Thermostable Newcastle disease virus (NDV) vaccines have been widely used in areas where a “cold-chain” is not reliable. However, the molecular mechanism of NDV thermostability remains poorly understood. In this work, we constructed chimeric viruses by exchanging viral fusion (F) and/or hemagglutinin-neuraminidase (HN) genes between the heat-resistant strain HR09 and thermolabile strain La Sota utilizing a reverse genetic system. The results showed that only chimeras with HN derived from the thermostable virus exhibited a thermostable phenotype at 56°C. The hemagglutinin (HA) and neuraminidase (NA) activities of chimeras with HN derived from the HR09 strain were more thermostable than those containing HN from the La Sota strain. Then, we used molecular dynamics simulation at different temperatures (310 K and 330 K) to measure the HN protein of the La Sota strain. The conformation of an amino acid region (residues 315–375) was observed to fluctuate. Sequence alignment of the HN protein revealed that residues 315, 329, and 369 in the La Sota strain and thermostable strains differed. Whether the three amino acid substitutions affected viral thermostability was investigated. Three mutant viruses based on the thermolabile strain were generated by substituting one, two or three amino acids at positions 315, 369, and 329 in the HN protein. In comparison with the parental virus, the mutant viruses containing mutations S315P and I369V possessed higher thermostablity and HA titers, NA and fusion activities. Taken together, these data indicate that the HN gene of NDV is a major determinant of thermostability, and residues 315 and 369 have important effects on viral thermostability.

耐高温的新城疫病毒（NDV）疫苗已广泛用于“冷链”不可靠的地区。然而，对NDV热稳定性的分子机理仍知之甚少。在这项工作中，我们通过利用反向遗传系统在耐热菌株HR09和不耐热菌株La Sota之间交换病毒融合（F）和/或血凝素神经氨酸酶（HN）基因来构建嵌合病毒。结果表明，仅具有衍生自热稳定病毒的HN的嵌合体在56℃下表现出热稳定表型。含有来自HR09菌株的HN的嵌合体的血凝素（HA）和神经氨酸酶（NA）活性比含有La Sota菌株的HN的嵌合体更热稳定。然后，我们在不同温度（310 K和330 K）下使用分子动力学模拟来测量La Sota菌株的HN蛋白。观察到氨基酸区域（残基315-375）的构象发生波动。HN蛋白的序列比对表明，La Sota菌株和热稳定菌株中的315、329和369位残基不同。研究了三个氨基酸取代是否影响病毒的热稳定性。通过在HN蛋白的315、369和329位取代一个，两个或三个氨基酸，生成了三种基于热不稳定菌株的突变病毒。与亲本病毒相比，含有突变S315P和I369V的突变病毒具有更高的热稳定性和HA滴度，NA和融合活性。在一起