Detection Of Neutralizing Antibodies In SFTS Patients,Expression Of Glycoprotein Of SFTSV And Discovery Of A Novel Rickettsia

BackgroundSevere fever with thrombocytopenia syndrome(SFTS)is a natural focus disease mainly characterized by the clinical manifestations of fever and thrombocytopenia.Originally this disease was misdiagnosed as human granulocytic anaplasmosis(HGA)until that Doctor Yu isolated a novel bunyavirus named severe fever with thrombocytopenia syndrome bunyavirus(SFTSV)from a serum of a patient in 2009.SFTSV is a member of the genus of Phlebovirusin the unyavirida ffamily.It is single-strand,negative-sensed segmented RNA virus.The genome of SFTSV consists of three segments,large(L),medium(M)and small(S)segments encoded different structural and nonstructural proteins.M segment code the glycoprotein precursor that generates two glycoproteins(Gn and Gc)by hydrolysis of protease in cells.Gn and Gc transfer to the envelope of virus after processed in endoplasmic reticulum and Golgi apparatus.Ticks mainly Haemaphysalis longicornis are the vector and host of SFTSV.They can transmit SFTSV to humans by biting.The minimum infection rate(MIR)of host questing ticks was 0.2%-5.4%.The nucleic acid of SFTSV has also been detected from some other arthropod such asLeptotrombidlum sutellare,Echinolaelaps echidninus and gadfly.Antibodies against SFTSV in domestic animals such as goats,cattle,dogs,chicken and pigs have been detected,but if they are the amplifying hostsstill need to be further investigated.The clinical manifestation of SFTS patients is nonspecific with flu-like symptoms,including fever,chill,fatigue,anorexia and vertigo.Abnormal laboratory examination includes thrombocytopenia,leukopenia,and increase ofaspartate aminotransferase(AST),creatine kinase(CK)and lactate dehydrogenase(LDH).The course of SFTS was divided into incubation period,pyretogenic stage,multiple organ failure(MOF)stage and convalescence.The specific SFTSV IgG antibody in convalescence patients could act as the evidence of virus infection.Up to now,23 provinces have reported 5,352 SFTS patients from 2011 to 2014 in China.The patients were concentrated in the eastern and central regions.In addition,SFTS patients have also reported in Japan,South Korea and Korea with a much higher fatality than that in China.The SFTS patients are mainly located in mountainous and hilly area.The onset time is generally from April to October.Most of the patients are middle-aged and elderly people,and almost all of them are farmers.SFTSV can also spread among people through contacting the blood,secretions or excretions,or aerosol of patients and the dead.The pathogenesis of SFTSV is not fully understood yet.Related studies have shown that SFTSV infection will causedysfunction in the immune system,and increase or decrease of various cytokines,which may be related to disease progression.Currently,the animal models of SFTSV are type I interferon receptor knockout(IFNAR-/-)mice,neonatal mice and mitomycin-treated mice.Animal models can be used to study the pathological changes that SFTSV causes in vivo.The spleen is recognized as the most severely affected organ by SFTSV.At present,there are no effective drugs and vaccines to treat the patients.Symptomatic and supportive treatment is the main method for patients.Therefore,disease prevention is critical,and the most important prevention measures are to avoid tick bites and speed up the development of vaccines.After infected with SFTSV virus,human bodies will produce neutralizing antibody that can neutralize SFTSV specifically.Detection of the level of the neutralizing antibodies in convalescent patients can assess the level of patients’ self-protection.Predicting the duration of the neutralizing antibodies in patients’ bodies can help to guide the vaccination program.For the Bunyavirus,glycoproteins play a crucial role in cell fusion,infection and immunity.They generally act as neutralizing epitopes to stimulate the body to produce neutralizing antibodies.Certain sites of the envelope glycoprotein can bind to cellular receptors,which can mediate the processes of adsorption and membrane fusion.Simultaneously,glycoprotein acts as a kind of