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Role of three tick species in the maintenance and transmission of Severe Fever with Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family, causing SFTS with high mortality rate. Haemaphysalis longicornis ticks has been demonstrated as a competent vector of SFTSV by experimental transmission study and field study. However, there has been query whether other tick species that infest human beings in the SFTS endemic regions are capable of transmitting the pathogen. Here by performing experimental transmission study, we compared the capable of transmitting SFTSV among Ixodes sinensis, Ixodes persulcatus and Dermacentor silvarum ticks. The transovarial transmission was seen in the I. sinensis ticks with a rate of 40%, but neither in I. persulcatus nor in D. silvarum ticks. I. sinensis ticks also have the ability to transmit SFTSV horizontally to uninfected mice at 7 days after feeding, but not for I. persalcatus or D. silvarum ticks. In the transstadial transmission of I. persulcatus and D. silvarum ticks, I. persulcatus ticks were tested negative from larvae to adults. But the D. silvarum ticks were tested positive from larvae to nymphs, with the positive rate of 100% (10/10) for engorged larval ticks and 81.25% (13/16) for molted nymphs. However, the mice bitten by SFTSV-infected D. silvarum nymphs were negative for SFTSV detection. Therefore, there is not enough evidence to prove the transstadial transmission of SFTSV in I. persalcatus and D. silvarum ticks.


Introduction
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease that was first recognized in 2009 in China [1], and subsequently reported in Korea [2,3], Japan [4], and Vietnam [5]. The case fatality rate ranged from 16% to 30% that differed among various studies as there is no specific therapy available [6,7,8,9,10]. The casative virus, previously know as SFTS virus [SFTSV]), was recently renamed as Huaiyangshan banyangvirus, by the International Committee on Taxonomy of Viruses [11], as it belongs to the genus Banyangvirus in the family Phenuiviridae. Haemaphysalis longicornis, a tick species with a wide distribution and broad host range, has been demonstrated to be a competent vector of SFTSV, evidenced by sucessful SFTSV transmission through bites on mice in the experimental study [12,13]. Epidemiological study also disclosed a strong association between H. longicornis tick presence and SFTS incidence, therefore corroborating the role of this tick specie in causing and transmitting human infection [14,15,16,17].
On the other hand, SFTSV were also detected in several other tick species, including Rhipicephalus microplus, Rhipicephalus sanguinensis, and Haemaphysalis concinna ticks in SFTS endemic regions [18,19]. In Korea, SFTSV has also been detected from H. longicornis, Haemaphysalis flava, Ixodes nipponensis, and Amblyomma testudinarium ticks in nature [3,20]. All these findings indicated that these ticks might be involve in the circulation of SFTSV in the nature, suggesting the possibility of other tick vectors in haboring and transmitting SFTSV. However, the mere detection or isolation of SFTSV does not guarintine their capacity of acting as competent vectors. The competency of transmitting the virus and causing human infection was determined by complicated factors, including the capacity of amplifying and transmitting SFTSV, adequate infection rate of SFTSV in these tick species, and their adequate contact frequency with human beings. In the current study, we performed an epidemiological study to delineate the correlation between the abundance of predominant tick species and the human incidence of SFTS on county level in SFTS endemic region, and conducted the experimental transmission study to compare the different capacity of transmitting SFTSV among Ixodes sinensis, Ixodes persulcatus, and Dermacentor silvarum ticks.

