Isolation and Identification of a Highly Divergent Kaeng Khoi Virus from Bat Flies (Eucampsipoda sundaica) in China

Background: Kaeng Khoi virus (KKV), which belongs to the genus Orthobunyavirus, family Perbunyaviridae, was originally isolated from the brain tissue of bats and may cause infection in humans. In this study, the KKV strain WDBC1403 was isolated from bat flies (Eucampsipoda sundaica), ectoparasites of the bat Rousettus leschenaultia, collected from Yunnan Province of China at the Sino-Burmese border. Methods and Results: The bat fly specimens were ground and inoculated in culture cells. The WDBC1403 strain was shown to induce cytopathic effects in Vero, baby hamster kidney (BHK-21), and Tb1Lu cells, but not in C6/36 cells; however, viral gene amplification was detected in the supernatants of C6/36 cells. Using electron microscopy, the virus was determined to be spherical, enveloped, and 80-90 nm in diameter; it was also shown to form plaques in BHK-21 cells and the titer reached 1 x 10(6.57) plaque-forming units/mL 24 h after infection. The phylogenetic analysis showed that WDBC1403 is a KKV strain, but is independent of the original KKV strain (PSC-19). Viral genome analysis revealed that the nucleotide and amino acid sequence identities of the S, M, and L segments of WDBC1403 with PSC-19 were 88.2% and 96.1%, 76.7% and 85.0%, and 78.3% and 88.9%, respectively. Two amino acids were removed at the end of the open reading frame of the M segment, and 47 nucleotides were removed in the 3 '-untranslated region of the M segment of the WDBC1403 strain compared with the PSC-19 strain. Conclusions: The WDBC1403 strain is a highly divergent KKV strain, suggesting that there are a variety of KKV strains that exhibit molecular differences. Moreover, because of the large variations in nucleotide and amino acid sequence in the M segment, which encodes an important membrane protein, further research on antigenicity and pathogenicity in humans and animals is needed.