J Bacteriol Virol.  2004 Dec;34(4):339-353.

Construction of A Stable Full-Length cDNA Clone of Japanese Encephalitis Virus Strain SA14-14-2 Using Low Copy Number Plasmid

• Min KI
• Kim YM
• Choo MS
• Baek SY
• Kim JO
• Ryu SR
• Min BS
• Kim Y
• Park MK
• Byeon WH
• Hur SJ

Abstract

Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.

Keyword

Japanese encephalitis virus; Full-length infectious cDNA clone; SA14-14-2

MeSH Terms

Asian Continental Ancestry Group*
Cell Culture Techniques
Clone Cells*
DNA
DNA, Complementary*
Encephalitis Virus, Japanese*
Encephalitis, Japanese*
Escherichia coli
Family Characteristics
Genetic Markers
Genome
Humans
Kinetics
Parents
Plasmids*
Replication Origin
Reverse Genetics
RNA
RNA Viruses
RNA, Viral
Vaccines, Synthetic
Virulence
DNA
DNA, Complementary
Genetic Markers
RNA
RNA, Viral
Vaccines, Synthetic