Replication-competent influenza A viruses expressing a red fluorescent protein

Nogales, Aitor; Baker, Steven F.; Martínez-Sobrido, Luis

doi:10.1016/j.virol.2014.12.006

Cited by 76 publications

(207 citation statements)

References 67 publications

(122 reference statements)

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“…Influenza A/Puerto Rico/8/1934 (PR8) H1N1 virus (26) was grown in MDCK cells as previously described (27,28). Viral titers were determined by an immunofocus assay and are presented as fluorescence-forming units (FFU) per milliliter (27,28).…”

Section: Cell and Virusesmentioning

confidence: 99%

“…To engineer the recombinant PR8 virus NS and M split segments (NSs and Ms, respectively), overlapping PCR and standard molecular biology techniques were used to introduce the modifications into the ambisense pDZ-NS or pDZ-M viral rescue plasmid (26). The modified plasmids, named pDZ-NSs and pDZ-Ms, respectively, contain the NS1 (NS segment) or M1 (M segment) open reading frames (ORFs) without stop codons or splice acceptor sites, followed by the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site (ATNFSLLKQAGDVEE NPGP) and the entire sequence of the NEP (NS segment) or M2 (M segment) ORF (27,29). The sequences of the modified pDZ-NSs and -Ms plasmids were confirmed by sequencing, and the plasmids were used for virus rescue.…”

Section: Cell and Virusesmentioning

confidence: 99%

“…Virus rescues were performed as previously described (27,28,30). Briefly, cocultures of 293T and MDCK cells (in a 6-well plate format) were cotransfected in suspension, using the Lipofectamine 2000 reagent, with 1 g of each of the ambisense plasmids.…”

Section: Cell and Virusesmentioning

confidence: 99%

“…At 3 to 4 days postinfection, recombinant viruses were plaque purified in 6-well plates and scaled up in MDCK cells (28). Stocks were titrated by the immunofocus assay on MDCK cells (27,28). Viral rescues and titrations were performed and stocks were produced at 33°C.…”

Section: Cell and Virusesmentioning

confidence: 99%

“…After 1 h of virus adsorption at room temperature, the cells were washed, overlaid with DMEM containing 0.3% BSA and TPCK-treated trypsin, and incubated at 33°C, 37°C, or 39°C. At the indicated times postinfection, tissue culture supernatants were collected and viral titers were determined by the immunofocus assay (27,28). The mean value and standard deviation (SD) were calculated using Microsoft Excel software.…”

Section: Reverse Transcription-pcr (Rt-pcr)mentioning

confidence: 99%

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Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

Nogales

DeDiego

Topham

et al. 2016

J Virol

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Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease which is most effectively prevented through vaccination. Segments 7 (M) and 8 (NS) of the influenza virus genome encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing M or NS viral segments individually or modified M or NS viral segments combined in which the overlapping open reading frames of matrix 1 (M1)/M2 for the modified M segment and the open reading frames of nonstructural protein 1 (NS1)/nuclear export protein (NEP) for the modified NS segment were split by using the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at nonpermissive high temperatures, whereas high viral titers could be obtained at permissive low temperatures (33°C). Furthermore, viruses containing the M split segment were highly attenuated in vivo, while they retained their immunogenicity and provided protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and that such attenuated viruses represent an excellent option as safe, immunogenic, and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of the M segment to encode M1 and M2 from two separate, nonoverlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product. Influenza viruses are enveloped pathogens that belong to the Orthomyxoviridae family and contain a segmented genome of eight single-stranded RNA molecules with negative polarity (1). Influenza virus infections cause both seasonal epidemics and occasional pandemics when novel viruses are introduced into humans (2). Despite comprehensive vaccination programs, the World Health Organization (WHO) estimates that the global disease burden from influenza results in 1 billion infections, 3 million to 5 million cases of severe disease, and between 300,000 and 500,000 deaths annually (3). Therefore, infection with influenza virus poses a threat to human health and results in significant negative economic impacts on society every year (4). The public health concerns posed by influenza viruses are aggravated by their efficient transmission and the limited antiviral therapeutic options (5). Hence, vaccination remains our best medical intervention to protect humans against influenza virus (6), even though the effectiveness of current vaccines is suboptimal (7). To date, the U.S. Food and Drug Administration (FDA) approves three types of influenza virus vaccines for human use: inactivated vir...

