|
1
|
Siegers JY, Ferreri L, Eggink D, Veldhuis Kroeze EJB, te Velthuis AJW, van de Bildt M, Leijten L, van Run P, de Meulder D, Bestebroer T, Richard M, Kuiken T, Lowen AC, Herfst S, van Riel D. Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets. PLoS Pathog 2023; 19:e1011214. [PMID: 36897923 PMCID: PMC10032531 DOI: 10.1371/journal.ppat.1011214] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 03/22/2023] [Accepted: 02/16/2023] [Indexed: 03/11/2023] Open
Abstract
Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.
Collapse
Affiliation(s)
- Jurre Y. Siegers
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| | - Lucas Ferreri
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America
| | - Dirk Eggink
- Department of Medical Microbiology, Amsterdam UMC, Amsterdam, The Netherlands
| | | | - Aartjan J. W. te Velthuis
- Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
| | | | - Lonneke Leijten
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| | - Peter van Run
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| | | | - Theo Bestebroer
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| | - Mathilde Richard
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| | - Thijs Kuiken
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| | - Anice C. Lowen
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America
| | - Sander Herfst
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| | - Debby van Riel
- Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands
| |
Collapse
|
|
2
|
Wang W, An X, Yan K, Li Q. Construction and Application of Orthogonal T7 Expression System in Eukaryote: An Overview. Adv Biol (Weinh) 2023; 7:e2200218. [PMID: 36464626 DOI: 10.1002/adbi.202200218] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2022] [Revised: 10/17/2022] [Indexed: 12/12/2022]
Abstract
The T7 system is an orthogonal transcription-system, which is characterized by simplicity, higher efficiency, and higher processivity, and it is used for protein or mRNA synthesis in various biological-systems. In comparison with prokaryotes, the construction of the T7 expression system is still on-going in eukaryotes, but it shows greatly applicable prospects. In the present paper, development of T7 expression system construction in eukaryotes is reviewed, including its construction in animal (mammalian cells, trypanosomatid protozoa, Xenopus oocytes, zebrafish), plant, and microorganism and its application in vaccine production and gene therapy. In addition, the innate challenges of T7 expression system construction in eukaryote and its potential application in vaccine production and gene therapy are discussed.
Collapse
Affiliation(s)
- Wenya Wang
- College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, P. R. China
| | - Xiaoyan An
- College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, P. R. China
| | - Kun Yan
- College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, P. R. China
| | - Qiang Li
- Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, P. R. China
| |
Collapse
|
|
3
|
Malik T, Klenow L, Karyolaimos A, Gier JWD, Daniels R. Silencing Transcription from an Influenza Reverse Genetics Plasmid in E. coli Enhances Gene Stability. ACS Synth Biol 2023; 12:432-445. [PMID: 36716395 PMCID: PMC9942234 DOI: 10.1021/acssynbio.2c00358] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into Escherichia coli. Using GFP as a reporter, we demonstrate that E. coli expresses the target genes in the RG plasmid at low levels. Incorporating lac operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two lac operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in E. coli. Viruses rescued from the lac operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in E. coli helps to maintain the correct influenza gene sequence and that the lac operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between lac operators can be used for reducing DNA segment instability in any plasmid that is propagated in E. coli which express the Lac repressor.
Collapse
Affiliation(s)
- Tahir Malik
- Division
of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
| | - Laura Klenow
- Division
of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
| | - Alexandros Karyolaimos
- Department
of Biochemistry and Biophysics, Stockholm
University, 10691 Stockholm, Sweden
| | - Jan-Willem de Gier
- Department
of Biochemistry and Biophysics, Stockholm
University, 10691 Stockholm, Sweden
| | - Robert Daniels
- Division
of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States,
| |
Collapse
|
|
4
|
Lu G, Zheng F, Ou J, Yin X, Li S. Investigating Influenza Virus Polymerase Activity in Feline Cells Based on the Influenza Virus Minigenome Replication System Driven by the Feline RNA Polymerase I Promoter. Front Immunol 2022; 13:827681. [PMID: 35693765 PMCID: PMC9185166 DOI: 10.3389/fimmu.2022.827681] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2021] [Accepted: 04/08/2022] [Indexed: 12/03/2022] Open
Abstract
Emerging influenza virus poses a health threat to humans and animals. Domestic cats have recently been identified as a potential source of zoonotic influenza virus. The influenza virus minigenome replication system based on the ribonucleic acid (RNA) polymerase I (PolI) promoter is the most widely used tool for investigating polymerase activity. It could help determine host factors or viral proteins influencing influenza virus polymerase activity in vitro. However, influenza virus polymerase activity has never been studied in feline cells thus far. In the present study, the feline RNA PolI promoter was identified in the intergenic spacer regions between adjacent upstream 28S and downstream 18S rRNA genes in the cat (Felis catus) genome using bioinformatics strategies. The transcription initiation site of the feline RNA PolI promoter was predicted. The feline RNA PolI promoter was cloned from CRFK cells, and a promoter size of 250 bp contained a sequence with sufficient PolI promoter activity by a dual-luciferase reporter assay. The influenza virus minigenome replication system based on the feline RNA PolI promoter was then established. Using this system, the feline RNA PolI promoter was determined to have significantly higher transcriptional activity than the human and chicken RNA PolI promoters in feline cells, and equine (H3N8) influenza virus presented higher polymerase activity than human (H1N1) and canine (H3N2) influenza viruses. In addition, feline myxovirus resistance protein 1 (Mx1) and baloxavir were observed to inhibit influenza virus polymerase activity in vitro in a dose-dependent manner. Our study will help further investigations on the molecular mechanism of host adaptation and cross-species transmission of influenza virus in cats.
