Related Experiment Videos
The detection and quantification of feline immunodeficiency provirus in peripheral blood mononuclear cells using the
M Lawson1, J Meers, L Blechynden
1School of Biological and Environmental Sciences, Murdoch University, Australia.
Abstract:
The polymerase chain reaction method (PCR) was used to detect feline immunodeficiency virus proviral DNA in peripheral blood mononuclear cells (PBMC) of a group of 8 experimentally infected cats. The proportion of PBMC containing provirus was determined from 6 to 32 weeks post inoculation (p.i.) by performing PCR on serially diluted samples of PBMC. Primers from the p15 and p24 regions of the gag gene were used and Southern hybridization using an end-labelled probe was required to confirm primer-specific products. Provirus was detected in 5 of 8 cats by 6 weeks p.i. in 50000 PBMC, and in all 8 infected cats by 8 weeks p.i. Provirus was not detected in PBMC from any of 3 FIV negative cats. The proportion of PBMC containing provirus in individual cats ranged from 1 in 70 to 1 in 99600 PBMC. There was no significant decline over time in the proportion of PBMC containing provirus. Sequencing of a segment (287 bases) of the gag region of a West Australian FIV isolate (T90) revealed only slight nucleotide divergence from the North American Petaluma and PPR isolates and wider divergence from the Japanese TM2 clone.
Insights
Feline immunodeficiency virus (FIV) proviral DNA was detected in cats using the polymerase chain reaction (PCR) method. Provirus was found in most infected cats within eight weeks, with stable proportions over time.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
Background:
- Feline immunodeficiency virus (FIV) is an important pathogen in domestic cats.
- Accurate detection of FIV proviral DNA is crucial for diagnosis and management.
Purpose of the Study:
- To establish and validate a polymerase chain reaction (PCR) method for detecting FIV proviral DNA in feline peripheral blood mononuclear cells (PBMC).
- To quantify the proviral DNA load in experimentally infected cats over time.
Main Methods:
- Experimental infection of 8 cats with FIV.
- Detection of FIV proviral DNA in serially diluted PBMC using PCR with gag gene primers (p15 and p24).
- Confirmation of PCR products by Southern hybridization.
- Sequencing of a segment of the FIV gag gene from a West Australian isolate.
Main Results:
- Provirus was detected in 5 of 8 cats by 6 weeks post-inoculation and in all 8 by 8 weeks.
- No provirus was detected in 3 FIV-negative control cats.
- The proportion of PBMC containing provirus ranged from 1 in 70 to 1 in 99,600 and remained stable over 32 weeks.
- The West Australian FIV isolate showed slight nucleotide divergence from North American isolates.
Conclusions:
- PCR is a sensitive method for detecting FIV proviral DNA in feline PBMC.
- FIV proviral DNA is detectable early post-infection and persists at stable levels.
- Genetic analysis reveals regional variations in FIV isolates.