The patient was a 69-year-old male plasterer, who resided in Daishan Island in Southeast China. On July 30, 2014, the patient was bitten by a tick on the right upper quadrant of the abdomen. After 2 days, the patient developed a 39.0 °C fever with fatigue and nausea, and was admitted to a local clinic. Leukopenia was noted. The blood specimen collected on admission tested positive for SFTSV using reverse transcription-polymerase chain reaction (RT-PCR) assay [10] at the Centers for Disease Control and Prevention (CDC).
After 5 days of antibiotics and ribavirin treatment, the patient remained feverish, and was transferred to Zhoushan Hospital, which is a regional medical center, on August 6. Upon arrival at Zhoushan Hospital, the patient had a 38.8 °C fever, but was conscious. A visible tick bite mark appeared on right upper quadrant of the patient’s abdomen. The patient complained about the tenderness on two lymph nodes in right groin upon physical examination. A dark red pimple-like bump was located on the lower left chest, and was punctured with a needle. The surviving tick was captured from the bump. The computed tomography (CT) of the patient’s chest revealed calcified pleural nodules on the lower lobe of the right lung and inflammation on the upper lobe of both lungs.
The laboratory tests revealed that the alanine and aspartate aminotransferase levels were elevated to 247 U/L and 987 U/L on day 10, respectively. Furthermore, the lactic dehydrogenase and creatine kinase levels continuously increased to 3211 U/L and 3860 U/L, respectively. However, the creatinine, blood urea nitrogen and serum albumin levels remained normal. The white blood cell count progressively decreased during the course of the patient’s illness. The platelet count was persistently low, which was accompanied by activated partial thromboplastin times (APTTs). The serum viral load, which was determined by qRT-PCR (PrimeScript RT-PCR Kit, Takara Bio Inc., Japan), remained steady at 9.0 × 104 copies/μl until the patient died. The qRT-PCR was performed using primers and probes specific to a conserved region of the SFTSV S segment: Forward primer P3: 5′-ACT CTC TGT GGC AAG ATG CCT TCA-3′; Reverse primer: P4: 5′-AGT TCA CAG CTG CAT GGA GAG GAT-3′, the probe: 5′(FAM)-AAT GTG AAG ATG CGT GGA GCC AGC AA(TAMARA)-3′. The RNA standard was prepared with the S-segment plasmid transcribed by T7 RNA polymerase (Thermo Scientific™, Shanghai, China), according to manufacturer’s instructions. The RNA quantification was carried out using a ND-2000c spectrophotometer (NanoDrop, Wilmington, USA). The standard curves for the SFTSV qRT-PCR were developed with 106, 105, 104, 103, 102 and 10 copies/μL RNA standards.
On the 10th day, the patient suddenly experienced convulsions and obnubilation. The findings from the multi-slice CT scan of the patient’s brain was unremarkable. The patient was transferred to the intensive care unit, and received tracheal intubation with artificial ventilation. The patient died of multiple organ failure on the following day (August 11, 2014).
The viral RNA was isolated from the second blood sample collected on August 6 using the QIAamp MinElute Virus spin kit (QIAGEN, Hilden, Germany), and RT-PCR (PrimeScript RT-PCR Kit, Takara Bio Inc., Japan) was performed. The full length of all three genome segments (L, medium [M] and small [S]) were obtained by overlapping the polymerase chain reaction (PCR) with the primers (see Additional file 1) [7]. The amplified PCR products were directly sequenced (ABI Prism 3100 genetic analyzer; Applied Biosystems, Foster City, CA, USA). The generated sequences were assembled using the DNASTAR 6.0 software. The complete sequences of the L, M and S segments were deposited in GenBank (Accession: KR017844, KR0178863, MT236315, MT236316, MT236317 and MT236318).
The tick that was isolated from the patient was identified as Haemaphysalis longicornis by morphology. The tick was kept alive for 6 months in a dry plastic tube, which was placed in an incubator at 25 °C with 90% relative humidity. The tick was molecularly confirmed as Haemaphysalis longicornis by sequencing the cytochrome c oxidase 1 gene. The RNA was isolated from the tick using a QIAamp MinElute Virus spin kit (QIAGEN, Hilden, Germany). The RT-PCR assay revealed that the SFTSV was RNA positive. Then, the resultant PCR fragments were sequenced, and the sequences of all three SFTSV genome segments were obtained.
Consequently, the whole genomic sequences of these two SFTSV isolates were established. These two isolates shared 100% homology in both the L and S segments (Figs. 1 and 2), and 99.9% homology in the M segment (Fig. 3). A difference from C to T at nt948 and another difference from T to A at nt1713 were detected in the human isolate, when compared to the tick isolate. Phylogenetic analysis was performed using all whole genome sequences from the NCBI database with the MEGA7 software [11]. Maximum likelihood (ML) trees were reconstructed using the bootstrap method, with 100 replications. Six SFTSV genotypes were classified (A-F) [12]. Both isolates in the present study belonged to genotype B, with high bootstrap values.
