| BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a new natural foci disease and was first reported in 2009, which was caused by a new bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV). SFTSV is a single-stranded negative RNA virus, including three RNA segments:L segment, M segment and S segment.SFTS is mainly distributed in the rural areas of the Eastern, Central, and Northeastern China. During the epidemical season (April in Central and Northeastern of China to September), SFTSV is epidemic among farmers over 50 years with a mortality rate of 12%-30%. The main clinical symptoms include fever, leukopenia and thrombocytopenia, general malaise, fatigue, headache, muscle aches, nausea and vomiting. Most cases are sporadic, and some individual clustered cases were also reported. Now it has been confirmed that SFTSV could be transmitted by the blood and secretions of the patients.So far, the studies ofpathogen, epidemiological characteristics, clinical diagnosis and treatment have made some progress, but there is no clear conclusion about the ecological cycle of the SFTSV. Many studies have reported detection of SFTSV in Haemaphysalis longicornis, Rhipicephalus microplus, and Amblyomma testudinarium. H. longicornis is the dominant tick species in China, and current research indicated that ticks, especially H. longicornis, were the major vector and reservoir of SFTSV.In recent years, many emerging and reemerging tick-borne diseases have posed a great threat to the public health. We studied the ecological cycle of the SFTSV, detected tick-borne bacterial pathogens including Anaplasma and Ehrlichia in ticks, and analyzed the seroprevalence of Anapalsma and SFTSV in healthy population.Purposes1. To examine the prevalence of tick-borne SFTSV, Ehrlichia and Anapalsma infection in different developmental stages of H. longicornis ticks from vegetation and to study the role ofH. longicornis in the transmission of SFTSV.2. To establish the animal infection model of SFTSV. to explore the role of H. longicornis in the transmission cycle of SFTSV.3. To express SFTSV NP inprokaryotic system for detection of SFTSV antibody in the healthy population.4. To determine whether SFTSV can be transmitted by H. longicornis and determine whether H. longicornis is the vector and reservoir of SFTSV.5. To determine the seroprevalence of SFTSV and Anaplasma in healthy population in Penglai County, Shangdong Province.Method1. Ticks were collected from vegetation by flagging from June to July in 2013 in Jiaonan County, Shandong Province, and in May of 2015 in Daishan County, Zhejiang Province, respectively.2. Using nested PCR to analyze the prevalence of SFTSV infection among H. longicornis ticks collected from vegetation, the positive PCR products were cloned and sequenced to build phylogenetic tree with Clustal X2.1, MegaAlign, Mega 6.0 software.3. Real-time PCR was used to detect the viral load of SFTSV in infected mice.4. Viral infectious dose was detemined with plaque forming assay.5. SFTSV IgG of SFTSV-infected mice were detected with ELISAand IFA.6. SFTSV NP gene was amplified by PCR and cloned into pET32a vector, which was transformed into BL21 (DE3) to express the recombinant NP. The recombinant NP was purified and tested for its antigenicity with Western Blot.7.628 healthy persons were randomly selected between November of 2015 and January of 2016 in Penglai County, Shandong Province. Sera were collected from the 628 persons to study the prevalence of Anaplasma and SFTSV antibodies by ELISA.Results1. Prevelence of tick-borne diseases1.1 Prevelence of SFTSV in ticksWe collected 3,300 ticks from vegetation in Jiaonan County, Shandong Province. The ticks were morphologically identified and molecularly confirmed as H. longicornis by sequencing the mitochondrial 16S rRNA gene.The prevalence of SFTSV infection was 0.2%(8/3,300) among ticks of all stages. The prevalence of SFTSV infection was 0% among 120 larvae,0.1%(2/1,620) among nymphs, and 0.4% (6/1,560) among adult ticks. Phylogenetic analysis showed that all sequences of SFTSV amplified from ticks were clustered with SFTSV from Shandong Province and other places in China.1.2 Prevelence of Ehrlichia in ticksA total of 2,460 ticks collected from Jiaonan and Daishan counties were used in this study. Ehrlichia was amplified with 16S rRNA, git A and groEL gene primers by nested PCR. The minimal Ehrlichia infection rate of ticks was 0.4%(3/780) for Daishan County, Zhejiang Province and 0% for Jiaonan County, Shandong Province. DNA sequence analysis revealed the homology of the Ehrlichia species from the Chinese ticks to the known Ehrlichia species was 89.6-93.8 %for the groEL gene and 68.5-90.4% for the glt A gene. E. ewingii is the closest species related to the Ehrlichia species from the ticks with both groEL and gltA genes. The phylogenetic analysis based on 16S rRNA gene sequences showed that the Ehrlichia species from ticks were tightly clustered together with uncultured Ehrlichia species from Xinjiang Province, China, and E.ewingii, but were distantly