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Section: Cell and Virusesmentioning

confidence: 99%

Section: Cell and Virusesmentioning

confidence: 99%

Section: Cell and Virusesmentioning

confidence: 99%

Section: Cell and Virusesmentioning

confidence: 99%

Section: Reverse Transcription-pcr (Rt-pcr)mentioning

confidence: 99%

See 3 more Smart Citations

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

Nogales

DeDiego

Topham

et al. 2016

J Virol

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Aryl and Arylalkyl Substituted 3‐Hydroxypyridin‐2(1H)‐ones: Synthesis and Evaluation as Inhibitors of Influenza A Endonuclease

et al. 2019

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Seasonal influenza infections are associated with an estimated 250–500 000 deaths annually. Resistance to the antiviral M2 ion‐channel inhibitors has largely invalidated their clinical utility. Resistance to neuraminidase inhibitors has also been observed in several influenza A virus (IAV) strains. These data have prompted research on inhibitors that target the cap‐snatching endonuclease activity of the polymerase acidic protein (PA). Baloxavir marboxil (Xofluza®), recently approved for clinical use, inhibits cap‐snatching endonuclease. Resistance to Xofluza® has been reported in both in vitro systems and in the clinic. An X‐ray crystallographic screening campaign of a fragment library targeting IAV endonuclease identified 5‐chloro‐3‐hydroxypyridin‐2(1H)‐one as a bimetal chelating agent at the active site. We have reported the structure–activity relationships for 3‐hydroxypyridin‐2(1H)‐ones and 3‐hydroxyquinolin‐2(1H)‐ones as endonuclease inhibitors. These studies identified two distinct binding modes associated with inhibition of this enzyme that are influenced by the presence of substituents at the 5‐ and 6‐positions of 3‐hydroxypyridin‐2(1H)‐ones. Herein we report the structure–activity relationships associated with various para‐substituted 5‐phenyl derivatives of 6‐(p‐fluorophenyl)‐3‐hydroxypyridin‐2(1H)‐ones and the effect of using naphthyl, benzyl, and naphthylmethyl groups as alternatives to the p‐fluorophenyl substituent on their activity as endonuclease inhibitors.

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Generation, characterization, and protective ability of mouse monoclonal antibodies against the HA of A (H1N1) influenza virus

Wang

Yang

Xiao

et al. 2022

Journal of Medical Virology

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Influenza virus infections pose a continuous threat to human health. Although vaccines function as a preventive and protective tool, they may not be effective due to antigen drift or an inaccurate prediction of epidemic strains. Monoclonal antibodies (mAbs) have attracted wide attention as a promising therapeutic method for influenza virus infections. In this study, three hemagglutinin (HA)-specific mAbs, named 2A1, 2H4, and 2G2, respectively, were derived from mice immunized with the HA protein from A/Michigan/45/2015(H1N1). The isolated mAbs all displayed hemagglutination inhibition activity and the 2G2 mAb exhibited the strongest neutralization effect. Two amino acid mutations (A198E and G173E), recognized in the process of selection of mAb-resistant mutants, were located in antigenic site Sb and Ca1, respectively. In prophylactic experiments, all three mAbs could achieve 100% protection in mice infected with a lethal dose of A/Michigan/45/2015(H1N1).A dose of 1 mg/kg for 2H4 and 2G2 was sufficient to achieve a full protective effect.Therapeutic experiments showed that all three mAbs could protect mice from death if they received the mAb administration at 6 h postinfection, and 2G2 was still protective after 24 h. Our findings indicate that these three mAbs may have potential prevention and treatment value in an H1N1 epidemic, as well as in the study of antigen epitope recognition.

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Replication-competent influenza A viruses expressing a red fluorescent protein

Cited by 76 publications

References 67 publications

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

Aryl and Arylalkyl Substituted 3‐Hydroxypyridin‐2(1H)‐ones: Synthesis and Evaluation as Inhibitors of Influenza A Endonuclease

Generation, characterization, and protective ability of mouse monoclonal antibodies against the HA of A (H1N1) influenza virus

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