scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’

2001 ◽  
Vol 82 (5) ◽  
pp. 1013-1025 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Garry Luke ◽  
Amit Mehrotra ◽  
Xuejun Li ◽  
Lorraine E. Hughes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Peptide Bond ◽  
Open Reading Frame ◽  
Translation Product ◽  
Reading Frame ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Green Fluorescent

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.

scholarly journals Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4

2021 ◽  
Vol 12 ◽  
Author(s):  
Qing Pan ◽  
Yu Zhang ◽  
Aijing Liu ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Virus Genome ◽  
Open Reading Frames ◽  
Vaccine Vector ◽  
Economic Losses ◽  
Foreign Gene ◽  
The Novel ◽  
Hydropericardium Syndrome ◽  
Fowl Adenovirus ◽  
Green Fluorescent

Severe hepatitis-hydropericardium syndrome (HHS) associated with a novel viral genotype, fowl adenovirus 4 (FAdV-4), has emerged and widely spread in China since 2015, causing severe economic losses to the poultry industry. We previously reported that the hexon gene is responsible for pathogenicity and obtained a non-pathogenic hexon-replacement rHN20 strain; however, the lack of information about the non-essential regions for virus replication limits the development of a FAdV-4 vector. This study first established an enhanced green fluorescent protein (EGFP)-indicator virus based on the FAdV-4 reverse genetic technique, effective for batch operations in the virus genome. Based on this, 10 open reading frames (ORFs) at the left end and 13 ORFs at the right end of the novel FAdV-4 genome were deleted separately and identified as non-essential genes for viral replication, providing preliminary insertion sites for foreign genes. To further improve its feasibility as a vaccine vector, seven combinations of ORFs were successfully replaced with EGFP without affecting the immunogenicity of the vector backbone. Finally, a recombinant rHN20-vvIBDV-VP2 strain, expressing the VP2 protein of very virulent infectious bursa disease virus (vvIBDV), was rescued and showed complete protection against FAdV-4 and vvIBDV. Thus, the novel FAdV-4 vector could provide sufficient protection for HHS and efficient exogenous gene delivery. Overall, our findings systemically identified 23 non-essential ORFs for FAdV-4 replication and seven foreign gene insertion regions, providing valuable information for an in-depth understanding of the novel FAdV-4 pathogenesis and development of multivalent vaccines.

scholarly journals Bluetongue Virus VP6 Acts Early in the Replication Cycle and Can Form the Basis of Chimeric Virus Formation

2009 ◽  
Vol 83 (17) ◽  
pp. 8842-8848 ◽  
Author(s):  
Eiko Matsuo ◽  
Polly Roy
Keyword(s):  
Bluetongue Virus ◽  
Reverse Genetics ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Core Protein ◽  
Foreign Gene ◽  
Chimeric Rna ◽  
Green Fluorescent ◽  
A Minor

ABSTRACT A minor core protein, VP6, of bluetongue virus (BTV) possesses nucleoside triphosphatase, RNA binding, and helicase activities. Although the enzymatic functions of VP6 have been documented in vitro using purified protein, its definitive role in BTV replication remains unclear. In this study, using a recently developed T7 transcript-based reverse genetics system for BTV, we examined the importance of VP6 in virus replication. We show that VP6 is active early in replication, consistent with a role as part of the transcriptase or packaging complex, and that its action can be provided in trans by a newly developed complementary cell line. Furthermore, the genomic segment encoding VP6 was mutated to reveal the cis-acting sequences required for replication or packaging, which subsequently enabled the construction of a chimeric BTV expressing enhanced green fluorescent protein. These data confirm that one of the 10 genome segments of BTV can be replaced with a chimeric RNA containing the essential packaging and replication signals of BTV and the coding sequence of a foreign gene.

