Marek's disease virus (MDV), a herpesvirus, and avian leucosis virus subgroup J (ALV-J), a retrovirus, were used for experimental coinfection of chickens. Chimeric molecules having sequences of both viruses were detected by the hotspot–combined polymerase chain reaction (HS-cPCR) system. The detection of chimeric molecules provided evidence for avian retroviral inserts in the herpesvirus genome. The persistence of chimeric molecules on in vivo passage served to indicate the infectivity of the recombinant virus. The evaluation of formation and persistence of the chimeric molecules was performed in two trials involving three in vivo passages. The chimeric molecules were identified according to the primer sets, their product length, and pattern. The persistence of chimeric molecules on in vivo passages served as an indication of their ability to replicate in and infect chickens.
In the first experimental passage, MDV and ALV-J prototype strains, MD11 and HC-1, were intraperitoneally (IP) injected into 1-day-old chicks. The second trial included two passages. Passage II chicks were injected IP and passage III chickens were in contact with the chickens of passage II. For passage II, enriched white blood cells from blood samples of chickens from the first trial that had chimeric molecules were injected IP into 1-day-old chicks. For passage III, uninfected chicks were included together with the infected chicks.
Synthesis evidence for the various species of chimeric molecules was assessed in the tissues of birds of the second trial. DNA was extracted from blood and feathers and analyzed by the hotspot–combined PCR and by pulsed field gel electrophoresis. To overcome the limits of detection, three amplification assays followed by hybridization of the products to specific viral probes were conducted. A variety of chimeric molecules were detected in low concentrations. Five species of chimeric molecules were characterized in blood, tumors, and feathers. Chimeric molecules were detected in 18 of 36 dually infected birds from the first trial and in 14 of 21 dually infected birds from the second trial. The findings show that, in four out of seven groups of the second trial, the chimeric molecule species persisted on passage.