| 研究実績の概要 |
The molecular analysis of Human respiratory syncytial virus (HRSV) in the Philippines 2012-2013 has been published in PLOS ONE. Among HRSV-A, ON1 strain with 72 nucleotide duplication in the G gene emerged, increased, and became the predominant strain with higher evolutionary rate than the NA1 strain with non-duplication while BA9 strain of HRSV-B were circulating during that time. Moreover, emerging of GB2 strains of HRSV-B might share the original ancestor with other GB2 strains circulating in Asia.
Whole genome sequencing of both types (the duplication type is ON1 strain; the non-duplication type is NA1 strain) was done by using the isolate strains representing of 7 ON1 and 6 NA1 strains in order to elucidate evolutionary characteristics by analyzing the complete genomic sequences of HRSV-A strains isolated in the Philippines and from other parts of the world. The highest number of mutation was observed in G gene. Interestingly, Y598H found in L protein of Philippine ON1 strains were observed in ON1 strains over the world (85%) while no observed in the other genotypes (GA1, GA2, GA5, and GA7). This finding may contribute to a better understanding of genomic diversity of HRSV-A circulating in the Philippines and all over the world.
Regarding to reverse genetics of HRSV, we successfully constructed the four kinds of helper plasmids which are important for replicating of HRSV-BAC vector after transfecting into host cells.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
During this time, I keep trying hard to construct the HRSV-BAC without 72 nucleotide duplication in G gene representing as NA1 strain. I designed to split the whole-length of NA1 strain to 8 fragments (A-H) and cloned each fragment into pGEM-T Easy vector which is the shuttle vector to obtain the whole-length cDNA clones. Then, each fragment will be cloned sequentially in the following order: H, G, C, D, F, E, B, and A in the correct site of the BAC vector (pBeloBAC11) to protect whole-length cDNA cut by restriction enzymes in the other site and generate the HRSV-BAC without 72 nucleotide duplication. Until now, I am cloning the third fragment and keep going on it. I am suffering with many problems. For examples, the bigger size of BAC vector carrying the large insert, the more difficult of cloning into competent cells. BAC vector, a single copy vector, can be time-consuming to propagate for getting the high yield of BAC vector. The big size of BAC DNA constructs are constructs are easily sheared and degraded during manipulation. Even I am suffering with many problems, I will try my best as much as I can.
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| 今後の研究の推進方策 |
If I get the HRSV-BAC without 72 nucleotide duplication, I will modify the sequence at the 2nd hypervariable region of G gene to generate the HRSV-BAC with 72 nucleotide duplication. Then, I will try to generate the BAC DNA constructs carrying the reporter gene can be quantitative detected by using Luciferase assays. After that, I will try to co-transfection of HRSV-BAC vector and helper plasmids into host cells to get the chimeric viruses. The chimeric viruses that I get will be tested for plaque assay, growth kinetics, G protein expression which compare between the chimeric RS virus without 72 nucleotide duplication and the chimeric RS virus with 72 nucleotide duplication.
For plaque assay, I optimized the condition of plaque assay for determining titers of HRSVs by using the ON1 or NA1 isolates for testing further with the chimeric viruses. Now, I am trying to compare the growth kinetics between ON1 and NA1 isolates to get the basic information about growth kinetics before I get the chimeric viruses.
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