Dissemination Mechanism And Human Monoclonal Antibody Of Severe Fever With Thrombocytopenia Syndrome Virus

BackgroundSevere fever with thrombocytopenia syndrome virus(SFTSV)is a newly discovered phlebovirus in Bunyaviridae family.SFTSV is a single-stranded negative RNA virus,including three segments L,M and S.Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTSV is an emerging tick-borne hemorrhagic fever.The major clinical symptoms of SFTS are non-specific including fever,headache,fatigue,muscle soreness,general malaise,nausea and vomiting,severe cases of multiple organs failure or even death.The case fatality of SFTS is up to 30%,on average 12%.SFTS has been reported in most parts of China,Korea and Japan.SFTSV is primarily transmitted by tick bite,especially Haemaphysalis longicornis tick.Person to person transmission of SFTS through contact with infected patient's blood or mucus has been occasionally reported.Tick is a blood-sucking vector and can be widely distributed in animals.Small mammals were the major hosts of larvae and nymphs of H.longicornis tick,but the role of small mammals such as rodents and shrews as animal host of SFTSV is not well defined.SFTSV was likely originated in Huaiyangshan region in central China,but transmission route among China,Japan and South Korea in East Asia is still unknown.There is no specific drug or effective vaccine for SFTS,only broad-spectrum anti-viral treatment and symptomatic supportive care are used for the patients.For severe patients,neutralizing antibody treatment may be an effective treatment.The analysis of SFTS epidemiological characteristics and transmission route,and research of antibody drugs are of great significance to enrich the knowledge of the pathogen,epidemiological theory,prevention and control of disease of SFTS and diagnosis and treatment of SFTS.ObjectiveTo investigate the role of small mammals in SFTSV transmission and to provide the theoretical basis for the prevention and control of the disease,SFTSV infection status of small mammal animals in natural state were analyzed.To investigate the origin and possible transmission route of SFTSV,and to provide important clues for the prediction of SFTS epidemic trend,genetic evolution analysis of SFTSV genome were performed.To find special treatment for SFTS patients,research of human monoclonal antibody targeted to SFTSV glycoprotein was performed.Methods1.Samples collection and testing(1)Small mammals(rodents and shrews)were collected in rural areas of Huangdao district(formally Jiaonan County)of Qingdao City in Shandong Province,from January to August in 2013.Rodents and shrews were trapped once every month using mousetraps with peanuts bait.The traps were set before sunset in rural areas surrounding farmlands and collected in the morning.Blood samples were collected from the heart of rodents and shrews using absorbing paper strips.The spleens and livers of the rodents and shrews were collected aseptically and frozen at-80? until use.Animal sera were tested for total antibodies(IgG and IgM)to SFTSV using a double antigen sandwich ELISA Kit.Animal spleens were homogenized with plastic pestles and total RNA was extracted from each animal spleen and was used as a template for RT-PCR amplification of SFTSV M and S segments.The amplification result was sequenced and compared with the NCBI database to determine whether it is SFTSV sequence.SFTSV sequences obtained in this study were uploaded to the NCBI database,to get the GenBank accession numbers.According to the results of ELISA and PCR amplification,the seroprevalence,SFTSV RNA positive rate and infection rate were calculated.(2)The obtained sequence was compared with the reference strain sequence obtained from NCBI.Phylogenetic tree was constructed by using the neighbor-joining(NJ)method with software Mega 5.02.2.SFTSV Genetic evolution analysis(1)We analyzed only SFTSV that had complete genome sequences for three segments in GenBank(http://www.ncbi.nlm.nih.gov/genbank).The information of each SFTSV strain was collected including GenBank number,host,collecting location and collection date.The phylogenetic trees used to analyze the molecular evolution were constructed using the maximum likelihood method based on the Kimura 2-parameter model using the MEGA.(2)For reassortment detection,the concatenated complete genome sequences were aligned using the multiple alignment program Clustal X and analyzed with Recombination Detection Program version 3.44(RDP3).Combined with the phylogenetic analysis,secondary screening was performed from the reassortment events analyzed by RDP3.