scholarly journals Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

2008 ◽  
Vol 89 (2) ◽  
pp. 389-396 ◽  
Author(s):  
Garth M. Funston ◽  
Susanna E. Kallioinen ◽  
Pablo de Felipe ◽  
Martin D. Ryan ◽  
Richard D. Iggo
Keyword(s):  
Fluorescent Protein ◽  
Burst Size ◽  
Foot And Mouth Disease ◽  
Human Adenovirus ◽  
Peptide Bond ◽  
Fusion Partner ◽  
Reading Frame ◽  
Mouth Disease Virus ◽  
Green Fluorescent ◽  
Protein Ix

Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip formation of a peptide bond at the junction of the 2A and downstream sequences, leading to the production of two proteins from a single open reading frame. Adenoviral protein IX is a minor capsid protein that has been used to display foreign peptides on the surface of the capsid. We have used 2A sequences from the foot-and-mouth disease virus (FMDV) and porcine teschovirus 1 (PTV-1) to express protein IX (pIX) and green fluorescent protein (GFP) from pIX–2A–GFP fusion genes in an oncolytic virus derived from human adenovirus 5. GFP was efficiently expressed by constructs containing either 2A sequence. Peptide bond skipping was more efficient with the 58 aa FMDV sequence than with the 22 aa PTV-1 2A sequence, but the virus with the FMDV 2A sequence showed a reduction in plaque size, cytopathic effect, viral burst size and capsid stability. We conclude that ribosome skipping induced by 2A sequences is an effective strategy to express heterologous genes in adenoviruses; however, careful selection or optimization of the 2A sequence may be required if protein IX is used as the fusion partner.

scholarly journals IN VITROTRANSPLANTATION OF GENETICALLY MODIFIED CELLS TO THE TENDON SURFACE

2008 ◽  
Vol 11 (02) ◽  
pp. 81-87
Author(s):  
Paulus J. J. Couvreur ◽  
Chunfeng Zhao ◽  
Stephen Murphy ◽  
Peter C. Amadio
Keyword(s):  
Fluorescent Protein ◽  
Control Group ◽  
Transfected Cells ◽  
Gfp Fluorescence ◽  
Tendon Cells ◽  
New Zealand White Rabbits ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
In Vitro Transfection

The objective of this paper was to study in vitro transfection of tendon cells and adherence of transfected cells to different tendon surfaces. Achilles tendon fibroblasts from 2-month-old New Zealand white rabbits were cultured to confluence, after which the cells were transfected by an adenovirus carrying either the β-galactosidase reporter gene or the green fluorescent protein (GFP) gene at multiplicities of infection (MOIs) of 50, 100, or 500. Two days later, the cells were transplanted onto the surfaces of rabbit Achilles, peroneus brevis, flexor profundus, and extensor longus tendons. The tendons were assessed by X-gal staining after 9 days, and by GFP fluorescence at 7, 14, and 21 days. Twenty percent to 50% of the treated cells stained for β-galactosidase at an MOI of 500. The GFP-labeled cells showed nearly 100% fluorescence at an MOI of 50. No positive cells were visible in the control group. The β-galactosidase and GFP-expressing cells remained viable for as long as 3 weeks. It is possible to introduce foreign genes into rabbit tendon cells, transplant the cells onto tendon surfaces, and maintain viability of the cell/tendon construct for several weeks.

scholarly journals A Borna Disease Virus Vector for Expression of Foreign Genes in Neurons of Rodents

2007 ◽  
Vol 81 (13) ◽  
pp. 7293-7296 ◽  
Author(s):  
Urs Schneider ◽  
Andreas Ackermann ◽  
Peter Staeheli
Keyword(s):  
Disease Virus ◽  
Fluorescent Protein ◽  
Foreign Gene ◽  
Borna Disease Virus ◽  
Highly Active ◽  
The Central Nervous System ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
A Site ◽  
Borna Disease

ABSTRACT An expression cassette for green fluorescent protein was successfully inserted at a site near the 5′ end of the genome of Borna disease virus (BDV). When introduced into a mutant virus with highly active polymerase, the foreign gene was strongly expressed in neurons of infected rats. Since BDV can establish long-term persistence in the central nervous system of rodents, it may be used to engineer efficient vectors for specific delivery of foreign genes into highly differentiated neurons.

