Molecular detection of feline herpesvirus by means Polymerase Chain Reaction
Creators: name: CARLOS NAVARRO
name: PAULINA MACIAS
Publisher: name: figshare
Year: 2018
Subjects: subject: 70712 Veterinary Virology
subject: FOS: Veterinary sciences
subject: FOS: Veterinary sciences

description: <b>Introduction</b> Feline herpes virus type 1 (VHF-1) was isolated in 1957 by Crandell and Maurer . It is a DNA virus linear double strand containing approximately 134 kilobases (kb). VHF-1 is widely distributed in the world, with approximately 90% of cats being seropositive to the virus. VHF-1 belongs to the order <i>Herpesvirales</i>, family <i>Herpesviridae</i>, subfamily <i>Alphaherpesvirinae</i> and genus <i>Varicellovirus</i>. The viruses of this subfamily are characterized by being highly species-specific, have a short replicative cycle of 24 hours, labile to the environment and common disinfectants, possess a marked tropism to the epithelia and generate latency in neural tissue. <b>Material and Methods</b> This research was carried out in the Virology and Microbiology Laboratories of the Department of Animal Preventive Medicine of the Faculty of Veterinary and Animal Sciences of the University of Chile with its own funding. 1. Samples Samples from 11 domestic cats under one year of age were used, without previous vaccinations, with clinical signs as conjunctivitis uni or bilateral, mucopurulent nasal and ocular discharge, paroxysmal sneezing, blepharospasm and / or corneal ulcer, typical of acute infection with VHF-1. Subsequently, each of them underwent a swab with a dry sterile swab on the ventral conjunctival mucosa of one or both affected eyes. The Feligen® lyophilized triple-feline attenuated vaccine from the Virbac Laboratory was used as a positive control, which was resuspended in 500 uL of the diluent recommended by the manufacturer. As a negative control, a swab with a sterile swab was obtained from each eye of a cat clinically free of feline herpes since childhood and without vaccinations. 2. Detection of the Thymidine Kinase (TK) gene in VHF-1 by n-PCR a) Nested PCR: The primers in this first reaction (PCR1) amplify a fragment of 383 base pairs (bp) and were commissioned to the company Bioscan® for its preparation: VHF-1 1: 5’ –GCATTTACATAGAT GGTGCCT – 3’ and VHF-1 25’ –ATATCTTGCGAGTGGGAAACAG – 3’ . In the second reaction (PCR2), a second pair of primers was used to amplify an internal segment of DNA obtained in PCR1 and with a size of 224 pairs of DNA. bases (13): VHF-1 35’ – CTTACTACTTCCCAGAACC – 3’ and VHF-1 45’ – GTTCCTCACATACAACTTTC – 3’. Reaction mixture: 15 uL of the 2X PCR master mix commercial kit (Taq DNA polymerase, MgCl2 and the deoxyribonucleotides trifostatos), 1 uL of sample and 5 uL of each specific splitter, in a final volume of 26 uL were used. b) DNA amplification: Both the first and the second reaction of the nested PCR (PCR1 and PCR2) are governed by the same amplification protocol: after the initial denaturation at 94ºC for 4 minutes, a PCR sequence of 35 cycles (denaturation: 94ºC for 1 minute, alignment: 55ºC for 1 minute, extension: 72ºC for 1 minute). Subsequently, a final elongation stage at 72ºC for 8 minutes. c) Visualization of amplified products by electrophoresis in 2% agarose gel carried out at 90 V for 45 minutes. After electrophoresis, the gel was incubated in ethidium bromide (0.5 μg / mL) (Fermelo®) for 45 minutes and then placed in a transilluminator of ultraviolet light (Transiluminator UVP ®) and photographed. 3. Determination of percentage of nucleotide identity with respect to data from GenBank®. Analysis. Using the open-access online program Clustal Omega the sequences delivered by Genytec were aligned to achieve a consensus sequence that was then compared with the fragment of the Thymidine Kinase from the feline herpes virus of GenBank® (access number E12463 .1).