(3)To determine the molecular evolutionary rate and estimate the time to the most recent common ancestor(TMRCA)for each of the data sets,Bayesian coalescent phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo(MCMC)method available in the BEAST package v 1.8.0.The HKY +G nucleotide substitution model and a flexible non-parametric Bayesian skyride coalescent model were used.To test the strength of temporal signal in these data,which is essential to the estimation of substitution rates,we repeated the BEAST analysis(using identical parameters)on a data set in which sampling times were randomized on the tips.The HPDs of these randomized sequences were then compared with those of the real data.Based on the reassortment and the TMRCA,the temporal and spatial dynamics of SFTSV were speculated.3.Expression of human monoclonal antibody and biological activity study(1)The transmembrane amino acid sequences of SFTSV glycoprotein precursor was predicted by protein analysis software to ensure the extracellular domains.The extracellular domains of SFTSV glycoproteins Gn and Gc were cloned to the eukaryotic expression vector.The glycoproteins Gn and Gc were expressed in mammalian cell and purified.(2)Human peripheral blood mononuclear cells were isolated from the patient's peripheral blood and the memory B cells targeting to SFTSV glycoprotein were isolated by flow cytometry.The antibody variable region gene sequences were amplified from the memory B cells by RT-PCR and PCR.To form the antibody light and heavy chain complete sequences,the variable region and the constant region were jointed by overlap PCR.Then the complete antibody light and heavy chain were cloned to the eukaryotic expression vector to build the recombinant eukaryotic expression plasmid.The recombinant plasmids containing the antibody light and heavy chain were co-transfected into HEK 293T mammalian cells.Western Blot was used to select the light and heavy chain group which can express the antibody.The selected antibodies were expressed to test biological activities,including protein binding activity by ELISA,virus binding activity by indirect immunofluorescence,and neutralizing activity by plaque reduction neutralization assay.Results1.Species composition and infection status of capture animals(1)We collected 774 small mammals Jiaonan County,Huangdao District from January to August in 2013,including brown rat(Rattus norvegicus)(157/774,20.3%),house mouse(Mus musculus)(183/774,23.6%),Chinese hamster(Cricetulus barabensis)(135/774,17.4%),stripped field mouse(Apodemus agrarius)(188/774,24.3%),greater long-tailed hamster(Cricetulus tyiton)(18/774,2.3%),and Asian house shrews(Suncus murinus)(93/774,12.0%).376 small mammals were collected indoors,including brown rat(151/376,40.2%),house mouse(172/376,45.7%),and Asian house shrews(53/376,14.1%).398 small mammals were collected outdoors,including brown rat(6/398,1.5%),house mouse(11/398,2.8%),Chinese hamster(135/398,33.9%),stripped field mouse(188/398,47.2%),greater long-tailed hamster(18/398,4.5%)and Asian house shrews(40/398,10.1%).(2)From the 774 small mammals,755 effective heart blood samples were collected,which include 156 brown rat,182 house mouse,125 Chinese hamster,186 stripped field mouse,17 greater long-tailed hamster and 89 Asian house shrews.SFTSV total antibodies were detected by ELISA,and 9 samples were positive,including 2 house mouse,1 Chinese hamster,2 stripped field mouse,and 4 Asian house shrews.SFTSV seroprevalence was 1.2%for rodents and Asian house shrews.SFTSV seroprevalence was higher in Asian house shrews(4.5%,4/89)than rodents(0.8%,6/666)(P=0.014).From the 774 small mammals,517 effective spleen samples were collected,which include 116 brown rat,103 house mouse,83 Chinese hamster,129 stripped field mouse,9 greater long-tailed hamster and 77 Asian house shrews.SFTSV RNA was amplified by nested RT-PCR,and 5 samples were positive,including 1 brown rat,1 house mouse,1 Chinese hamster,and 2 Asian house shrews.SFTSV seroprevalence was 1.2%for rodents and Asian house shrews.The PCR positive rate of SFTSV was 1.0%for rodents and Asian house shrews.The PCR positive rate was higher in Asian house shrews(2.6%,2/77)than in rodents(0.7%,3/440),but,there was no significantly difference between rodents and Asian house shrews(P=0.162).The PCR positive rate was 4.7%(4/86)in March,1.7%(1/62)in June,and 0.0%in other 6 months.The PCR positive rate was higher in March than other months(P=0.043).The total infection rate was 1.9%(14/755)in all the tested samples,with 1.2%in rodents and 6.7%in Asian house shrews.The infection rate was higher in Asian house shrews than rodents(P=0.003),suggesting that Asian house shrews were more important than rodents in SFTSV transmission.The