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Background This study determined whether the naturally attenuated, thermotolerant Newcastle disease vaccine virus I-2 could acquire virulence after five in vivo passages through SPF chickens. Methods Study design was to international requirements including European Pharmacopoeia, Ph. Eur., v9.0 04/2013:0450, 2013. I-2 Working Seed (WS) was compared with five-times-passaged I-2 WS (5XP WS) in intracerebral pathogenicity index (ICPI), F o cleavage site sequencing and Safety tests. Results The first passage series used a 50% brain: 50% tracheal tissue challenge homogenate and was unsuccessful as I-2 was not detected after the fourth passage. A second passage series used 10% brain: 90% tracheal tissue homogenates. I-2 was isolated from tracheal tissue in each passage. However harvested titres were below the minimum challenge level (10 7 EID 50 ) specified for the ICPI and Safety tests, possibly reflecting I-2’s inherently low pathogenicity (interestingly caecal tonsils yielded significant titres). Given this the WS and 5XP WS comparisons proceeded. ICPI values were 0.104 and 0.073 for the WS group and the 5XP WS group respectively confirming that I-2, whether passaged or not, expressed low pathogenicity. F 0 amino-acid sequences for both WS and 5XP WS were identified as 112 R-K-Q-G-R-↓-L-I-G 119 and so compatible with those of avirulent ND viruses. In safety, no abnormal clinical signs were observed in both groups except for two chicks in the 5XP WS group, where one bird was withdrawn due to a vent prolapse, and another bird died with inconclusive necropsy results. Conclusions: These data, the issue of low passage titres with little or no virus isolation from brain tissues and the genomic copy approach suggest a need to amend Ph. Eur. v9.0 04/2013:0450, 2013 for naturally attenuated, low pathogenicity vaccine viruses such as I-2. These results add to the literature and field data demonstrating that Newcastle Disease vaccine virus I-2 is safe for use.

Newcastle disease vaccine virus I-2 fails to acquire virulence during repeated passage in vivo