Collapse
Affiliation(s)
- Gang Lu
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Feiyan Zheng
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Jiajun Ou
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Xin Yin
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin, China
- *Correspondence: Xin Yin, ; Shoujun Li,
| | - Shoujun Li
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, Guangzhou, China
- Guangdong Technological Engineering Research Center for Pet, Guangzhou, China
- *Correspondence: Xin Yin, ; Shoujun Li,
| |
Collapse
|
|
5
|
Ding S, Greenberg HB. Perspectives for the optimization and utility of the rotavirus reverse genetics system. Virus Res 2021; 303:198500. [PMID: 34331991 DOI: 10.1016/j.virusres.2021.198500] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Revised: 06/27/2021] [Accepted: 06/30/2021] [Indexed: 11/21/2022]
Abstract
Following Kobayashi and colleagues' seminal paper in 20171, in the past four years the rotavirus (RV) field has witnessed a burst in research and publications based on the use of a fully plasmid-based RV reverse genetics systems and subsequent modifications2,3. However, in most cases, the rotaviral strain under interrogation has been the prototypic simian RV SA11-L2 strain (G3P[2]). Of note, a variety of other weakly-to-modestly replication-competent animal and human RV strains, bioluminescent and fluorescent reporter viruses, and clinical isolates of human RVs have proved hard or impossible to rescue using the original reverse genetics system2,4, highlighting a critical need to further enhance the recovery efficiency and expand the rescue tool kit. A number of further modifications of the initial reverse genetics system have enabled the rescue of other RV strains such as the human RV KU and CDC-9 strains, and a murine RV D6/2-like strain4,5. Here, we discuss future possible modifications of existing RV reverse genetics systems to further increase efficiency based on past experience with the improvement of influenza A virus recovery. The development of RV to accommodate the insertion and expression of heterologous sequences has substantial potential in the design of next-generation RV vaccine candidates and enteric viral vectors.
Collapse
Affiliation(s)
- Siyuan Ding
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.
| | - Harry B Greenberg
- Department of Medicine, Division of Gastroenterology and Hepatology, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA; VA Palo Alto Health Care System, Department of Veterans Affairs, Palo Alto, CA, USA.
| |
Collapse
|
|
6
|
Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells. Viruses 2021; 13:v13091723. [PMID: 34578303 PMCID: PMC8471996 DOI: 10.3390/v13091723] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2021] [Revised: 08/24/2021] [Accepted: 08/27/2021] [Indexed: 11/17/2022] Open
Abstract
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.
Collapse
|
|
7
|
Neumann G. Influenza Reverse Genetics-Historical Perspective. Cold Spring Harb Perspect Med 2021; 11:cshperspect.a038547. [PMID: 31964649 DOI: 10.1101/cshperspect.a038547] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The generation of wild-type, mutant, and reassortant influenza viruses from viral cDNAs (reverse genetics) is now a basic molecular virology technique in many influenza virus laboratories. Here, I describe the original RNA polymerase I reverse genetics system and the modifications that have been developed in past years. Together, these technologies have made possible many advances in basic and applied influenza virology that would not have been otherwise attainable, including the revival and study of extinct influenza viruses, the rapid characterization of emerging influenza viruses, the generation of conventional influenza vaccines, and the development of novel influenza vaccines.
Collapse
Affiliation(s)
- Gabriele Neumann
- Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA
| |
Collapse
|
|
8
|
Establishment of a Reverse Genetics System for Influenza D Virus. J Virol 2020; 94:JVI.01767-19. [PMID: 32102883 DOI: 10.1128/jvi.01767-19] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Accepted: 02/03/2020] [Indexed: 12/22/2022] Open
Abstract
Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures.IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.
Collapse
|
|
9
|
Development and Characterization of a Reverse-Genetics System for Influenza D Virus. J Virol 2019; 93:JVI.01186-19. [PMID: 31413133 DOI: 10.1128/jvi.01186-19] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2019] [Accepted: 08/07/2019] [Indexed: 01/23/2023] Open
Abstract
Influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range and a broad geographical distribution. Recent IDV outbreaks in swine along with serological and genetic evidence of IDV infection in humans have raised concerns regarding the zoonotic potential of this virus. To better study IDV at the molecular level, a reverse-genetics system (RGS) is urgently needed, but to date, no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven-plasmid-based system and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV minigenome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein (RNP) complex, resulting in either attenuated or replication-incompetent viruses. Finally, by using the minigenome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3' conserved noncoding region in IDV and influenza C virus (ICV) resulted in the inefficient cross-recognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a minigenome replication assay and a robust reverse-genetics system that can be used to further study replication, tropism, and pathogenesis of IDV.IMPORTANCE Influenza D virus (IDV) is a new type of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Increased outbreaks in pigs and serological and genetic evidence of human infection have raised concerns about potential IDV adaptation in humans. Here, we have developed a plasmid-based IDV reverse-genetics system that can generate infectious viruses with replication kinetics similar to those of wild-type viruses following transfection of cultured cells. Further characterization demonstrated that viruses rescued from the described RGS resembled the parental viruses in biological and receptor-binding properties. We also developed and validated an IDV minireplicon reporter system that specifically measures viral RNA polymerase activity. In summary, the reverse-genetics system and minireplicon reporter assay described in this study should be of value in identifying viral determinants of cross-species transmission and pathogenicity of novel influenza D viruses.
Collapse
|
|
10
|
Molouki A, Nagy A. Rescue of recombinant Newcastle disease virus: a promising vector with two decades of intensive vaccine research. Future Virol 2019. [DOI: 10.2217/fvl-2019-0063] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Two decades have passed since the first reverse genetics system for the rescue of recombinant Newcastle disease virus was developed. Since then, the recombinant Newcastle disease virus vector has shown promising results as a safe and potent vector for development of many vaccines for both avian and human use. Herein, we review several technical topics that would be useful to further understanding of this technology. First, the effect of using helper plasmids encoding proteins belonging to strains other than the full-length cDNA and the possible incorporation of these expressed proteins into progeny virus will be discussed. Then, we will discuss the effect of removal of additional G residues from the T7 initiation sequence and finally, we will review different ways to improve rescue efficiency.