Determination of major tick species in the SFTS endemic regions
All laboratory-confirmed cases reported from 2010-2018 in China were retrieved from the Chinese Information System for Diseases Control and Prevention (CISDCP). Based on the data of the CISDCP, totally 25 provinces had reported the incidence of human SFTS cases. We analysed the distribution of the predominant tick species in China by checking literatures reporting the occurrence ticks species in China. Briefly, five main electronic databases (PubMed, ISI Web of Science, China WanFang database, China National Knowledge Infrastructure, and Chinese Scientific Journal Database) were searched for studies published between Jan, 1950 and May, 2017, using the following keywords: ("Tick" or "Ticks") and "China" in all fields. We also checked the references in retrieved articles to reach more relevant articles. The information was collected using a standard form, including study date, study area at the county level, tick species identified, laboratory methods, and detection results for tickborne pathogens. Based on literature review, H. longicornis, R. microplus, D. silvarum, I. persulcatus and I. sinensis ticks were determined to be the top 5 commonly seen tick species that bites human beings in China. The geographical distribution of the five tick species and the SFTS laboratory-confirmed cases from 2010 to 2018 in China are shown on the map (S1 Fig), which was created in ArcGIS 9.2 software (ESRI Inc., Redlands, CA, USA). The R. microplus ticks is the main ectoparasite of cattle and considered to be the most important external parasite impacting the cattle industry in the world. There are currently no reports of biting human beings by R. microplus ticks [21,22]. Our laboratory had established colonies of H. longicornis, I. sinensis, I. persulcatus and D. silvarum ticks, which were used for the subsequent experimental transmission study. H. longicornis tick, which has been proved to be a competent vector of SFTSV, was evaluated in the current study as a positive control.

SFTSV strain and culturing
The SFTSV strain WCH/97/HN/China/2011 used in this study was isolated from a human patient in Xinyang City, Henan province, China in 2011 [23], and maintained in Vero E6 Cell line with complete Dulbecco's modified eagle's medium (DMEM) with 10% fetal bovine serum, and 10U/mL penicillin and streptomycin. After the viral titer was determined by plaque assay, the virus was harvested for artificial infection of ticks by microinjection.

Tick colony and experimental animals
I. sinensis, I. persulcatus and D. silvarum ticks were collected by flagging on vegetation in Zhejiang province, Heilongjiang Province, and Inner Mongolia Autonomous Region, China in 2017, respectively. We established SFTSV-free tick colonies in our laboratory from engorged female ticks. Briefly, the adults of D. silvarum ticks were allowed to feed on specific-pathogen free female New Zealand white rabbits weighting 2-3 kg, and the other ticks were allowed to feed on specific-pathogen free female BALB/c mice weighing 10-12 g. All the animals used in this study were supplied by the Laboratory Animal Center of Academy of Military Medical Sciences (Beijing, China). The fully engorged females were kept individually in an Intelligent Climate Cabinet (Saife Company, Ningbo City, China) with a relative humidity of 95 ± 5% at 26˚C until they laid eggs. In each of tick species, 10 batches (30 eggs in each batch) of eggs were randomly sampled to screen for SFTSV by real-time reverse transcription PCR (rRT-PCR) assays as described [12], along with the corresponding engorged females. The eggs from the groups with both the mother tick and the filial eggs detected as SFTSV-negative were incubated to larvae. The larvae and the following nymphs were fed on BALB/c mice, and the molted adults were subjected to the trial.

Artificial infection of ticks with SFTSV
For the artificial infection of adult ticks, the SFTSV-free colony of each tick species were infected with 1 μl SFTSV virus culture (3.65×10 6 PFU/ml), or the same volume of phosphate buffered saline (PBS), by microinjection protocol through its anal pore as described [12]. The ticks which remained alive and active after injection were maintained till to lay eggs and further molt to larvae.
The artificial infection of larvae and nymphs was conducted through feeding on SFTSVinfected mice. Brief, two-week old mice were intraperitoneally inoculated with 200 μl of virus solution (3.65×10 6 PFU/ml) or the same volume of PBS, and the infected mice were subsequently used to feed the ticks for 3-5 days until engorged [13].

