scholarly journals A One-Plasmid System To Generate Influenza Virus in Cultured Chicken Cells for Potential Use in Influenza Vaccine

2009 ◽  
Vol 83 (18) ◽  
pp. 9296-9303 ◽  
Author(s):  
Xiangmin Zhang ◽  
Wei Kong ◽  
Shamaila Ashraf ◽  
Roy Curtiss
Keyword(s):  
Influenza Virus ◽  
Reverse Genetics ◽  
Transfection Efficiency ◽  
Limiting Factor ◽  
Viral Rnas ◽  
Viral Yield ◽  
Plasmid Transfection ◽  
Dual Promoters ◽  
Plasmid Size ◽  
Potential Use

ABSTRACT Influenza virus has a set of ribonucleoproteins (RNPs) consisting of viral RNAs, influenza virus polymerase subunits, and nucleoprotein. Intracellular reconstitution of the whole set of RNPs via plasmid transfection results in the generation of influenza virus. By the use of reverse genetics and dual promoters, we constructed a 23.6-kb eight-unit plasmid that contains all the required constituents to generate influenza virus in chicken cells. Our “one-plasmid” system generated higher titers of influenza virus in chicken cells than the “eight-plasmid” system, enabling a simpler approach for generating vaccine seeds. Our study identified plasmid size as a potential limiting factor affecting transfection efficiency and hence the influenza viral yield from chicken cells.

scholarly journals An Adenovirus Vector-Mediated Reverse Genetics System for Influenza A Virus Generation

2007 ◽  
Vol 81 (17) ◽  
pp. 9556-9559 ◽  
Author(s):  
Makoto Ozawa ◽  
Hideo Goto ◽  
Taisuke Horimoto ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Transfection Efficiency ◽  
Adenovirus Vector ◽  
Vero Cells ◽  
Efficient Gene ◽  
Cloned Cdna ◽  
Plasmid Transfection

ABSTRACT Plasmid-based reverse genetics systems allow the generation of influenza A virus entirely from cloned cDNA. However, since the efficiency of virus generation is dependent on the plasmid transfection efficiency of cells, virus generation is difficult in cells approved for vaccine production that have low transfection efficiencies (e.g., Vero cells). Here we established an alternative reverse genetics system for influenza virus generation by using an adenovirus vector (AdV) which achieves highly efficient gene transfer independent of cell transfection efficiency. This AdV-mediated reverse genetics system will be useful for generating vaccine seed strains and for basic influenza virus studies.

scholarly journals Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1723
Author(s):  
Soroth Chey ◽  
Juliane Maria Palmer ◽  
Laura Doerr ◽  
Uwe Gerd Liebert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Measles Virus ◽  
Reverse Genetics ◽  
Hammerhead Ribozyme ◽  
Polymerase Activity ◽  
T7 Promoter ◽  
Gene Delivery Vectors ◽  
Cloned Cdna ◽  
Dual Promoters

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

scholarly journals Neuraminidase Inhibitor Sensitivity and Receptor-Binding Specificity of Cambodian Clade 1 Highly Pathogenic H5N1 Influenza Virus

2011 ◽  
Vol 55 (5) ◽  
pp. 2004-2010 ◽  
Author(s):  
M. Naughtin ◽  
J. C. Dyason ◽  
S. Mardy ◽  
S. Sorn ◽  
M. von Itzstein ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Reverse Genetics ◽  
Binding Specificity ◽  
Neuraminidase Inhibitor ◽  
H5n1 Influenza Virus ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Receptor Binding Specificity ◽  
Pathogenic H5n1

ABSTRACTThe evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.

scholarly journals An improved method for the rescue of recombinant Newcastle disease virus

BioTechniques ◽  
2020 ◽  
Vol 68 (2) ◽  
pp. 96-100
Author(s):  
Pheik-Sheen Cheow ◽  
Tiong Kit Tan ◽  
Adelene Ai-Lian Song ◽  
Khatijah Yusoff ◽  
Suet Lin Chia
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Transfection Efficiency ◽  
Transfection Method ◽  
Helper Plasmids ◽  
Immunogenic Properties ◽  
Recombinant Newcastle Disease Virus

Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method.

scholarly journals Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Irina Baranovskaya ◽  
Mariia Sergeeva ◽  
Artem Fadeev ◽  
Renata Kadirova ◽  
Anna Ivanova ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Secondary Structure ◽  
Protein Expression ◽  
Rna Secondary Structure ◽  
Reverse Genetics ◽  
Early Stage ◽  
Influenza Virus Infection ◽  
Main Function ◽  
Ns1 Protein ◽  
Virus Strains

AbstractRNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82–148) NS RNA region influence NS1 protein expression.

scholarly journals Restriction of Viral Replication by Mutation of the Influenza Virus Matrix Protein