Collapse
Affiliation(s)
- Aidin Molouki
- Department of Avian Disease Research & Diagnostic, Razi Vaccine & Serum Research Institute, Agricultural Research Education & Extension Organization (AREEO), Karaj, Iran
| | - Abdou Nagy
- Department of Virology, Faculty of Veterinary Medicine, Zagazig University, Ash Sharqyiah 44519, Egypt
| |
Collapse
|
|
11
|
Viral Factors Important for Efficient Replication of Influenza A Viruses in Cells of the Central Nervous System. J Virol 2019; 93:JVI.02273-18. [PMID: 30867311 PMCID: PMC6532103 DOI: 10.1128/jvi.02273-18] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2018] [Accepted: 03/05/2019] [Indexed: 11/20/2022] Open
Abstract
Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections, and the frequency and severity differ between seasonal, pandemic, and zoonotic influenza viruses. However, little is known about the interaction of these viruses with cells of the CNS. Differences among seasonal, pandemic, and zoonotic influenza viruses in replication efficacy in CNS cells, in vitro, suggest that the presence of an alternative HA cleavage mechanism and ability to attach are important viral factors. Identifying these viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease. Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic influenza viruses. Little is known about the interaction between influenza A viruses and cells of the CNS; therefore, it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH) and astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells, which was associated with efficient attachment and infection. Seasonal H3N2 and to a lesser extent pandemic H1N1 virus replicated in a trypsin-dependent manner in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multibasic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate, replication. Taken together, our results showed that the MBCS and, to a lesser extent, the ability to attach are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative hemagglutinin (HA) cleavage mechanism and preference for α-2,3-linked sialic acids allowing efficient attachment contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses. IMPORTANCE Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections, and the frequency and severity differ between seasonal, pandemic, and zoonotic influenza viruses. However, little is known about the interaction of these viruses with cells of the CNS. Differences among seasonal, pandemic, and zoonotic influenza viruses in replication efficacy in CNS cells, in vitro, suggest that the presence of an alternative HA cleavage mechanism and ability to attach are important viral factors. Identifying these viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease.
Collapse
|
|
12
|
Abstract
Influenza virus epidemics are caused when seasonal influenza viruses (i.e., those circulating in humans) acquire mutations in the antigenic sites of the viral hemagglutinin (HA) protein that prevent the antibodies present in people from binding to the virus and blocking virus interaction with cellular receptors. To date, vaccination is the best protective option against seasonal influenza viruses. Because influenza viruses frequently acquire mutations in their antigenic sites, vaccine viruses need to be updated regularly. Here, we present an experimental system that allows the simulation of influenza virus evolution in the test tube. By using this system, we can identify antigenic variants that may emerge among natural influenza viruses in the near future. This information would help in the selection and prioritization of variants for vaccine production.
Collapse
Affiliation(s)
- Gabriele Neumann
- Department of Pathobiological Sciences, Influenza Research Institute, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Shufang Fan
- Department of Pathobiological Sciences, Influenza Research Institute, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Yoshihiro Kawaoka
- Department of Pathobiological Sciences, Influenza Research Institute, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA.
- Division of Virology, Department of Microbiology and Immunology, University of Tokyo, Tokyo, Japan.
- International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
| |
Collapse
|
|
13
|
A single-plasmid reverse genetics system for the rescue of non-segmented negative-strand RNA viruses from cloned full-length cDNA. J Virol Methods 2017; 248:187-190. [PMID: 28743584 DOI: 10.1016/j.jviromet.2017.07.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2017] [Revised: 07/18/2017] [Accepted: 07/18/2017] [Indexed: 11/23/2022]
Abstract
Reverse genetics systems for non-segmented negative-strand RNA viruses rely on co-transfection of a plasmid containing the full-length viral cDNA and helper plasmids encoding essential viral replication proteins. Here, a system is presented in which virus can be rescued from a single plasmid without the need for helper plasmids in cells infected with a host-restricted recombinant poxvirus that expresses T7 RNA polymerase. This approach relies on the insertion of T7 promoter sequences in the viral cDNA at positions that allow transcription of sub-genomic RNAs encoding essential viral replication proteins.
Collapse
|
|
14
|
Ghassemi F, Madadgar O, Roohvand F, Rasekhian M, Etemadzadeh MH, Boroujeni GRN, Langroudi AG, Azadmanesh K. Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines. Mol Biol 2017. [DOI: 10.1134/s002689331702011x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
|
|
15
|
Perez DR, Angel M, Gonzalez-Reiche AS, Santos J, Obadan A, Martinez-Sobrido L. Plasmid-Based Reverse Genetics of Influenza A Virus. Methods Mol Biol 2017; 1602:251-273. [PMID: 28508225 DOI: 10.1007/978-1-4939-6964-7_16] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Influenza A viruses have broad host range with a recognized natural reservoir in wild aquatic birds. From this reservoir, novel strains occasionally emerge with the potential to establish stable lineages in other avian and mammalian species, including humans. Understanding the molecular changes that allow influenza A viruses to change host range is essential to better assess their animal and public health risks. Reverse genetics systems have transformed the ability to manipulate and study negative strand RNA viruses. In the particular case of influenza A viruses, plasmid-based reverse genetics approaches have allowed for a better understanding of, among others, virulence, transmission, mechanisms of antiviral resistance, and the development of alternative vaccines and vaccination strategies. In this chapter we describe the cloning of cDNA copies of viral RNA segments derived from a type A influenza virus into reverse genetics plasmid vectors and the experimental procedures for the successful generation of recombinant influenza A viruses.
