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Viral genomic DNA was extracted from scab specimens, and PCR amplification was performed immediately by using the specific primers for FWPV P4b gene (P4b Fw1: 5'-GATAGAGGATCGTACATCCA-3'; and P4b Rv1: 5'-CATCTACTCATGACTGGCAA-3').
To investigate the possibility of an integration of an REV gene sequence into the FWPV genome, we designed another 2 sets of primers for the amplification of a partial REV env gene and the REV env-FWPV open reading frame 203, which contains the entire REV 3' long terminal repeat.
We further determined the pathogenesis of the FWPV isolate by experimentally infecting 18-day-old, 53-dayold, and 145-day-old SPF chickens.
The paraffin sections of scab samples from the SPF chickens inoculated with FWPV were positive not only for FWPV, tested by using a chicken anti-FWPV polyclonal antibody, but also for REV, tested by using a monoclonal antibody that specifically recognized the envelope protein of REV in the cytoplasm of stratified squamous epithelial cells of the folliculus pili by immunohistochemical assay (online Technical Appendix Figure 1).
Our investigation of an acute outbreak of the cutaneous form of fowlpox determined that the outbreak was caused by a novel type of FWPV that carried integrated REV genomic sequences.
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