antigen that can stimulate body to produce corresponding specific antibodies.These antibodies’ binding to the glycoprotein epitope block the sites of binding to cellular receptor,thereby,the glycoprotein will not mediate membrane fusion and thus cannot infect cells.Therefore,we intended to study the features and functions of the glycoprotein of SFTSV,and map the neutralizing epitopes of SFTSV,which is of great significance for the study of pathogenic mechanism and the design and development of vaccines.Mites are arthropod with a wide range and are closely related to human and animal diseases.Medical important mites include chigger mite,gamasid mite,scabies mites,dust mites,and demodex mites.Chiggermites can transmit scrub typhus that is caused byOrientia tsutsugamushi.Chigger mite Leptotrombidium scutellareis the reservoir and vector of O.tsutsugamushiin northern China.And the RNA of SFTSV has been detected inLeptotrombidium scutellare.This study is intended to detect SFTSV and other pathogens in chigger mitesharvested on wild rodents from Shandong Province.To understand the pathogenscarried bychigger mites of rodents in Shandong Province we performed PCR and RT-PCR analysis for Rickettsia,O.tsutsugamushi,SFTSV and Hantavirus in chigger mites collected on rodent ears.Objective1.To collect information of SFTS patients,and analysis its epidemiological characteristics.2.To detect the level of neutralizing antibodies in SFTS convalescent patients,and analysis the change rules with time.To predict the duration of neutralizing antibodies in SFTS convalescent patients,and guide the immunization programs of vaccine.3.To construct the expression vectors of SFTSV glycoprotein Gn,Gc and their fragments,and express and purify these proteins.4.To map the neutralizing epitopes of SFTSV,and help to design and develop the vaccines.5.To learn about the species and distribution of rodents in Qingdao,Shandong Province.6.To determine the pathogens carried by parasitic mites in rodents in Qingdao,Shandong Province.Methods1.The information of SFTS patients in 201 land 2012 was obtained from Yiyuan CountyDisease Prevention and Control Center,Zibo City,Shandong Province.We obtained convalescent sera from SFTS patients four times in November 2011,November 2012,July 2013 and June 2015,respectively.To confirm SFTS,the level of IgG in convalescent sera of SFTS patients were tested by double antigen sandwich enzyme linked immunosorbent assay(ELISA).2.SFTSV was cultured in Vero cells.After several passages,virus that could produce clear plaques was selected and used for the subsequent experiments.The titer of neutralizing antibodies inthe convalescent sera of the patients was determined using plaque reduction neutralization test(PRNT).The titer was indicated by the serum dilution that reduces the number of plaues by 50%in cells(PRNTso).3.Microsoft Excel 2010 was used forthe preliminary processing of data.SPSS 19.0 software was used for statistical analysis of data.The epidemiology three-dimensional distribution was analyzed,and the trend of the titer of neutralizing antibodies in patients’convalescent sera was analyzed.4.The duration of neutralizing antibodies was predicted using R 3.3.1 software by generalized estimating equations(GEE),binom package for calculating the titer of neutralizing antibodies and its 95%CI,geepack package for calculating the duration of neutralizing antibodies,and msm package for calculating the 95%CI of the duration of neutralizing antibodies by Delta method.5.The gene of glycoprotein Gn was divided into 4 fragments with equal length for each,and the gene of Gc was divided into 6 fragments with different lengths for each according to the boundaries of domains.Two adjacent segments overlap 30 bases.Corresponding primers were designed for to amplify each segment.The 6×His or Strep Ⅱ tag was added to everyfragment.The segment and full genes of Gn and Gcwere amplified by RT-PCR and ligated into pCAGGS and pET32a(+)vectors,respectivelyto construct eukaryotic and prokaryotic expression vectors for expression of the proteins.Western blot was used to detect the expressed protein.The expressed protein was purified by Ni-NTA column.6.In October 2015,wild