J Bacteriol Virol.  2020 Dec;50(4):263-272. 10.4167/jbv.2020.50.4.263.

Isolation and Molecular Characterization of Feline Herpesvirus 1 from Naturally Infected Korean Cats

• Yang DK
• Kim HH
• Park YR
• Yoo JY
• Choi SS
• Park Y
• An S
• Park J
• Kim J
• Kim HJ
• Lee J
• Hyun BH

• KMID: 2512146
• DOI: http://doi.org/10.4167/jbv.2020.50.4.263

Abstract

Feline herpesvirus type 1 (FHV-1) causes respiratory and ocular disease in cats. Although isolates of FHV-1 circulating in cats have been reported worldwide, Korean FHV-1 isolates and their features have not been reported thus far. We aimed to investigate the biological and molecular characterization of two FHV-1 isolates based on the nucleotide sequence of thymidine kinase (TK) and glycoprotein B (gB) gene. In total, 48 samples from 12 cats were prepared for virus isolation.For the diagnosis, virus isolation, indirect fluorescence assay (IFA), electron microscopy (EM), and polymerase chain reaction (PCR) and for the molecular characterization, cloning and sequencing were used. Based on many methods such as virus isolation with specific cytopathic effects, IFA, EM, and PCR, two isolates were confirmed as FHV-1 and they showed the highest viral titer (10^8.3 to 10^8.5 TCID₅₀ /mL) in the Crandell–Rees Feline Kidney cells at 48 h after inoculation, but did not grow in MDCK and Vero cells. The nucleotide and amino acid sequences of the full TK and gB gene of FHV191071 and FHV191072 isolates were determined and compared with those of other herpesvirus strains. Two isolates possessed the same nucleotide sequences belonging to FHV-1 group and had the highest similarity (99.9%) with the KANS-02 strain, which was isolated from shelter in USA in 2016. Two isolates were confirmed as FHV-1 and they will be a useful basic resource for evaluating current FHV-1 vaccine and developing diagnostic tools.

Keyword

Feline herpes virus 1; Virus isolation; Genetic characterization

Figure

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  Fig. 1 Identification of feline herpesvirus type 1 (FHV-1) based on cytopathic effects (CPE) and fluorescence assays. CPE in CRFK cells infected with FHV191071 and FHV191072 isolates at 48 h post-inoculation (A, B); specific fluorescence observed in CRFK cells infected with FHV191071 and FHV191072 (D, E); and normal CRFK cells (C and F). Six pictures are magnified 200 times and scale bars indicate 100 μm.

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  Fig. 2 Growth kinetics of FHV191071 and FHV191072 isolates, according to harvest time in CRFK cells and viral titers of two FHV isolates propagated in six cell types to examine cell tropism. The isolates showed maximum titers of 108.5 and 108.3 TCID50/mL in CRFK cells at 48 h post-inoculation (A). Mean viral titers of the two isolates ranged from 104.3 to 108.4 TCID50/mL in four cell types, while neither isolate grew in MDCK or Vero cells (B).

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  Fig. 3 Viral particles of FHV191071 and FHV191072 isolates purified with sucrose gradient centrifugation (A, C) and in the nuclei of infected CRFK cells, as observed by electron microscopy (B, D). Scale bars indicate 100 nm (A, C) and 500 nm (B, D).

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  Fig. 4 Six primer sets targeting the thymidine kinase (TK) and glycoprotein B (gB) genes of the FHV191071 isolate were used for polymerase chain reaction (PCR). PCR products of expected sizes confirmed that the FHV191071 isolate was feline herpesvirus type 1. M, 1 kb ladder; PCR products of the TK (lanes 1-3) and gB (lanes 4-6) genes of the FHV191071 isolate.

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  Fig. 5 Phylogenetic trees based on the thymidine kinase (TK) (A) and glycoprotein B (gB) genes of animal herpesviruses. In both phylogenetic trees, the FHV191071 and FHV191072 isolates belong to the feline herpesvirus type 1 group.

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  Fig. 6 Alignment of amino acid sequences of TK proteins of the FHV191071 and FHV191072 isolates and the KANS-02 strain. The amino acid threonine at position 341 in the TK protein of the Korean FHV isolates was replaced by isoleucine (solid box). A potential N-linked glycosylation site (N-X-S/T) is indicated by the box with a dashed line.

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  Fig. 7 Changes in mean body weight in mice inoculated with FHV191071 and FHV191072 isolates intraperitoneally. No mice lost weight during the 10-day experimental protocol.

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