ALV-J strain 733 was isolated from commercial layer hens and stored after replication in DF-1 cells (American Type Culture Collection, Manassas, VA) in our lab in China in 2012 . The strain shows the typical myelocytomatosis, but no contamination of reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian reovirus (ARV), chicken infectious anemia virus (CIAV), and Marek’s disease virus (MDV) by indirect immunofluorescence assay (IFA), reverse transcription polymerase chain reaction (RT-PCR), and dot-blot hybridization. There were 104 TCID50 in each 0.1 mL of DF-1 cellular supernatant, which was diluted with PBS 1:10 before inoculating chick embryos.
The infection experiment of ALV-J in cocks
SPF chicken embryos were obtained from the SPAFAS Co. (Jinan, China; a joint venture with Charles River Laboratory, Wilmington, MA, USA) and SPF chickens were normally hatched in our lab. Approximately 100 SPF chicken embryos were divided into 2 groups. All 11-day old chick embryos were intravenously inoculated with ALV-J of 1000 TCID50 (50 chicken embryos, group 1). The chicken embryos from the control group were inoculated with PBS (50 chicken embryos, group 2). For group 1, all hens were weeded out after eggs hatched and cocks were kept in a separate isolator. For group 2, all chickens were raised in another separate isolator. Blood samples were aseptically collected from all chickens in heparinized tubes at the age of 4, 8, 16, 20, and 24 weeks. For ALV viremia detection, DF-1 cells were inoculated with plasma samples from the chickens.
Isolation and identification of the ALV-J in seminal fluid
Six cocks, which showed positive persistent viremia and negative viremia, were chosen for the collection of seminal fluid from group 1 after being raised to 24 weeks of age in isolators. The resulting seminal fluid was diluted 1:10 with Gibco Dulbecco Modified Eagle medium (DMEM, Life Technologies, Carlsbad, CA). The samples were then centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was removed and filtered through a 0.22 μm filter (EMD Millipore, Billerica, MA), which was used to inoculate the DF-1 cells. The cells were cultured for 2 h at 37 °C, and the supernatant was replaced with fresh medium containing 1% foetal bovine serum . The cells were incubated for an additional 7 d, and blind passages were performed for 2 generations over a total period of 21 d.
A 100 μL aliquot of cell culture supernatant was analysed for the presence of p27 from ALV using the Avian Leukosis Virus Antigen Test Kit (IDEXX Laboratories, Westbrook, ME). The ALV-positive supernatant samples were stored at −80 °C. The ALV-positive cells were fixed in an acetone-ethanol (3:2) bath for 5 min, and analysed using IFA with the JE9 anti-ALV-J monoclonal antibody  and an ALV-A/B antiserum , as previously described. Primary antibody reactivity was detected using a fluorescein isothiocyanate-labelled anti-mouse IgG antibody (Sigma-Aldrich, Saint Louis, MO). A drop of 50% glycerol was added to the coverslip, and the cells were observed using a fluorescence microscope.
Detection of ALV-J infection in hens inseminated with the ALV-J infected semen
Semen samples were collected from six cocks with persistent viremia and mixed for artificial insemination of 12 SPF hens as the experimental group after the hens started producing eggs. Another 12 SPF hens were artificially inseminated with the semen collected from four cocks with no ALV-J infection as the control group. All hens of the two groups were bled once a week for 6 weeks after insemination. Plasma and serum were prepared for each individual hen for virus isolation in cell cultures or ALV-J antibody tests with ELISA antibody detection kits (IDEXX Laboratories, Westbrook, ME). At the same time, cloaca swabs were collected for each hen for p27 antigen detection with the Avian Leukosis Virus Antigen Test Kit (IDEXX Laboratories, Westbrook, ME). The eggs were collected to detect the presence of the p27 antigen from the egg albumen. Eggs were collected 1–3 weeks after insemination and hatched.
Determination of ALV-J infection in progeny chicks
Anticoagulated blood samples from each chicken were collected at the age of 1 d. Subsequently, it took 1–3 weeks to inoculate DF-1 cells in order to conduct virus isolation tests as previously described. The cloacal swabs and meconium were analysed for the presence of the p27 antigen using the Avian Leukosis Virus Antigen Test Kit (IDEXX Laboratories), according to the manufacturer’s instructions. All samples collected were analysed in duplicate.
Amplification and sequence analysis of viral RNA
The viral RNAs were extracted from the ALV strain 733, semen plasma samples, and ALV-J isolated from the progeny chicks using the Viral RNA Kit (Omega Bio-Tek, Doraville, CA). The purified RNAs were used for ALV detection by RT-PCR. The primers used for the amplification of the gp85 cDNA from the ALV isolate were designed based on previous studies of representative ALV strains. The primers were as follows: F: 5′-GATGAGGCGAGCCCTCTCTTTG-3′; R: 5′-TGTTGGGAGGTAAAATGGCGT-3′. The PCR products were separated by electrophoresis on a 1% agarose gel. The gp85 cDNA bands were purified from the gel using the EZNA Gel Extraction Kit (Omega), and ligated into the PMD-18 T plasmid (Takara Bio, Shiga, Japan). The vector was used to transform competent DH5α Escherichia coli. The sequence of the gp85 cDNA was determined by a commercial service (Invitrogen, Shanghai, China). At least three independent RT-PCR experiments were performed for each sample to ensure the accuracy of the results. The sequence alignment was performed using with the Clustal application in the MegAlign program of DNAStar, version 7.01, software suite (DNAStar, Madison, WI, USA).