Materials and Reagents

1. Vero cells (acquired from ATCC, CCL-81 )
   
2. SFTSV from patients sera
   
3. Dulbecco’s modified Eagle medium (DMEM) + 4.5 g/L D-glucose with L-Glutamine (Life Technology, catalog number: C11965500BT )
   
4. Fetal bovine serum (FBS) (Life Technologies, Gibco^®, catalog number: 10091148 )
   
5. Penicillin streptomycin (PS) (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
6. Antibody against nuclear protein (NP) of SFTSV
   Note: Produced in rabbit by the Antibody Facility of our institute (Sun et al., 2014), the antibody was affinity purified, 1 mg/ml, the working concentration is 5 μg/ml.
   
7. TRIzol reagent (Life Technologies, Invitrogen^TM, catalog number: 15596-026 )
   
8. Reverse Transcriptase M-MLV (RNase H) [Takara Biotechnology (Dalian), catalog number: D2639A ]
   
9. SYBR Premix Ex TaqTM Perfect Real Time [Takara Biotechnology (Dalian), catalog number: DRR039A ]
   
10. Methanol
    
11. 4',6-diamidino-2-phenylindole (DAPI)
    
12. Alexa Fluor 488 goat anti-rabbit (Life Technologies, Invitrogen^TM, catalog number: A11008 )
    
13. Hipure Plasmid Filter Midiprep Kit (Life Technologies, catalog number: K210005 )
    
14. Maintaining cell culture (see Recipes)
    
15. Infection cell culture (see Recipes)
    
16. Mounting media (see Recipes)
    

Equipment

1. T25 flask (25 cm²) (BD, Falcon^TM, catalog number: 353108 )
   
2. Tissue culture plate, 24-well flat bottom (BD, Falcon^TM, catalog number: 353047 ) or 48-well plate (BD, Falcon^TM, catalog number: 353078 )
   
3. 25-cm bottles
   
4. 37 °C, 5% CO₂ incubator
   
5. 7500 fast Real Time PCR System (Life Technologies, Applied Biosystems^®)
   
6. Zeiss LSM510 Meta laser scanning confocal microscope
   

Procedure