scholarly journals Characterization of Rose rosette virus and development of reverse genetic system for studying virus accumulation and movement in whole plants

2019 ◽  
Author(s):  
Jeanmarie Verchot ◽  
Venura Herath ◽  
Cesar D. Urrutia ◽  
Mathieu Gayral ◽  
Kelsey Lyle ◽  
...  
Keyword(s):  
Infectious Clone ◽  
Systemic Infection ◽  
Genetic System ◽  
Open Reading Frame ◽  
Reverse Genetic System ◽  
Reverse Genetic ◽  
Glycoprotein Precursor ◽  
Reading Frame ◽  
Negative Strand ◽  
Rose Rosette

ABSTRACTRose rosette virus (RRV) is an Emaravirus, a negative-sense RNA virus with a 7-segmented genome that is enclosed by a double membrane. While the genome sequences of many emaraviruses are reported, there is negligible information concerning virus replication and movement in host plants. Computational methods determined that RNA1 encoded the RNA dependent RNA polymerase (RdRp), RNA2 encoded glycoprotein precursor, and the RNA3 encoded the nucleocapsid (N), all share significant homologies with similar proteins of the Orthobunyavirus family. The RRV terminal UTR sequences are complementary and share significant identity with the UTR sequences of Bunyamwera virus. We report a minireplicon system and a full length infectious clone of RRV, which are the first for any emaravirus species. The photoreversible fluorescent iLOV protein was used to replace the RNA5 open reading frame (R5-iLOV). We demonstrate that agro-infiltration of Nicotiana benthamiana leaves to deliver RNA1, RNA3, and R5-iLOV cDNAs led to iLOV expression. A mutation was introduced into the RdRp active site and iLOV expression was eliminated. Delivery of four segments or seven segments of the RRV infectious clone produced systemic infection in N. benthamiana and rose plants. iLOV was also fused to the glycoprotein precursor (R2-iLOV). Using confocal microscopy, the R2-iLOV was seen in spherical bodies along membrane strands inside N. benthamiana epidermal cells. This new technology will enable future research to functionally characterize the RRV proteins, to study the virus-host interactions governing local and systemic infection, and examine the subcellular functions of the Gc.IMPORTANCERRV has emerged as a severe threat to cultivated roses, causing millions of dollars in losses to commercial producers. The majority of the viral gene products have not been researched or characterized until now. We constructed a minireplicon system and an infectious clone of the seven-segmented RRV genome that is contained in a binary vector and delivered by Agrobacterium. This technology has been slow to develop for viruses with negative-strand RNA genomes. It has been especially tricky for plant viruses with multicomponent negative-strand RNA genomes. We report the first reverse genetic system for a member of the genus Emaravirus, Rose rosette virus (RRV). We introduced the iLOV fluorescent protein as a fusion to the Gc protein and as a replacement for the open reading frame in genome segment 5. This game-changing reverse genetic system creates new opportunities for studying negative-strand RNA viruses in plants.

scholarly journals Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants

2020 ◽  
Vol 33 (10) ◽  
pp. 1209-1221 ◽  
Author(s):  
Jeanmarie Verchot ◽  
Venura Herath ◽  
Cesar D. Urrutia ◽  
Mathieu Gayral ◽  
Kelsey Lyle ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Rna Virus ◽  
Infectious Clone ◽  
Systemic Infection ◽  
Genetic System ◽  
Polymerase Activity ◽  
Full Length ◽  
Reverse Genetic System ◽  
Reading Frame ◽  
Rose Rosette

Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology.

scholarly journals Single-Amino-Acid Substitutions in Open Reading Frame (ORF) 1b-nsp14 and ORF 2a Proteins of the Coronavirus Mouse Hepatitis Virus Are Attenuating in Mice

2005 ◽  
Vol 79 (6) ◽  
pp. 3391-3400 ◽  
Author(s):  
Steven M. Sperry ◽  
Lubna Kazi ◽  
Rachel L. Graham ◽  
Ralph S. Baric ◽  
Susan R. Weiss ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Genetic System ◽  
Open Reading Frame ◽  
Reverse Genetic System ◽  
Reverse Genetic ◽  
Wild Type ◽  
Reading Frame ◽  
Cdna Fragments

ABSTRACT A reverse genetic system was recently established for the coronavirus mouse hepatitis virus strain A59 (MHV-A59), in which cDNA fragments of the RNA genome are assembled in vitro into a full-length genome cDNA, followed by electroporation of in vitro-transcribed genome RNA into cells with recovery of viable virus. The “in vitro-assembled” wild-type MHV-A59 virus (icMHV-A59) demonstrated replication identical to laboratory strains of MHV-A59 in tissue culture; however, icMHV-A59 was avirulent following intracranial inoculation of C57BL/6 mice. Sequencing of the cloned genome cDNA fragments identified two single-nucleotide mutations in cloned genome fragment F, encoding a Tyr6398His substitution in open reading frame (ORF) 1b p59-nsp14 and a Leu94Pro substitution in the ORF 2a 30-kDa protein. The mutations were repaired individually and together in recombinant viruses, all of which demonstrated wild-type replication in tissue culture. Following intracranial inoculation of mice, the viruses encoding Tyr6398His/Leu94Pro substitutions and the Tyr6398His substitution alone demonstrated log10 50% lethal dose (LD50) values too great to be measured. The Leu94Pro mutant virus had reduced but measurable log10 LD50, and the “corrected” Tyr6398/Leu94 virus had a log10 LD50 identical to wild-type MHV-A59. The experiments have defined residues in ORF 1b and ORF 2a that attenuate virus replication and virulence in mice but do not affect in vitro replication. The results suggest that these proteins serve roles in pathogenesis or virus survival in vivo distinct from functions in virus replication. The study also demonstrates the usefulness of the reverse genetic system to confirm the role of residues or proteins in coronavirus replication and pathogenesis.

