the need for ‘helper’ plasmids that express viral proteins independently (Elliott, 2012). …”

Section: Resultsmentioning

confidence: 99%

Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe

Elliott

Blakqori

Knippenberg

et al. 2013

Journal of General Virology

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Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.

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“…Minigenome systems have been described for a number of orthobunyaviruses, and comprise a negative-sense genome analogue encoding a reporter gene that is packaged into ribonucleoprotein complex, transcribed and replicated by co-expressed viral N and L proteins, leading to measurable reporter activity (Elliott, 2012). After confirmation of the nucleotide sequences, the ORFs in each segment are amplified by PCR and subcloned into the pTM1 expression vector (Moss et al, 1990).…”

Section: Establishment Of An Orov Minigenome Systemmentioning

confidence: 99%

“…However, much less is known about the general molecular biology of OROV or virus-host interactions. To facilitate such investigations we intended to develop a reverse genetics system for OROV, as has been reported for other orthobunyaviruses (Elliott, 2012), including two Simbu group viruses: Akabane virus (Ogawa et al, 2007) and Schmallenberg virus (Elliott et al, 2013;Varela et al, 2013). When we produced cDNA clones of the OROV genome segments, we noticed several discrepancies between the viral sequences we obtained and the sequences in the database, notably that the S segment contained an additional 204 nt.…”

Section: Introductionmentioning

confidence: 95%

Establishment of a minigenome system for Oropouche virus reveals the S genome segment to be significantly longer than reported previously

Acrani

Tilston-Lunel

Spiegel

et al. 2015

Journal of General Virology

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Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared with sequences in the database. Most notable were that the S segment contained an additional 204 nt at the 39 end and that there was a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL-9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins, and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By cotransfecting a cDNA to the viral glycoproteins, virus-like particles were generated that packaged a minigenome and were capable of infecting naive cells.

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Bunyavirus Reverse Genetics and Applications to Studying Interactions with Host Cells

Cited by 5 publications

References 95 publications

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe

Establishment of a minigenome system for Oropouche virus reveals the S genome segment to be significantly longer than reported previously

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