Ruan Baoyang, Zhang Xiaorong, Zhang Chengcheng, Du Pengyu, Meng Chengcheng, Guo Mengjiao, Wu Yantao, Cao Yongzhong
College of Veterinary Medicine, Yangzhou University, Yangzhou, China.
The Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou, China.
Front Microbiol. 2020 Sep 22;11:560482. doi: 10.3389/fmicb.2020.560482. eCollection 2020.
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.
耐热新城疫病毒（NDV）疫苗已在“冷链”不可靠的地区广泛使用。然而，NDV耐热性的分子机制仍知之甚少。在这项工作中，我们利用反向遗传系统，通过在耐热株HR09和热不稳定株La Sota之间交换病毒融合（F）和/或血凝素神经氨酸酶（HN）基因构建嵌合病毒。结果表明，只有源自耐热病毒的HN嵌合体在56°C时表现出耐热表型。源自HR09株的HN嵌合体的血凝素（HA）和神经氨酸酶（NA）活性比含有La Sota株HN的嵌合体更耐热。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株的HN蛋白。观察到一个氨基酸区域（残基315 - 375）的构象波动。HN蛋白的序列比对显示，La Sota株和耐热株中的残基315、329和369不同。研究了这三个氨基酸取代是否影响病毒耐热性。通过在HN蛋白的315、369和329位取代一个、两个或三个氨基酸，基于热不稳定株产生了三种突变病毒。与亲本病毒相比，含有S315P和I369V突变的突变病毒具有更高的耐热性、HA滴度、NA和融合活性。综上所述，这些数据表明NDV的HN基因是耐热性的主要决定因素，残基315和369对病毒耐热性有重要影响。
