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. 2022 Oct 25;14(11):2338.
doi: 10.3390/v14112338.

Biological Significance of Dual Mutations A494D and E495K of the Genotype III Newcastle Disease Virus Hemagglutinin-Neuraminidase In Vitro and In Vivo

Xiaolong Lu  1 , Tiansong Zhan  1 , Kaituo Liu  1 , Yu Chen  1 , Zenglei Hu  1 , Jiao Hu  1 , Min Gu  1 , Shunlin Hu  1 , Xiaoquan Wang  1 , Xiaowen Liu  1   2 , Xiufan Liu  1   2   3
Affiliations

Biological Significance of Dual Mutations A494D and E495K of the Genotype III Newcastle Disease Virus Hemagglutinin-Neuraminidase In Vitro and In Vivo

Xiaolong Lu et al. Viruses. .

Abstract

As a multifunctional protein, the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is involved in various biological functions. A velogenic genotype III NDV JS/7/05/Ch evolving from the mesogenic vaccine strain Mukteswar showed major amino acid (aa) mutations in the HN protein. However, the precise biological significance of the mutant HN protein remains unclear. This study sought to investigate the effects of the mutant HN protein on biological activities in vitro and in vivo. The mutant HN protein (JS/7/05/Ch-type HN) significantly enhanced the hemadsorption (HAd) and fusion promotion activities but impaired the neuraminidase (NA) activity compared with the original HN protein (Mukteswar-type HN). Notably, A494D and E495K in HN exhibited a synergistic role in regulating biological activities. Moreover, the mutant HN protein, especially A494D and E495K in HN, enhanced the F protein cleavage level, which can contribute to the activation of the F protein. In vitro infection assays further showed that NDVs bearing A494D and E495K in HN markedly impaired the cell viability. Simultaneously, A494D and E495K in HN enhanced virus replication levels at the early stage of infection but weakened later in infection, which might be associated with the attenuated NA activity and cell viability. Furthermore, the animal experiments showed that A494D and E495K in HN enhanced case fatality rates, virus shedding, virus circulation, and histopathological damages in NDV-infected chickens. Overall, these findings highlight the importance of crucial aa mutations in HN in regulating biological activities of NDV and expand the understanding of the enhanced pathogenicity of the genotype III NDV.

Keywords: Newcastle disease virus; biological significance; genotype III; hemagglutinin-neuraminidase protein; vaccine strain.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the mutants used in this study. (A) Three NDV strains were used as model viruses in this study, including the vaccine strain rMukteswar, the vaccine variant strain rJS/7/05/Ch, and the dual-site mutant strain rMukHN494 + 495JS. rMukHN494 + 495JS was the rMukteswar derivative bearing the 494 and 495 aa of rJS/7/05/Ch. The aa mutations in the HN protein of NDV are marked with yellow triangles, including P438S in NP, and N19S, S29T, M145T, V266I, A494D, and E495K in HN. (B) Schematic representation of the aa mutations ranging from position 490 to 500 of different HN expression plasmids. Residues in blue and yellow indicate amino acids from Mukteswar and JS/7/05/Ch, respectively. (C) Constructed clones were identified by Sanger sequencing. The sequences of key mutation sites were displayed in red frames using the SnapGene 3.2.1 software.
Figure 2
Figure 2
Biological activities of HN proteins at the protein level. HAd (A) and NA (B) activities were determined after transfection with each HN protein expression plasmid. (C) The fusion promotion activity of different HN proteins was measured following co-transfection with each HN protein expression plasmid and the F protein plasmid. Images were taken under a microscope at ×200 magnification. The syncytia formation is indicated by white arrows. Bar indicates 200 µm. All values are normalized to the expression of the values for pCA-MukHN, which was set at 100%. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Figure 3
Figure 3
Analysis of expression efficiency of different HN proteins. Different HN protein expression plasmids were transfected into Vero cells, followed by incubating with the HN protein primary antibody and corresponding secondary antibody. Images were then taken by a fluorescent microscope at ×100 magnification. Bar indicates 100 µm. IFA results were evaluated by the ImageJ software. ns indicates no significance.
Figure 4
Figure 4
HAd and NA activities of NDVs bearing different HN proteins. Vero cells were infected with three viruses at 0.1 MOI for 24 h, and then detected for the HAd activity (A) and the NA activity (B), respectively. HAd and NA activities of NDV are normalized to the expression of the values for rMukteswar, which was set at 100%. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Figure 5
Figure 5
Syncytium formation and hemolytic activities of NDVs bearing different HN proteins. (A) Vero cells were infected with three viruses at 0.1 MOI for 24 h, and then detected for the syncytia formation. Images were taken under a microscope at ×200 magnification. White arrows were used to mark the syncytia formation. Bar indicates 200 µm. (B) The hemolytic activity was compared among the parental and recombinant viruses. Statistical significance was compared with that of rMukteswar. All values are normalized to the expression of the values for rMukteswar, which was set at 100%. * p < 0.05, *** p < 0.001, and **** p < 0.0001.
Figure 6
Figure 6
Evaluation of F protein cleavage activities of NDVs bearing different HN proteins. (A) Three viruses were inoculated into CEF cells at 0.1 and 1 MOI, respectively. The amounts of HN, F0, and F1 proteins were detected by Western blotting and then the cleavage-promotion activity of different HN proteins was analyzed. (B) The gray bands were evaluated by the ImageJ software. The F1/HN ratio at each infected group was normalized to that of the rMukteswar group. * p < 0.05.
Figure 7
Figure 7
Evaluation of cell viability and virus replication ability in NDV-infected CEF cells. Three viruses were inoculated into CEF cells at 0.1 and 1 MOI, respectively. (A) Reduction of cell viability in NDV-infected CEF cells at 3, 6, 12, 24, 36, and 48 hpi. (B) Growth characteristics of the three viruses were determined during 3–48 hpi. All values were compared with that of rMukteswar. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Figure 8
Figure 8
Pathogenicity of NDVs bearing different HN proteins. (A) Clinical signs were observed and recorded once daily. Clinical scores of chickens were calculated according to degree of severity. (B) Clinical symptoms of diseased chickens. rJS/7/05/Ch-infected chickens at 5 dpi (left) and rMukHN494 + 495JS-infected chickens at 6 dpi (right). Both rJS/7/05/Ch- and rMukHN494 + 495JS-infected chickens showed serious clinical symptoms, including breathing difficulty, lethargy, and decreased appetite. (C) The mRNA levels of the NDV NP gene in peripheral blood of chickens at 1, 3, and 5 dpi. All values were compared with that of rMukteswar. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 9
Figure 9
Histopathology of the lung, spleen, and thymus at 4 dpi. The obvious lesions are marked with black arrows. Magnification 200×, scale bar 100 μm.
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Grants and funding

This study was supported by the National Natural Science Foundation of China: 31873021, by the Earmarked Fund for China Agriculture Research System: CARS-40, and by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).