Collapse
Affiliation(s)
- Daniel R Perez
- Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA.
| | - Matthew Angel
- Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA
| | - Ana Silvia Gonzalez-Reiche
- Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA
| | - Jefferson Santos
- Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA
| | - Adebimpe Obadan
- Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA, 30602, USA
| | - Luis Martinez-Sobrido
- Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, Rochester, NY, 14642, USA
| |
Collapse
|
|
16
|
Horie M, Sassa Y, Iki H, Ebisawa K, Fukushi H, Yanai T, Tomonaga K. Isolation of avian bornaviruses from psittacine birds using QT6 quail cells in Japan. J Vet Med Sci 2015; 78:305-8. [PMID: 26346745 PMCID: PMC4785123 DOI: 10.1292/jvms.15-0372] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Avian bornaviruses (ABVs) were recently discovered as the causative agents of proventricular dilatation
disease (PDD). Although molecular epidemiological studies revealed that ABVs exist in Japan, no Japanese
isolate has been reported thus far. In this study, we isolated four strains of Psittaciform 1
bornavirus from psittacine birds affected by PDD using QT6 quail cells. To our knowledge, this is
the first report to isolate ABVs in Japan and to show that QT6 cells are available for ABV isolation. These
isolates and QT6 cells would be powerful tools for elucidating the fundamental biology and pathogenicity of
ABVs.
Collapse
Affiliation(s)
- Masayuki Horie
- Transboundary Animal Diseases Research Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan
| | | | | | | | | | | | | |
Collapse
|
|
17
|
Zhang X, Curtiss R. Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid. Virol J 2015; 12:95. [PMID: 26093583 PMCID: PMC4495709 DOI: 10.1186/s12985-015-0321-5] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Accepted: 06/08/2015] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse PolI-driven plasmid for efficient production of influenza virus. RESULTS A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 10(5)-10(6) 50% tissue culture infective dose (TCID50)/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin-Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method. CONCLUSIONS An all-in-one plasmid and a 3-plasmid murine PolI-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust "1 + 2" approach to generate influenza vaccine seed virus.
Collapse
Affiliation(s)
- Xiangmin Zhang
- Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA. .,Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy/Health Sciences, Wayne State University, Detroit, MI, USA.
| | - Roy Curtiss
- Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA. .,School of Life Science, Arizona State University, Tempe, AZ, 85287, USA. .,Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, PO Box 110880, Gainesville, FL, 32611-0880, USA.
| |
Collapse
|
|
18
|
Finch C, Li W, Perez DR. Design of alternative live attenuated influenza virus vaccines. Curr Top Microbiol Immunol 2015; 386:205-35. [PMID: 25005928 DOI: 10.1007/82_2014_404] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/16/2023]
Abstract
Each year due to the ever-evolving nature of influenza, new influenza vaccines must be produced to provide protection against the influenza viruses in circulation. Currently, there are two mainstream strategies to generate seasonal influenza vaccines: inactivated and live-attenuated. Inactivated vaccines are non-replicating forms of whole influenza virus, while live-attenuated vaccines are viruses modified to be replication impaired. Although it is widely believed that by inducing both mucosal and humoral immune responses the live-attenuated vaccine provides better protection than that of the inactivated vaccine, there are large populations of individuals who cannot safely receive the LAIV vaccine. Thus, safer LAIV vaccines are needed to provide adequate protection to these populations. Improvement is also needed in the area of vaccine production. Current strategies relying on traditional tissue culture-based and egg-based methods are slow and delay production time. This chapter describes experimental vaccine generation and production strategies that address the deficiencies in current methods for potential human and agricultural use.
Collapse
Affiliation(s)
- Courtney Finch
- Department of Veterinary Medicine, College Park and Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD, USA
| | | | | |
Collapse
|
|
19
|
Edenborough K, Marsh GA. Reverse genetics: Unlocking the secrets of negative sense RNA viral pathogens. World J Clin Infect Dis 2014; 4:16-26. [DOI: 10.5495/wjcid.v4.i4.16] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2014] [Revised: 08/29/2014] [Accepted: 09/24/2014] [Indexed: 02/06/2023] Open
Abstract
Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cDNA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed with reference to new technologies that may be utilized to improve reverse genetic approaches.
Collapse
|
|
20
|
RNA virus reverse genetics and vaccine design. Viruses 2014; 6:2531-50. [PMID: 24967693 PMCID: PMC4113782 DOI: 10.3390/v6072531] [Citation(s) in RCA: 72] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2014] [Revised: 06/18/2014] [Accepted: 06/19/2014] [Indexed: 12/22/2022] Open
Abstract
RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines.
Collapse
|
|
21
|
Construction of a Minigenome Rescue System for Measles Virus, AIK-c Strain. IRANIAN JOURNAL OF BIOTECHNOLOGY 2014. [DOI: 10.5812/ijb.18002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
|
|
22
|
Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site. mBio 2014; 5:e01070-14. [PMID: 24917596 PMCID: PMC4056550 DOI: 10.1128/mbio.01070-14] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011. During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition. Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for severe outbreaks in both commercial and backyard poultry, causing considerable economic losses and regular zoonotic transmissions to humans. Vaccination is used increasingly to reduce the burden of HPAI H5N1 virus in poultry. Influenza viruses can escape from recognition by antibodies induced upon vaccination or infection through genetic changes in the hemagglutinin protein. The evolutionary patterns and molecular basis of antigenic change in HPAI H5N1 viruses are poorly understood, hampering formulation of optimal vaccination strategies. We have shown here that HPAI H5N1 viruses in Indonesia diversified into multiple antigenic variants, that antigenic differences were due to one or a very few substitutions near the receptor binding site, and that the molecular basis for antigenic change was remarkably similar to that for seasonal human influenza viruses. These findings have consequences for future vaccination and surveillance considerations and contribute to the understanding of the antigenic evolution of influenza viruses.
Collapse
|
|
23
|
Interaction between hantavirus nucleocapsid protein (N) and RNA-dependent RNA polymerase (RdRp) mutants reveals the requirement of an N-RdRp interaction for viral RNA synthesis. J Virol 2014; 88:8706-12. [PMID: 24850733 DOI: 10.1128/jvi.00405-14] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Viral ribonucleocapsids harboring the viral genomic RNA are used as the template for viral mRNA synthesis and replication of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Here we show that hantavirus nucleocapsid protein (N protein) interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain at the N terminus of N protein. Similarly, the N protein binding pocket is located at the C terminus of RdRp. We demonstrate that an N protein-RdRp interaction is required for RdRp function during the course of virus infection in the host cell.
