Rapid differentiation of vaccine and field strains of avian metapneumovirus of subtype b by restriction enzyme digestion of PCR products

Live vaccines predominantly control avian metapneumovirus (AMPV) infection in the poultry industry but these can persist after application and revert to virulence. Sequencing of the G gene of the prevailing subtype B VCO3 vaccinal strain identified a unique nucleotide substitution (A→G, nucleotide in position 91, G gene), which fortuitously introduced an MseI restriction endonuclease site within the amplicon obtained from a popular AMPV diagnostic RT- nested PCR. The study reports the development of a method which uses the restriction enzyme digestion of PCR products as a method for rapid differentiation between AMPV subtype B vaccine or vaccine-derived strains and field strains. Two Italian subtype B AMPV isolates and the VCO3 vaccine were used to test the restriction enzyme digestion protocol. The amplicons obtained from these strains by RT-nested PCR were incubated with restriction enzyme MseI for two hours at 65 °C and analyzed by agarose gel electrophoresis. After enzymatic digestion field strains remained uncut showing an amplicon of 360bp, while the vaccine strain was cut resulting in a smaller band of 320bp detectable by gel electrophoresis, and a band of 40 bp, not detectable. The developed protocol was subsequently used for a field epidemiological study. Forty-one AMPVs of subtype B, detected in Italy from 2001 to 2011, were collected. After RT-nested PCR and enzymatic digestion, 8 out 41 strains revealed a vaccine origin, while the others were field strains. The method herein described for rapid differentiation of vaccine and field strains of AMPV of subtype B is applicable in geographic areas where the specific vaccine included in the study is used and might be a good alternative to sequencing, which although provides further information, requires more time and higher costs.