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Sequence comparison of three infectious molecular clones of RD-114 virus
Sayumi Shimode1, Rokusuke Yoshikawa, Shigeki Hoshino
1Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan.
Abstract:
RD-114 virus is a replication-competent feline endogenous retrovirus. RD-114 virus contaminates several feline and canine live attenuated vaccines and the issue of contamination of RD-114 virus in vaccines should be solved. To date, three infectious molecular clones (pSc3c, pCRT1, and pRD-UCL) have been reported. In this study, we sequenced the entire nucleotide sequence of pRD-UCL and compared the nucleotide sequences of the three infectious molecular clones. As a result, these three infectious clones were nearly identical with each other in gag-pol and env coding regions. These data support the notion that the active locus of infectious RD-114 virus is single in the feline genome. The length of long terminal repeat (LTR) of pCRT1 was 47 bp shorter than those of pSc3c and pRD-UCL. The 47-bp sequence named direct repeat A (DR-A) was duplicated in the U3 region in pSc3c and pRD-UCL. Although several potential enhancer binding sites are present in the DR-A, there was no significant difference in promoter activities between the LTRs of pRD-UCL and pCRT1 in two human cell lines. We also analyzed the splicing pattern of the RD-114 virus by reverse transcription-polymerase chain reaction and confirmed that RD-114 virus is a simple retrovirus. The data presented here will provide basic information about RD-114 virus to solve the contamination issue in live attenuated vaccines.
Insights
RD-114 virus, a feline retrovirus, contaminates vaccines. Comparing three clones revealed near-identical coding regions, suggesting a single active locus. This research aids in resolving vaccine contamination issues.
Area of Science:
- Virology
- Molecular Biology
- Genomics
Background:
- RD-114 virus is a replication-competent feline endogenous retrovirus.
- Contamination of RD-114 virus in live attenuated vaccines for felines and canines is a significant issue.
- Three infectious molecular clones (pSc3c, pCRT1, pRD-UCL) of RD-114 virus have been previously reported.
Purpose of the Study:
- To sequence and analyze the pRD-UCL molecular clone of RD-114 virus.
- To compare the nucleotide sequences of three reported infectious molecular clones of RD-114 virus.
- To provide foundational data for addressing RD-114 virus contamination in vaccines.
Main Methods:
- Full nucleotide sequencing of the pRD-UCL molecular clone.
- Comparative sequence analysis of pSc3c, pCRT1, and pRD-UCL.
- Reverse transcription-polymerase chain reaction (RT-PCR) to analyze splicing patterns.
- Promoter activity assays of LTRs in human cell lines.
Main Results:
- The gag-pol and env coding regions of the three clones were nearly identical, supporting a single active locus for RD-114 virus in the feline genome.
- The long terminal repeat (LTR) of pCRT1 was 47 bp shorter than pSc3c and pRD-UCL due to the absence of a direct repeat A (DR-A) sequence.
- No significant difference in promoter activity was observed between the LTRs of pRD-UCL and pCRT1, despite potential enhancer sites in DR-A.
- RT-PCR confirmed RD-114 virus exhibits a simple retrovirus splicing pattern.
Conclusions:
- The genetic similarity across coding regions suggests a single origin for infectious RD-114 virus.
- Understanding LTR variations and promoter activity is crucial for assessing viral replication and potential contamination.
- This study provides essential molecular information to help resolve RD-114 virus contamination issues in live attenuated vaccines.
