scholarly journals Interaction between hexon and L4-100K determines virus rescue and growth of hexon-chimeric recombinant Ad5 vectors

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Jingyi Yan ◽  
Jianing Dong ◽  
Jiaxin Wu ◽  
Rui Zhu ◽  
Zhen Wang ◽  
...  
Keyword(s):  
Virus Rescue

scholarly journals Multiple polymerase acidic (PA) I38X substitutions in influenza A(H1N1)pdm09 virus permit polymerase activity and cause reduced baloxavir inhibition

2020 ◽  
Author(s):  
Jeremy C Jones ◽  
Philippe N Q Pascua ◽  
Walter N Harrington ◽  
Richard J Webby ◽  
Elena A Govorkova
Keyword(s):  
Inhibitory Activity ◽  
Influenza A ◽  
Antiviral Drug ◽  
Polymerase Activity ◽  
Resistance Marker ◽  
Complex Activity ◽  
Replication Complex ◽  
Virus Rescue ◽  
Influenza A H1n1 ◽  
Targeted Approach

Abstract Background Baloxavir marboxil is an antiviral drug that targets the endonuclease activity of the influenza virus polymerase acidic (PA) protein. PA I38T/M/F substitutions reduce its antiviral efficacy. Objectives To understand the effects of the 19 possible amino acid (AA) substitutions at PA 38 on influenza A(H1N1)pdm09 polymerase activity and inhibition by baloxavir acid, the active metabolite of baloxavir marboxil. Methods Influenza A(H1N1)pdm09 viral polymerase complexes containing all 19 I38X AA substitutions were reconstituted in HEK293T cells in a mini-replicon assay. Polymerase complex activity and baloxavir inhibitory activity were measured in the presence or absence of 50 nM baloxavir acid. Results Only three substitutions (R, K, P) reduced polymerase activity to <79% of I38-WT. When compared with the prototypical baloxavir marboxil resistance marker T38, 5 substitutions conferred 10%–35% reductions in baloxavir acid inhibitory activity (M, L, F, Y, C) and 11 substitutions conferred >50% reductions (R, K, S, N, G, W, A, Q, E, D, H), while two substitutions (V, P) maintained baloxavir acid inhibitory activity. Conclusions Most PA 38 substitutions permit a functional replication complex retaining some drug resistance in the mini-replicon assay. This study provides a targeted approach for virus rescue and analysis of novel baloxavir marboxil reduced-susceptibility markers, supports the consideration of a broader range of these markers during antiviral surveillance and adds to the growing knowledge of baloxavir marboxil resistance profiles.

An improved method for infectious bursal disease virus rescue using RNA polymerase II system

2007 ◽  
Vol 142 (1-2) ◽  
pp. 81-88 ◽  
Author(s):  
Xiaole Qi ◽  
Yulong Gao ◽  
Honglei Gao ◽  
Xiaoyun Deng ◽  
Zhigao Bu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Disease Virus ◽  
Infectious Bursal Disease Virus ◽  
Infectious Bursal Disease ◽  
Improved Method ◽  
Virus Rescue ◽  
Bursal Disease

scholarly journals Construction, Rescue, and Characterization of Vectors Derived from Ovine Atadenovirus

2003 ◽  
Vol 77 (22) ◽  
pp. 11941-11951 ◽  
Author(s):  
Peter Löser ◽  
Christian Hofmann ◽  
Gerald W. Both ◽  
Wolfgang Uckert ◽  
Moritz Hillgenberg
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Human Factor ◽  
Insertion Site ◽  
Factor Ix ◽  
Vector System ◽  
Virus Rescue ◽  
Human Factor Ix

ABSTRACT Gene transfer vectors derived from ovine atadenovirus type 7 (OAdV) can efficiently infect a variety of mammalian cells in vitro and in vivo to deliver and express transgenes. However, early OAdV vectors were designed on human mastadenovirus principles prior to the complete characterization of OAdV genes and transcripts. The distinctive arrangement of the OAdV genome has suggested ways to improve OAdV vector design and utility. We therefore developed a cosmid-based approach that allows efficient construction of recombinant ovine atadenovirus genomes in which the transgene is inserted at one of three sites. Viruses were rescued by transfection of viral DNA into a new ovine fetal skin fibroblast producer cell line, HVO156. The suitability of the three insertion sites was compared with respect to virus rescue efficiency, gene expression levels, and genetic stability of the vectors. We found that one vector with a transgene inserted at site 1, between the pVIII and fiber genes, was unstable. Only one vector that carried a transgene at site 2, near the right end of the genome, together with a nearby deletion was rescued. In contrast, several vectors with different transgenes inserted in site 3, between the E4 and RH transcription units, were repeatedly rescued, and these vectors were stable over at least four passages. Transgene orientation in site 3 had only little effect on expression. Finally, a vector carrying a human factor IX cDNA at site 3, when administered intravenously, produced nearly physiological levels of human factor IX in mice. The availability of an efficient method for vector construction and the identification of a new insertion site for virus rescue and gene expression substantially enhance the utility of the OAdV vector system.

