Monitoring of Newcastle Disease Virus Vaccine Strain Replication in Embryonated Chicken Eggs by Reverse Transcription-Polymerase Chain Reaction

The knowledge of virus and replication kinetics plays a key role in developing a vaccine. This study aimed to monitor the replication process and determine the best harvesting time of the Newcastle disease virus (NDV) V4 vaccine strain in the allantoic fluids of specific pathogen-free (SPF)-embryonated chicken eggs (ECEs) by reverse transcription-polymerase chain reaction (RT-PCR), hemagglutination (HA), and egg infective dose 50% (EID50) tests. For this purpose, the V4 vaccine strain of the virus was intra-allantoically inoculated into 96 10-day-old SPF-ECEs at the rate of 0.1 mL/ECE. The allantoic fluids of the inoculated eggs were collected from six eggs at six-hour intervals up to 96 hours post-infection (hpi). The harvested suspensions were confirmed to contain the NDV by the mentioned serologic and molecular techniques. Based on the results, the virus was first detected at 36 hpi in ECEs by RT-PCR. The peak of HA and EID50 titers in allantoic fluids started at 42 hpi, and the titers were at the highest level until the end of the experiment. The results indicated that the best virus harvesting time for the NDV V4 vaccine strain in ECEs is between 42-60 hpi. These findings pave the way for adequate and enhanced production rate, immunogenicity, and cost-related parameters in the V4 Newcastle vaccine development.