Transmission of Severe Fever with Thrombocytopenia Syndrome Virus by Haemaphysalis longicornis Ticks, China

We demonstrate maintenance and transmission of severe fever with thrombocytopenia syndrome virus by Haemaphysalis longicornis ticks in the larva, nymph, and adult stages with dissemination in salivary gland, midgut, and ovarian tissues. The H. longicornis tick is a competent vector to transmit this virus in both transovarial and transstadial modes.

the following nymphs were fed on Balb/C mice, and the molted adults were subjected to the trial.
The transmission cycle of SFTSV in H. longicornis ticks was simulated following the procedures shown in Technical Appendix Figure 1, with each step described as follows.

Artificial Infection of Ticks with SFTSV by Microinjection
Adult female H. longicornis ticks from the aforementioned SFTSV-free colony were infected with SFTSV by the microinjection protocol developed by Kocan et al. (2) with modification. We injected 1 L of virus culture (5.9×10 5 copies/mL) into each tick through its anal pore with a microsyringe (1 inch, 33 gauge needle) under a dissecting stereomicroscope (Technical Appendix Figure 2). We injected the same volume of phosphate-buffered saline (PBS) into ticks that were used as the control group. The ticks that were crawling and active after injection were maintained in an Intelligent Climate Cabinet (Saife Company, Ningbo City, China) with a relative humidity of 95 ± 5% at 22°C.

Transmission Cycle of SFTSV in Ticks
Two weeks after injection, the female ticks were fed on Balb/C mice so we could investigate transovarial transmission. 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. Larvae and subsequent nymphs were allowed to feed on Balb/C mice until fully engorged and molt to nymphs and adults. At each developmental stage, ticks were starved for 3 weeks between molting and the next feeding. We tested the derived nymphs and adults for SFTSV to evaluate the efficiency of transstadial transmission.

Detection of SFTSV in Ticks of Different Developmental Stages
We subjected ticks of different developmental stages to real-time PCR and RT-PCR to determine their SFTSV infection status. We extracted RNA from egg pools (60/pool), larva pools (50/pool), nymph pools (5/pool) (Technical Appendix Table 2 We prepared the positive control standard (nt. 1456-1557 of the SFTS virus segment S sequence, reference sequence GenBank accession no. KC505134) as described previously (4).
We prepared serial dilutions from 10 8 to 10 3 copies/mL in diethylpyrocarbonate-treated water and stored them in RNase-free tubes at 80°C.
We performed quantification of SFTSV as described earlier in the 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Absolute RNA quantification was included in every assay and was generated by using RNA transcripts produced earlier. Standard curves included 5 dilutions and 3 replicate wells for each dilution. All samples were quantified in 3 replicate wells. Levels of SFTSV RNA concentrations were expressed as copies/mL.

Detection of SFTSV in Saliva and Hemolymph of Adult Ticks
To prepare the molted adult ticks to salivate, we allowed them to engorge partially by feeding on Balb/C mice. Saliva was collected (Technical Appendix Figure 3, panel A) from the engorged ticks as described previously (5). After saliva collection, we obtained hemolymph samples from each tick by clipping a front leg and placing the tip of a glass micropipette to the wound (Technical Appendix Figure 3, panel B). The saliva from 5 ticks in each group was Page 4 of 10 pooled and the hemolymph from the same 5 ticks was pooled, mixed with 140 L PBS, and subjected to the RNA extraction procedure using the QIAamp Viral RNA Mini Kit (QIAGEN).
We then applied the RNA to RT-PCR assay, for which positive amplicons were sequenced as mentioned earlier.

IFA Detection of SFTSV in Ticks
Twelve days following injection with SFTSV, we embedded the whole bodies of the ticks in paraffin and cut them longitudinally at a cryostat (Leica CM 3050; Leica Microsystems, Wetzlar, Germany). We put the frozen slices on glass slides and subjected them to immunofluorescent assay (IFA) for SFTSV detection.
We then selected 10 females at random from the SFTSV and control groups and individually dissected their guts, salivary glands, and ovaries under sterile conditions using a dissecting microscope. After marginal cuts and scutum removal with lancets, we carefully removed the hemolymph around the tissues with filter papers and replaced it with sterilized PBS 3 times. We placed the tick tissues on glass slides and subjected them to IFA for SFTSV detection.
We soaked sheet glasses with prepared tissues in PBS with 5% skim milk to deparaffinize. We incubated the slices at 37°C for 1 hour with mAb that was previously prepared (6) in PBS with 0.05% Tween20. We used serum from Balb/C mice asa negative control.
Following triple washing with PBS and 1 final washing with distilled water, we incubated the slices at 37°C for 30 minutes with fluorescence-conjugated goat antimouse antibodies (Zhongshanjinqiao, Beijing, China) at 1:100 dilution with Evans blue. We washed the slices in PBS 3 times and finally washed them with distilled water, visualizing with an Olympus BX51 Microscope until dried.

Detection of SFTSV in Mice
We collected serum samples from the mice 3 times (before tick feed, 1 week after tick engorgement, and 3 weeks after tick repletion) and extracted RNA using a QIAamp Viral RNA mini kit and detection by real-time PCR, as described earlier. By using the viral antigen of the SFTSV patient source from the Vero E6 cell line, we detected specific IgG against SFTSV by indirect IFA, as previously described. We measured antibody titers with serum dilution starting at 1:16 and then serially 2-fold to determine endpoint titers.