Transmission cycle of SFTSV in ticks
The transovarial transmission of SFTSV in I. sinensis, I. persulcatus and D. silvarum ticks and the transstadial transmission of SFTSV in I. persulcatus and D. silvarum ticks were conducted following the procedures (Fig 1). Thirty adults for each three tick species that were microinjected with SFTSV cell culture dilution, were used as SFTSV group, and thirty adults for each three tick species microinjected with PBS were used as control group. Two weeks after Thirty adults for each three tick species that were microinjected with SFTSV cell culture dilution (3.65×10 6 PFU/ml) were used as SFTSV group. Thirty adults for each three tick species microinjected with PBS were used as control group. (B) Transstadial Transmission. To determine transstadial transmission of SFTSV by two tick species, we fed larvae and nymphs on SFTSV-infected mice in the SFTSV group and fed larvae and nymphs on PBS-injected mice in the control group. In larval or nymph acquisition feeding, the engorged larvae or engorged nymphs were tested for SFTSV by RT-PCR before or after molting.
https://doi.org/10.1371/journal.pntd.0008368.g001 injection, the female ticks of I. sinensis and I. persulcatus ticks were fed on BALB/c mice, and the female ticks of D. silvarum ticks were fed on New Zealand white rabbits. The engorged female ticks were maintained until they laid eggs, which were allowed to hatch to larvae under the same conditions as described earlier. We screened subsequent larvae for SFTSV infection to assess the efficiency of transovarial transmission.
To determine transstadial transmission of SFTSV by two tick species, we fed larvae and nymphs on SFTSV-infected mice in the SFTSV group and fed larvae and nymphs on PBSinjected mice in the control group. In larval or nymph acquisition feeding, the engorged larvae or engorged nymphs were tested for SFTSV by rRT-PCR before or after molting. At each developmental stage, ticks were starved for 3 weeks between molting and the next feeding, each period of development of four tick species shown in S1 Table.

Detection of SFTSV in ticks of different developmental stages
Ticks of different developmental stages were subjected to SFTSV detection by performing rRT-PCR. Briefly, total RNAs were extracted from egg pools (60/pool), larva pools (50/pool), engorged larva pools (10/pool), nymph pools (3/pool), engorged nymph pools (3/pool), and individual adult tick, and were then analyzed for SFTSV by rRT-PCR with specific primers and probes. The virus load was determined using rRT-PCR targeting the same gene segments as described [12]. The viral RNA was prepared as a positive control.

Detection of SFTSV in animals
Serum samples were collected from the mice or rabbits 3 times (before tick feed, 1 week after tick engorgement, and 3 weeks after tick repletion). Total RNA was extracted from the sera by using a QIAamp Viral RNA mini kit, and subsequently used for detection of SFTSV by rRT-PCR as mentioned above. Using the viral antigen of the SFTSV strain WCH/97/HN/ China/2011 from the Vero E6 cell line, we detected specific IgG against SFTSV by IFA (Indirect Immunofluorescence Assay), as described [12]. We measured antibody titers with serum dilution starting at 1:20. Samples from uninfected animals were taken at the same time for negative control use.

Carrying days of SFTSV in adult ticks
Before the formal experiment, 60 female ticks from SFTSV-free colony of H. longicornis, I. sinensis, I. persalcatu and D. silvarum ticks by microinjection of SFTSV, five ticks were taken from each tick species to detect for SFTSV RNA on 0, 3, 6, 9, 12, 15, 18 and 21 days post injection. SFTSV genome was detected in the adult ticks for as long as 21 days in H. longicornis ticks, 18 days in I. sinensis ticks, 9 days in I. persalcatus ticks, and 6 days in D. silvarum ticks.

Positive rate of SFTSV in adult ticks
For I. sinensis, I. persalcatu, D. silvarum and H. longicornis ticks, 60 female ticks of each tick species from an SFTSV-free colony were randomly and equally grouped into experimental and control group. Five days post injection, 6 live SFTSV-infected ticks for each tick species were screened for SFTSV infection. All (6/6) I. sinensis ticks, 66.7% (4/6) of I. persalcatu ticks, 33.3% (2/6) of D. silvarum ticks and 100% (6/6) of H. longicornis ticks were positive for detection of SFTSV RNA. Sequencing analysis based on the same gene segments as previously described [12] demonstrated that the virus derived from each of the infected tick species matched that of the inoculated strain. None of the negative control group had SFTSV detected (S2 Table).

Transovarial transmission of SFTSV by ticks
Twelve live female ticks of D. silvarum ticks were fed on New Zealand white rabbits (4 ticks per rabbit), and twelve live female ticks of each of the other three tick species were fed on BALB/c mice (4 ticks per mouse) until the ticks detached from the animal. The engorged females were harvested and maintained to lay eggs (S3 Table). All the 15 pools of eggs laid by 5 infected I. sinensis ticks (3 pools from each tick) were SFTSV RNA positive. All 15 pools of eggs laid by 5 infected I. persalcatus ticks (3 pools from each tick) were SFTSV RNA negative. All 15 pools of eggs laid by 5 infected D. silvarum ticks (3 pools from each tick) were SFTSV RNA negative. None of the egg pools from the control group was positive (Table 1) (Table 1).