Collapse
|
|
24
|
Koel BF, Burke DF, Bestebroer TM, van der Vliet S, Zondag GCM, Vervaet G, Skepner E, Lewis NS, Spronken MIJ, Russell CA, Eropkin MY, Hurt AC, Barr IG, de Jong JC, Rimmelzwaan GF, Osterhaus ADME, Fouchier RAM, Smith DJ. Substitutions Near the Receptor Binding Site Determine Major Antigenic Change During Influenza Virus Evolution. Science 2013; 342:976-9. [DOI: 10.1126/science.1244730] [Citation(s) in RCA: 407] [Impact Index Per Article: 33.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
|
|
25
|
Mostafa A, Kanrai P, Ziebuhr J, Pleschka S. Improved dual promotor-driven reverse genetics system for influenza viruses. J Virol Methods 2013; 193:603-10. [PMID: 23886561 DOI: 10.1016/j.jviromet.2013.07.021] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2013] [Revised: 07/01/2013] [Accepted: 07/10/2013] [Indexed: 12/25/2022]
Abstract
Reverse genetic systems for influenza A virus (IAV) allow the generation of genetically manipulated infectious virus from a set of transfected plasmid DNAs encoding the eight genomic viral RNA segments (vRNA). For this purpose, cDNAs representing these eight vRNA segments are cloned into specific plasmid vectors that allow the generation of vRNA-like transcripts using polymerase I (Pol I). In addition, these plasmids support the transcription of viral mRNA by polymerase II (Pol II), leading to the expression of viral protein(s) encoded by the respective transcripts. In an effort to develop this system further, we constructed the bi-directional vector pMPccdB. It is based on pHW2000 (Hoffmann et al., 2000b) but contains additionally (i) the ccdB gene whose expression is lethal for most Escherichia coli strains and therefore used as a negative selection marker and (ii) more efficient AarI cloning sites that flank the ccdB gene on either side. Furthermore, we used a modified one-step restriction/ligation protocol to insert the desired cDNA into the respective pMPccdB vector DNA. Both the use of a negative selection marker and an improved cloning protocol were shown to facilitate the generation of genetically engineered IAV as illustrated in this study by the cloning and rescue of the 2009 pandemic isolate A/Giessen/6/2009 (Gi-H1N1).
Collapse
Affiliation(s)
- Ahmed Mostafa
- Institute of Medical Virology, Justus Liebig University Giessen, BFS, Schubertstrasse 81, 35392 Giessen, Germany; Virology Laboratory, Environmental Research Division, National Research Center, 12311 Dokki, Giza, Egypt.
| | | | | | | |
Collapse
|
|
26
|
A simple and fast system for cloning influenza A virus gene segments into pHW2000- and pCAGGS-based vectors. Arch Virol 2013; 158:2049-58. [PMID: 23615869 DOI: 10.1007/s00705-013-1697-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2013] [Accepted: 03/11/2013] [Indexed: 11/27/2022]
Abstract
The reverse genetics system for influenza A viruses described by Hoffmann et al. (Virology 267(2):310-317, 2000, Proc Natl Acad Sci USA 97(11):6108-6113, 2000, ArchVirol 146(12):2275-2289, 2001) is one of the most commonly used. However, this cloning strategy is rather time-consuming and lacks a selection marker to identify positive clones carrying viral genes. We report here the optimization of the cloning protocol of viral genes into pHW2000 by (i) introducing a selection marker and (ii) simplifying the cloning strategy: now the cloning reaction takes only a few minutes and, in addition, is independent of internal restriction sites for BsmBI/Esp3I, BsaI or AarI. In order to accelerate the whole cloning protocol for the generation of recombinant viruses, we first introduced a lacP/Z-element (lac-promoter/lacZα-fragment) between the two BsmBI sites of pHW2000 to allow selection of positive clones by blue/white screening. Then we optimized the digestion/ligation-protocol: In our system, enzymatic digestion and ligation of PCR products into the vector is performed in a single "one-tube" reaction. Due to this strategy, time and material consumption is reduced by a great amount, as vector and cDNA do not have to be digested and purified prior to the ligation. Therefore, this one-tube reaction yields positive clones with high efficiency and fidelity, again saving time and material, which were formerly required for screening and analyzing clones. Finally, to add more versatility to the system, we also created an entry vector based on TA-cloning. This entry vector provides several advantages: inserted genes can easily be modified, e.g., by site-directed mutagenesis or tag attachment, and then subcloned into pHW2000 or other plasmids containing a similar cloning site (e.g., our modified pCAGGS-Esp-blue) by the same rapid and reliable one-tube reaction protocol described here. In fact, the presented protocol is suitable to be adapted to other reverse genetics systems (e.g., those for members of the order Mononegavirales or the family Bunyaviridae) or cloning of genes in general.
Collapse
|
|
27
|
Song MS, Baek YH, Pascua PNQ, Kwon HI, Park SJ, Kim EH, Lim GJ, Choi YK. Establishment of Vero cell RNA polymerase I-driven reverse genetics for Influenza A virus and its application for pandemic (H1N1) 2009 influenza virus vaccine production. J Gen Virol 2013; 94:1230-1235. [PMID: 23486669 DOI: 10.1099/vir.0.051284-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The constant threat of newly emerging influenza viruses with pandemic potential requires the need for prompt vaccine production. Here, we utilized the Vero cell polymerase I (PolI) promoter, rather than the commonly used human PolI promoter, in an established reverse-genetics system to rescue viable influenza viruses in Vero cells, an approved cell line for human vaccine production. The Vero PolI promoter was more efficient in Vero cells and demonstrated enhanced transcription levels and virus rescue rates commensurate with that of the human RNA PolI promoter in 293T cells. These results appeared to be associated with more efficient generation of A(H1N1)pdm09- and H5N1-derived vaccine seed viruses in Vero cells, whilst the rescue rates in 293T cells were comparable. Our study provides an alternative means for improving vaccine preparation by using a novel reverse-genetics system for generating influenza A viruses.
