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.

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.

scholarly journals Organ-Specific Attenuation of Murine Hepatitis Virus Strain A59 by Replacement of Catalytic Residues in the Putative Viral Cyclic Phosphodiesterase ns2

2009 ◽  
Vol 83 (8) ◽  
pp. 3743-3753 ◽  
Author(s):  
Jessica K. Roth-Cross ◽  
Helen Stokes ◽  
Guohui Chang ◽  
Ming Ming Chua ◽  
Volker Thiel ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Genetic System ◽  
Single Amino Acid ◽  
Reverse Genetic System ◽  
Embryonic Cells ◽  
Wild Type ◽  
Murine Hepatitis Virus ◽  
Recombinant Viruses ◽  
Organ Specific

ABSTRACT The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-amino-acid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.

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.

Characterization and Reverse Genetic Establishment of Cattle Derived Akabane Virus in China

2021 ◽  
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 ◽  
L Segment ◽  
Genome Manipulation ◽  
Constructed Plasmids

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

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 Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

2014 ◽  
Vol 89 (6) ◽  
pp. 3026-3037 ◽  
Author(s):  
Benjamin Brennan ◽  
Ping Li ◽  
Shuo Zhang ◽  
Aqian Li ◽  
Mifang Liang ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Far East ◽  
Gastrointestinal Symptoms ◽  
Case Fatality ◽  
The United States ◽  
Cdna Clones ◽  
Wild Type ◽  
Content Type ◽  
Recombinant Viruses ◽  
Severe Fever

ABSTRACTSevere 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 thatSFTS virusrepresents a new lineage within thePhlebovirusgenus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the familyBunyaviridae. 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.

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
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