Detection and localization of naturally transmitted avian leukosis subgroup J virus in egg-type chickens by in situ PCR hybridization

Avian leukosis virus (ALV) subgroup J (ALV-J) is an exogenous ALV and causes myeloid leukosis in meat-type chickens. We have previously reported the isolation and identification of ALV-J in commercial layer flocks from 12 farms in northern China. In this report, we further characterized this virus by in situ polymerase chain reaction (PCR) hybridization in various affected organs of chickens from six of the 12 farms. A routine method for hybridization of nucleic acid uses radioactive probe, such as a p(32)-labelled probe. We found that the non-radioactive digoxigenin (DIG) probe is sensitive enough to detect the nucleic acid of virus in chicken tissues. We used a pair of published primers (H5/H7) specific to the gp85 envelope gene and 3' region of pol gene of prototype ALV-J strain HPRS-103. The total RNA extracted from tumour, bone marrow, oviduct, liver and spleen of the diseased chickens from six commercial flocks, and cDNA was successfully amplified. Using the primers and cDNA, we obtained an ALV-J-specific cDNA probe of 545 bp in length by PCR. In situ PCR with H5/H7 primers was carried out in the paraffin sections from tissues of the diseased chickens, followed by in situ hybridization using the DIG-labelled cDNA probe. Positive hybridization signals were detected in the cytoplasm of paraffin sections of tumours and other organ tissues. The intensity of the signals was documented using an image analysis system measuring integral optical density (IOD). The IOD values for tissue sections treated by in situ PCR hybridization are significantly higher than that by in situ hybridization alone (P < 0.01). These data taken together suggest that in situ PCR hybridization is a more sensitive technique for detection of ALV-J in tissue sections.