Collapse
Affiliation(s)
- Min-Suk Song
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| | - Yun Hee Baek
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| | - Philippe Noriel Q Pascua
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| | - Hyeok-Il Kwon
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| | - Su-Jin Park
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| | - Eun-Ha Kim
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| | - Gyo-Jin Lim
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| | - Young-Ki Choi
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Republic of Korea
| |
Collapse
|
|
28
|
Ortiz-Riaño E, Cheng BYH, Carlos de la Torre J, Martínez-Sobrido L. Arenavirus reverse genetics for vaccine development. J Gen Virol 2013; 94:1175-1188. [PMID: 23364194 DOI: 10.1099/vir.0.051102-0] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1.
Collapse
Affiliation(s)
- Emilio Ortiz-Riaño
- Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
| | - Benson Yee Hin Cheng
- Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
| | - Juan Carlos de la Torre
- Department of Immunology and Microbial Science, Scripps Research Institute, La Jolla, CA 92037, USA
| | - Luis Martínez-Sobrido
- Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
| |
Collapse
|
|
29
|
Engelhardt OG. Many ways to make an influenza virus--review of influenza virus reverse genetics methods. Influenza Other Respir Viruses 2012; 7:249-56. [PMID: 22712782 PMCID: PMC5779834 DOI: 10.1111/j.1750-2659.2012.00392.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
Methods to introduce targeted mutations into a genome or, in the context of virology, into a virus are subsumed under the term reverse genetics (RG). Influenza viruses are important human pathogens that continue to surprise us. The development of RG for influenza viruses has greatly expanded our knowledge about influenza virus and enabled researchers to generate influenza viruses with rationally designed genotypes. Currently, a wide array of influenza virus RG methods is available. These can all be traced to fundamental principles essential in any RG system for negative-strand RNA viruses. This review gives an overview of these principles and of the multitude of RG methods, categorising them by technical characteristics.
Collapse
Affiliation(s)
- Othmar G Engelhardt
- Division of Virology, National Institute for Biological Standards and Control, Health Protection Agency, Potters Bar, UK.
| |
Collapse
|
|
30
|
Abstract
The ability to modify influenza viruses at will has revolutionized influenza research. Reverse genetics has been used to generate mutant or reassortant influenza viruses to assess their replication, virulence, pathogenicity, host range, and transmissibility. Moreover, this technology is now being used to generate approved influenza virus vaccines and develop novel vaccines to combat seasonal and (future) pandemic influenza viruses. Several variations of the original system have been established, all of which are considerably robust and efficient.
Collapse
Affiliation(s)
- Gabriele Neumann
- Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | | | | |
Collapse
|
|
31
|
Hoenen T, Groseth A, de Kok-Mercado F, Kuhn JH, Wahl-Jensen V. Minigenomes, transcription and replication competent virus-like particles and beyond: reverse genetics systems for filoviruses and other negative stranded hemorrhagic fever viruses. Antiviral Res 2011; 91:195-208. [PMID: 21699921 DOI: 10.1016/j.antiviral.2011.06.003] [Citation(s) in RCA: 92] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2011] [Revised: 06/02/2011] [Accepted: 06/08/2011] [Indexed: 12/27/2022]
Abstract
Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research.
Collapse
Affiliation(s)
- Thomas Hoenen
- Laboratory of Virology, Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA; Department of Virology, Philipps University Marburg, Marburg, Germany
| | | | | | | | | |
Collapse
|
|
32
|
Martina BEE, van den Doel P, Koraka P, van Amerongen G, Spohn G, Haagmans BL, Provacia LBV, Osterhaus ADME, Rimmelzwaan GF. A recombinant influenza A virus expressing domain III of West Nile virus induces protective immune responses against influenza and West Nile virus. PLoS One 2011; 6:e18995. [PMID: 21541326 PMCID: PMC3082541 DOI: 10.1371/journal.pone.0018995] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2010] [Accepted: 03/21/2011] [Indexed: 12/11/2022] Open
Abstract
West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 105 TCID50 Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.
Collapse
|
|
33
|
Feng H, Wei D, Nan G, Cui SJ, Chen ZN, Bian H. Construction of a minigenome rescue system for Newcastle disease virus strain Italien. Arch Virol 2011; 156:611-6. [PMID: 21207083 DOI: 10.1007/s00705-010-0898-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2010] [Accepted: 12/16/2010] [Indexed: 02/04/2023]
Abstract
Newcastle disease virus (NDV) Italien, a velogenic strain, is an oncolytic virus that is considered to be a potential agent for antitumor viral therapy. We constructed three helper plasmids expressing the NP, P and L genes of NDV Italien based on the eukaryotic expression plasmid pcDNA3.1(+). The minigenome consisting of the 3' leader and 5' trailer regions of NDV Italien flanking a reporter gene encoding firefly luciferase was constructed to examine the efficacy of the three helper plasmids in viral genome replication and transcription. After co-transfection of BSR-T7/5 cells with the three helper plasmids and the minigenome plasmid, replication of minigenome RNA was evaluated by determining luciferase activity. In the minigenome rescue system, expression of the reporter gene was detected. Our results indicate that the three proteins NP, P, and L are correctly expressed and can assemble into a functional ribonucleoprotein complex that effectively directs the transcription of minigenome RNA.
Collapse
Affiliation(s)
- Hao Feng
- Cell Engineering Research Centre and Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, No.17 Changle West Road, Xi'an, China
| | | | | | | | | | | |
Collapse
|
|
34
|
Lim HS, Vaira AM, Domier LL, Lee SC, Kim HG, Hammond J. Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology 2010; 402:149-63. [PMID: 20381827 DOI: 10.1016/j.virol.2010.03.022] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2010] [Revised: 02/07/2010] [Accepted: 03/14/2010] [Indexed: 11/27/2022]
Abstract
We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.
