Enhanced pathogenicity by up-regulation of A20 after avian leukemia subgroup a virus infection

Abstract

Avian leukemia virus subgroup A (ALV-A) infection slows chicken growth, immunosuppression, and tumor occurrence, causing economic loss to the poultry industry. According to previous findings, A20 has a dual role in promoting and inhibiting tumor formation but has rarely been studied in avians. In this study, A20 overexpression and shRNA interference recombinant adenoviruses were constructed and inoculated into chicken embryos, and ALV-A (rHB2015012) was inoculated into 1-day-old chicks. Analysis of body weight, organ index, detoxification, antibody production, organ toxin load, and Pathological observation revealed that A20 overexpression could enhance ALV-A pathogenicity. This study lays the foundation for subsequent exploration of the A20-mediated tumorigenic mechanism of ALV-A.

Keywords: A20; ALV-A; chicken; pathogenic; recombinant adenovirus.

Figure 1

A20 recombinant adenovirus and ALV-A vaccination schedule. Ten-day-old chicken embryos were inoculated with A20 recombinant adenovirus, and one-day-old chickens were inoculated with ALV-A.

Figure 2

Constructing A20 recombinant adenovirus. (A) A20 gene amplification. (B) Identifying pshuttle-IRES-hrGFP2-A20 positive colonies. (C) Identifying Ad-pshuttle-IRES-hrGFP2-A20 positive colonies. (D) The plasmid Ad-pshuttle-IRES-hrGFP2-A20 has been digested with restriction endonuclease PacI. (E) A20 single-stranded shRNA anneals to double-stranded. (F) The plasmid Ad-pshuttle-H1-A20 shRNA is digested with restriction endonuclease PacI. (G) The effect of A20 adenovirus in the chicken embryo kidney. (H) The effect of A20 adenovirus in the chicken embryo liver.

Figure 3

Effects of A20 on ALV-A rHB2015012 in vitro replication. (A) A20 recombinant adenoviruses capable of overexpressing and interfering with A20 production in DF-1 cells. (B) A20 promotes the replication of ALV-A rHB2015012 in DF-1 cells. a, b, c, and d indicate differences, with the same letter indicating no difference and different letters indicating a difference.

Figure 4

Net gain in animal body weight. There was a net increase in the weight of animals in the four groups. PBS group had the largest weight gain, different from others. After A20 overexpression, ALV-A rHB2015012 group had the smallest body weight. Growth was suppressed in A20-disrupted group, although body weight was higher than in rHB2015012 group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Animal experimental organ index. (A–E) 7-, 14-, 21-, 28- and 35-day organ index. ALV-A rHB2015012 infection in A20-overexpressing chickens promotes the occurrence of hepatomegaly symptoms but does not promote or inhibit heart, spleen, lung, kidney and bursa. (F) Animal experiment group legend. a, b, c, and d indicate differences, with the same letter indicating no difference and different letters indicating a difference.

Figure 6

ALV-A infection status, viremia and viral shedding. (A) Identifying ALV-A infection by blood DNA PCR. (B) Viremia after ALV-A infection was detected by ELISA. (C) Detecting viral shedding after ALV-A infection by anal swab p27 group-specific antigen ELISA.

Figure 7

Antibody production after ALV-A infection. (A) Gp85 antibody production after ALV-A infection. (B) p27 antibody production after ALV-A infection.

Figure 8

The viral loads in liver, spleen and kidney after ALV-A infection was determined by Western blotting. (A) Liver viral loads. (B) Spleen viral loads. (C) Kidney viral loads. a, b, and c indicate differences, with the same letter indicating no difference and different letters indicating a difference.

Figure 9

The viral loads in liver, spleen and kidney after ALV-A infection was determined by q-PCR. (A–C) Liver, spleen, and kidney viral loads. (D–F) Expression of A20 in liver, spleen, and kidney. a, b, c, and d indicate differences, with the same letter indicating no difference and different letters indicating a difference.

Figure 10

Pathological observations (H,E) of ALV-A infection at 100 days (400×). (A,E,I) Liver, spleen and kidney tissue sections in PBS group. ALV-A rHB2015012 infection caused liver congestion (B), and tissue congestion was relieved after A20 expression was interfered with (C), while A20 overexpression aggravated congestion with lymphocyte infiltration (D). ALV-A rHB2015012 infection caused spleen congestion (F), and tissue congestion was relieved after A20 expression was interfered with (G), while A20 overexpression aggravated congestion with myeloma-like cells (H). ALV-A rHB2015012 infection caused kidney congestion (J), and tissue congestion was relieved after A20 expression was interfered with (K), while A20 overexpression aggravated congestion (L).