Transstadial transmission of SFTSV by three tick species
As I. persalcatus and D. silvarum ticks were negative in hateched larvae, we further performed transstadial transmission of SFTSV by artificially infecting larvae. For I. persalcatu, D. silvarum and H. longicornis ticks, 400 larvae ticks negative for SFTSV of each tick species were artificially infected with SFTSV. In larval acquisition feeding, the engorged larvae were tested for SFTSV by rRT-PCR before and after molting (S4 Table). For I. persulcatus ticks, the engorged larval pools show a positive rate of 55% (11/20), and the molted nymphs was negative (0/20).  (Table 2). These results indicated that transstadial transmission of SFTSV from larvae to nymphs occurred for H. longicornis and D. silvarum ticks, but not for I. persulcatus ticks. The I. sinensis ticks were not tested for the transstadial transmission from larvae to nymphs due to inadequate tick number. For the transstadial transmission study from nymph to adult, 100 negative nymphs of I. persalcatus, D. silvarum and H. longicornis ticks were artificially infected with SFTSV. Similarly, the engorged larvae were tested for SFTSV by rRT-PCR before and after molting (S4 Table). For I. persalcatus and D. silvarum ticks, 10 pools of engorged nymphs of each tick species were all found to be positive for SFTSV and none of the adult ticks of each tick species derived from the nymphs were infected with SFTSV. For H. longicornis ticks, the positive rate was 100% (10/ 10) for engorged nymphs pools and 50% (10/20) for molted adult ticks. Control group of three tick species were also tested negative ( Table 2). These results indicated no transstadial transmission of SFTSV from nymphs to adult ticks for either I. persulcatus or D. silvarum ticks. The I. sinensis ticks were not tested for the transstadial transmission from nymph to adult due to inadequate tick number.

Transmission of SFTSV to animals by ticks
During the transovarial transmission experiment, a total of 18 BALB/c mice and 6 New Zealand white rabbits were used for feeding ticks. For the injected adult tick feeding, 1 of 3 BALB/ c mice fed by the SFTSV-infected adult I. sinensis ticks was positive for detection of SFTSV RNA 7 days after the ticks detached. All the 3 mice bitten by the SFTSV-infected adult I. persulcatus ticks and the 3 rabbits bitten by the SFTSV-infected adult D. silvarum ticks were negative. As a positive control, 3 of 3 BALB/c mice fed by the SFTSV-infected adult H. longicornis ticks was positive for SFTSV RNA detection 7 days after the ticks detachment. The animals bitten by ticks from the control group were also negative. We used IFA to test serum samples from the mice collected before and 3 weeks after detachment of ticks at different developing stages; the animals having positive result for SFTSV RNA detection showed seroconversion against SFTSV (Table 3, Fig 2) During the transstadial transmission experiment, a total of 16 BALB/c mice and 2 New Zealand white rabbits were used for feeding ticks. For the nymph ticks feeding. The 2 mice bitten by the positive nymphs of D. silvarum ticks were negative. None of 2 mice bitten by the nymphs of I. persulcatus ticks were positive. As a positive control, 2 of 2 BALB/c mice fed by the nymphs of H. longicornis ticks was positive. For the adult ticks feeding, the animals bitten by the adults of D. silvarum and I. persulcatus ticks were negative. The animals bitten by the adults of H. longicornis ticks was positive. The animals bitten by ticks from the control group were negative for SFTSV detection. The animals having positive result for SFTSV RNA detection showed seroconversion against SFTSV (Table 4).

