Genetic and biological characterization of tick-borne encephalitis virus isolated from wild rodents in southern Hokkaido, Japan in 2008

Abstract

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. A recent epizootiological survey indicated that endemic foci of TBEV have been maintained in the southern part of Hokkaido until recently. In this study, we sought to isolate TBEV from wild rodents in the area. One virus, designated Oshima 08-As, was isolated from an Apodemus speciosus captured in Hokuto in 2008. Oshima 08-As was classified as the Far Eastern subtype of TBEV and formed a cluster with the other strains isolated in Hokkaido from 1995 to 1996. Thirty-six nucleotide differences resulted in 12 amino acid changes between Oshima 08-As and Oshima 5-10 isolated in 1995. Oshima 08-As caused high mortality and morbidity in a mouse model compared with Oshima 5-10. Although similar transient viral multiplication in the spleen was observed in the mice infected with Oshima 08-As and Oshima 5-10, greater viral multiplication with an inflammatory response was noted in the brains of mice infected with Oshima 08-As than those infected with Oshima 5-10. These data indicate that a few naturally occurring mutations affect the pathogenicity of the Oshima strains endemic in the southern part of Hokkaido.

FIG. 1.

Geographical location of the study areas.

FIG. 2.

Detection of tick-borne encephalitis virus (TBEV)-specific antigen and RNA. Baby hamster kidney (BHK) cells were inoculated with the brain homogenate of a sick mouse inoculated with the spleen homogenate of wild rodents. Inoculated (A) and mock-treated (B) cells were stained using anti-tick-borne flavivirus antibodies. (C) TBEV-specific products were amplified by RT-PCR from cells infected with the brain homogenate of the sick mouse (lane 1). TBEV-infected (lane 2) and mock-treated (lane 3) cells were used as positive and negative controls, respectively.

FIG. 3.

Phylogenetic tree of the tick-borne encephalitis virus (TBEV) strains The tree was constructed using 1488 nucleotides of the viral E gene and Omsk hemorrhagic fever virus (OHFV) as the outgroup. Horizontal distances are proportional to the minimum number of nucleotide differences. The numbers beside the branches are bootstrap values. Accession numbers are shown after the virus strains.

FIG. 4.

Comparison of the growth curves of Oshima 08-As and Oshima 5–10. A monolayer of baby hamster kidney (BHK) cells was infected with each virus at a multiplicity of infection (MOI) of 0.01. At each time point, the medium was harvested and virus titers were determined using a plaque assay in BHK cells.

FIG. 5.

The survival of mice inoculated with Oshima 08-As and Oshima 5–10. Mice were inoculated subcutaneously with 10² to 10⁶ plaque-forming units (pfu) of each virus and were monitored for 28 days.

FIG. 6.

Virus replication in organs. Mice were infected with 1000 pfu of Oshima 08-As or Oshima 5–10. Virus titers in spleen (A) and brain (B) at the indicated days after infection were determined using plaque assays. Error bars represent the standard deviation (SD) (n=3).

FIG. 7.

Histopathological features in the brain at 10 days post-infection. B6 mice were infected with 103 pfu of the Oshima 08-As (A and B) or Oshima 5–10 (C and D) strain. TBEV antigens were detected using E protein-specific antibodies (lower columns: B and D).