scholarly journals Rescue of Infectious Rift Valley Fever Virus Entirely from cDNA, Analysis of Virus Lacking the NSs Gene, and Expression of a Foreign Gene

2006 ◽  
Vol 80 (6) ◽  
pp. 2933-2940 ◽  
Author(s):  
Tetsuro Ikegami ◽  
Sungyong Won ◽  
C. J. Peters ◽  
Shinji Makino
Keyword(s):  
Protein Expression ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Reverse Genetics ◽  
Rift Valley Fever Virus ◽  
Foreign Gene ◽  
Deletion Mutants ◽  
Luciferase Gene ◽  
Valley Fever ◽  
Virus Rescue

ABSTRACT Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.

scholarly journals Rescue of Infectious Rotavirus Reassortants by a Reverse Genetics System Is Restricted by the Receptor-Binding Region of VP4

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 363
Author(s):  
Alexander Falkenhagen ◽  
Marno Huyzers ◽  
Alberdina A. van Dijk ◽  
Reimar Johne
Keyword(s):  
Receptor Binding ◽  
Reverse Genetics ◽  
Genome Segment ◽  
Future Research ◽  
Binding Region ◽  
Virus Propagation ◽  
Virus Rescue ◽  
Plasmid Transfection ◽  
Strain Dependent ◽  
Receptor Binding Region

The rotavirus species A (RVA) capsid contains the spike protein VP4, which interacts with VP6 and VP7 and is involved in cellular receptor binding. The capsid encloses the genome consisting of eleven dsRNA segments. Reassortment events can result in novel strains with changed properties. Using a plasmid-based reverse genetics system based on simian RVA strain SA11, we previously showed that the rescue of viable reassortants containing a heterologous VP4-encoding genome segment was strain-dependent. In order to unravel the reasons for the reassortment restrictions, we designed here a series of plasmids encoding chimeric VP4s. Exchange of the VP4 domains interacting with VP6 and VP7 was not sufficient for rescue of viable viruses. In contrast, the exchange of fragments encoding the receptor-binding region of VP4 resulted in virus rescue. All parent strains and the rescued reassortants replicated efficiently in MA-104 cells used for virus propagation. In contrast, replication in BSR T7/5 cells used for plasmid transfection was only efficient for the SA11 strain, whereas the rescued reassortants replicated slowly, and the parent strains failing to produce reassortants did not replicate. While future research in this area is necessary, replication in BSR T7/5 cells may be one factor that affects the rescue of RVAs.

scholarly journals Enhanced Measles Virus cDNA Rescue and Gene Expression after Heat Shock

1999 ◽  
Vol 73 (5) ◽  
pp. 3560-3566 ◽  
Author(s):  
Christopher L. Parks ◽  
Robert A. Lerch ◽  
Pramila Walpita ◽  
Mohinderjit S. Sidhu ◽  
Stephen A. Udem
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Measles Virus ◽  
Vero Cells ◽  
Helper Virus ◽  
Cdna Clones ◽  
Virus Rescue ◽  
Virus Cdna ◽  
Hsp72 Expression ◽  
Genomic Cdna

ABSTRACT Rescue of negative-stranded RNA viruses from full-length genomic cDNA clones is an essential technology for genetic analysis of this class of viruses. Using this technology in our studies of measles virus (MV), we found that the efficiency of the measles virus rescue procedure (F. Radecke et al., EMBO J. 14:5773–5784, 1995) could be improved by modifying the procedure in two ways. First, we found that coculture of transfected 293-3-46 cells with a monolayer of Vero cells increased the number of virus-producing cultures about 20-fold. Second, we determined that heat shock treatment increased the average number of transfected cultures that produced virus another two- to threefold. In addition, heat shock increased the number of plaques produced by positive cultures. The effect of heat shock on rescue led us to test the effect on transient expression from an MV minireplicon. Heat shock increased the level of reporter gene expression when either minireplicon DNA or RNA was used regardless of whether complementation was provided by cotransfection with expression plasmids or infection with MV helper virus. In addition, we found that MV minireplicon gene expression could be stimulated by cotransfection with an Hsp72 expression plasmid, indicating that hsp72 likely plays a role in the effect of heat shock.

scholarly journals Development of an improved polykaryon-based influenza virus rescue system

2012 ◽  
Vol 12 (1) ◽  
Author(s):  
Vincent Bourret ◽  
Jon Lyall ◽  
Mariette F Ducatez ◽  
Jean-Luc Guérin ◽  
Laurence Tiley
Keyword(s):  
Influenza Virus ◽  
Virus Rescue ◽  
Rescue System
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