Figure 1

Figure 1. Migratory bird flyways and distribution of Ixodes persulcatus ticks in study of transboundary movement of Yezo virus via ticks on birds, Japan, 2020–2021. Distribution of I. persulcatus...

We conducted this research under approval by the animal research review board of Yamashina Institute for Ornithology, Chiba, Japan (approval nos. 2020-004 and 2021-002). We collected ticks infesting birds that mostly fly from Sakhalin and the Kuril Islands to Hokkaido, Japan. We collected the ticks during October 2–12, 2020, and October 2–13, 2021, in Lake Kutcharo, Hamatonbetsu Town, and Lake Furen, Nemuro City, Japan (Figure 1). We analyzed YEZV genes in the ticks to clarify virus spread. Because migratory birds at these locations in autumn are considered to have just arrived from Sakhalin (Sakhalin route) and the Kuril Islands (Kuril Islands route) (Figure 1), it is likely that the ticks crossed the sea attached to the birds.

We collected 2,534 ticks from 15 species of birds in October 2020 and 2021 (Appendix Tables 1, 2). All ticks were morphologically identified and pooled according to their species, life stage, and host species. Each pool consisted of <10 larval or 5 nymphal ticks (Appendix).

We examined 2,323 Ixodes persulcatus ticks (1,727 larvae and 596 nymphs), 203 I. pavlovskyi ticks (169 larvae and 34 nymphs), 1 I. turdus tick (nymph), 3 Haemaphysalis megaspinosa ticks (larvae), and 4 H. concinna ticks (3 larvae and 1 nymph) by using quantitative reverse transcription PCR (Appendix). Of those, 8 pools of I. persulcatus ticks were positive for YEZV RNA; no pools for the other tick species were YEZV positive (overall minimum infection rate 0.3%) (Table 1). Seven of 8 positive pools showed low cycle threshold (Ct) values of 22.08–30.08; 1 pool had a high Ct value of 39.19. Seven pools with low Ct values were used for further analysis. Ticks in all positive pools were I. persulcatus collected from black-faced buntings (Emberiza spodocephala) (Table 2). Five pools were collected in Lake Furen, Nemuro City, Hokkaido, and the other pools were collected in Lake Kutcharo, Hamatonbetsu Town, Hokkaido. Results indicated that ticks with YEZV were transferred by migratory birds along both the Sakhalin and Kuril Islands routes.

In I. persulcatus ticks, the minimum infection rate of YEZV was 0.2% in larvae and 0.8% in nymphs (Table 1). YEZV has been reported in 3 adult tick species, H. megaspinosa, I. ovatus, and I. persulcatus, found on vegetation in Hokkaido (1). In addition, 4 YEZV–positive pools consisted of only unfed ticks (Appendix Table 3), indicating that I. persulcatus might transmit Yezo virus.

We sequenced 7 complete genomes of YEZV: YEZV/tick/BT-1821/Japan/2020, YEZV/tick/BT-1826/Japan/2020, YEZV/tick/BT-1844/Japan/2020, YEZV/tick/BT-1864/Japan/2020, YEZV/tick/BT-1968/Japan/2020, YEZV/tick/BT-2135/Japan/2021, and YEZV/tick/BT-2155/Japan/2021 (Appendix) and deposited those sequences into the DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp) (Table 2). For 1 pool that had a high Ct value, YEZV/tick/BT-1822/Japan/2020, we could not determine the complete genome sequence; however, we amplified a short fragment of the S segment by nested reverse transcription PCR and deposited that sequence in the DNA Data Bank of Japan (Table 2).

We analyzed the nucleotide and amino acid sequences of the RNA-dependent RNA polymerase, glycoprotein precursor, and nucleoprotein among YEZV strains and compared those sequences to Sulina virus IxriSL16–01 sequences (10) (Appendix Tables 4–6). Sequence identity between YEZV strains was 93.0%–100.0% at the nucleotide level and 99.2%–100.0% at the amino acid level.

Figure 2

Figure 2. Phylogenetic analysis of Yezo virus strains in study of transboundary movement of Yezo virus via ticks on migratory birds, Japan, 2020–2021. Trees were constructed by using the maximum-likelihood method in...

We also performed phylogenetic analysis of YEZV strains by using the nucleotide sequences of the open reading frames of the L, M, and S segments (Figure 2). All tick-derived strains detected in this study belonged to the same cluster as the strains obtained from patients in Hokkaido, YEZV/human/HH003-2020/Japan/2020 and YEZV/human/HH008-2017/Japan/2017 (1). In addition, our tick-derived strain, YEZV/tick/BT-2135/Japan/2021, and the tick-derived strain from Heilongjiang, China, YEZV/tick/T-HLJ02/People’s Republic of China/2021, were reassortment strains; both were generated through reassortment with the strains found in patients in Hokkaido, Japan. We also confirmed the events of genetic reassortment by using a recombination detection program (A. Nishino and K. Maeda, unpub. data), which suggested that YEZV might be transferred between China and Hokkaido, Japan.

We successfully detected YEZV in I. persulcatus ticks from migratory birds (E. spodocephala buntings) flying to Hokkaido, Japan, from Sakhalin and the Kuril Islands (5,11). The distribution of I. persulcatus ticks partially overlapped with the flyway of E. spodocephala buntings passing through Hokkaido in autumn (12-15). Together with the possible genetic reassortment event between YEZV strains from Japan and China, this finding indicated that YEZV might be carried by migratory birds from other countries to Japan and from Hokkaido to other prefectures in Japan. To elucidate the distribution area and transmission routes of YEZV, further surveillance of YEZV infection should be conducted in ticks, birds, and wild and domestic animals.