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Molecular diagnostics and phylogenetic analysis of feline immunodeficiency virus env gene in naturally infected cats in Croatia

Feline immunodeficiency virus (FIV) is an important viral infection of domestic cats, which occurs worldwide. Like other members of the genus Lentivirus, FIV causes a lifelong infection after the integration of provirus into host cell genome. The virus is mainly transmitted by parenteral inoculation of the virus present in saliva and blood through bites and fight wounds. The aim of this study was to determine persistent FIV infection by proving the specific nucleotide sequences of the provirus env gene as well as to determine the phylogenetic relationships of FIV sequences identified in Croatia. The research was conducted on 14 FIV-positive cats. The infection was confirmed by using the commercially available IDEXX SNAP Combo Plus test. The test detected a FIVspecific antibody to viral antigen gp40 in the blood samples. Prior to molecular testing, DNA was extracted from EDTA-treated cat whole blood using the QIAamp DNA Mini Kit. The proviral env gene was amplified with primers FIV-7316F (5ʹ - ATA CCA AAA TGT GGA TGG TG - 3ʹ) and FIV-7868R (5ʹ - TGC AAG ACC AAT TTC CAG CA - 3ʹ). An agarose gel electrophoresis was performed to visualise PCR product. All 14 positive PCR products were sequenced in both directions by using Macrogen Europe. A persistent infection was proved in 14 FIV serologically positive cats by using the stated molecular method. Phylogenetic analyses of the multiplied sequence of FIV proviral env gene, along with the sequences of the env gene from the gene bank, identified the phylogenetic affiliation of all 14 isolates from the territory of the Republic of Croatia to the viral subtype B. In addition to belonging to the viral B subtype, the isolates were phylogenetically shown to correlate with other isolates from the nearest geographic region. The majority of the nucleotide sequences obtained within this research show the highest resemblance with the isolates from Austria. Although phylogenetically all 14 nucleotide sequences of the proviral env gene belong to the B subtype, their mutual heterogeneity was also observed.

molecular, env gene, FIV, Croatia

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