Quantitative analysis of maedi-visna virus DNA load in peripheral blood monocytes and alveolar macrophages.
Viral load may be an important indicator of disease progression in sheep infected with maedi-visna virus (MVV). To assess this variable accurately in MVV-infected sheep, a quantitative competitive-polymerase chain reaction (QC-PCR) was developed. A conserved region of the MVV pol gene was selected. The RT-PCR MVV pol product was cloned and mutagenised in vitro by PCR to produce a competitor template reduced in length from 217 to 192 bp, but which retained the original flanking MVV pol PCR primers. The competitor template was quantified accurately and in an optimised QC-PCR protocol serial dilutions of this template were co-amplified with known amounts of sample DNA. MVV DNA levels in peripheral blood monocytes and alveolar macrophages from MVV-infected sheep (n=12) were assessed by QC-PCR. Viral DNA load in alveolar macrophages was significantly higher than that in peripheral blood monocytes when the animals were compared overall. A comparison was also made between alveolar macrophages from the lungs of seropositive animals with or without histopathological evidence of pulmonary lesions. The load of MVV DNA in alveolar macrophages was low in sheep without histopathological evidence of lesions in the lung. In contrast, in alveolar macrophages from sheep with histopathological lesions in the lung, there was a significantly higher level of MVV DNA. The correlation of MVV load with pulmonary lesions suggests that infected alveolar macrophages play a key role in the pathogenesis of this lymphoid interstitial pneumonia.
Zhang
Z
Z
Department of Veterinary Pathology, University of Edinburgh, Edinburgh, UK. zhidong.zhang@bbsrc.ac.uk
Watt
N J
NJ
Hopkins
J
J
Harkiss
G
G
Woodall
C J
CJ
D000818:Animals; D004279:DNA, Viral / Q000032:analysis / Q000097:blood; D015684:Gene Products, pol / Q000235:genetics; D008168:Lung / Q000473:pathology; D016676:Macrophages, Alveolar / Q000821:virology*; D009000:Monocytes / Q000821:virology*; D011021:Pneumonia, Progressive Interstitial, of Sheep / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012756:Sheep; D019562:Viral Load; D014790:Visna-maedi virus / Q000235:genetics / Q000502:physiology*
D004279:DNA, Viral; D015684:Gene Products, pol