| ObjectiveTo provide a scientific basis for prevention and control of SFTS(severe fever with thrombocytopenia syndrome), epidemiology characteristics of SFTS, laboratory diagnostic methods of SFTS and possible animal hosts and vectors were investigated.Methods1. Laboratory diagnosis, clinical features and epidemiological studies of SFTS patients. Acute sera were collected from24suspected patients with SFTS in Yiyuan County, Shandong Province from June to November in2011. Their convalescent sera were collected after three months. We also recorded their basic datas, clinical information and laboratory test results. Sera were tested by Enzyme-linked immunosorbent assay (ELISA) for total antibodies to SFTSV, at the same time, followed the detection of SFTSV RNA with RT-PCR. RT-PCR primers were designed from the sequences of L, M, and S segments of SFTSV genome by using Primer Premmier5.0and Oligo6.0softwares (LCYY/L、MCYY/M、SCYY/S). Six primers were obtained from references(LW/LN MW/MN、SW/SN) in order to amplify the sequences of SFTSV. SFTSV RNA detection rate between different primers were also compared. SFTSV RANs were amplified by RT-PCR and sequenced. According to the diagnostic standard,22patients were confirmed SFTS. Then we studied epidemilogical characteristics and clinical features of them. At the same time, the relationship between the detection rate and serum acquisition time were compared.2. Seroprevalence of SFTSV in healthy persons. Sera were collected from healty persons and a questionnaire regarding age, sex, history of illness, tick exposure, occupation was taken from each participant. The sera were tested for antibody to SFTSV using double antigen sandwich ELISA.3. SFTSV RNA, antibodies detection and virus isolation from goats’sera. Venous blood were collected from free-roaming goats in SFTS epidemic areas in Yiyuan County and sera were separated conventionally. Goats sera were tested by ELISA for antibodies to SFTSV, and SFTSV was isolated from them with cells DH82.4.SFTSV RNA detection and virus isolation from ticks and eggs. The free ticks were collected manually with white cloth from the grassland, and the parasitic ticks were collected from the domestic animals by handing searching. All ticks were collected into the same tube by the areas, then they were identificated morphologically. Part of the engorging ticks lay their eggs in the laboratory. All the ticks were grinded on the ice, and then followed by detection of SFTSV RNA with RT-PCR and isolation of SFTSV with DH82cells.5. SFTSV RNA and antibodies test in rodents. Rodents were traped in field from SFTSV endemic areas in Shandong Province. Rodent pecies and gerder were identified, then spleens were harvested from all rodents in the field and blood sample was collected from heart using absorbing paper strip, All spleens were grinded on the ice, and then followed by detection of SFTSV with RT-PCR. At the same time, blood samples were tested by ELISA for antibodies to SFTSV.Results1.SFTS laboratory diagnosis.54.2%(13/24) of acute sera and85.7%(18/21) of convalescent sera were positive for antibodies to SFTSV, respectively.50%(12/24) specimen were detected SFTSV RNA. We can confirme22patients from24suspected patients. RT-PCR results showed that the SFTSV detection rate was different between primers. For the same segment, the RT-PCR detection rate was varied with different primers. S segment was most frequently amplified with one primer pair (SCYY/S), L-segment was amplified occasionally and M segment was never amplified.Sera SFTSV RNA detection within7days was64.5%and between8to14days was37.5%. The difference was not statistically significant (P>0.05).2. Clinical features and epidemiological characteristics of SFTS patients. Mainly clinical characters were fever and gastrointestinal symptoms. Every patients had fever. Routine blood tests occur thrombocytopenia and (or) white blood cells reduce. Their onset time concentrated in May to October, peaked in August. There were14male and8female, aged from40-80years old.22confirmed patients were all farmers.90.9%patients raised cattle and27.3%had clear history of tick bite. There was no significant gender differences between different age groups.3.Age of the healthy persons distribed from20to89, the median age was54and the ratio of male to female was1:1.6. There were no significant difference on gender distribution among different age groups. They were all farmers.79.7%of healthy people raise cattle, and they all had not the clear history of tick bite.0.8%(2/237) sera from healthy people were positive for antibodies to SFTSV and antibody titer was above128.4.85.6%(161/188) goats sera were positive for antibodies to SFTSV and sera antibody titer between1:32to1:1024and SFTSV were not isolated. We can not detect SFTSV RNA by RT-PCR from27goats sera which were negative for antibodies to SFTSV.5.All the ticks we collected were Haemaphysalis longicornis.450free adult ticks and2000nymphal ticks were collected and SFTSV RNA were not detected by PCR.517parasitic ticks were collected from goats.0.2(1/492) parasitic ticks could detect SFTSV RNA. SFTSV RNA were not detected in eggs producted by25engorging ticks.6.A total of93rodents were collected include Apodemus agrarius (59/93), Cricetulus tyiton (3/93), Rattus flavipectus (17/93), Rattus norvegicus (11/93) and Shrew (3/93). The results of detecting SFTSV RNA from rodents’spleen were all negative. ELISA showed that none of the rodents was positive to SFTSV antibodies.Conclusions1. Healthy people exists SFTSV infection in Yiyuan County, Shandong Province In SFTS epidemic areas, clinical fever with thrombocytopenia and (or) leukopenia should consider SFTS infection firstly and take timely antiviral treatment when SFTS is prevalent.2. RT-PCR detected SFTSV RNA in serum from SFTS patients in Yiyuan County, primers SCYY/S were priority selection. 3.Goat was probable animal host of SFTSV and ticks might infect SFTSV through blood. It was uncertain that SFTSV transmited vertically by ticks and did not find rodents infec or carry SFTSV. |
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