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

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.

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

10.21203/rs.3.rs-547132/v1 ◽

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.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.03153-14 ◽

2014 ◽

Vol 81 (4) ◽

pp. 1210-1224 ◽

Cited By ~ 8

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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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

Journal of Virology ◽

10.1128/jvi.79.6.3391-3400.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3391-3400 ◽

Cited By ~ 70

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.

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Reverse Genetic Systems for Pseudomonas aeruginosa Leviphages

Methods and Protocols ◽

10.3390/mps2010022 ◽

2019 ◽

Vol 2 (1) ◽

pp. 22

Author(s):

Jae-Yeol Lee ◽

Se-Jeong Ahn ◽

Chanseop Park ◽

Hee-Won Bae ◽

Eun Sook Kim ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Life Cycle ◽

Rna Viruses ◽

Genetic System ◽

Rna Polymerases ◽

Host Cells ◽

Reverse Genetic System ◽

Reverse Genetic ◽

T7 Promoter ◽

Genetic Systems

Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.

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Generation of duck Tembusu virus using a simple reverse genetic system in duck embryo fibroblast cells

Journal of Virological Methods ◽

10.1016/j.jviromet.2021.114385 ◽

2021 ◽

pp. 114385

Author(s):

Feng Hu ◽

Tong Zhu ◽

Xiaozhen Guo ◽

Kexiang Yu ◽

Xiuli Ma ◽

...

Keyword(s):

Genetic System ◽

Reverse Genetic System ◽

Fibroblast Cells ◽

Reverse Genetic ◽

Duck Embryo Fibroblast ◽

Duck Tembusu Virus ◽

Duck Embryo ◽

Tembusu Virus

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

Pathogens ◽

10.3390/pathogens9110948 ◽

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.

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