JMTV, the first segmented Flavivirus to be identified, has changed the public understanding of the structure of Flavivirus genomes with its genome characterization, while revealing an unexpected evolutionary link between non-segmented and segmented RNA virus genomes [20]. The genomic structure of segmented RNA viruses makes these viruses more susceptible to genetic recombination during evolution, resulting in the emergence of novel genomic combinations and viral strains [21]. At the same time, the greater susceptibility to genetic recombination also makes prevention of segmented viruses such as JMTV more difficult [22]. In addition, JMTV has a widely spread host spectrum and has been detected not only in a variety of ticks such as R. microplus, H. longicornis, Haemaphysalis campanulata, but in D. melanogaster, and two species of mosquitoes. It was also found in many mammals, including sheep, cattle, and bats [23]. This suggested that the transmission route of the virus in nature may be more complex. Besides, JMTV has been shown to be associated with viremia in humans and animals, with infected individuals showing clinical symptoms ranging from mild to severe [16, 24, 25]. Therefore, it is imperative to enhance the epidemiological surveillance and pathogenicity investigation of the virus to elucidate the distribution characteristics and pathogenic mechanism of JMTV, to avoid potential significant public emergency arising from the virus.

In this study, the positive rate of JMTV in ticks collected from sheep was 8.45%. This result indicated a high prevalence of JMTV in engorged ticks, compared to that of our previous study (1.4%) in the same region [18]. This result may be due to several scenarios. Firstly, all the ticks in this study were swollen and engorged adult ticks, while in previous studies, most of the ticks were unfed ticks from grasslands. This indicated that domesticated animals could enhance virus replication or play a role in amplifying hosts during virus transmission, which also validated the role of domestic animals in the life cycle of JMTV [26]. Secondly, the method of calculating the positive rate adopted in the two studies was different. In the present study, the true carrying rate of JMTV in ticks was calculated by testing each tick for viral RNA, whereas in the previous study, the authors predicted the carrying rate by the minimum infection rate (MIR), which may be a lower value than the reality. Besides, JMTV was also detected in sheep blood samples collected and the prevalence was high, maintaining at about 15%. This suggested that the sampling area involved in this study was a JMTV endemic area, where sheep were grazed on pastures or hills during the day freely, resulting in their heavy infestation by ticks carried with JMTV. Meanwhile, this result is also similar to the findings of Guo et al. [23]. They found that the prevalence of JMTV in the blood of bats in the Neixiang area of Henan Province could reach 14.2%. It suggests that JMTV has a high prevalence in mammals of Henan Province and the potential of cross-species transmission that could be transmitted and reproduced in different species of animals. In addition, Qin et al. detected JMTV RNA and related antibodies in the serum of yellow cattle in Hubei, and Guo et al. detected JMTV RNA in rodents in Zhejiang, which further suggests that the host range and geographic distribution of the virus may be broader than currently known [10, 23].

There are several main transmission routes of tick-borne viruses that have been demonstrated, including horizontal (host to tick or tick to host), transstadial (from one tick stage to next), transovarial (from female ticks to their progenies), venereal (tick mating), and co-feeding [27]. Co-feeding is a mode of pathogen transmission for a wide range of tick-borne diseases where susceptible ticks can acquire infection from co-feeding with infected ticks on the same hosts. Studies have reported that D. reticulatus ticks were capable of tick-to-tick non-viraemic transmission of TBEV to the Haemaphysalis inermis nymphs during co-feeding on the same animal under experimental conditions [28]. According to our results, we speculated that this transmission pathway was of great significance to the survival and dissemination of JMTV in nature. In the present study, 38 groups of corresponding RNA samples were obtained from ticks and sheep serum samples, and JMTV was detected simultaneously in sheep serum and corresponding ticks among 4 groups, indicating that the potential viremia might promote the spread of the virus between tick and animal host. Nevertheless, not all ticks from JMTV RNA-positive sheep were detected as carrying JMTV, probably because there were too few copies of this virus below the detection threshold of qRT-PCR due to insufficient blood intake. Otherwise, JMTV was detected positive in some ticks of 4 groups of samples, but negative in the corresponding sheep blood, and this inconsistency may be explained by the fact that ticks carrying JMTV failed to replicate efficiently and cause high levels of viraemia in the host animals for various reasons. These results suggested that there might be a potential link of viral infection between sheep and ticks in JMTV-endemic areas. Moreover, JMTV may existed and be transmitted among ticks and many kinds of other domestic animals (e.g., cattle, pigs, dogs, etc.) within the endemic area.

Vero, Vero E6, BHK-21, C6/36 cells were used for the isolation and characterization of JMTV in this study. Unfortunately, live virus strains that could be stably passaged were not obtained. Despite the fact that the JMTV RNA were detected in the supernatants of these cells in the initial two generations, the viral load was low and absent in the third generation of cell supernatants. Available studies indicated that JMTV was isolated in IRE/CTVM19 cells and could be stably passed on; however, propagation in mammalian cell lines is restricted [29]. This suggests that JMTV was able to grow robustly in tick lineage cells, therefore, cells from other tick sources could be considered for virus isolation.

Eventually, phylogenetic analysis showed that these 9 strains of JMTV in this study were closely related to the isolates found in several provinces of China and shared high homology with each other, indicating that JMTV from different regions had a high degree of similarity. This further demonstrated that JMTV was relatively evolutionarily conserved, which was also consistent with our previous findings. Importantly, JMTV was detected simultaneously in sheep and their attached individual ticks with more than 99.6% nucleotide identity and the closest evolutionary relationship. This suggested that the animal and tick infections were most likely caused by the mutual transmission of the same strain. Through sequence comparison and evolutionary analysis, we further explored the evolutionary relationship among different virus strains. Our findings indicate that domestic animals probably played an important role in the JMTV transmission cycle and also further enriched the evidence for the mode of transmission of JMTV.

In conclusion, our study enriched important evidence for the prevalence of JMTV in Henan Province, and suggested that R. microplus may play an important role in the transmission of JMTV to sheep, which further deepened our understanding of the mechanisms and pathways of JMTV transmission.