Thermostability study of virulent Newcastle disease viruses isolated in Southern Angola

Abstract

Newcastle disease (ND) is endemic in Angola. Several outbreaks of ND occurred in small backyard flocks and village chickens with high mortality in the southern provinces of the country, Cunene, Namibe and Huíla, in 2016 and 2018. In those years, 15 virulent ND virus (NDV) strains were isolated and grouped within subgenotype 2 of genotype VII (subgenotype VII.2). We now present a study on the thermostability of the isolates, aiming at the selection of the most thermostable strains that, after being genetically modified to reduce their virulence, can be adapted to the production of vaccines less dependent on cold chain and more adequate to protect native chickens against ND. Heat-inactivation kinetics of haemagglutinin (Ha) activity and infectivity (I) of the isolates were determined by incubating aliquots of virus at 56 °C for different time intervals. The two isolates from Namibe province showed a decrease in infectivity of 2 log10 in ≤ 10 min, therefore belonging to the I-phenotype, but while the NB1 isolate from 2016 maintained the Ha activity up to 30 min and was classified as thermostable virus (I-Ha+), the Ha activity of the 2018 NB2 isolate decreased by 2 log2 in 30 min, being classified as a thermolabile virus (I-Ha-). Of the 13 NDV isolates from Huíla province, 10 isolates were classified as thermostable, eight with phenotype I+Ha+ and 2 with phenotype I-Ha+. The other three isolates from this province were classified as thermolabile viruses (I-Ha-).Contribution: This study will contribute to the control and/or eradication of Newcastle disease virus in Angola. The thermostable viral strains isolated from chickens in the country can be genetically manipulated by reverse genetic technology in order to reduce their virulence and use them as a vaccine in the remote areas of Angola.

Keywords: Angola; Newcastle disease virus; haemagglutinin activity; infectivity; thermostability.

FIGURE 1

Location where samples were collected from chickens in Huíla, Namibe and Cunene provinces for virus screening. The number of samples are indicated in black, while numbers in red correspond to NDV positive samples.

FIGURE 2

Heat-inactivation kinetics of Ha activity (▲) and infectivity (●) of the NDV isolates determined at 56 °C. The thermostability of NDV isolates is shown as the mean time required for 2 log₂ and 2 log₁₀ decreases in Ha activity and infectivity, respectively. The rate constants K(Ha) and K(I) are indicated as a measure of the exponential decline in activity of Ha and infectivity, respectively. Data shown represented the average of two independent experiments (Mean ± s.d., n = 2). The lentogenic strain Ulster 2C of NDV, known as thermostable, was also included. (a) AAvV-1/chicken/AO-HL1/2018; (b) AAVV-1/chicken/AO-HL2/2018; (c) AAVV-1/chicken/AO-HL3/2018; (d) AAVV-1/chicken/AO-HL4/2018; (e) AAvV-1/chicken/AO-HL5/2018; (f) AAvV-1/chicken/AO-HL6/2018; (g) AAvV-1/chicken/AO-HL7/2018; (h) AAvV-1/chicken/AO-HL8/2018; (i) AAvV-1/chicken/AO-HL9/2018; (j) AAvV-1/chicken/AO-HL10/2018; (k) AAVV-1/chicken/AO-HL11/2018; (l) AAVV-1/chicken/AO-HL12/2018; (m) AAVV-1/chicken/AO-HL13/2018; (n) AAVV-1/chicken/AO-NB1/2016; (o) AAVV-1/chicken/AO-NB2/2018 and (p) ULSTER 2C.