Residues 315 and 369 in HN Protein Contribute to the Thermostability of Newcastle Disease Virus

Abstract

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.

Identification of an additional N-glycosylation site and Thermostable mutations within the Hemagglutinin-Neuraminidase gene of the Newcastle disease virus belonging to the VII.1.1 subgenotype.

“…2, the results demonstrated mutations S315P, I369V, and V369A among the VII.1.1 isolates. These results are consistent with the recent study [23], which revealed that residues 315, 329, and 369, especially mutations S315P and I369V, could signi cantly enhance the viral thermostability, HA, and NA activities.…”

Avian Paramyxovirus Type 1 in Egypt: Epidemiology, Evolutionary Perspective, and Vaccine Approach

“…Due to the intensive unplanned vaccination in Egypt, selection/vaccination pressure could explain the occurrence of such aa substitutions in the epitopes of Egyptian NDV HN overtime, which in turn may affect, to different levels, the vaccine efficacy, induced immunity, and virus shedding in birds as a result of antigenic variability (108). HN protein is a determinant viral factor for thermostability, as mutant NDV viruses (S315P and I369V) were more thermostable and possessed more HA titers and NA fusion activity (109).…”

Generation and Evaluation of Recombinant Thermostable Newcastle Disease Virus Expressing the HA of H9N2 Avian Influenza Virus

“…To achieve this goal, a thermostable NDV strain is necessary. Previously, a thermostable NDV HR09 strain, which could stay infectious for more than 100 min under 56 °C, was attenuated using a reverse genetic system in our lab [ 15 , 16 , 41 ]. Compared with the widely used La Sota strain, the attenuated rAHR09 strain could efficiently reduce the NDV virus shedding and provide full protection against the challenge of NDV [ 15 ].…”

“…Previously, one thermostable NDV was isolated in our lab, designated HR09, which could remain infectious for more than 1 hour under 56 °C treatment [ 15 ]. Through exchanging the proteins of thermostable HR09 and the La Sota strain, it was found that the residues 315 and 369 in the HN protein were responsible for the thermostability of HR09 [ 16 ]. The thermostable HR09 strain, which belongs to genotype VIII, is a mesogenic NDV strain.…”