rodents were captured by the snap trap in hills in Huangdao District,Qingdao City,Shandong Province.The captured animals weredissected,and the earsand spleens were collected.Mites in the ears were separated under the microscope with a brush and tweezers,classified according to body featuresas previouslyreported.7.DNA and RNA of mites and rodents’spleenswereextracted.The primers of mite mitochondrial DNA and related pathogenswere designed.The positive PCR products with expected sizes were purified from agarose gel after electrophoresis,ligated with T Vector pMD 19(Simple)and transformed into DH5a competent cells.Single colonies were picked and sequenced on both strands.The sequences were compared with those in GenBank database.8.SPSS 19.0 software was used for statistical processing of data.χ2test method was used for statistics inference ofmite infection rate of different rodentspecies.P<0.05 was considered to indicate a statistically significant difference.The sequence was edited and processed by using Seqman and EditSeq software,and the positive gene sequences were used for phylogenetic analysisrespectively.The corresponding Rickettsia sequences were downloaded from GenBank,and aligned for multiple sequence alignments by Clustal X software.Phylogenetic analysis was performed using the Neighbor-joining(NJ)method and the Maximum likelihood(ML)method to confirm the evolutionary relationship between the novelRickettsia and other species.9.Homogenized mite tissues that were PCR positiveto Rickettsiawas inoculated into Vero cells and cultured for 7 days.DNA of the cells were extracted andamplified for Rickettsiaby nested PCR.Results1.Detection of neutralizing antibody level and prediction of its duration time in sera of SFTS patients.1.1 A total of 33 laboratory confirmed SFTS patients were occurred in Yiyuan County,Zibo City,Shandong Province in 2011 and 2012 with 2 deaths.With patients refusing to donate blood samples,we collectedconvalescent sera from 25 SFTS patients including 19 patients in 2011 and 6 patientsin 2012.A total of 72 convalescent sera were collected from the patients in four follow-up visits.1.2 All patients’ sera have different levels of IgG antibodies by the double antigen sandwich ELISA tests,confirming that all patients have previously been infected with SFTSV.1.3 A SFTSV strain with clear and stable plaques was picked up by passage in Vero cells.The titer of the virus was determined to be PFU=1.25×106/ml by plaque formation assay.1.4 Neutralizing plaque reduction test was established to detect neutralizing antibody titers.All patients had different levels of neutralizing antibodies in their sera.The neutralizing antibody titer(PRNT50)fluctuated in the range of 1:5-1:640.1.5 Combined the data of neutralizing antibody levels in 25 patients over several years,there was a gradual decline trend in geometric mean titer(GMT)of neutralizing antibodies over time,but 2 of the patients had a higher third serum antibody titer than the first time.1.6 The titers of PRNT50=1:10 and 1:20 were used as the protective threshold of neutralizing antibody respectively.After calculated by the generalized estimating equation,when PRNT50=1:20was used as the threshold,the duration of neutralizing antibody was 6.7 years(95%CI:5.1-3.3years),and when PRNT50=1:10 was used as the threshold,the duration of neutralizing antibody was 9.2 years(95%CI:7-11.4 years).Compared with the male patients,the titer of neutralizing antibody in femalepatientslasted 2 yearlonger.There was no significant difference in the trend of decline over time among different age groups.2.Expression and purification of Gn,Gc and their fragments of SFTSV.2.1 The eukaryotic expression vectors of pCAGGS-Gn-strep,pCAGGS-Gc-strep,pCAGGS-Gn1-strep,pCAGGS-Gn2-strep,pCAGGS-Gn3-strep,pCAGGS-Gn4-strep,pCAGGS-Gc1-strep,pCAGGS-Gc2-strep,pCAGGS-Gc3-strep,pCAGGS-Gc4-strep,pCAGGS-Gc5-strep and pCAGGS-Gc6-strepwere successfully constructed and then transfected into 293T cells to express target protein.Except that the full length Gn and Gc proteins were expressed in the secretion,none of the segments were expressed successfully.2.2 The prokaryotic expression vectorsof