Emergence and Phylogenetic Analysis of a Getah Virus Isolated in Southern China

Abstract

Getah virus (GETV) has caused many outbreaks in animals in recent years. Monitoring of the virus and its related diseases is crucial to control the transmission of the virus. In the summer of 2018, we conducted routine tests on clinical samples from different pig farms in Guangxi province, South China, and isolated and characterized a GETV strain, named GX201808. Cytopathic effects were observed in BHK-21 cells inoculated with GX201808. The expression of E2 protein of GETV could be detected in virus-infected cells by indirect immunofluorescence assays. Electron microscopic analysis showed that the virus particles were spherical and ~70 nm in diameter with featured surface fibers. The multistep growth curves showed the virus propagated well in the BHK-21 cells. Molecular genetic analysis revealed that GX201808 belongs to Group 3, represented by Kochi-01-2005 isolated in Japan in 2005, and it clustered closely with the recently reported Chinese strains isolated from pigs, cattle, and foxes. A comparison of the identities of nucleotides and amino acids in the coding regions demonstrated that the GX201808 showed the highest amino acid identity (99.6%) with the HuN1 strain, a highly pathogenic isolate resulting in an outbreak of GETV infection in swine herds in Hunan province in 2017. In the present study, GETV was identified and isolated for the first time in Guangxi province of southern China, suggesting that future surveillance of this virus should be strengthened.

Keywords: Getah virus; emergence; genetic analysis; isolation; phylogenetic analysis.

Figure 1

Isolation and identification of the Getah virus (GETV) GX201808 strain grown in baby hamster kidney (BHK-21) cells. (A) Cytopathic effects (CPEs) in BHK-21 cells infected with the GETV GX201808 strain. Mock-infected and virus-infected BHK-21 cells were observed at 36 hpi. (B) GETV GX201808 strain was identified in infected BHK-21 cells using immunofluorescence assay (IFA) at 24 hpi. The infected BHK-21 cells were fixed and stained using an anti-GETV-E2 PcAb against the GETV E2 protein and goat anti-rabbit H&L IgG. Images were taken using a 20x objective. (C) Plaque morphology of GETV GX201808 on BHK-21 cells. Monolayers of BHK-21 cells in six-well plates were infected with GETV GX201808. The cell monolayers were overlaid with 1% agarose and stained with crystal violet at 48 hpi. (D) Electron microscopic examination of morphology of GETV particles. BHK-21 cells were infected with GETV GX201808, and the precipitated viruses from the supernatants were processed for electron microscopy. (E) The growth of GETV GX201808 at a multiplicity of infection (MOI) of 1 on BHK-21 cells. The viral titers were determined as TCID₅₀, and the values obtained were the means of three independent experiments.

Figure 2

Phylogenetic analysis of GETV GX201808 with the available complete GETV genomes from the GenBank. The phylogenetic trees were generated using the neighbor-joining method implemented in the program MEGA 6.0. Bootstrap values are expressed as a percentage based on 1,000 replications. The strain obtained in this study is indicated by a closed triangle. (A) Diagrammatic representation of a tree based on the complete E2 gene nucleotide sequences of GETVs. (B) Diagrammatic representation of a tree based on the complete genome sequence of GETV GX201808 and the available complete GETV genomes from the GenBank.