Collapse
Affiliation(s)
- Hyoun-Sub Lim
- USDA-ARS, Plant Sciences Institute, Molecular Plant Pathology Laboratory, 10300 Baltimore Avenue, Beltsville, MD 20705, USA.
| | | | | | | | | | | |
Collapse
|
|
35
|
Molecular determinants of adaptation of highly pathogenic avian influenza H7N7 viruses to efficient replication in the human host. J Virol 2009; 84:1597-606. [PMID: 19939933 DOI: 10.1128/jvi.01783-09] [Citation(s) in RCA: 125] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Two viruses isolated during the highly pathogenic avian influenza (HPAI) H7N7 virus outbreak in The Netherlands in 2003, one isolated from a person with conjunctivitis and one from a person who died as the result of infection, were used for an in vitro study of influenza A virus pathogenicity factors. The two HPAI H7N7 viruses differed in 15 amino acid positions in five gene segments. Assays were designed to investigate the role of each of these substitutions in attachment and entry, transcription and genome replication, and virus production and release as determined by hemagglutinin (HA), polymerase proteins, nonstructural protein 1 (NS1), and neuraminidase (NA). These in vitro studies confirmed the roles of the E627K substitution in basic polymerase 2 (PB2) and the A143T substitution in HA in pathogenicity observed in a mouse model previously. However, the in vitro studies identified a contribution of acidic polymerase (PA) and NA to the efficient replication in human cells of the fatal case virus, despite the fact that these are rarely marked as determinants of pathogenicity in in vivo studies. With the exception of PB2 E627K, all substitutions contributing to enhanced replication of the fatal case virus in vitro were present in poultry viruses prior to transmission to the human fatal case, indicating that viruses with enhanced replication efficiency in the mammalian host can be generated in poultry. Thus, detailed in vitro analyses of mutations facilitating replication of avian influenza viruses in mammalian cells are important to assess the zoonotic risk posed by these viruses and, in addition, highlight the value of in vitro studies to complement animal models.
Collapse
|
|
36
|
Bouloy M, Flick R. Reverse genetics technology for Rift Valley fever virus: current and future applications for the development of therapeutics and vaccines. Antiviral Res 2009; 84:101-18. [PMID: 19682499 PMCID: PMC2801414 DOI: 10.1016/j.antiviral.2009.08.002] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2009] [Revised: 07/22/2009] [Accepted: 08/06/2009] [Indexed: 11/30/2022]
Abstract
The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.
Collapse
Affiliation(s)
- Michele Bouloy
- Institut Pasteur, Unité de Génétique Moléculaire des Bunyavirus, 25 rue du Dr Roux, 75724 Paris Cedex, France
| | - Ramon Flick
- BioProtection Systems Corporation, 2901 South Loop Drive, Suite 3360, Ames, IA 50010-8646, USA
| |
Collapse
|
|
37
|
van der Vries E, Jonges M, Herfst S, Maaskant J, Van der Linden A, Guldemeester J, Aron GI, Bestebroer TM, Koopmans M, Meijer A, Fouchier RAM, Osterhaus ADME, Boucher CA, Schutten M. Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase. J Clin Virol 2009; 47:34-7. [PMID: 19857993 PMCID: PMC7185517 DOI: 10.1016/j.jcv.2009.09.030] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2009] [Revised: 09/22/2009] [Accepted: 09/25/2009] [Indexed: 10/31/2022]
Abstract
BACKGROUND Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. OBJECTIVES We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. STUDY DESIGN Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. RESULTS 121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. CONCLUSIONS We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.
Collapse
Affiliation(s)
- E van der Vries
- Erasmus MC, Department of Virology, 's Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands
| | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
38
|
Simultaneous one-tube full-length amplification of the NA, NP, M, and NS genes of influenza A viruses for reverse genetics. J Virol Methods 2009; 159:308-10. [PMID: 19406155 DOI: 10.1016/j.jviromet.2009.04.020] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2008] [Revised: 04/02/2009] [Accepted: 04/21/2009] [Indexed: 11/21/2022]
Abstract
Reverse genetics of influenza A viruses has expedited increasingly basic research and vaccine development. Target-primed plasmid amplification using full-length PCR amplicons as inserts was established previously for strain-independent and rapid cloning of all eight influenza A virus genes. This method involves separate amplification of each viral gene using segment-specific primers. Four different primer pairs are required for PCR amplification of the neuraminidase gene depending on the subtype. In order to reduce the number of necessary PCRs, a pair of primers with truncated 3' ends was designed in the present study. This primer pair permitted reliable amplification of the NA, NP, M, and NS genes in one tube whose products can be separated subsequently by their sizes. Full-length amplicons can be generated with this one primer pair from the NA genes of all nine subtypes. By avoiding separate assays for several viral genes, this parallel PCR steps up rapid universal cloning of influenza A virus genes further.
Collapse
|
|
39
|
Koudstaal W, Hartgroves L, Havenga M, Legastelois I, Ophorst C, Sieuwerts M, Zuijdgeest D, Vogels R, Custers J, de Boer-Luijtze E, de Leeuw O, Cornelissen L, Goudsmit J, Barclay W. Suitability of PER.C6® cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics. Vaccine 2009; 27:2588-93. [DOI: 10.1016/j.vaccine.2009.02.033] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2008] [Revised: 02/02/2009] [Accepted: 02/11/2009] [Indexed: 11/16/2022]
|
|
40
|
Abstract
Recent outbreaks of highly pathogenic avian influenza A virus infections (including those of the H5N1 subtype) in poultry and in humans (through contact with infected birds) have raised concerns that a new influenza pandemic will soon occur. Effective vaccines against H5N1 virus are therefore urgently needed. Reverse genetics-based inactivated vaccines have been prepared according to WHO recommendations and licensed in several countries following their assessment in clinical trials. However, the effectiveness of these vaccines in a pandemic is not guaranteed. We must therefore continue to develop alternative pandemic vaccine strategies. Here, we review the current strategies for the development of H5N1 influenza vaccines, as well as some future directions for vaccine development.
