Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks

Abstract

At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the São Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to the TCO and CEO vaccines. The two Group II isolates could not be distinguished from one of the CEO vaccines. The nucleotide and amino acid sequence analyses discriminated between the Brazilian and non-Brazilian isolates, as well as between the TCO and CEO vaccines. Sequence analysis of the TK gene enabled classification of the field isolates (Group I) as virulent and non-vaccine. This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain.

Acknowledgements

The authors would like to thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, grant number 05/56165-1) for financial support. They also thank the Biovet, Fort Dodge and Merial Laboratories (Brazil) for the provision of embryonated eggs, and thank CDA–SAA, São Paulo for the field sample and vaccine strains they sent.