pET32a-s-Gn,pET32a-s-Gc,pET32a-s-Gn1,pET32a-s-Gn2,pET32a-s-Gn3,pET32a-s-Gn4,pET32a-s-Gc1,pET32a-s-Gc2,pET32a-s-Gc3,pET32a-s-Gc4,pET32a-s-Gc5 and pET32a-s-Gc6 were successfully constructed and expressed in soluble format low temperature of 16℃.However,the proteinscould not be successfully purified.3.Detection of pathogens in rodent parasite mites.3.1 In October 2015,a total of 32 rodents were captured in Huangdao District,Qingdao City,Shandong Province.The dominant species were Apodemus agrarius(53.13%,17/32)and Mus musculus(28.13%,9/32).Fifteen rodents had mites in their ears with the mite carrying rate of 46.88%.There was no significant difference in the mitecarrying rate among different rodent species.The number of mites in each of 15 rodents varied from 3 to 54,with a total of 319 chigger mites.The chigger index was 9.97.AfterDNA was extracted from these mites,PCR amplification and DNA sequencing indicated the the mites wereLeptotrombidium scutellare.3.2 The sequences of 16S rRNA,glt A,ompB and 17 kD gene of Rickettsia were successfully amplified in the mite DNA.The minimum infection rate(MIR)of the Rickettsia in the mites was 9/319,that was 2.8%.BLAST analysis indicated that the 4 PCR products amplified from mitescorrespondare highy homologous to Rickettsia genes(96.5%～99.4%).3.3 The sequences ofO.tsutsugamushi,SFTSV and Hantaan viruswere not amplified from the mite tissues.Rickettsiawerenot amplified by PCR in spleen DNA of the rodents.3.4 Phylogenetic trees were constructed by using individual Rickettsia genes and concatenated sequence of the 4 genes.Phylogenetically the ompB gene sequence of the Rickettsia sp from mite was most closely related to Rickettsia sp.TwKM02 and was located on the same branch of the phylogenetic tree with a support rate of 92%.The 16S rRNA gene sequence was most closely related to R.australis.The 17 kD gene sequence was most closely related to R.akari,and located in the same branch of the phylogenetic tree with a support rate of 62%.The gltA gene sequence is most closely related to R.australis.The concatenated sequencewas most closely related to R.australis and R.akari and located on the same branchof the evolutionary tree.The Rickettsia from mite was not identical any knownRickettsia species,indicating that itwas a novelRickettsiaand we named Candidatus Rickettsia leptotrombidium after the name of chigger mite.3.5 Rickettsia was not detected in cell cultures,indicating that the novelRickettsia was not successfully isolated.Conclusions1.The SFTSV strain that produces clear plaques was picked up and a well-established PRNT method for detecting serum neutralizing antibody in SFTS patients was established.2.A total of 25 SFTS patients were investigated in Yiyuan County.The convalescent sera ofeach patient were collected for 3 to 4 years.The initial follow-up and the detection of 4-year serum neutralizing antibody levels and trends in SFTS patients were helpful for the evaluation of human anti-SFTSV immune ability.3.Statistical methods were used to predict the duration of protective neutralizing antibodies in SFTS patients for the first time,which provided a theoretical basis for the vaccine immunization program.The length of duration of neutralizing antibody was different in gender.The neutralizing antibody in female lasted longer than in male,suggesting gender differences in the immune system.Sexual effects should be considered when medications and vaccinations are taken.4.The eukaryotic and prokaryotic expression vectors of SFTSV glycoprotein Gn,Gc and their segments were successfully constructed and the recombinant proteins of SFTSV glycoprotein Gn,Gc and their segments were expressed.The immunogenicity of the recombinant protein of each segment was the important basis for development of SFTSV subunit vaccine.5.A novelRickettsia was detected in the parasitcal mites in Qingdao,Shandong Province.It had a closest evolutionary relationship withR.australis and R.akari.Rickettsia DNA was negative in the spleens of rodents,indicating that the rodent cannot be infect by thisRickettsia or was not in therickettsemia period.6.Orientia tsutsugamushi,SFTSV and Hantaan viruses were not detected in the mites.The noveliRickettsiawas not successfully separated.