Replication of non‐defective reticuloendotheliosis viruses in the avian embryo assayed by PCR and immunofluorescence

Abstract

SUMMARYCongenital transmission of reticuloendotheliosis virus (REV) is known to occur when one of the parents in either tolerantly infected or viraemic; however little attention has been paid to replication of the virus within the embryo itself. In this study five non-defective and one replication-defective strain of REV were inoculated into the yolk sac of 6-day-old embryonated eggs. With the exception of one non-defective strain, the others induced embryonic deformation or lesions that were apparent on the day of hatching. Replicative integration of the provirus was determined for several tissues of each embryo by the polymerase chain reaction employing a set of primers specific for the long terminal repeat. Indirect immunofluorescent detection of whole virus was performed on the same tissues in parallel. A variation in replication was found among the strains and between embryos inoculated with the same strain of virus, therefore, the results for each virus strain were expressed as cumulative values. The liver and spleen were the most commonly affected organs, while the bursa of Fabricius and thymus supported viral replication to a lesser degree. Detection of proviral DNA in blood was evidence that the chicks had been congenitally infected. Such an infection would likely lead to tolerance. Isolates REV-S and REV-D were found to replicate to the highest extent, and they also caused lesions in the commercial type of embryonated eggs, while REV-S and mainly the REV-T prototype were pathogenic for the SPF embryonated eggs.

A duplex real-time polymerase chain reaction assay for the simultaneous detection of long terminal repeat regions and envelope protein gene sequences of Reticuloendotheliosis virus in avian blood samples

“…12 In comparison, molecular techniques such as polymerase chain reaction (PCR) have been used in the diagnosis of various infectious diseases and are considered one of the best alternatives to conventional assays because of their sensitivity, specificity, and reproducibility. 15,17 Conventional gel-based PCR tests, 5,6,10 as well as a nested PCR assay, 2 have been developed for routine REV diagnostics; however, these methods require manipulations post-PCR amplification including gel electrophoresis, gel staining steps, or transfer of amplified products to secondary tubes for further amplification. These methods can be time consuming and are prone to carryover contamination.…”