Discussion
The current study supported that in addition to H. longicornis ticks, I. sinensis ticks also served as an efficient vector capable of transovarial transmitting SFTSV, therefore posing as a potential threat in causing the circulation of SFTSV. In contrast, D. silvarum and I. persalcatus ticks might not serve as an efficient vector of transmitting SFTSV.
Experimental transstadial transmission of D. silvarum ticks for SFTSV RNA was positive in the larvae to nymph routes, but neither in the nymph to adult route nor in transovarial transmission. The mice bitten by SFTSV-positive nymphs of D. silvarum ticks were negative. There is not enough evidence to prove the transstadial transmission of SFTSV in D. silvarum ticks. We speculate that the possible reason is the time from larval to nymph molting stage of D. silvarum ticks was short. SFTSV in nymph of D. silvarum ticks was only the residue of engorged larvae, but no SFTSV proliferation in nymph of D. silvarum ticks. Further studies are needed to confirm the transstadial transmission of SFTSV in D. silvarum ticks. This presented as the first experimental studies on evaluating other tick species than H. longicornis ticks to serve as vectors of the SFTSV.
Since the discovery of SFTSV, much interest has been put to identify the potential transmission vectors of the pathogen, but mostly via the epidemiological study to perform the molecular test of SFTSV RNA in ticks. A previous study screening H. longicornis ticks showed that SFTSV was detected in 4.93% of the ticks in Hubei and Henan provinces in central China [18] and 2.2% of the ticks in Shandong province [24] in eastern China. Except for H. longicornis, SFTSV was detected in 0.61% of R. microplus ticks in central China [18]. In addition, a recent investigation in Xinjiang Uygur Autonomous Region found the infection with SFTSV in D. nuttalli and Hyalomma asiaticum ticks [25]. Besides China, SFTSV was detected in 4.77% of H. longicornis ticks, 1.15% of H. flava ticks, and 20% in A. testudinarium ticks in South Korea [20]. SFTSV was also detected in I. nipponensis ticks in South Korea [3]. A study in Japan suggested that SFTSV positivity rates were considered to be very low in ticks and viral loads were also very limited, as SFTSV was not detected in 2222 adult and nymph ticks collected from vegetation [26]. These results suggested that several tick species might act as vectors for SFTSV, however, the presence of viral RNA in ticks does not verify that the tick can transmit the virus. Here in addition to H. longicornis ticks, we have tested the most commonly seen tick species in SFTS endemic regions for their role in transmitting SFTSV. Here a differential capacity of transmitting SFTSV was displayed. Among the tested ticks, I. sinensis was shown to be the only competent one, with transovarial transmission competency of SFTSV that was next only to H. longicornis ticks. SFTSV RNA was detected in eggs and larvae of I. sinensis ticks. And one mouse fed by the SFTSV-infected adult I. sinensis ticks became infected, evidenced by both detection of SFTSV RNA and seroconversion. However, the transstadial transmission from larval to nymph and from nymph to adult was not accomplished, due to the low quality of colonies that were kept in the laboratory. Still, the current available results provided adequate evidence that the I. sinensis ticks played roles in maintaining and transmitting SFTSV among natural environment. According to the epidemiological study, I. sinensis tick was predominantly distributed in the middle China, as well as Anhui, Zhejiang, and Fujian provinces, where both H. longicornis and I. sinensis ticks were abundant during the SFTS epidemic season, both could act as reservoirs and vectors for the transmission of SFTSV in the nature. Until recently, there has been no report on the detection of SFTSV in I. sinensis ticks, probably due to its low density in local endemic region, thus more efforts should be put on this tick species, in order to enhance the understanding on this tick species.
In the previous paper [12], we have shown that SFTSV was disseminated in ovaries and salivary glands, indicating the infected H. longicornis ticks could transovarially and transstadially transmit SFTSV successfully. In this study, in addition SFTSV could be transmitted by H. longicornis ticks in both transovarial and transstadial way. Moreover, the maintenance of SFTSV genetic sequences in the adult H. longicornis ticks were determined to last as long as 21 days, significantly longer than those of other three tick species (18 days in I. sinensis ticks, 9 days in I. persalcatus ticks, and 6 days in D. silvarum ticks). Long carrying days of SFTSV in adults of H. longicornis ticks laid the foundation of transmission of SFTSV.
In addition to H. longicornis ticks, we demonstrated that I. sinensis ticks also served as an efficient vector capable of transstadial transmitting SFTSV by experimental study. Less important role of SFTSV transmission was implicated for D. silvarum and I. persalcatus ticks. Further study of tick-host-pathogen interactions are needed to explore the effect of abiotic factors on SFTSV transmission during the animal experiment.  No. ticks fed on each mouse or rabbit