Differentiation of Avian Poxvirus Strains on the Basis of Nucleotide Sequences of 4b Gene Fragment

Abstract

Investigations for detection and differentiation of nine avian poxviruses (APVs) were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis (REA) and further nucleotide sequence analysis. With one primer set, which framed a region within the fowl poxvirus 4b core protein gene, we were able to detect APV-specific DNA from 19 tested strains and isolates belonging to five defined Avipoxvirus species and four previously undefined isolated species. PCR results revealed no recognizable differences in size of amplified fragments among the different APVs. REA of PCR products with MseI and EcoRV allowed us to differentiate most of the tested avipox species. Nucleotide sequence analysis of the amplified fragments showed a nucleotide similarity of 72%-100% among the different species. Phylogenetic analysis documented five distinguishable sequence clusters in accordance with results obtained by REA. PCR in combination with REA and sequencing of the amplified fragments is a rapid and effective diagnostic system, and it is a new approach to refine epidemiologic studies of APV infections.

Use of polymerase chain reactions to detectMycoplasma gallisepticum,Mycoplasma imitans,Mycoplasma iowae,Mycoplasma meleagridisandMycoplasma synoviaein birds of prey

“…The samples selected had been positive for mycoplasmal DNA using a Mycoplasma genusspecific PCR. DNA was originally extracted from the swabs using the QIAmp † DNA Mini Kit (Qiagen, Hilden, Germany) using the manufacturer's instructions, and as modified by Lüschow et al (2004). Briefly, 200 ml sample (swab diluted in phosphate-buffered saline buffer) was mixed with 20 ml proteinase K and 200 ml lysis buffer (AL).…”

Worldwide Phylogenetic Relationship of Avian Poxviruses

“…Comparative analysis of genomic sequences is the most informative and reliable method for comparing closely related viral genomes, so a definite phylogeny will have to await additional genome sequencing. The relationships of avian poxviruses isolated from free-ranging birds have been analyzed using DNA sequences of the 4b core protein coding genomic region (21,(24)(25)(26)(27). Until recently, the significant divergence among avipoxviruses impeded the efforts to identify other pan-genus PCR primers.…”

First phylogenetic analysis of Avipoxvirus (APV) in Brazil

“…The closest turkeys avipoxvirus 4b virion core protein sequences found in GenBank ® , in the subclade A1, were from Italy (GQ180212) and Germany (AY530304), which were identical to our samples on the level of nucleotide and amino acid sequence. The turkey APV DNA sequenced here appears to be closely related to a falcon APV (AM050376) isolate from United Arab Emirates (Jarmin et al 2006), a turkey (AM050387) isolate from United Kingdon (Jarmin et al 2006) and an ostrich (AY530305) without any isolation location defined (Lüschow et al 2004), and all of them belonging to A2 clade. The closest turkey APV sequence found in A2 clade was from Italy (GQ180212), and varied 9% on the amino acid level.…”

“…Hence, the amplification of a 578-base pair (bp) region of P4b, a highly conserved gene of APV, is commonly used to diagnose APV infections, which is highly conserved amongst all poxviruses (Binns et al 1989). The P4b gene has already been reported in other phylogenetic studies of APV to distinguish between clades A, B, C, A1-4 and B1-2 (Lüschow et al 2004, Weli et al 2004, Jarmin et al 2006). In addition, conventional and real time PCR techniques provide results more rapidly than virus isolation.…”

“…This also suggests the possible introduction of APV strains from overseas with the introduction of exotic bird species such as passerines (Ha et al 2011). Previous work showed that an APV isolate from a free-ranging bird presented a genetic similarity with FWPV and turkeypox virus (Lüschow et al 2004;Ha et al 2011).…”