Expression of Gn protein of severe fever with thrombocytopenia syndrome virus and its immunogenicity / 中国生物制品学杂志

@#Objective To express the Gn protein of severe fever with thrombocytopenia syndrome virus(SFTSV) through adeno-associated virus 9(AAV9) expression system and evaluate its immunogenicity.Methods SFTSV Gn gene was inserted into viral vector pAAV-CMV-FH and the recombinant plasmid was transfected into HEK293T cells to obtain recombinant virus AAV9-Gn.The expression of Gn protein was determined by immunofluorescence and Western blot.Eighteen fernale BALB/c mice were randomly divided into three groups:Mock group(serum-free DMEM),AAV9-GFP group(1 × 10~(11) vg) and AAV9-Gn group(1 × 10~(11) vg),all of which were injected intramuscularly into the right hind limb at a dose of 100 μL per mouse.The body mass,diet,behavior and mental state of mice in each group were monitored continuously for 21 d,and the change rate of body mass was calculated;At 2,4,8 and 16 weeks after immunization,the levels of SFTSV neutralizing antibody in serum of mice in each group were detected by fluorescent reduction neutralization test(FRNT),and the levels of specific IgGl and IgG2a in serum of mice in AAV9-Gn group were detected by ELISA.Results After incubation with specific antibody,Vero cells transfected with AAV9-Gn showed specific green fluorescence under fluorescence microscope,and had specific binding to mouse anti-SFTSV Gn monoclonal antibody,and the specific binding bands were found at a relative molecular mass of about 61 000.The body mass of the three groups showed an increasing trend,there was no significant difference between the three groups(F=0.158—2.621,P> 0.05),and the diet,behavior and mental state were normal.At 2,4,8 and 16 weeks after immunization,the titer of SFTSV neutralizing antibody in serum of mice in AAV9-Gn group was significantly higher than that of Mock group and AAV9-GFP group(H=13.332—14.538,each P <0.001),and the titer peak appeared at 8 weeks;The level of specific IgG1 in serum of mice was significantly higher than that of IgG2a(F=4.373—12.975,each P <0.05) at different time points.Conclusion SFTSV Gn protein can be expressed correctly through AAV9 expression system,and has low toxicity to mice with good immunogenicity,which is expected to be a candidate component of SFTSV vaccine.

Analysis of microbial diversity in the midgut of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus / 中国热带医学

@#Abstract: Objective　To investigate the composition and diversity of midgut microbial community of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus (SFTSV)． Methods　The midgut DNA of three group Haemaphysalis longicornis infected with SFTSV was extracted, and the 16S rDNA gene of the sample was sequenced by HiSeq platform. The composition and diversity of endosymbiotic microbial community were clarified by OTU cluster analysis and alpha diversity analysis. Results　The midgut microbial clusters of the three groups infected with SFTSV were 143, 113, 163 OTUs respectively; the sparsity curve and abundance grade curve showed that the data had sufficient sequencing depth, and the midgut of Haemaphysalis longicornis infected with SFTSV was rich in microbial composition, but the species distribution was uneven. The analysis of microbial community composition showed that Proteobacteria, Firmicutes and Actinobacteria were the main dominant bacteria at the phyla level. At the class level, Gammaproteobacteria, Bacilli, Betaproteobacteria and Actinomycetia were the main dominant bacteria. At the order level, Legionellales, Bacillales, Burkholderiales and Actinomycetales were the main dominant orders. At the family level, Coxiellaceae, Bacillaceae, Moraxellaceae and Rhodococcaceae were the main dominant families. At the genus level, the relative abundance of Coxiella was the highest, followed by Aeribaillus and Azonexus. Alpha diversity analysis showed that the average Shannon index was 139.67, the average Simpson index was 0.48, the average Chao index was 145.06, and the average ACE index was 147.11. Conclusions　The species diversity of intestinal microorganisms in Haemaphysalis longicornis infected with SFTSV is rich. The results provide a basis for further exploring the interaction between intestinal microbes of Haemaphysalis longicornis and SFTSV and developing new ideas for the prevention and control of ticks and tick-borne diseases.