scholarly journals Identification of the largest non-essential regions of the C-terminal portion in 3A protein of foot-and-mouth disease virus for replication in cell culture

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Pinghua Li ◽  
Xueqing Ma ◽  
Xingwen Bai ◽  
Pu Sun ◽  
Hong Yuan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Disease Virus ◽  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Nucleotide Sequencing ◽  
Foreign Gene ◽  
Terminal Portion ◽  
Mouth Disease Virus ◽  
Foot And Mouth

Abstract Background Recent study has shown that the C-terminal portion of 3A (amino acids (aa) 81–153) is not essential for foot-and-mouth disease virus replication in cell culture, however, the complete C-terminal portion (aa 77–153) of 3A is highly variable and prone to occur deletions and mutations, therefore, we presume that this region plays a very limited role and probablely is completely nonessential for virus viability. Methods In this study, to identify the largest non-essential region of the C-terminal portion in 3A for FMDV viability, several deletions containing aa 80–153, 77–153 and 76–153 of 3A protein were introduced into an FMDV full-length infectious cDNA clone pOFS by the overlapping extension PCR. Additionally, to explore the importance of the highly conserved residue 76 L of 3A for the FMDV of Cathay topotype, two mutants containing 3A L76I and 3A L76V were generated based on the 3A deletion mutant by point mutation. We also introduced the enhanced green fluorescent protein (eGFP) into one of the 3A deletion mutants by the extension PCR to investigate the genetic flexibility of 3A to express foreign genes. All linearized full plasmids were transfected into BSR/T7 cells to rescue infectious foot-and-mouth disease viruses. The rescused viruses were analyzed by RT-PCR, nucleotide sequencing, immunofluorescence assay and western blot and were characterized by plaque assays and one-step growth kinetics. Results The results demonstrated that the deletion of aa 80–153 and aa 77–153 and the substitutions of 3A L76I and 3A L76V did not affect the production of infectious virus, while the fusion of the eGFP gene to the C-terminus of 3A resulted in nonviable FMDV. Conclusions Our results firstly reported that the aa 77–153 rather than aa 81–153 of 3A protein was dispensable for FMDV replication in cell culture. This study is of great significance for development of FMD marker vaccine and foreign gene expression in the future.

scholarly journals The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences

2001 ◽  
Vol 82 (5) ◽  
pp. 1027-1041 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Lorraine E. Hughes ◽  
Garry Luke ◽  
Heidi Mendoza ◽  
Edwin ten Dam ◽  
...  
Keyword(s):  
Disease Virus ◽  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Reading Frame ◽  
Naturally Occurring ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Green Fluorescent ◽  
Translation Systems

The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins ‘cleaving’ at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to β-glucuronidase (GUS) – forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (∼90%) were in the form of the ‘cleavage’ products GUS and [GFP2A]. Alternative models have been proposed to account for the ‘cleavage’ activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring ‘2A-like’ sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that ‘cleavage’ occurred in constructs in which all the candidate nucleophilic residues were substituted – with the exception of aspartate-12. This residue is not, however, conserved amongst all functional ‘2A-like’ sequences. ‘2A-like’ sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial α-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial α-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the Picornaviridae. Taken together, these data provide additional evidence that neither FMDV 2A nor ‘2A-like’ sequences are autoproteolytic elements.

scholarly journals An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene

2007 ◽  
Vol 88 (4) ◽  
pp. 1196-1205 ◽  
Author(s):  
Erwin van den Born ◽  
Clara C. Posthuma ◽  
Kèvin Knoops ◽  
Eric J. Snijder
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Heterologous Gene Expression ◽  
Equine Arteritis Virus ◽  
Expression Vectors ◽  
Wild Type Virus ◽  
Foreign Gene ◽  
Replicase Gene ◽  
Green Fluorescent

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP–nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.

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