Isolation and characterization of a novel coltivirus from Haemaphysalis concinna ticks in Northeastern China

Coltiviruses are spherical, non-enveloped viruses with 12 double-stranded RNA segments, belonging to the family Spinareoviridae, and predominantly transmitted by ticks. This study isolated and characterized a novel coltivirus, designated Woodland tick reovirus (WLTRV), from Haemaphysalis concinna ticks collected in Helong City, Jilin Province, in Northeastern China.

SW-13 and Vero cells were used to isolate WLTRV through three blind passages, while seven mammalian cell lines assessed viral growth. Viral morphology was observed by electron microscopy. Next-generation sequencing, 5' and 3' rapid amplification of cDNA ends were used to determine WLTRV whole genome sequences, and phylogenetic methods were used to characterize WLTRV. A real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect WLTRV RNA in ticks.

WLTRV grew and exerted cytopathic effects in human (SW-13 and 293T) and mouse (BHK-21 and N2A) cell lines, revealing its potential to infect mammals. Phylogenetic analysis based on RNA-dependent RNA polymerase sequences classified WLTRV within the genus Coltivirus, with a close evolutionary relationship to Tarumizu tick virus. The nucleotide and amino acid sequence homologies between WLTRV and Tarumizu tick virus across the 12 segments analyzed ranged from approximately 44.79% to 69.09% and 33.73% to 75.60%, respectively. WLTRV shared conserved the 5'-terminal (GACA^A/_U ^U/_A) and 3'-terminal (UGCAGUC) consensus sequences of the genus Coltivirus genomes. Electron microscopy revealed WLTRV as spherical (diameter ∼80 nm), non-enveloped, and morphologically consistent with coltiviruses. Among the 4,717 ticks collected from six towns in the Yanbian Korean Autonomous Prefecture, WLTRV RNA was only detected in H. concinna (0.95% virus-carrying rate) but not in Haemaphysalis japonica, Haemaphysalis longicornis, Ixodes persulcatus, and Dermacentor silvarum.

This study represents the first isolation and identification of WLTRV from H. concinna in the Yanbian Korean Autonomous Prefecture, providing new insights into the genetic diversity and evolution of the genus Coltivirus.