Three patients (cases A, B, and C) from Hubei Province, China, were admitted to Wuhan Union Hospital from April 2015 to June 2016. The personal information, clinical features, and results of laboratory tests were summarized in Table 1. All three patients were farmers over 50 years old; two were from Macheng City (cases A and C) and one was from Guangshui City (case B). Case B reported that he was bitten by an unknown wild insect 2 weeks before the onset of disease. All patients had fever, headache, fatigue, myalgia, nausea, vomiting, and diarrhea when they presented in the hospital 4-5 days after illness onset. They had leukocytopenia and thrombocytopenia. Moreover, severe proteinuria (2 + to 3 +) and hematuria (3 +) was observed, and serum biochemistry tests revealed elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK). Because patients came from epidemic areas of SFTS and presented typical symptoms and abnormal laboratory results, they were suspected to have been infected by SFTSV. SFTSV RNA was further detected by qRT-PCR from serum samples (Table 1), confirming the occurrence of SFTS in these patients. After treatment, cases A and C recovered and were discharged on the 21th day post illness onset. However, the condition of case B worsened as he developed more severe neurologic symptoms (blurred mind and hyperspasmia), respiratory symptoms (rales in lung and dyspnea), and hemorrhagic manifestations (bleeding in mouth and impaired coagulation). He was transferred to the intensive care unit (ICU) three days after admission. Because his family decided to quit therapy, case B was discharged after spending one day in ICU. Case B died after 10 days post illness onset.
Case A Case B Case C Personal information Age and gender 52, female 59, male 67, male Occupation Farmer Farmer Farmer Admission day May 13, 2015 (the 4th day after illness onset) June 21, 2015 (the 5th day after illness onset) April 7, 2016 (the 5th day after illness onset) Location Macheng Guangshui Macheng Outcome Survived Fatal Survived Clinical manifestation on admission Fever Headache Fatigue Myalgia Yes♮ Yes Yes Yes Yes (38 ℃) Yes Yes Yes Yes (39.5 ℃) Yes Yes Yes Gastrointestinal symptoms Diarrhea Diarrhea Abdominal pain Nausea Vomiting Diarrhea Abdominal pain Respirotory symptoms Rales in lung Rales in lung Dyspnea Rales in lung Neurologic symptoms Headache Headache Blurred mind Limb tremor Headache Blurred mind Blood counts WBC (× 109/L) 3.37↓ 1.97↓ 2.16↓ PLT (× 109/L) 40↓ 16↓ 21↓ Urine routine test Proteinuria +++ ++ +++ Hematuria +++ +++ +++ Serum biochemistry ALT (U/L) 75↑ 91↑ 78↑ AST (U/L) 353↑ 291↑ 291↑ LDH (U/L) 4309↑ 4602↑ 192 CK (U/L) 1502↑ 1096↑ 666↑ CK-MB (U/L) 9.5 3.7 0.9 Laboratory tests qRT-PCR (TCID50/mL) 9.74 × 104 3.41 × 105 4.99 × 105 Virus isolation HBMC16 HBGS13 HBMC5 Note: WBC, white blood cells; PLT, platelet count; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactate dehydrogenase. CK-MB, creatine kinase isoenzyme. aThe accurate body temperature of the patient is uncertain. Clinical parameters out of the normal range are shown in bold numbers with up or down arrows.
Table 1. Personal information, clinical features, and laboratory tests for SFTS patients
Serum samples collected from the three SFTS patients collected on the first day of admission were incubated with Vero cells. After three passages, the supernatants were harvested. RT-PCR was performed, detecting S, M, and L segments in all three samples (data not shown). Blastn comparisons showed that the sequences of the PCR products had very high similarity (95%-99%) to SFTSV strains from Henan Province (data not shown). Furthermore, SFTSV NP expression was detected in Vero cells by IFA (Figure 1A). Virus particles were visualized in supernatants of each sample by negative staining EM analysis, presenting a typical morphology of bunyavirus as enveloped spherical particles with an average diameter of~100 nm (Figure 1B). Taken together, these results identified new SFTSV strains isolated from serum samples of three patients with SFTS; these strains were designated as HBMC16_human_2015 (case A), HBGS13_human_2015 (case B), and HBMC5_human_ 2016 (case C).
Figure 1. Isolation of the three new SFTSV strains in cell culture. (A) Detection of viral protein expression in Vero cells. SFTSV NP expression in Vero cells was detected by immunostaining using IFA after three generations of blind passaging. Cells showing green fluorescence were infected by the three new SFTSV strains. Bars, 1 mm. (B) Visualization of virus particles in cell culture supernatants by negative staining EM analysis. Bars, 200 nm.
One-step growth curve analysis was performed to characterize SFTSV growth properties in vitro. The three isolates showed similar growth properties, with yields of progeny viruses increasing rapidly beginning at 12 h post infection (p.i.) and reached a plateau at 72 h p.i. (P > 0.05; Figure 2).
Figure 2. One-step growth curves of three new SFTSV strains.Vero cells were inoculated with an MOI of 10 TCID 50 units/cell. Supernatants were harvested at 6, 12, 24, 48, 72, and 96 h p.i. Virus titers were determined by end-point dilution assays. The tests were performed in triplicate. Bars, standard deviations.
The complete sequences of HBMC5 (accession numbers: KY440769, KY440770, and KY440771), HBMC16 (accession numbers: KY440775, KY440776, and KY440 777), and HBGS13 (accession numbers: KY440772, KY440773, and KY440774) were sequenced and deposited in GenBank. All reported SFTSV genomes from GenBank can be classified into five genotypes: the C1, C2, C3, C4 and J clades (Shi et al., 2016). Phylogenetic trees based on the complete sequences of S, M, and L segments showed that HBMC5 and HBMC16 together with other strains from Hubei Province (HB29, HB154, HB155, and HB156) clustered into the C3 clade and were most closely related to two strains from Henan Province (HNXY_212 and HNXY_327) (Figure 3). In contrast, HBGS13 belonged to the C2 clade and clustered with other strains from Henan Province (Figure 3).
Figure 3. Phylogenetic analysis of the three new strains.The ML trees were constructed based on the complete sequences of S (A), M (B), and L (C) segments. The three isolates in this study are labeled with black solid circles. The isolates from Hubei Province are highlighted in red solid characters.
Diagnosis of SFTSV infection in patients with typical symptoms of SFTS
Isolation of novel SFTSV strains from serum samples of the three patients
Phylogenetic analysis of the three strains
Primers Sequences (5'-3') Locations of primers S segment S1F acacaaagacccccttcatttrg 1-23 S1R ACACAAAGAACCCCCAAAAAAGGA 1722-1744 M segment M1F acacaragacggccaacaatga 1-22 M1R TCTCCCAGTTGTGAYGCATTCCTTC 1767-1791 M2F GGCAACCAWGATGATGTTAGGAT 1597-1619 M2R ACACAAAGACCGGCCAACACT 3358-3378 L segment L1 F acacaragacgcccagatgrac 1-22 L1 R GAGACCACTGRACCACATTRCTG 1549-1571 L2F GTGTCAATCTTGTTGGAAAARGCAT 1463-1487 L2R GAGCTTYGAGACGAAATARGAC 3313-3314 L3F GGTTGAAGTCAGCCCGYAGTCT 2913-2934 L3R ATTCTRACTACTTGGCTTATGGTGG 4800-4824 L4-F TTAGGGAGAGAAACATTGTCAGGAG 4485-4509 L4-R acacaaagaccgcccagatctta 6346-6368
Table S1. Primers used to sequence the complete genome of SFTSV isolates