scholarly journals Development of a Reverse Genetic System to Generate Recombinant Chimeric Tacaribe Virus that Expresses Junín Virus Glycoproteins

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 948
Author(s):  
Sabrina Foscaldi ◽  
María Eugenia Loureiro ◽  
Claudia Sepúlveda ◽  
Carlos Palacios ◽  
María Belén Forlenza ◽  
...  
Keyword(s):  
Hemorrhagic Fever ◽  
Genetic System ◽  
Reverse Genetic System ◽  
Dependent Manner ◽  
Reverse Genetic ◽  
Junin Virus ◽  
Tacaribe Virus ◽  
Growth Properties ◽  
Intracellular Expression ◽  
Junín Virus

Mammarenaviruses are enveloped and segmented negative-stranded RNA viruses that comprise several pathogenic members associated with severe human hemorrhagic fevers. Tacaribe virus (TCRV) is the prototype for the New World group of mammarenaviruses and is not only naturally attenuated but also phylogenetically and antigenically related to all South American pathogenic mammarenaviruses, particularly the Junín virus (JUNV), which is the etiological agent of Argentinian hemorrhagic fever (AHF). Moreover, since TCRV protects guinea pigs and non-human primates from lethal challenges with pathogenic strains of JUNV, it has already been considered as a potential live-attenuated virus vaccine candidate against AHF. Here, we report the development of a reverse genetic system that relies on T7 polymerase-driven intracellular expression of the complementary copy (antigenome) of both viral S and L RNA segments. Using this approach, we successfully recovered recombinant TCRV (rTCRV) that displayed growth properties resembling those of authentic TCRV. We also generated a chimeric recombinant TCRV expressing the JUNV glycoproteins, which propagated similarly to wild-type rTCRV. Moreover, a controlled modification within the S RNA 5′ non-coding terminal sequence diminished rTCRV propagation in a cell-type dependent manner, giving rise to new perspectives where the incorporation of additional attenuation markers could contribute to develop safe rTCRV-based vaccines against pathogenic mammarenaviruses.

scholarly journals Establishment of an efficient reverse genetic system of Mumps virus S79 from cloned DNA

2019 ◽  
Vol 15 (5) ◽  
pp. 499-505 ◽  
Author(s):  
Duo Zhou ◽  
Meng-Ying Zhu ◽  
Yi-Long Wang ◽  
Xiao-Qiang Hao ◽  
Dong-Ming Zhou ◽  
...  
Keyword(s):  
Genetic System ◽  
Mumps Virus ◽  
Reverse Genetic System ◽  
Reverse Genetic

scholarly journals Characterization and reverse genetic establishment of cattle derived Akabane virus in China

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Dongjie Chen ◽  
Di Wang ◽  
Fang Wei ◽  
Yufang Kong ◽  
Junhua Deng ◽  
...  
Keyword(s):  
Genetic System ◽  
Reverse Genetic System ◽  
Akabane Virus ◽  
Reverse Genetic ◽  
Wild Type ◽  
T7 Promoter ◽  
Cytopathic Effects ◽  
The World ◽  
L Segment ◽  
Genome Manipulation

Abstract Background Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Construction of a reverse genetic system for porcine astrovirus

2018 ◽  
Vol 163 (6) ◽  
pp. 1511-1518 ◽  
Author(s):  
Yifeng Qin ◽  
Qingli Fang ◽  
Huan Liu ◽  
Chengyuan Ji ◽  
Ying Chen ◽  
...  
Keyword(s):  
Genetic System ◽  
Reverse Genetic System ◽  
Reverse Genetic ◽  
Porcine Astrovirus

scholarly journals Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

2014 ◽  
Vol 81 (4) ◽  
pp. 1210-1224 ◽  
Author(s):  
Daniela Toro-Ascuy ◽  
Carolina Tambley ◽  
Carolina Beltran ◽  
Carolina Mascayano ◽  
Nicolas Sandoval ◽  
...  
Keyword(s):  
Atlantic Salmon ◽  
Cytopathic Effect ◽  
Genetic System ◽  
Reverse Genetic System ◽  
Reverse Genetic ◽  
Wild Type ◽  
Internal Transcribed Spacer Region ◽  
Content Type ◽  
Recombinant Viruses ◽  
Infectious Salmon Anemia

ABSTRACTInfectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genusIsavirus, familyOrthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV901_09and rISAVrS6-NotI-HPRcontaining a NotI restriction site and rISAVS6/EGFP-HPRharboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 105PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.

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