| [Background]A novel virus, designated SFTS bunyavirus, was isolated from patients who presented with fever, thrombocytopenia, leukocytopenia, and mμltiorgan dysfunction by the institute for viral disease control and prevention, China CDC, in2010. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. The viral genome contains three single-stranded negative RNA segments, namely L, M and S segments. The L segment contains6368nucleotides with one open reading frame encoding2084amino acids. The M segment contains3378nucleotides with one open reading frame encoding1073amino acid precursors of glycoproteins (Gn and Gc).The S segment contains1744nucleotides of ambisense RNA encoding two proteins, the N and NSs proteins, in opposite orientations, separated by a54-bp intergenic region. The sequences of lOnt at the ends of the viral genome are highly conserved, same with other phlebovirus and could form a panhandle structure.So far, the cases of this disease had been found in Henan, Hubei, Shandong, Anhui, Liaoning and Jiangsu provinces, which were presented as highly sporadic and mainly located in the rural fields of the mountainous and hilly areas. This disease mostly occurred in the spring and summer, slightly differences may exist among different regions. Human are generally susceptible to this novel virus, living or working in the hills, mountains and forests, as well as tourists going to such areas have higher risk of infection. The route of transmission of the virus is still unknown, while tick biting had been reported in part of cases. In addition, there were recently reported that exposure to the acute phase blood of critically ill patients may get the possibility of human to human transmission, more attention and personal protection should be considered by family members of patients and health workers. Bunyavirus usually have weak resistance to acid, and could be rapidly inactivated by heat, ether, sodium deoxycholate, common disinfectants, and ultraviolet irradiation.Members of the family Bunyaviridae are found worldwide, and some are significant pathogens in animals, or in the case of the genus Tospovirus, plants. Most are spread through sylvatic transmission cycles susceptible vertebrate hostsand hematophagous arthopods, including mosquitos, phlebotomine flies, and ticks. Viruses in the genus Hantavirus are unique among the Bunyaviridae in that they do not infect insect vectors. Instead, hantaviruses are maintained in nature through persistent, mostly benign infection of their natural rodent hosts. In addition, an alternative route of nonvectored transmission has been reported for some nairoviruses and phleboviruses, including Crimean-Congo hemorrhagic fever virus and Rift Valley fever virus. These viruses can also be spread by exposure to infected animal tissues or body fluids.The model of transmission cycle of SFTSV in nature is still unclear. In our study, samples like vector specimens and animal serum were collected from infected areas. viral RNA and serum antibodies were tested, virus in RNA positive samples were isolated and whole-genome were sequenced and aligned, the dynamics of viral replication and antigenicity of different strains were also analyzed to interpret the biology and genetic characteristics of the virus from different aspects, and the possibility of vector and livestock as the viral vectors and reservoirs could be.To understand the biological characteristics of this novel virus, physical and chemical sensitive experiments and drug sensitive test were also carried out,which could also supply basic data for future work.[Objectives]1. To explore the prevalence situation of the SFTS virus in ticks and animal hosts.2. To analyze the etiology and molecular characteristics of the novel virus isolated from different specimens.3. To understand the biological characteristics of the novel virus and provide basic evidence for further research.[Methods]1. Ticks and animal serum samples were collected from affected areas, and detected by real time quantitative PCR and double antibody sandwich ELISA.2. Virus was isolated by cell culture techniques from viral RNA positive samples and were identified by real time quantitative PCR and indirect immuno fluorescence assay (IFA).3. Isolated virus were sequenced by RT-PCR amplification, homology comparison and phylogenetic analysis were then carried out.4. The replication features of the virus were observed through Growth dynamics experiment, and the antigenic characteristics of different strains were tested by cross neutralization test and indirect immunofluorescence assay.5. The virus inactivated condition was determined by inactivation experiments, the result of which could provide a theoretical basis for the study of inactivated vaccine.6. Physicochemical characteristics of the novel virus were tested by different physical and chemical factors.7. The effect of ribavirin against the novel virus had been tested through in vitro experiment.