infection rate was 2.4%(9/372)in host animals indoors and 1.3%outdoors.There was no significant difference between them.The infection rate was 9.8%(5/51)in Asian house shrews indoors,which was higher than rodents indoors 1.2%(4/321)(P=0.003).Compared with the infection rate in Asian house shrews outdoors 2.6%(1/38),the infection rate indoors was higher(P=0.001),which suggested that Asian house shrews indoors were more important than outdoors in SFTSV transmission.2.Genetic evolution analysis(1)In our analysis,122 SFTSV strains with complete genome sequences were selected,with a time span from 2010 to 2014.Among the 122 SFTSV strains selected,108 strains were from China(61 from Henan,21 from Jiangsu,8 from Shandong,7 from Liaoning,4 from Hubei,4 from Anhui,and 3 from Zhejiang),8 from Japan and 6 from South Korea with year ranging from 2010 to 2014.Phylogenetic analysis showed that all SFTSV strains were classified into 5 lineages in each segment,named A,B,C,D and E.(2)The concatenated sequences was analyzed by RDP4,suggesting 14 reassortment events in SFTSV complete genomes.Combined with the phylogenetic analysis,secondary screening was performed from the reassortment events analyzed by RDP4.Six strains were identified as reassortment events,which were strains LN2012-14,LN2012-34,LN2012-41,LN2012-42,LN2012-58 and AHL.The dN/dS values of the four genes ranged from 0.026 to 0.092,which indicated that SFTSV was under purifying selection.Two sites(37,1033)in glycoprotein were identified as being under strong positive selection.(3)The average evolutionary rate of each complete genome was different,which was estimated by the Bayesian time-scaled phylogenetic analysis.For the L segment,the mean rate was 4.16E-4 substitutions per site per year(s/s/y)ranging from 8.99E-5 to 9.72E-4(with 95%highest posterior densities,HPD).For the M segment,the mean rate was 6.76E-4 s/s/y(95%HPD= 3.92E-4 to 1.00E-3 s/s/y).The S segment had a mean rate of 1.09E-3 s/s/y(95%HPD= 5.43E-4 to 1.60E-3 s/s/y).Temporal and spatial transmission routes were primarily confirmed using the Bayesian analysis based on the 122 complete concatenated genomes from China,Japan and South Korea.The extant SFTSV originated 42(95%HPD=20 to 87)years ago in the Huaiyang Mountain area in central China encompassing Henan,Hubei,Anhui,and Jiangsu provinces.Around 37(95%HPD=18 to 77)years ago,the virus moved to Shandong from Henan Province,and the virus was transmitted from Jiangsu to Liaoning Province in Northeastern China about 33(95%HPD=16 to 67)year ago.SFTSV spread to Zhoushan islands of Zhejiang Province,China,Jeju island of South Korea,and Japan approximately 31(95%HPD=13 to 61)year ago,and there was mutual transmission among these areas.3.Expression of human monoclonal antibody and biological activity study(1)The recombinant eukaryotic expression plasmids pCAGGS-SP-Gn-Strep ?and pCAGGS-SP-Gc-Strep ? were obtained which can express the ectodomain of SFTSV glycoprotein Gn and Gc in mammalian cell HEK 293T.The Gn and Gc ectodomain were expressed and purified by affinity chromatography.In the supernatant of cells,Gn was present as a dimer and multimer,but the majority of Gc was present as a monomer,and a small portion was present in the form of a dimer.(2)Seven groups of light and heavy chain plasmids were selected by Western Blot that could express the antibody,and the antibodies were labeled Ab03,Ab05,Ab52,Ab62,Ab63,Ab64 and Ab71,respectively.Among them,6 monoclonal antibodies(Ab05,Ab52,Ab62,Ab63,Ab64 and Ab71)had the binding activity for SFTSV glycoprotein Gn,3 monoclonal antibodies(Ab05,Ab63 and Ab71)had virus binding activity for SFTSV,none monoclonal antibody had neutralization activity for SFTSV.Conclusion1.In natural conditions,small mammals such as rodents and insectivorous insect shrews can infect and carry SFTSV,which may be the storage host of SFTSV and play an important role in the spread of the virus.Compared with rodents,shrews play a more important role in the diffusion of SFTSV.2.SFTSV was under purifying selection with low evolution rate,but the virus had a high frequency of reassortment,which may be the main force for SFTSV evolution.SFTSV may originate from the Huaiyang Mountain area in central China and then spread to eastern and northeastern China,Japan and South Korea.3.At pH 8.0,SFTSV glycoprotein Gn was polymer,and glycoprotein Gc was mostly present as a monomer and a small portion was a dimer.Seven human monoclonal antibodies were successfully expressed and purified.Among them,6 monoclonal antibodies had the binding activity of SFTSV glycoprotein Gn,3 strains had SFTSV virus binding activity and none had neutralization activity.