W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Department of Otolaryngology - Head and Neck Surgery, Johns Hopkins Outpatient Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Vaccine. 2017 Dec 4;35(48 Pt B):6691-6699. doi: 10.1016/j.vaccine.2017.10.018. Epub 2017 Oct 25.
The influenza A virus components of the live, attenuated influenza vaccine (LAIV) encode the HA and NA gene segments from a circulating virus strain and the remaining gene segments from the cold-adapted master donor virus, A/Ann Arbor/6/1960 (H2N2). The master donor virus imparts at least three phenotypes: temperature-sensitivity (ts), attenuation (att), and cold-adaption (ca). The genetic loci responsible for the att and ts phenotypes of LAIV were mapped to PB1, PB2, and NP by reverse genetics experiments using immortalized cell lines. However, some in vivo studies have demonstrated that the M segment, which acquired an alanine (Ala) to serine (Ser) mutation at M2 position 86 during cold adaption - a mutation found in no other influenza A virus strain - contributes to the att phenotype. Prior studies have shown this region of the M2 cytoplasmic tail to be critical for influenza virus replication. Using reverse genetics, we demonstrate that certain amino acid substitutions at M2 positions 83 and 86 alter the replication of influenza A/Udorn/307/72 (H3N2). Importantly, substitution of a Ser at M2 position 86 reduces A/Udorn/307/72 replication in differentiated primary human nasal epithelial cell (hNECs) cultures, but does not considerably affect replication in MDCK cells. When a Ser was substituted for Ala at M2 86 in LAIV, the virus replicated to higher titers and with faster kinetics in hNEC cultures, implicating this amino acid change as contributing to LAIV attenuation. Increased replication also resulted in increased production of IFN-λ. These data indicate the LAIV associated Ser mutation at M2 position 86 contributes to the att phenotype and is associated with a differential regulation of interferon in LAIV infection.
流感 A 病毒成分的活疫苗（LAIV）减毒编码血凝素（HA）和神经氨酸酶（NA）基因片段从一个流行的病毒株和剩余的基因片段从冷适应主供体病毒，A/安纳堡/6/1960（H2N2）。主供体病毒赋予至少三个表型：温度敏感性（ts），衰减（att）和冷适应（ca）。用传代细胞系进行反向遗传学实验，将 LAIV 的 att 和 ts 表型的遗传基因座定位到 PB1、PB2 和 NP。然而，一些体内研究表明，M 片段在冷适应过程中获得了 M2 位置 86 处的丙氨酸（Ala）到丝氨酸（Ser）的突变 - 在其他流感 A 病毒株中未发现的突变 - 有助于 att 表型。先前的研究表明，M2 细胞质尾巴的这一区域对于流感病毒的复制至关重要。我们使用反向遗传学证明，M2 位置 83 和 86 的某些氨基酸取代改变了流感 A/Udorn/307/72（H3N2）的复制。重要的是，M2 位置 86 处 Ser 的取代降低了 A/Udorn/307/72 在分化的原代人鼻上皮细胞（hNECs）培养物中的复制，但对 MDCK 细胞中的复制没有明显影响。当 LAIV 中的 M2 86 处的 Ala 被 Ser 取代时，病毒在 hNEC 培养物中的复制滴度更高，动力学更快，这表明这种氨基酸变化有助于 LAIV 衰减。增加的复制也导致 IFN-λ 的产生增加。这些数据表明，LAIV 相关的 M2 位置 86 处的 Ser 突变有助于 att 表型，并与 LAIV 感染中干扰素的差异调节有关。
