リバースジェネティクス法を用いた新遺伝子型ＲＳウイルス（ＯＮ１）の増殖能の解析

我在2013年、2014年和2015年的三个季节分析了菲律宾碧兰岛HRSV的分子特征。鼻咽拭子取自碧兰省医院重症肺炎住院患儿。采用实时定量聚合酶链式反应(PCR)对样本进行筛选。采用常规聚合酶链式反应和G基因第二高变区(HVR)测序方法进行基因分型。在1240份检测样本中，检出HRSV阳性356份(28.7%)，其中HRSV-A 183份(51.4%)，HRSV-B 173份(48.6%)。进一步将HRSV-A分为两种基因型：NA1和on1。NA1和ON1在2013年是共同循环的，只有ON1在2014年仍然在循环。2014年发现了一种新的on1变异株，它在系统发育上与其他on1菌株不同。这些新的ON1病毒在G基因的第二个HVR中存在三个独特的氨基酸替换(K212R、K213G和K223G)，这在其他国家还没有发现。除两份标本鉴定为GB2外，其余均为BA9型。2013、2014两季均为A、B两型同时流行，但2015年季节检出的病毒以B型为主。G基因第2个HVR的大部分氨基酸位点处于纯化/负选择状态。本研究为菲律宾地区HRSV的分子遗传学研究奠定了基础。此外，我去年还在菲律宾制作了甲型HRSV病毒全基因组序列的手稿。

I analyzed molecular characterization of HRSV in Biliran Island, Philippines over three seasons in 2013, 2014, and 2015. Nasopharyngeal swabs were collected from hospitalized children with severe pneumonia at the Biliran Provincial Hospital. Samples were screened by using real-time polymerase chain reaction (PCR). Genotypes were determined using conventional PCR and sequencing of the 2nd hypervariable region (HVR) of the G gene. Of 1,240 sample tested, 356 (28.7%) were positive for HRSV including 183 (51.4%) HRSV-A and 173 (48.6%) HRSV-B. HRSV-A were further divided into two genotypes; NA1 and ON1. Both NA1 and ON1 were co-circulating in 2013, only ON1 remained to be circulating in 2014. A novel ON1 variant, which was phylogenetically distinct from other ON1 strains, was detected in 2014. These novel ON1 viruses possess three unique amino acid substitutions (K212R, K213G, and K223G) in the 2nd HVR of the G gene, which had not been found in other countries. All HRSV-B viruses were identified as genotype BA9 except two samples identified as GB2. Both HRSV-A and -B were co-circulating in 2013 and 2014 seasons, but most of HRSV detected in 2015 season were HRSV-B. Most of amino acid sites of the 2nd HVR of the G gene were under purifying/negative selection. This study can build on the molecular characterization to contribute the advance of molecular genetics on HRSV in the Philippines. Moreover, I made the manuscript of complete genome sequences of HRSV-A in the Philippines that I did last year.