DISCUSSION
We show the metagenomic description of the RNA viruses present in ticks in NE China. Analysis of the transcriptomes of I. persulcatus , D. silvarum ,H. concinna , and H. japonica ticks demonstrated that these ticks harbored a wide diversity of RNA viruses, belonging to at least 8 families of Flaviviridae , Nairoviridae ,Phenuiviridae , Rhabdoviridae , Chuviridae ,Partitiviridae , Tombusviridae , and Solemoviridae . Abundant viruses of the families Flaviviridae ,Nairoviridae , Phenuiviridae , and Chuviridaedetected in the ticks were consistent with those of previous studies, suggesting that viruses of these families have a wide geographical distribution (Guo et al., 2020; Harvey et al., 2019; Klimentov et al., 2020; C. X. Li et al., 2015; Pettersson et al., 2017; Sakamoto et al., 2016; Sameroff et al., 2019; M. Shi et al., 2016; Temmam, Bigot, et al., 2019; Tokarz et al., 2018; Y. Zhang et al., 2021).
Previous metagenomic analysis of ticks in Heilongjiang has revealed viral contigs annotated to South Bay virus (SBV), blacklegged tick phlebovirus (BTPV), deer tick Mononegavirales-like virus (DTMV), and Jingmen tick virus (JMTV) (Meng et al., 2019). Due to lack of whole genome sequence of these viruses, it is difficult to accurately define their classification. In this study, we obtained the complete genome of 16 viral species, including 3 flaviviruses (ALSV, TBEV and BLTV-4), 4 nairoviruses (SGLV, JANV, BJNV, and YCNV), 4 phleboviruses (MV, KV, STPV, and OPTV), 3 rhabdoviruses (THRV-1, 2, and 3), and 2 chuviruses (NUMV and YCMV), showing an extensive diversity of RNA viruses in ticks in northeastern China.
There were significantly differences in tick viromes among CBM, XXAM, and DXAM. In this study, ALSV, TBEV, and THRV2-3 were only detected in DXAL, while MJPV, YCNV, YCMV, and FTLV were identified in CBM and XXAL. Nairovirus SGLV was detected in DXAM, while its closely related virus JANV was detected in CBM and XXAM; Rhabdovirus THRV1 tended to evolve into two genotypes: one was distributed in DXAM, the other distributed in CBM and XXAM. Additionally, the genetic differences of TBEV also supports the occurrence of geographical barriers of tick-borne viruses in northeastern China.
Segmented flaviviruses have recently been reported as emerging tick-borne viruses, with a wide distribution in Asia, Africa, Europe, Central America, and South America (Guo et al., 2020; Temmam, Bigot, et al., 2019). Of them, Jingmen tick virus (JMTV) and ALSV are associated with the febrile illness in tick-bitten patients in northeastern China. JMTV has been found in various vertebrates, including cattle, sheep, rodents, and non-human primate, showing that the virus cocirculates between ticks and mammals (Guo et al., 2020); JMTV and ALSV have also been detected in mosquitoes (Qin et al., 2014; Z. D. Wang et al., 2019); however, the transmission modes of these viruses remain to be investigated. In this study, only I. persulcatus tick was tested ALSV-positive in DXAM, where ALSV patients have been recently found (Z. D. Wang et al., 2019), suggesting potential public health risk of ALSV infection and limited distribution of the emerging tick-borne virus in northeastern China. The pathogenicity of segmented flaviviruses in humans and animals needs to be further verified through animal infection models.
Interestingly, co-feeding transmission might affect the viromes in ticks collected from animals. In this study, two viral species, THRV1 and MKWV, identified in D. silvarum collected from cattle (Shulan), were mainly found in H. japonica and H. concinna , andI. persulcatus ticks, respectively. However, the two viruses were not detected in the questing D. silvarum ticks in Dunhua adjacent to Shulan. As cattle can act as hosts of ticks, such as D. silvarum , H. japonica , H. concinna , and I. persulcatus ticks, and co-feeding transmission may be an efficiency way of viral transmission in ticks (Gargili et al., 2017; Kazimirova et al., 2017; Moraes-Filho, Costa, Gerardi, Soares, & Labruna, 2018). D. silvarum collected from cattle here may contract the viruses fromHaemaphysalis sp . or I. persulcatus ticks by co-feeding transmission, but not the actual vector of these viruses. Thus, it is suggested to investigate tick virome diversity using questing ticks instead of ticks collected from animals. Moreover, further studies should be focus on the vector competence of ticks for the transmission of the identified viruses, which may further confirm the roles of different tick species for virus transmission.
The Bunyavirales order includes important human pathogens, such as Crimean-Congo hemorrhagic fever virus in the Nairoviridae family, and SFTSV and Valley fever virus in the Phenuiviridae family, whose genome are negative single-stranded RNA of small (S), medium (M), and large (L) segments, encoding structural nucleoprotein (NP), glycoprotein precursor (GPC), and RNA-dependent RNA polymerase (L) proteins, respectively. Recently, several bi-segmented viruses without the M gene have been found in nairoviruses (such as Gakugsa tick virus and Norway nairovirus 1) and phleboviruses (such as Tacheng tick virus 2) (Klimentov et al., 2020; C. X. Li et al., 2015; Pettersson et al., 2017; Sakamoto et al., 2016). We also identified four viruses, including YCNV and BJNV within the Nairoviridae family, and STPV together with OTPV in the Phenuiviridae family. However, we did not find the M gene encoding the glycoproteins. The possible reason may be the substantially genetical diversity of these viruses from the reference viruses (Dong et al., 2021; Y. C. Wang et al., 2021).
We also found several viral species closely related to plant viruses, including JPLV1 in the Partitiviridae family, FTLV in theTombusviridae family, and ISAV1, XTAV1, and JLLV2 in theSolemoviridae family. Compared with viruses in theFlaviviridae , Nairoviridae and Phenuiviridaefamilies, plant-related viruses identified here may have low pathogenicity to humans or mammals.
There are some limitations to the present study. Although some tick-borne viruses associated with diseases in humans or mammals, including TBEV, ALSV, SGLV, BJNV, and NUMV, were detected in this study, some other pathogenic viruses, such as severe fever with thrombocytopenia syndrome virus (SFTSV) (H. Liu et al., 2016; X. Zhang et al., 2021), lymphocytic choriomeningitis virus (LCMV) (Zhang et al., 2018), Nairobi sheep disease virus (NSDV) (Gong et al., 2015), and Jingmen tick virus (JMTV) (Jia et al., 2019) identified in previous studies, were not detected here, indicating that larger tick samples and wider sampling sites are necessary to characterize the tick-borne viruses in NE China. Some novel viruses had close relationship with TBVs of public health significance. For example, JANV and YCNV were genetically related to SGLV and BJNV, respectively, which have been shown to be associated with febrile diseases, indicating the potentially pathogenic to humans and animals of these novel viruses. As in thePhenuiviridae family, however, although MKWV does not detected in tick-bitten patients, it can grow in a human-derived cells and mice, and its nonstructural proteins can suppress the anti-innate immune responses (Matsuno et al., 2018), suggesting the potential public health significance of MKWV and its closely related virus (MJPV), and the necessity of epidemiology studies on tick-bitten patients, livestock, and even wild animals.