Collapse
|
|
41
|
Stech J, Stech O, Herwig A, Altmeppen H, Hundt J, Gohrbandt S, Kreibich A, Weber S, Klenk HD, Mettenleiter TC. Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification. Nucleic Acids Res 2008; 36:e139. [PMID: 18832366 PMCID: PMC2588516 DOI: 10.1093/nar/gkn646] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available. Here, we describe a cloning method universal for any influenza A virus strain which is independent of restriction sites. It is based on target-primed plasmid amplification in which the insert provides two megaprimers and contains termini homologous to plasmid regions adjacent to the insertion site. For improved efficiency, a cloning vector was designed containing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. Using this method, we generated complete sets of functional gene segments from seven influenza A strains and three haemagglutinin genes from different serotypes amounting to 59 cloned influenza genes. These results demonstrate that this approach allows rapid and reliable cloning of any segment from any influenza A strain without any information about restriction sites. In case the PCR amplicon ends are homologous to the plasmid annealing sites only, this method is suitable for cloning of any insert with conserved termini.
Collapse
Affiliation(s)
- Jürgen Stech
- Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
42
|
Habjan M, Penski N, Spiegel M, Weber F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus. J Gen Virol 2008; 89:2157-2166. [PMID: 18753225 DOI: 10.1099/vir.0.2008/002097-0] [Citation(s) in RCA: 126] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae, genus Phlebovirus, and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 5' triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I. Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses.
Collapse
Affiliation(s)
- Matthias Habjan
- Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany
| | - Nicola Penski
- Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany
| | - Martin Spiegel
- Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany
| | - Friedemann Weber
- Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany
| |
Collapse
|
|
43
|
Billecocq A, Gauliard N, Le May N, Elliott RM, Flick R, Bouloy M. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses. Virology 2008; 378:377-84. [PMID: 18614193 DOI: 10.1016/j.virol.2008.05.033] [Citation(s) in RCA: 96] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2008] [Revised: 05/28/2008] [Accepted: 05/30/2008] [Indexed: 01/06/2023]
Abstract
Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.
Collapse
Affiliation(s)
- Agnès Billecocq
- Institut Pasteur, Unité de Génétique Moléculaire des Bunyavirus, 25 rue du Dr Roux, 75724 Paris Cedex, France
| | | | | | | | | | | |
Collapse
|
|
44
|
Establishment of canine RNA polymerase I-driven reverse genetics for influenza A virus: its application for H5N1 vaccine production. J Virol 2007; 82:1605-9. [PMID: 18045936 DOI: 10.1128/jvi.01876-07] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
In the event of a new influenza pandemic, vaccines whose antigenicities match those of circulating strains must be rapidly produced. Here, we established an alternative reverse genetics system for influenza virus using the canine polymerase I (PolI) promoter sequence that works efficiently in the Madin-Darby canine kidney cell line, a cell line approved for human vaccine production. Using this system, we were able to generate H5N1 vaccine seed viruses more efficiently than can be achieved with the current system that uses the human PolI promoter in African green monkey Vero cells, thus improving pandemic vaccine production.
Collapse
|
|
45
|
Wang Z, Duke GM. Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells. Virol J 2007; 4:102. [PMID: 17956624 PMCID: PMC2241602 DOI: 10.1186/1743-422x-4-102] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2007] [Accepted: 10/23/2007] [Indexed: 12/13/2022] Open
Abstract
Background Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells. Results The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 106 to 107 plaque forming units per ml by 5 to 7 days post-transfection. Conclusion This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.
Collapse
Affiliation(s)
- Zhaoti Wang
- MedImmune, 297 North Bernardo Avenue, Mountain View, CA 94043, USA.
| | | |
Collapse
|
|
46
|
Crescenzo-Chaigne B, van der Werf S. Rescue of influenza C virus from recombinant DNA. J Virol 2007; 81:11282-9. [PMID: 17686850 PMCID: PMC2045542 DOI: 10.1128/jvi.00910-07] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2007] [Accepted: 07/30/2007] [Indexed: 01/08/2023] Open
Abstract
The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3' and 5' noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 10(3) and 10(4) PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.
Collapse
Affiliation(s)
- Bernadette Crescenzo-Chaigne
- Unité de Génétique Moléculaire des Virus Respiratoires, URA CNRS 1966, EA 302 Université Paris Diderot, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | | |
Collapse
|
|
47
|
Abstract
Newly optimized reverse genetics techniques have allowed influenza researchers to generate recombinant influenza viruses expressing mutant viral proteins, as well as foreign proteins. Approaches include the insertion of noninfluenza epitopes and polypeptides into viral glycoproteins, foreign open reading frames as additional segments, and the fusion of independent proteins into viral genes encoding glycoproteins or the nonstructural protein 1. These genetically engineered viruses have been demonstrated to be good viral vectors for mounting B- and T-cell responses and are attractive candidates for vaccine development. As the molecular biology of influenza viral infection is more fully understood, influenza vectors can be concurrently manipulated to produce designed chimeric viruses, unveiling the possibility of a prosperous future with cheap, effective and safe vaccines against different human diseases.
Collapse
Affiliation(s)
- Luis Martínez-Sobrido
- Mount Sinai School of Medicine, Department of Microbiology, Emerging Pathogens Institute, 1 Gustave L Levy Place, Box #1124, NY 10029, USA
| | - Adolfo García-Sastre
- Mount Sinai School of Medicine, Department of Microbiology, Emerging Pathogens Institute & Department of Medicine, Division of Infectious Diseases, 1 Gustave L Levy Place, Box #1124, NY 10029, USA
| |
Collapse
|