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

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.

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

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.

Evaluation of Limit of Detection and Range of Quantitation for RT-PCR, Real-Time RT-PCR and RT-PCR-ELISA Detection of Bovine Viral Diarrhoea Virus Contamination in Biologics Derived from Cell Cultures

Risk of viral contamination is one of major concerns common to all biologics derived from cultivated cells. Bovine viral diarrhoea virus (BVDV) has widely been known as a contaminant of cell culture-derived vaccines. The objective of the study was to assess the limit of detection and range of quantitation of the detection methods for BVDV using a reverse transcription-polymerase chain reaction (RT-PCR) assay, real-time RT-PCR assay, and RT-PCR-ELISA. One milliliter of cell culture supernatant containing 106.5+/-0.2 median tissue culture infectious dose (TCID50)/ml of BVDV NADL strain was subjected to RNA isolation. The isolated RNA was 10-fold serially diluted and each diluted sample (10-1 to 10-6) was subjected to RT-PCR on a GeneAmpR PCR System 9700 and/or LightCycler(TM). The amplified products were analyzedly (1) agarose gel electrophoresis for RT-PCR assay, (2) melting curve analysis for real-time RT-PCR assay (in this case a program is automatically linked to amplification step), and (3) ELISA using capture and detection probes for RT-PCR-ELISA. The limit of detection of the 3 assay methods was equally estimated to be 316 TCID50/ml of starting virus culture supernatant subjected to the assay. The quantitation range of real-time RT-PCR assay and RT-PCR-ELISA was estimated to be from 3.16x105 to 3.16x102 TCID50/ml of starting virus culture supernatant. The overall results suggested that the 3 assay methods for BVDV detection can be reliably applied to evaluate BVDV contamination in biologics derived from cell cultures.

Development of One-step Real-time Reverse Transcription-polymerase Chain Reaction in Combination with Automated RNA Extraction for Detection and Quantitation of Hepatitis A Virus

One-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using the MagNA Pure LC and LightCycler(TM) system was developed and validated for the detection and quantitation of hepatitis A virus (HAV) RNA. The assay was evaluated using in-house synthetic HAV RNA standard. The real-time RT-PCR assay could quantitate a dynamic range of HAV RNA standard between 10(2) and 10(8) copies per reaction. The regression coefficient of the standard curve was an 0.99. The detection limit of the assay was 31.3 RNA copies per reaction. The coefficient variations (CVs) of the assay in combination with automated RNA extraction were less than 1.91% in both intra- and inter-assay. The real-time RT-PCR assay for quantitative detection of HAV would serve a useful method for improving the safety of biological products.

Study on Variation of Endemic Mumps Viruses in Korea

No Abstract Available.