Terminal Genome Sequences of the Soft Tick Bunyavirus

The complete genome sequence of the soft tick bunyavirus (STBV) was obtained using the Sanger sequencing technique. Comparison with other viral sequences revealed that STBV has unique sequences in the terminal regions that are highly conserved among the genus Orthonairovirus.

T he soft tick bunyavirus (STBV) was originally isolated by Oba et al. (1) from a pool of soft ticks, Argas vespertilionis, collected in feces beneath bat colonies located in a human habitat in Japan (2). A next-generation sequencing analysis revealed that the STBV genome consists of three negative-strand RNA segments (1). The virus is most closely related to Keterah virus isolated in Malaysia (3), which is a member of the species Keterah orthonairovirus of the genus Orthonairovirus of the family Nairoviridae. The sequence of STBV deposited in GenBank (accession no. LC027465 to LC027467) lacked the 3= and 5= terminal sequences in each segment (24 and 20 nucleotides in segment L, 24 and 17 nucleotides in segment M, and 228 and 23 nucleotides in segment S at the 3= and 5= termini, respectively). Previously, it was recognized that each segment of Orthonairovirus genomes has nine complementary terminal consensus sequences, 3= terminus AGAGUUUCU and 5= terminus AGAAACUCU (4). However, Kuhn et al. (3) reported that several recently identified Orthonairovirus members have consensus regions that differ by one or two nucleotides. Here, we determined the complete genome sequence, including the terminal regions of STBV, and compared the terminal sequences with those of other Orthonairovirus members.
STBV was passaged three times in Vero cells and once in BME/CTVM2 cells (5). The viral RNA was extracted from the culture supernatant using a High Pure viral RNA kit (Roche Applied Science, Mannheim, Germany). To obtain DNA corresponding to the viral terminal regions, rapid amplification of cDNA ends (RACE) (6) was performed. Virus-specific primers used in RACE were as follows: CCCCAGTAATCATCTCTC for the L segment, TCTCTGTGTCCACTGTTC for the M segment, and GAAGCAGAGAGAGTTGCT for the S segment at the 3= termini and GGACTAATCTAATCTGCGGC for the L segment, GGAACACCGCAGTACTATCT for the M segment, and GGCTTCTACCTGCACTAACA for the S segment at the 5= termini. The amplified products were directly used for sequencing without cloning using Sanger's method (7) with an Applied Biosystems 3500xL genetic analyzer (Thermo Fisher Scientific) according to the manufacturer's protocol. The complete sequences of the STBV L, M, and S segments were deposited in GenBank under accession no. LC495731, LC495732, and LC495733, respectively.
Although a few differences were detected in the L segment (3 nucleotides) and S segment (13 nucleotides), the determined sequences were almost the same as those in the previously reported sequences, except for the termini (1). The terminal 9-nucleotide sequences of STBV were not the same as those of the consensus sequences of the genus Orthonairovirus (4), and noncomplementary nucleotide pairings at the fifth-mostterminal nucleotide were observed in the L and M segments (Table 1). We observed the same terminal sequences in the Issyk-Kul virus (ISKV) propagated in SW-13 cells by applying the strategy for sequencing described above (GenBank accession no. LC495734 to LC495736); terminal nucleotides of ISKV were identical to those reported by Atkinson et al. using the virus propagated in a suckling mouse (8). Thus, the terminal sequence appears to well reflect the reported phylogenetic tree (3). Similar terminal sequences with one or two mismatched pairings were observed for STBV-related viruses, e.g., Qalyub virus and Dera Ghazi Khan virus (Table 1).
In summary, a full-genome sequence, including the termini of STBV was determined using Sanger's method. The STBV has the same terminal sequences as ISKV. Determination of terminal sequences of viral genomes might accelerate the classification of Orthonairovirus members.
Data availability. The sequences determined in the present study have been deposited under the accession no. LC495731, LC495732, and LC495733 (STBV) and LC495734, LC495735, and LC495736 (ISKV).