Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
J Virol. 2011 Feb;85(4):1834-46. doi: 10.1128/JVI.01648-10. Epub 2010 Dec 1.
The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.
H5N1 高致病性禽流感（HPAI）病毒在鸡体内的致病性的分子基础在很大程度上仍然未知。H5N1 A/鸡/山口/2004 病毒（CkYM7）在鸡的巨噬细胞和血管内皮细胞中快速复制，导致突然死亡而无发热或明显病变，而 H5N1 A/鸭/横滨/ aq10/2003 病毒（DkYK10）引起高热、严重的大体病变和延长的死亡时间，尽管这两种病毒的氨基酸序列有 98%的同源性。为了探索这种致病性差异的分子基础，对来自这些 HPAI 病毒的一系列 8 种单基因重配病毒进行了比较，以研究其在鸡体内的致病性。在 CkYM7 背景下具有 DkYK10 的 NP 或 PB2 基因的两种重配病毒与其他重配病毒或 CkYM7 相比，致病性降低。相反，在 DkYK10 背景下具有 CkYM7 的 NP 或 PB2 基因的重配病毒（rgDkYK-PB2(Ck)、rgDkYK-NP(Ck)）复制迅速，达到更高的滴度，并且伴随着巨噬细胞更快、更频繁的凋亡。rgDkYK-NP(Ck) 和 rgDkYK-PB2(Ck) 重配病毒在鸡胚成纤维细胞（CEFs）中的复制速度也比 rgDkYK10 更快，但这些病毒的复制与 CkYM7 和 DkYK10 在鸭胚成纤维细胞中的复制相似。比较了具有 NP(Dk) 中单个氨基酸取代的 7 种 rgDkYK10 突变体的致病性，表明 NP(Ck) 中的第 105 位缬氨酸是导致其在鸡中致病性增加的原因。NP(Ck)、NP(105V) 和 PB2(Ck) 增强了 DkYK10 在 CEFs 中的聚合酶活性。这些结果表明，NP 和 PB2 都有助于 H5N1 HPAI 病毒在鸡中的高致病性，NP 第 105 位的缬氨酸可能是禽流感病毒从鸭适应到鸡的决定因素之一。