2002 ◽  
Vol 76 (24) ◽  
pp. 13055-13061 ◽  
Author(s):  
Teresa Liu ◽  
Zhiping Ye
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Zinc Finger ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Reverse Genetics ◽  
Rna Binding ◽  
Viral Assembly ◽  
Zinc Finger Motif

ABSTRACT The matrix protein (M1) of influenza virus plays an essential role in viral assembly and has a variety of functions, including association with influenza virus ribonucleoprotein (RNP). Our previous studies show that the association of M1 with viral RNA and nucleoprotein not only promotes formation of helical RNP but also is required for export of RNP from the nucleus during viral replication. The RNA-binding domains of M1 have been mapped to two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105, which is also involved in RNP-binding activity. To further understand the role of the RNP-binding domain of M1 in viral assembly and replication, mutations in the coding sequences of RKLKR and the zinc finger motif of M1 were constructed using a PCR technique and introduced into wild-type influenza virus by reverse genetics. Altering the zinc finger motif of M1 only slightly affected viral growth. Substitution of Arg with Ser at position 101 or 105 of RKLKR did not have a major impact on nuclear export of RNP or viral replication. In contrast, deletion of RKLKR or substitution of Lys with Asn at position 102 or 104 of RKLKR resulted in a lethal mutation. These results indicate that the RKLKR domain of M1 protein plays an important role in viral replication.

scholarly journals Potential use of a chemical leaching reject from a kaolin industry as agricultural fertilizer

2007 ◽  
Vol 31 (5) ◽  
pp. 939-946
Author(s):  
Fabiana Rodrigues Ribeiro ◽  
Fernando Barboza Egreja Filho ◽  
José Domingos Fabris ◽  
Wagner da Nova Mussel ◽  
Roberto Ferreira Novais
Keyword(s):  
Paper Industry ◽  
Soil Samples ◽  
Limiting Factor ◽  
Metallic Zinc ◽  
Chemical Leaching ◽  
Industrial Processing ◽  
Industrial Residue ◽  
Potential Use ◽  
Agricultural Fertilizer ◽  
Soil Fertilizer

The industrial refining of kaolin involves the removal of iron oxides and hydroxides along with other impurities that cause discoloration of the final product and depreciate its commercial value, particularly undesirable if destined to the paper industry. The chemical leaching in the industrial processing requires treatments with sodium hyposulfite, metallic zinc, or sulfuric and phosphoric acids, in order to reduce, dissolve and remove ferruginous compounds. To mitigate the environmental impact, the acidic effluent from the leaching process must be neutralized, usually with calcium oxide. The resulting solid residue contains phosphorous, zinc, and calcium, among other essential nutrients for plant growth, suggesting its use as a macro and micronutrient source. Samples of such a solid industrial residue were used here to evaluate their potential as soil fertilizer in an incubation greenhouse experiment with two soil samples (clayey and medium-textured). The small pH shift generated by applying the residue to the soil was not a limiting factor for its use in agriculture. The evolution of the concentrations of exchangeable calcium, and phosphorous and zinc extractability by Mehlich-1 extractant during the incubation period confirms the potential use of this industrial residue as agricultural fertilizer.

scholarly journals The PA Subunit of the Influenza Virus Polymerase Complex Affects Replication and Airborne Transmission of the H9N2 Subtype Avian Influenza Virus

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 40 ◽  
Author(s):  
Mengchan Hao ◽  
Shaojie Han ◽  
Dan Meng ◽  
Rong Li ◽  
Jing Lin ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Guinea Pigs ◽  
Influenza A ◽  
Reverse Genetics ◽  
Species Barrier ◽  
Airborne Transmission ◽  
H9n2 Subtype ◽  
Influenza A H1n1

The polymerase acidic (PA) protein is the third subunit of the influenza A virus polymerase. In recent years, studies have shown that PA plays an important role in overcoming the host species barrier and host adaptation of the avian influenza virus (AIV). The objective of this study was to elucidate the role of the PA subunit on the replication and airborne transmission of the H9N2 subtype AIV. By reverse genetics, a reassortant rSD01-PA was derived from the H9N2 subtype AIV A/Chicken/Shandong/01/2008 (SD01) by introducing the PA gene from the pandemic influenza A H1N1 virus A/swine/Shandong/07/2011 (SD07). Specific pathogen-free (SPF) chickens and guinea pigs were selected as the animal models for replication and aerosol transmission studies. Results show that rSD01-PA lost the ability of airborne transmission among SPF chickens because of the single substitution of the PA gene. However, rSD01-PA could infect guinea pigs through direct contact, while the parental strain SD01 could not, even though the infection of rSD01-PA could not be achieved through aerosol. In summary, our results indicate that the protein encoded by the PA gene plays a key role in replication and airborne transmission of the H9N2 subtype AIV.

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