Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: Influence of mismatches onquantification

Lentiviruses are associated not only with immunodeficiency but also with ma lignancies. The mechanisms involved in tumorigenesis are still not fully un derstood. Cats infected with feline immunodeficiency virus (FIV) in the wil d represent one model in which the role of viral load in the pathogenesis c an be studied, since tumors, especially lymphomas, are quite often observed in cats infected with FIV. To be able to compare the viral load data among cats infected with different FIV isolates, the method used to obtain the v iral load has to be unaffected by isolate-specific differences. This is esp ecially true for the real-time polymerase chain reaction (PCR), a new metho d for viral load determination, since nucleotide sequence mismatches have b een used for allelic discrimination with this method. To investigate the in fluence of these mismatches on PCR efficiency, we have used an FIV-specific real-time PCR and determined the influence of nucleotide sequence variatio n in several characterized FIV isolates as well as unknown isolates from na turally infected cats. We could demonstrate that minor mismatches, such as point mutations in the primer or the probe region, decrease overall PCR eff iciency but do not abolish the quantification, in contrast to major mismatc hes of three or four nucleotides, which lead to complete inhibition of the realtime PCR detection. Based on these results, it will be possible to desi gn real-time PCR systems allowing the quantification of a broad range of is olates, which is a prerequisite for the investigation of the impact of vira l load in tumorigenesis.