US Army Medical Research Unit-Kenya, Village Market, 00621 Nairobi, Kenya.
J Infect Dis. 2012 Dec 15;206 Suppl 1:S46-52. doi: 10.1093/infdis/jis586.
Among influenza viruses, type A viruses exhibit the greatest genetic diversity, infect the widest range of host species, and cause the vast majority of cases of severe disease in humans, including cases during the great pandemics. The hemagglutinin 1 (HA1) domain of the HA protein contains the highest concentration of epitopes and, correspondingly, experiences the most intense positive selection pressure.
We sought to isolate and genetically characterize influenza A virus subtype H1N1 (A[H1N1]) circulating in Kenya during 2007-2008, using the HA1 protein.
Nasopharyngeal swab specimens were collected from patients aged ≥ 2 months who presented to 8 healthcare facilities in Kenya with influenza-like illness. We tested specimens for seasonal influenza A viruses, using real-time reverse-transcription polymerase chain reaction (RT-PCR). Viruses were subtyped using subtype-specific primers. Specimens positive for seasonal A(H1N1) were inoculated onto Madin-Darby canine kidney cells for virus isolation. Viral RNAs were extracted from isolates, and the HA1 gene was amplified by RT-PCR, followed by nucleotide sequencing. Nucleotide sequences were assembled using BioEdit and translated into amino acid codes, using DS Gene, version 1.5. Multiple sequence alignments were performed using MUSCLE, version 3.6, and phylogenetic analysis was performed using MrBayes software.
We found that, similar to A/Brisbane/59/2007 (H1N1)-like virus, which was included in the southern hemisphere vaccine for the 2009 influenza season, all 2007 Kenyan viruses had D39N, R77K, T132V, K149R, and E277K amino acid substitutions, compared with A/Solomon Islands/3/2006 (H1N1)-like virus, a component of the southern hemisphere vaccine for the 2008 influenza season. However, the majority of 2008 viruses from Kenya also had R192K and R226Q substitutions, compared with A/Solomon Islands/3/2006 (H1N1)-like virus. These 2 changes occurred at the receptor binding site. The majority of the 2008 Kenyan isolates contained N187S, G189N, and A193T mutations, which differed from A/Brisbane/59/2007 (H1N1)-like virus. The A193T substitution is involved in binding the sialic acid receptor. Phylogenetically, the 2008 Kenyan isolates grouped into 2 clusters. The main cluster contained viruses with N187S and A193T changes; residue 187 is involved in receptor binding, whereas residue 193 is at antigenic site Sb.
Overall, the major genetic variations that occurred in seasonal A(H1) viruses either affected receptor binding or altered epitopes at the immunodominant sites. These genetic variations in seasonal A(H1N1) isolated in Kenya during 2007-2008 highlight the importance of continuing surveillance and characterization of emerging drift variants of influenza virus in Africa.
在流感病毒中，A型病毒表现出最大的遗传多样性，感染宿主物种的范围最广，并且在人类中引起绝大多数严重疾病病例，包括大流行期间的病例。血凝素 1（HA1）蛋白的 HA 蛋白包含最高浓度的抗原表位，相应地，经历了最强烈的正选择压力。
我们试图使用 HA1 蛋白分离和遗传特征化 2007-2008 年在肯尼亚循环的甲型流感病毒亚型 H1N1（A[H1N1]）。
从年龄≥2 个月的患有流感样疾病的 8 家肯尼亚医疗机构就诊的患者中采集鼻咽拭子标本。我们使用实时逆转录聚合酶链反应（RT-PCR）检测季节性流感 A 病毒。使用亚型特异性引物对病毒进行亚型鉴定。对季节性 A（H1N1）阳性的标本进行接种，在 Madin-Darby 犬肾细胞中进行病毒分离。从分离物中提取病毒 RNA，通过 RT-PCR 扩增 HA1 基因，然后进行核苷酸测序。使用 BioEdit 组装核苷酸序列，并使用 DS Gene，版本 1.5 将其翻译成氨基酸密码子。使用 MUSCLE，版本 3.6 进行多序列比对，并使用 MrBayes 软件进行系统发育分析。
我们发现，与包括在 2009 年流感季节南半球疫苗中的 A/Brisbane/59/2007（H1N1）样病毒相似，所有 2007 年肯尼亚病毒都具有与 A/Solomon Islands/3/2006（H1N1）样病毒相比的 D39N、R77K、T132V、K149R 和 E277K 氨基酸取代，A/Solomon Islands/3/2006（H1N1）样病毒是 2008 年南半球疫苗的一部分。然而，与 A/Solomon Islands/3/2006（H1N1）样病毒相比，大多数 2008 年肯尼亚病毒也具有 R192K 和 R226Q 取代。这两个变化发生在受体结合位点。大多数 2008 年肯尼亚分离株含有 N187S、G189N 和 A193T 突变，与 A/Brisbane/59/2007（H1N1）样病毒不同。A193T 取代涉及与唾液酸受体的结合。系统发育分析表明，2008 年肯尼亚分离株分为 2 个聚类。主要聚类包含具有 N187S 和 A193T 变化的病毒；残基 187 参与受体结合，而残基 193 位于抗原位点 Sb。
总的来说，季节性 A（H1）病毒中发生的主要遗传变异要么影响受体结合，要么改变免疫显性位点的抗原表位。2007-2008 年在肯尼亚分离的季节性 A（H1N1）中的这些遗传变异突出表明，继续监测和描述非洲流感病毒新出现的漂移变体的重要性。
