Detection and differentiation of chicken anemia virus isolates by using the polymerase chain reaction

Abstract

Complementary oligonucleotide primers which flank a 675-bp DNA fragment encompassing part of the putative gene for the capsid protein of chicken anemia virus (CAV) were used for the enzymatic amplification of CAV DNA by the polymerase chain reaction (PCR). Application of a dot blot hybridization assay by using a 32P-labeled cloned CAV DNA probe allowed PCR product amplified from as little as 0.1 fg of the target DNA sequence to be detected. When it was used for PCR amplification, DNA extracted from thymus tissue by a guanidine isothiocyanate-based method proved to be more efficient than that extracted by methods involving phenol or boiling. DNAs specified by 14 CAV isolates originating in the United Kingdom, Ireland, Germany, Sweden, the United States, Japan, and Australia were amplified. Restriction endonuclease analysis of the PCR-amplified DNAs with the enzymes HaeIII, HinfI, and HpaII indicated that the 14 CAV isolates can be assigned to seven groups, with isolates from different countries usually exhibiting the greatest number of restriction site differences.

A hypervariable region in VP1 of chicken infectious anemia virus mediates rate of spread and cell tropism in tissue culture

“…The CIA-1 promoter region contains only four direct repeats in contrast to the five repeats found in Cux-1(C). Evidence indicates that the wild-type configuration is four repeats, and in some isolates a fifth repeat has been selected for by repeated passage in culture (13,24). This results in an overall length of 2,298 bp for CIA-1 in comparison with 2,319 bp for Cux-1(C).…”

“…Although these viruses are grouped together on the basis of a common genome form, no similarities in amino acid composition, open reading frame (ORF) arrangement, or transcriptional machinery have been identified (15). Recently, the 2.3-kb genome of the Cux-1 strain (27) was sequenced (14) and found to have three partially overlapping ORFs, potentially encoding proteins of 51.6 (VP1), 24.0 (VP2), and 13.6 (VP3) kDa. No relationship with other viruses has been established at the amino acid level, and at present CIAV is considered to be a unique infectious agent.…”

Inhibition and facilitation of nucleic acid amplification

“…Nevertheless, if whole cells are loaded into the reaction tube and released DNA fails to be sufficiently separated from structural and DNA-binding proteins by boiling, PCR inhibition may result. Extraction by boiling alone has been noted to reduce sensitivity, due to the mechanism described above or poor lysis efficiency, and may give rise to spurious bands in some cases (41) or prevent amplification altogether (117). The high concentration of salt in Listeria selective media was found to be the reason for unlysed cells and false-negative reactions by the Accuprobe DNA probe test (86), and PCR may be similarly affected.…”

Detecção do virus da anemia das galinhas em coinfecção com o vírus doença infecciosa bursal em frangos

“…For over a year, a commercial broiler farm located 11 , astrovirus 12 , CAV 13 and avian coronavirus 14 . CAV was surveyed by a PCR aimed to amplify a 675-bp fragment of the VP1 protein gene 13 .…”

“…For over a year, a commercial broiler farm located 11 , astrovirus 12 , CAV 13 and avian coronavirus 14 . CAV was surveyed by a PCR aimed to amplify a 675-bp fragment of the VP1 protein gene 13 . Positive CAV was detected in the examined birds, but early presentation of clinical symptoms, as well as absence of some CAV classic lesions, such as bone marrow aplasia, suggest that this particular CAV strain may be a nonclassical one.…”