Establishment and evaluation of a genome-wide multiplex PCR sequencing method for severe fever with thrombocytopenia syndrome virus based on high-throughput sequencing technology / 中华微生物学和免疫学杂志

Objective:To establish a sequencing method for the genome of severe fever with thrombocytopenia syndrome virus (SFTSV) based on next-generation sequencing (NGS).Methods:SFTSV RNA was extracted from serum samples of patients with severe fever with thrombocytopenia syndrome. SFTSV-specific primers were designed using Primer 5.0 software. A multiplex PCR method was constructed and used to amplify the nucleotide sequence of SFTSV. Whole-genome sequencing was performed on the NGS platform.Results:The whole genes of SFTSV isolates in 28 serum samples were amplified by the multiplex PCR with a coverage over 94%. Sequencing and phylogenetic analysis of those strains revealed that the predominant strains ( n=20) belonged to genotype A, followed by genotypes B ( n=4) and E ( n=3). Conclusions:A high-throughput sequencing method for SFTSV based on multiplex PCR was established in this study. This method was characterized by high specificity and good quality and could improve the sequencing efficiency.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection

ABSTRACT Introduction: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. Methods: To develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. Results: The MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. Conclusions: The MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Establishment of a high throughput sequencing technology for detection of SFTS virus genome / 中华实验和临床病毒学杂志

Objective@#The objective of this study was to establish a next generation sequencing (NGS) method for severe fever with thrombocytopenia syndrome virus(SFTS).@*Methods@#SFTS virus RNA was extracted from the patient serum inoculated and isolated by Vero cells. Two methods of random primer sequencing and oligo(dT) beads selection sequencing were used for library construction. The libraries were built based on the best amplification and purification conditions. Whole genome sequencing was performed on NGS platform.@*Results@#There were significant differences in data of 3 virus between the two methods.The sample was sequenced by random primer sequencing showed low coverage and depth. However, three samples were sequenced by oligo(dT) beads selection showed coverage was over 99% and depth was over 900.The alignment rate of database from NCBI was more than 90%. The initial detection quality of this method was 300ng RNA.@*Conclusions@#In this study, we used the method of oligo(dT) beads selection to build libraries, and established a SFTS virus genome detection based on next generation sequencing.

Effects of severe fever with thrombocytopenia syndrome virus infection on the expression of TLR2 and inflammatory cytokines in mouse peritoneal macrophages / 医学研究生学报

Objective The secretion level of inflammatory factors is closely related to the severity of severe fever with thrombocytopenia syndrome (SFTS). This paper mainly discussed the effect of SFTSV infection on the expression of Toll-like receptor 2（TLR2） and inflammatory factors in macrophages in mice. Methods The expression of TLR2 andTNF-α, IFN-β, IL-6, IL-10 and other inflammatory factors were observed at 0, 6, 12, 24, 36, 48, 72 hours after SFTSV infected mice macrophages by qPCR, Western blot and ELISA. Results QPCR results showed that the mRNA levels of TNF-α at 0, 6, 12, 24, 36, 48, 72 h were 0.85±0.14, 15.23±2.45, 28.67±1.59, 94.52±7.05, 55.86±1.82, 23.55±6.15, 9.76±2.03, and the mRNA levels of IFN-β were 0.93±0.21, 1.55±0.33, 14.51±1.98, 55.16±3.64, 26.57±1.49, 9.22±0.51, 5.18±1.06. QPCR results showed that the mRNA levels of TNF-α and IFN-β produced by abdominal macrophages in the infected mice showed a trend of first increasing (the highest at the 24 hour) and then decreasing, and the difference of TNF-α and IFN-β mRNA levels at other time points was statistically significant compared with that at the 0 hour (P<0.05). However, IL-6 and IL-10 mRNA levels continued to increase, and the difference at other time points was statistically significant (P<0.05). ELISA results showed that the expression of four inflammatory factors showed a trend of gradual increase: TNF-α 0 and 72 h were (38.31±4.25, 140.41±23.45) pg/mL; IFN-β 0th and 72th were (17.56±0.66, 1084.93±111.42) pg/mL; IL-6 protein 0 and 72 h were (113.30±0.07, 2302.32±134.09) pg/mL; IL-10 protein 0 and 72 h were (515.00±21.21, 2590.40±226.19) pg/mL, respectively, with significant increases at the 24, 36, 48 and 72 hours compared with that at the 0 hour (P<0.05). The expression of TLR2 mRNA generated by mouse peritoneal macrophages showed a trend of first increasing and then decreasing, and increased to the highest level at 24 h, and the difference between each time point and 0 h was statistically significant (P<0.01). The expression of TLR2 gradually increased after infection with time extension. Conclusion SFTSV infection can up-regulate the expression of TLR2 in macrophages, thereby leading to the increased secretion of the cytokines.

