Phosphorylated abacavir analogue (ABC-1) has ameliorative action against Newcastle disease virus induced pathogenesis in chicken

Abstract

Newcastle disease virus (NDV) causes huge economic loss to the poultry industry due to high mortality and morbidity. The present study aimed to assess the protective role of novel phosphorylated analogue ABC-1 in vivo in NDV-infected chickens through the inhibition of fusion protein. Both NDV-induced oxidative damage and protective role of novel phosphorylated ABC-1 were evaluated in vital organs such as the liver and lung of chickens. Enzyme linked immunosorbent assay (ELISA) results showed that protein oxidation and nitration levels were significantly raised in NDV-infected tissues compared to healthy controls, whereas these levels were reduced significantly (P < 0.05) in birds treated with phosphorylated compounds compared to the NDV-infected group alone. Additional investigation with double immunofluorescence showed that the large amount of immuno colocalization and Western blot analysis also confirmed this observation through its band pattern in NDV-infected birds compared to healthy birds, whereas these alterations were reduced in treatment with novel phosphorylated ABC-1. The expression of fusion glycoprotein was studied by immuno colocalization, PCR, and flow cytometry, and results demonstrated that the novel phosphorylated analogues reduced the expression of fusion glycoprotein. These results put forth that novel phosphorylated ABC-1 protects chickens from NDV-induced pathogenesis, protein oxidation/nitration, and exerts potent antiviral activity.

Keywords: Newcastle disease virus; fusion protein; oxidative stress; phosphorylated compound.

Figure 1

The protein carbonyl levels in lung and liver tissues from control, NDV‐infected, and NDV + ABC‐1‐treated chicken were measured by ELISA. Values expressed are mean and SD of triplicates. *P < 0.05 between the control group and the NDV‐infected group. #P < 0.05 between NDV‐infected and treatment groups.

Figure 2

The nitrated protein levels in control, NDV‐infected, and NDV + ABC‐1‐treated chicken were measured in the lung and liver by ELISA. Values expressed are mean and SD of triplicates. *P < 0.05 between the control group and the NDV‐infected group. #P < 0.05 between the NDV‐infected group and NDV + ABC‐1 groups.

Figure 3

Photomicrographs of double immunofluorescence stained liver tissues with anti‐DNP and α‐SMA to detect oxidized protein. (A–C) Anti‐DNP, (D–F) α‐SMA, and (G–I) colocalized. A considerable amount of immunocolocalized cells in liver was observed (H) when compared to control (G) and treated birds (I). Scale bar () = 50 µm.

Figure 4

Photomicrographs of double immunofluorescence stained lung tissues with anti‐DNP and anti‐clara primary antibodies. (A–C) Anti‐DNP, (D–F) anti‐clara, and (G–I) colocalized. A considerable amount of immunocolocalized (oxidized protein) cells in lung was observed (H) when compared to control (G) and treated birds (I). Scale bar () = 50 µm.

Figure 5

Photomicrographs of double immunofluorescence stained liver tissues with anti‐nitrotyrosine and α‐SMA. (A–C) Anti‐nitrotyrosine, (D–F) α‐SMA, and (G–I) colocalized. (H) A large amount of nitrated protein due to NDV infection, whereas (I) treated bird showed a lesser number of colocalized cells than control bird (G). Scale bar () = 50 µm.

Figure 6

Photomicrographs of double immunofluorescence stained lung tissues with anti‐nitrotyrosine and anti‐clara primary antibodies. (A–C) Anti‐nitrotyrosine, (D–F) anti‐clara, and (G–I) colocalized. (H) Tissue from NDV‐infected animal showed more number of colocalized cells than that of (I) treated bird. (G) Control bird shows only few nitrated cells. Scale bar () = 50 µm.

Figure 7

Western blot analysis of protein nitration and oxidation in control, NDV‐infected, and NDV + ABC‐1‐treated chickens. The antibodies used to measure nitrated proteins are primary antibody (mouse anti‐nitrotyrosine, 1:1,000) and secondary antibody (rabbit antimouse, 1:5,000), respectively, and protein oxidation was detected using the antibodies: primary antibody (rabbit anti‐DNP, 1:1,000 dilution) and secondary antibody (goat anti‐rabbit, 1:5,000), respectively. The NDV‐infected group animal shows an increased intensity of bands compared to control, whereas the band intensity was decreased in the treated animals. (A) Protein nitration, (B) protein oxidation, and (C and D) quantification of band density by image j software.

Figure 8

Photomicrographs of immunocolocalization of fusion protein in liver tissues. (A–C) α‐SMA, (D–F) anti‐F mAb, and (G–I) colocalized. A considerable amount of immunocolocalized cells in liver was observed (H) when compared to control (G) and treated birds (I). Scale bar () = 50 µm.

Figure 9

Photomicrographs of immunocolocalization of fusion protein in lung tissues. (A–C) Anti‐clara, (D–F) anti‐F mAb, and (G–I) colocalized. A considerable amount of immunocolocalized cells in lung was observed (H) when compared to control (G) and treated birds (I). Scale bar () = 50 µm.

Figure 10

Quantification of immunocolocalized photomicrographs. (A) Protein nitration, (B) protein oxidation, and (C) Fusion protein. Fluorescence density values were raised in the NDV‐infected alone group in comparison to control animals. The levels in treated animals were brought down with novel phosphorylated compounds ABC‐1.

Figure 11

Viral detection in liver and lung tissue of experimentally infected and treated animals by PCR. Control chicken was uninfected and represents a healthy group, whereas the NDV‐infected group is experimentally infected and NDV + ABC‐1 represents the treatment group. The intensity of bands decreased upon treating with ABC‐1 compared to NDV‐infected liver and lung tissue. GAPDH was used as internal control for the experiment.

Figure 12

Characterization of NDV viral fusion protein in liver and lung cell population. Cells were sorted on the basis of labeling with anti‐F mAb 8E12A8C3 (1:5,000; CVI, Lelystad) primary antibody and AF‐350 secondary antibody. Liver: (A) Control and (B) NDV infected, and (C) ABC‐1 treated. NDV fusion protein level make up 17.8% in infected and 10.3% ABC‐1 based on population values of fusion protein. Lung: (D) Control, (E) NDV infected, and (F) ABC‐1 treated. NDV fusion protein level make up 23.5% in infected, and 7.8% in ABC‐1 based on population values of fusion protein.