Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand

Abstract

Feline immunodeficiency virus (FIV) affects domesticated housecats all over the world, with a significant diversity among the five different subtypes (A, B, C, D & E). FIV has been identified as an endemic disease with a prevalence that varies among geographic locations. In New Zealand the prevalence ranges from 6,8% in clinically healthy cats to 20,9-27,3% in cats with clinical symptoms. There has not been a lot of research on subtype C and because of the high level of diversity even within subtypes, it is very plausible that studies on overseas isolates may not accurately represent the situation in New Zealand. This research determined whether naturally infected New Zealand cats that tested positive for FIV ELISA were really infected with FIV and subtyped the sequence. In total 20 cats who tested positive on FIV with the ELISA were used in this research. Blood from these cats was taken and examined by a Gag PCR and an Env PCR reaction. The Gag PCR failed, so further research was done only on the Env gene. From the 20 samples that were collected, 17 samples were tested positive on the FIV Env PCR. Among the 17 samples analysed from PCR FIV positive cats, subtype C was present in 5 samples and subtype A was prevalent in 1 sample. Although not enough samples were subtyped to make a final conclusion, these present study results add to the finding of other studies that the predominant FIV subtype in New Zealand appears to be subtype C and A. Also determination of the optimal time from infection to harvest of the supernatant with the most produced virus by FIV infected peripheral blood mononuclear cells (PBMC’s) was performed. Healthy PBMC’s were cultured together with FIV infected PBMC’s, a media change was performed every 3-4 days and supernatant was frozen. RNA quantification of the supernatant was performed using a spectrophotometer. In 5 of the 7 samples the RNA concentration increased on day 18 and decreased after that. FIV infected PBMC cultivation without donor PBMC’s indicated that the highest level of RNA was 17 days after incubation. RNA quantification was also performed using different real time RT-PCR. It was found that one step real time RT-PCR wasn’t working on the RNA samples, but two step real time RT-PCR did. No time was left to get the real time RT PCR working for the viral RNA cell culture supernatants, so determination of the optimal time from infection to harvest of the supernatant with the most produced virus by FIV infected PBMC’s wasn’t performed.