Evaluation of two different enzyme-linked immunosorbent assay for severe fever with thrombocytopenia syndrome virus diagnosis

To develop the large scale serological assay for severe fever with thrombocytopenia syndrome virus (SFTSV) infection, we evaluated two different enzyme-linked immunosorbent assay (ELISA) methods using nucleocapsid protein (NP) and Gn proteins of CB1 (genotype B) SFTSV strains. The NP-based ELISA tests showed more sensitive with broad cross-reactivity between two different genotype A and B strains compared with those of Gn-based ELISA tests. However, Gn-based ELISA showed more genotype specificity and specificity. These result suggested that NP-based ELISA test could be applicable for general sero-prevalence studies of SFTSV infections, while Gn-based ELISA could be applicable for a certain specific genotype sero-prevalence study.

Intracellular location of severe fever with thrombocytopenia syndrome bunyavirus NP protein with correlative light and electron microscopy based on miniSOG / 中华实验和临床病毒学杂志

Objective@#To study the intracellular location and characteristic of SFTSV NP protein in different phases using mini singlet oxygen generator (miniSOG) labeling technique.@*Methods@#MiniSOG is a recently-invented genetically-encoded tag for EM. MiniSOG-fused SFTSV NP (NPSOG) gene was cloned by PCR, and inserted into pcDNA3.0 plasmid to form pTPL-NPSOG, which was used to transfect 293 cells. The transfected cells of different phases were fixed in 2.5% glutaraldehyde in situ, stained with DAB through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Intracellular location and characteristic of SFTSV NP protein in different phases were studied by observing the sections under transmission electron microscope.@*Results@#After transfecting the plasmid with NPSOG to 293 cells, NP protein was expressed in cytoplasm and peri nucleus, and gradually aggregated, which connected with endoplasmic reticulum and Golgi apparatus to form larger volume and irregular inclusion bodies in cytoplasm. No obvious subcellular structure changes were found.@*Conclusions@#The SFTSV nucleoprotein can be expressed separately to form inclusion bodies without the assistance of other viral proteins. The formation of inclusion bodies requires the directional movement and aggregation of a certain number of NP proteins, which may involve the interaction of NP protein and host organelles during this period.

Prevalence of severe fever with thrombocytopenia syndrome virus among ticks surveyed at Mt. Gwanak, Korea

This study was performed to investigate the distribution of ticks and the rate of infection with severe fever with thrombocytopenia syndrome (SFTS) virus in ticks collected at Mt. Gwanak and the Seoul National University campus, Korea. Ticks (n = 273) were collected from May to October and included 76 Haemaphysalis longicornis (4 adult females, 72 nymphs), 49 Haemaphysalis flava (9 adult females, 3 adult males, 37 nymphs), and 148 Haemaphysalis spp. larvae. SFTS virus detection was performed by using one-step RT PCR and nested PCR. The SFTS virus was detected in 7 samples (1 Haemaphysalis longicornis nymph, 3 Haemaphysalis flava nymphs, and 3 Haemaphysalis spp. larva). The overall minimum field infection rate was 2.6%, whereas the minimum field infection rates of adult, nymphal, and larval ticks were 0%, 3.2%, and 2.0%, respectively. For a more accurate indication of the prevalence of SFTS virus in Korea, further in-depth investigations of tick species and SFTS virus occurrence over a larger area and longer period are needed.

Severe Fever with Thrombocytopenia Syndrome Virus, Crimean-Congo Haemorrhagic Fever Virus, and Migratory Birds

Severe Fever with Thrombocytopenia Syndrome (SFTS) and Crimean-Congo Haemorrhagic Fever (CCHF) are tick-borne diseases belonging to the family Bunyaviridae. Since SFTS was first reported in China in 2009, the virus was isolated and confirmed in 2011, with additional reports of SFTSV expanding its geographic range from China to South Korea and Japan. CCHFV has the widest geographic distribution of any tick-borne virus, encompassing around 30 countries from eastern China through Asia, the Middle East, and southeastern Europe to Africa. During the past decade, CCHFV has emerged in new areas of Europe, Africa, the Middle East, and Asia and has increased in endemic areas. Migratory birds are considered to play a role in dispersing CCHFV vectors, and the virus. This review summarises SFTSV and CCHFV, highlighting the role of migratory birds in the transmission of tick-borne disease.