Chicken farm and samples
All chickens and samples were obtained from a breeding broiler chicken farm in Nanjing, China. An outbreak of ALV-J emerged before 2010 in this area. In this study, 1812 meconium samples were collected from hatched one-day-old chickens on the farm. The samples of cloacal swab, blood and spleen were collected from 10 chickens at 2 days of age, which meconium samples were positive in sELISA kit but negative in commercial ELISA kit. Blood samples were directly taken from the jugular vein. Spleen samples were taken from the chickens, which were first anesthetized by ether inhalation and then sacrificed by exsanguination. Other positive chickens were lately sent the laboratory to be euthanized. All the experiments complied with institutional animal care guidelines and were approved by the University of Yangzhou Animal Care Committee (Authorized XYSK(Su)2016–0020).
sELISA kit
The sELISA kit applied in China was developed using two mAbs (5D3 and 4F12) against the capsid protein p27 of ALV; this kit was generated and characterized in our previous work [5, 15, 16]. The two mAbs were purified with a protein G column. In the ELISA, the purified mAb 5D3 was used as the coating antibody, and the mAb 4F12 was labeled with HRP and used as the detection antibody. In brief, the purified mAb 5D3 was used to coat 96-well microplates at a concentration of 0.35 μg/mL in 0.1 M carbonate/bicarbonate buffer at pH 9.6 overnight at 4 °C and was then blocked with 5% skim milk in a 10 mM phosphate buffered saline (PBS) solution at pH 7.4 containing 0.05% Tween-20 (PBST) for 2 h at 37 °C. The plate was washed three times with PBST before adding 100 μL of sample. A noninfected DF1 cell supernatant sample and a purified recombinant p27 protein expressed in Escherichia coli were used as negative and positive controls, respectively. The plate was incubated for 1 h at 37 °C and washed three times. Then, 100 μL of HRP-labeled 4F12 diluted in PBST was added to the wells, and the plate was incubated at 37 °C for 1 h. After another 5 washes, 100 μL of freshly prepared TMB substrate solution was dispensed into each well. Color development occurred in the dark for 15 min, and the reaction was stopped by adding 100 μL of 1% sodium dodecyl sulfate (SDS). The absorbance at 650 nm was measured using an ELISA reader.
Commercial ELISA kit
Briefly, samples (blood or tissue) were inoculated into DF-1 cells, which were then incubated at 37 °C in a 5% CO2 incubator for 7 days. After the incubation, cell lysates were prepared by 2 freeze-thaw cycles and tested for an ALV group specific antigen (p27) by an antigen capture ELISA using anti-p27 antibody-coated plates (IDEXX Laboratories Pty. Ltd., Westbrook, USA) according to the manufacturer’s protocol. Other samples (meconium) could be tested directly. Samples (100 μL), including positive and negative controls, were added to the ELISA plate wells and incubated at room temperature for 60 min, followed by three to five washes with 350 μL/well washing buffer. Then, 100 μL of an HRP-conjugated rabbit antibody against p27 were added to all the wells, and the plate was incubated for another 60 min. After 3–5 washes, 100 μL of substrate solution was added to develop color for 15 min, and the reaction was stopped by adding 100 μL of stop solution to each well. The absorbance at 650 nm was measured using an ELISA reader.
Virus isolation
The chicken DF-1 cell line (an immortalized cell line of chicken embryonic fibroblasts, provided by Dr. Lucy Lee, USDA) was used for virus isolation (kept in our laboratory). The cells were grown in Dulbecco’s modified Eagle medium (DMEM) with 5% fetal bovine serum (FBS) and were maintained in DMEM with 1% FBS at 37 °C in a humidified atmosphere containing 5% CO2. Before inoculation, DF-1 cells were seeded in 96-well plates and cultured for 24 h. The following day, 60 μL of isolated lymphocytes or tissue homogenate supernatant and 120 μL of DMEM were added and incubated for 2.5 h before replacement with fresh DMEM maintenance medium containing 1% FBS. The plates were then incubated in a culture chamber for 7 days.
PCR
Viral DNA was prepared using the phenol-chloroform method [17]. The env gene of ALV was amplified using DNA extracted from lysed tissue samples as the template with the primers listed in Additional file 1: Table S1. In the PCR system, a 50-μL reaction volume was used, and the reaction mixture consisted of 5 μL of 10× LA Taq PCR buffer, 2 μL of each primer (25 pmol/μL), 0.5 μL of LA Taq DNA polymerase, 4 μL of 10 mM deoxyribonucleotide triphosphate mixture, 36.5 μL of ddH2O, and 2 μL of DNA template. The PCR parameters for the env gene were 94 °C for 5 min; 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min; and then 72 °C for 10 min. The PCR products were separated on a 1% DNA gel.
Sequence analysis of the viral genome
PCR products were sent to the Beijing Genomics Institute for sequencing. Multiple sequence alignment with other ALVs (Additional file 2: Table S2) was carried out using the sequence analysis software Lasergene7 (DNASTAR) and the NCBI BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree was constructed using the neighbor-joining method (MEGA 5.0).
Indirect IFA
Cells infected with or without virus were fixed with an acetone:ethanol solution (3:2) for 10 min after 7 days and washed once with PBS. After blocking with 1% BSA in PBS, the fixed cells were incubated with the mouse anti-ALV p27 mAb 5D3 for 45 min at 37 °C. After three washes with PBS, the cells were incubated with a FITC-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich, USA) for another 45 min. After three washes with PBS, the cells were observed under a fluorescence microscope.
