Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector

Zhang, Fuquan; Huang, Qingsheng; Ma, Wei; Jiang, Shaozhun; Fan, Yinglun; Zhang, Hongyi

doi:10.1016/s0166-0934(01)00331-7

Cited by 32 publications

(21 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous attempts to develop such a system (23,38,39,51), including our own, were thwarted by the instability of the cloned JEV cDNA. The system described in this paper will greatly facilitate the study of the molecular mechanisms of replication, virulence, and pathogenesis employed by the virus.…”

Section: Discussionmentioning

confidence: 99%

“…A variety of vector systems were used previously in attempts to assemble a full-length JEV cDNA (23,38,39,51), including a cosmid vector in E. coli (51). In all cases, the cloned cDNA became genetically unstable during its propagation in a host.…”

Section: Discussionmentioning

confidence: 99%

“…However, the construction of a full-length infectious cDNA clone for Japanese encephalitis virus (JEV) has been hampered, largely because of the genetic instability of the cloned cDNA. Despite extensive efforts (23,38,39,51), a genetically stable full-length infectious cDNA molecular clone for JEV does not exist.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

Yun

Kim

Rice

et al. 2003

J Virol

117

128

View full text Add to dashboard Cite

Japanese encephalitis virus (JEV) is a common agent of viral encephalitis that causes high mortality and morbidity among children. Molecular genetic studies of JEV are hampered by the lack of a genetically stable full-length infectious JEV cDNA clone. We describe here the development of such a clone. A JEV isolate was fully sequenced, and then its full-length cDNA was cloned into a bacterial artificial chromosome. This was then further engineered so that transcription of the cDNA in vitro would generate synthetic RNAs with authentic 5 and 3 ends. The synthetic RNAs thus produced were highly infectious in susceptible cells (>10 6 PFU/g), and these cells rapidly generated a high titer of synthetic viruses (>5 ؋ 10 6 PFU/ml). The recovered viruses were indistinguishable from the parental virus in terms of plaque morphology, growth kinetics, RNA accumulation, protein expression, and cytopathogenicity. Significantly, the structural and functional integrity of the cDNA was maintained even after 180 generations of growth in Escherichia coli. A single point mutation acting as a genetic marker was introduced into the cDNA and was found in the genome of the recovered virus, indicating that the cDNA can be manipulated. Furthermore, we showed that JEV is an attractive vector for the expression of heterologous genes in a wide variety of cell types. This novel reverse genetics system for JEV will greatly facilitate research into JEV biology. It will also be useful as a heterologous gene expression vector and will aid the development of a vaccine against JEV.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

Yun

Kim

Rice

et al. 2003

J Virol

117

128

View full text Add to dashboard Cite

show abstract

“…Long PCR amplification protocols were first developed by Barnes 26 and Cheng 27 to study large human and bacteriophage genes. Long PCR amplifications are now involved in numerous biological molecular studies: constructions of viral infectious clones [28][29][30] ; cloning and expression of large-protein genes 31 for structural or functional studies; replacement of the standard subcloning strategy 32 ; and site-directed mutagenesis 33,34 . Sequence determination of the cloned DNA is crucial to such applications for which LoPPS may be a convenient tool.…”

Section: Applications Of the Lopps Techniquementioning

confidence: 99%

Long PCR Product Sequencing (LoPPS): a shotgun-based approach to sequence long PCR products

et al. 2007

View full text Add to dashboard Cite

Here we describe a practical procedure for sequencing long PCR products. The method relies on ultrasonic shearing of PCR products, resulting in fragments 700-1,000 nt long. Termini are subsequently repaired to obtain blunt ends and 3¢ A-overhangs are added before TA cloning. A predetermined number of clones are sequenced using an insert-independent primer to obtain an overlapping contig covering the full length of the PCR product. This method is cost effective and enables the complete sequencing of any large PCR product in a high-throughput format. Processing of amplified DNA requires 3 h handling time prior to the ligation step, and the clone library is available 2 d later. The complete sequence information is obtained approximately 5 d after the PCR step, depending on the sequencing procedure adopted.

show abstract

“…A national JE vaccination program has successfully controlled the disease and swine vaccination can prevent disease in swine and help to reduce JE infection in humans [1][2][3] . Recently, molecular biology studies of this virus have provided a better way for the development of several viral vaccines [1][2][3][4][5][6][7][8][9] . The Japanese encephalitis virus (JEV) is a member of the genus fl avivirus in the family Flaviviridae.…”

Section: Introductionmentioning

confidence: 99%

Programmed Cell Death Induced by Japanese Encephalitis Virus YL Vaccine Strain or Its Recombinant Envelope Protein in Varied Cultured Cells

Chen

Chang²,

Stone³

et al. 2006

Intervirology

View full text Add to dashboard Cite

Objective: The Japanese encephalitis virus YL vaccine strain (JEV-YL) was investigated as regards its organ tropism and the role of recombinant envelope glycoprotein in the induction of apoptosis was explored. Methods: Threevaried cell lines (HepG2, Vero and C6) were infected with JEV-YL or transfected with eukaryotic expression plasmids (pcE, pcF1R2, pcF1R1 and pcF2R2) which contain different parts of the envelope gene and phenotypic properties were examined by flow cytometry and DNA fragmentation analysis. Results: After JEV-YL infection, smaller plaque was produced on HepG2 cells than on Vero cells, whereas no cytopathic effect was observed on C6 cells; moreover, by apoptosis and DNA fragmentation assays, the hallmark cytopathic effects were detected in HepG2 and Vero cells but not in C6 cells. Furthermore, cells used in our study transfected with recombinant core plasmid, pcE, which include full-length E gene but the deleted forms (pcF1R2, pcF1R1 and pcF2R2) did not have similar results as JEV-YLs. Conclusions: The JEV-YL vaccine strain had changed cell tropism to liver cells different from other virulent strains which have neural tropism, and in this study we proved that the transient-expressed entire E protein of JEV-YL could induce apoptosis and the mutations of E protein may change the organ tropism of JEV-YL.

show abstract

Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector

Cited by 32 publications

References 32 publications

Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

Long PCR Product Sequencing (LoPPS): a shotgun-based approach to sequence long PCR products

Programmed Cell Death Induced by Japanese Encephalitis Virus YL Vaccine Strain or Its Recombinant Envelope Protein in Varied Cultured Cells

Contact Info

Product

Resources

About