Live vaccines predominantly control avian metapneumovirus (AMPV) infection in the poultry industry but these can persist after application and revert to virulence (Catelli et al., 2006; Catelli et al., 2010; Lupini et al., 2011). Sequencing of the G gene of the prevailing subtype B VCO3 vaccinal strain (Cecchinato et al., 2010) 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 (Naylor et al., 1997). 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 (Figure 1). 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.
Rapid differentiation of vaccine and field strains of avian metapneumovirus of subtype b by restriction enzyme digestion of PCR products
LISTORTI, VALERIA;LUPINI, CATERINA;CATELLI, ELENA
2013
Abstract
Live vaccines predominantly control avian metapneumovirus (AMPV) infection in the poultry industry but these can persist after application and revert to virulence (Catelli et al., 2006; Catelli et al., 2010; Lupini et al., 2011). Sequencing of the G gene of the prevailing subtype B VCO3 vaccinal strain (Cecchinato et al., 2010) 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 (Naylor et al., 1997). 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 (Figure 1). 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