related to other species of Ehrlichia.1.3 Prevelence of Anaplasma in ticks from Jiaonan County, Shandong ProvinceUsing nested PCR with the specific primer 16S rRNA, the minimal Anaplasma infection rate in ticks from Shandong Province was 0.18%(3/1,680). The prevalence of Anaplasma infection was 0 among 120 larvae,0.1%(2/1,200) among nymphs, and 0.28%(1/360) among adult ticks.2. Ticks acquiring SFTSV through acquisition feeding on infected animalsEach stage of ticks was acquisition feeding on SFTSV infected mice to determine the minimum infection rate (MIR) of ticks (assuming that an infected pool of ticks contained 1 SFTSV-infected tick). The MIR was 18%(9/50) for engorged larval and 11.7%(14/120) for molted nymphs. In nymphal acquisition feeding,10 engorged nymphs were tested individually, and all were found to be infected with SFTSV; however, only 20%(2/10) of the adults ticks derived from the nymphs were infected with SFTSV; thirteen engorged adult ticks were tested individually for SFTSV, and 6/13 (46.2%) of them were infected with SFTSV as determined by RT-PCR.53.8%(7/13) of the larval pools were infected with SFTSV.3. Ticks transmission feeding for SFTSVFor study of ticks transmission of SFTSV,10 uninfected mice were fed upon by 10 nymphs molted from larvae fed on SFTSV-infected mice until engorged, viral RNA was detected in 40%(4/10) of the mice.20 adult ticks were fed individually on 20 naive mice until engorged, viral RNA was detected in 10.0%(2/20) of the mice. Real-time PCR results indicate that viral load of SFTSV-infected mice decreased in seven days, and the first day was maximized. According to real-time PCR results, the mice were infected 24 or 48 hours after H. longicornis bites. All SFTSV RT-PCR positive mouse serum samples were also positive by ELISAand IFAat various titers.3. Seroepidemiological study in healthy population in Penglai3.1 Prokayotic pET32a-NP expression vector was constructed successfully, SDS-PAGE result showed that the recombinant protein pET32a-NP mainly expressed as inclusion body protein. The recombinant NP protein was purified by Ni-chelating affinity chromatography.3.2 Seroprevalence of Anaplasma (AP) and SFTSV in healthy populationIn a total of 628 serum samples, AP positive serum was 33, the AP-IgG antibody positive rate was 5.25%; SFTSV positive serum was 22, and the SFTSV-IgG positive rate was 3.5%(22/628).3.2.1 Seroprevalence of AP in healthy population in PenglaiThe AP IgG antibody positive rate was 3.50%. The positive rate of AP-IgG antibody was 2.84%(6/211),3.85%(8/208) and 3.83%(8/209) in serum collected from Xiaomenjia Town, Daliuhang Town and Daxindian Town, respectively. There was no significant difference in seroprevalence among towns (χ2=0.41,p=0.82); the positive rate of AP-IgG antibody was 3.45%(11/319) for males and 3.56%(11/309) for females. There was no significant difference between genders (χ2=0.01,p=0.94); Different occupations were infected with AP and the antibody rates were not significantly different among occupation groups (χ2==3.01, p=0.15); each age group was infected with AP and the antibody positive rate was significantly different among different age groups (χ2=4.82,p=0.09).3.2.2 Seroprevalence of SFTSV in healthy population in PenglaiThe SFTSV-IgG positive rate was 5.25%. The positive rate of SFTSV-IgG was 5.69%(12/211),5.29%(11/208) and 4.78%(10/209) among Xiaomenjia Town, Daliuhang Town and Daxindian Town, respectively. There was no significant difference among towns (χ2=0.15,p=0.93); the positive rate of SFTSV-IgG antibody was 4.39%(14/319) for males and 6.15%(19/309) for females. There was no significant difference between genders (χ2=0.98, p=032); Different age and occupation were infected with SFTSV and the antibody positive rate was significantly different (χ2=4.75, p=0.09; χ2=3.67, p=0.03). The middle age is higher than other age groups. The positive rate of farmer in the occupation group was highest.Conclusion1. The prevalence of SFTSV, Anaplasma and Ehrlichia infection of H. longicornis ticks collected from vegetation was low, suggesting the existence of natural infection of SFTSV, A nap lasm a and Ehrlichia in H. longicornis ticks.2. Both molted nymph and molted adult ticks derived from larvae and nymph ticks fed on SFTSV-infected mice, became SFTSV positive, indicating that SFTSV could be acquired by ticks from animals and can be transstadially transmitted from larvae to nymph ticks, and from nymph to adult ticks.3. After feeding on SFTSV-infected mice, both engorged adult ticks and newly spawned larval ticks became SFTSV positive, indicating that adult female ticks could transovarially transmit SFTSV.4. Nymphs and adult ticks transmitted SFTSV to mice during feeding. We concluded that ticks were the vector and reservoir of SFTSV.5. In healthy population of Penglai County, Shandong Province, the high positive rate of the antibody of AP and SFTSV suggests that it is necessary to educate physicians in the area to diagnose Anaplasma and SFTSV infection. |
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