[Results]1. Specimen collection and SFTSV RNA detection140Ticks were collected from livestock including cattle, sheep and dog of22patients and their neighbors in Penglai and Laizhou City, Shandong province, the rate of animal taking with ticks is100%(with tick rate=ticks/checked animals). After Classification and identification, these collected ticks mostly classified to3species,91.4%for Haemaphysalis longicomis,5%for R.sangnineus and3.6%for Dermacentor silvarum respectively. The result of multiple quantitative PCR of suspension of ticks showed that, three Haemaphysalis longicomis are positive, two of them were collected from dogs kept by patients, and one was from sheep. The minimum infection rate of ticks was2.14%.106animal serum samples were collected from21cattles,50sheep and35dogs, the viral RNA positive rate were4.7%,6%and2.8%respectively.2. SFTSV antibody detection106animal serum samples were collected from21cattles,50sheep and35dogs, positive rates of total antibody were0%,60%and19%, and positive rate of indirect immunofluorescence antibody were0%,60%and19%respectively.3. Virus isolationBy three successive passages, three stains of virus were isolated and tested by indirect immunofluorescence assay assay from ticks, dog and goat serum collected from Laizhou city Shandong province respectively.4. Genetic analysisThree isolated stains of virus from tick, dog and goat serum were whole genome sequenced, compared to virus isolated from patients, the homology of nucleic acid sequence for S, M and L segments of the isolated virus were94.9%-99.3%,95.2%-99.6%,95.9%-98.9%for virus isolated from tick,95.2%-100%,95.6%-98.1%,96%-98.1%for virus isolated from dog serum,94.8%-98.2%,95.9%-97.5%,95.9%-98.1%for virus isolated from goat serum. By phylogenetic analysis, no significant link was found between various strains, it was suggested that the virus was highly conserved and clustered together, and there was no obvious geographical features in strains isolated from different provinces.5. Viral growth dynamicsBy observing the replication characteristics of the virus, no significant difference in growth trend were found among virus strains isolated from human and ticks, during1-3days, the virus replicated slowly, then in5-7days, the virus growth entered a logarithmic period, then reached to the plateau in7-10days, virus titers could reach to3*107TCID50/ml.6. Serological characteristicBy indirect immunofluorescence assay and neutralization test, no significant difference were found in antibody titer against to the same convalescent serum among virus strains isolated from human, sheep, dogs or ticks, the difference of all titers were less than4times, the result suggested that there were no significant differences in antigenicity of different virus strains isolated from different specimens.7. Physicochemical identificationBy physical and chemical experiments, the SFTS virus could be inactivated under conditions like60℃for30min, Formalin (1:2000), β-propiolactone (1:4000), and were sensitive to ether, chloroform, ultraviolet, PH3and acetone, the result of in vitro observation showed that ribavirin could effectively suppress viral replication.[Conclusions]1. It was the first time that isolated the novel virus from Haemaphysalis longicornis, sheep and dog serum, the possibility of ticks, sheep and dog as the novel virus’s vectors and reservoirs had been confirmed from the etiology aspect. Viral antibody had been detected in cattle, sheep, dog serum, the result showed the possibility of these animals as the hosts in serology.2. By full genome sequencing and homology analysis of virus strains isolated from Haemaphysalis ticks, sheep serum, dog serum and human serum, it showed that there were no significant differences in nucleic acid or amino acid sequences among these strains. Strains isolated from different areas and different specimens clustered together.3. It had been proved that there were no obvious differences in growth characteristics and antigenicity of virus strains isolated from Haemaphysalis ticks, sheep serum, dog serum and human serum by viral growth dynamic test, indirect immunofluorescence assay and neutralization assay.4. The result of sensitivity to physical and chemical experiments and susceptibility testing could supply basic data for the prevention and control of the virus and inactivated vaccine research. |
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