[Prokaryotic expression and purification of nucleoprotein of Guertu virus and its establishment of ELISA detection method].

Objective: To obtain purified protein antigen of guertu virus (GTV) nucleoprotein (NP) and establish a rapid and accurate enzyme-linked immunosorbent assay (ELISA) method for detection of GTV antibody. Methods: Codon optimized GTV NP encoding genes were synthesized, cloned into the pet32a (+) vector, and recombinant expression plasmids were constructed and transformed into BL21 (DE3). Recombinant protein (rNP) obtained from the optimized expression were purified over a Ni column and identified by SDS-PAGE and Western blot. The purified protein was used as the antigen to optimize the reaction conditions, and an indirect ELISA assay for GTV IgG antibody was developed and optimized, which was evaluated and initially applied. Results: The prokaryotic expression plasmid pet32a-NP was successfully constructed, the recombinant protein was highly expressed in E. coli in the form of inclusion bodies, the size was about 44 kD, and the results of Western blot indicated that the recombinant protein had good antigenicity with GTV positive serum. The optimized ELISA (GTV-rNP-iELISA) established in this study showed strong specificity, high sensitivity, and the coefficient of variation within and between batches is less than 10%, and has good repeatability; the detection results are consistent with the IFA detection results. Using the established ELISA method to detect 162 sheep sera from some regions of Xinjiang in 2017-2019, the total positive rate of antibodies was 39.8%. Conclusions: The GTV NP antibody detection ELISA method has good sensitivity, reproducibility, and specificity and has the potential to be a powerful tool for the diagnosis and serological investigation of GTV infection.

Evidence of Human Exposure to Tamdy Virus, Northwest China.

We report the isolation of Tamdy virus from Hyalomma asiaticum ticks in northwest China and serologic evidence of human Tamdy virus infection in the same region. These findings highlight the need to further investigate a potential causal relationship between Tamdy virus and febrile illnesses of unknown etiology in that region.

Molecular cloning, expression, and characterization of a Sophora alopecuroides lectin from Escherichia coli.

Sophora alopecuroides lectin (SAL) has been isolated from the seeds and confirmed to have antifungal and antitumor activities, and presently the preparation of the natural lectin was cumbersome, time-consuming, and the yield was relatively low for further analysis. In this study, the signal peptide of lectin, the modification sites, and the secondary structure were analyzed, and the three-dimensional structures of SAL were modeled. The gene of SAL was amplified by the reverse transcription polymerase chain reaction, and cloned into the pET-30a vector and expressed in Escherichia coli BL21(DE3) by the induction of isopropyl-beta-d-thiogalactopyranoside. Totally, 400 mg of recombinant SAL (rSAL) was purified from 1 l of bacterial culture through Ni-NTA agarose column and the purity reached 95%. The recombinant protein was further confirmed by western blot using rSAL-specific antibody. The biological activity analysis results showed that rSAL exclusively bound to d-galactose and had universal hemagglutinating activities to human A, B, O, and AB, and rabbit and mouse erythrocytes. rSAL also inhibited the growth of fungi, the proliferation of cancer cells, and the HIV-I reverse transcriptase activity. In conclusion, this study indicates that rSAL can be produced in large quantities in the prokaryotic expression system and the recombinant protein still retains the various biological activities, which will make the large-scale production of SAL recombinant protein at dramatically reduced cost possible.

Experimental study on the full cycle evolution of high-intensity atmospheric dc arc discharge from breakdown to extinguishment.

In this study, the spatiotemporal evolution of full cycle of high-intensity dc argon arc discharge at atmospheric pressure is investigated by using a transferred arc device, which is easy to be directly observed in the experiment. Combining the voltage and current waveforms with high-speed images, the full cycle evolution process of high-intensity atmospheric dc arc can be divided into five different stages: breakdown pulse stage, cathode heating stage, current climbing stage, stable arc discharge stage, and finally arc extinguishing stage. The characteristics of each different stage are analyzed in detail through the electrical properties, high-speed pictures, and spectroscopic measurements. The results show that the strong luminescence region develops from the vicinity of cathode and anode to the middle in the breakdown pulse stage, which is explained from the spatiotemporal evolution of distributions of excited argon atom and ions. The development velocity of emission intensity of argon ions is mainly determined by the dominant stepwise ionization process. Then the cathode heating stage appears with many bright and nonuniformly distributed light spots on the cathode surface, and the electron emission mechanism of cathode gradually changes to the thermionic emission as the surface temperature rises. With the increase of arc current, the discharge channel significantly expands, then becomes stable due to the increment of the Lorentz force. The characteristics of arc extinguishing stage are clarified in terms of the decay of charged particles density.

Epidemiological surveys revealed the risk of TAMV spill-over from ticks to hosts.

BACKGROUND: Tick-borne viral diseases have become an increasingly important public health concern. Tamdy virus (TAMV) is a tick-borne virus of the genus Orthonairovirus in the family Nairoviridae. While some studies have suggested that TAMV is a pathogen associated with human febrile illness, its epidemiology and the risk of TAMV spill-over remain poorly understood. METHODS: Ticks were collected in Xinjiang, China, and grouped into pools. RT-PCR assays were used to detect TAMV RNA in these pools. The seroprevalence of TAMV was investigated using Immunofluorescence assays, Western blotting, and Luciferase immunoprecipitation system (LIPS) assays. RESULTS: TAMV RNA was detected in 17 out of 363 tick pools, resulting in a minimum infection rate (MIR) of 4.7%. Hyalomma asiaticum and Dermacentor nuttalli were identified as major tick vectors of TAMV. Phylogenetic analysis demonstrated that TAMV strains from Xinjiang are closely related to strains from other countries. Seroprevalence studies showed that TAMV exposure has been occurring in Xinjiang since at least 2006. Antibody responses to TAMV were detected in 1.1% (26/2296) of animals, including domestic animals and wild rodents. The seropositivity rates were as follows: sheep (1.7%), dog (2.3%), Marmota monax (0.8%), Meriones meridianus (3.5%). CONCLUSIONS: The research findings reveal that TAMV can be transmitted by ticks to various animal species, posing a significant public health risk. The wide distribution of TAMV and its tick vectors emphasise the importance of early preparedness and control measures. This study highlights the necessity for maintaining vigilance in addressing emerging zoonotic diseases transmitted by ticks.

Reassortment and migration analysis of Crimean-Congo haemorrhagic fever virus.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus with high pathogenicity to humans. CCHFV contains a three-segment [small (S), medium (M) and large (L)] genome and is prone to reassortment. Investigation of identified reassortment events can yield insight into the evolutionary history of the virus, while migration events reflect its geographical dissemination. While many studies have already considered these issues, they have investigated small numbers of isolates and lack statistical support for their findings. Here, we consider a larger set of 30 full genomes to investigate reassortment using recombination methods, as well as two sets of partial S segments comprising 393 isolates, reflecting a broader geographical range, to investigate migration events. Phylogenetic analysis revealed that the S segment showed strong geographical subdivision, but this was less apparent in the M and L segments. A total of 16 reassortment events with 22 isolates were identified with strong statistical support. Migration analysis on the partial S segments identified both long- and short-range migration events that spanned the entire geographical region in which the CCHFV has been isolated, reflecting the complex processes associated with the dissemination of the virus.

Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae.

CecropinXJ is a cationic antimicrobial peptide originally isolated from the larvae of Bombyx mori. In this study, an antibacterial peptide gene of cecropinXJ was cloned into the pYES2/CT/α Factor expression vector and expressed in the Saccharomyces cerevisiae INVSc1 strain. Following an induction of recombinant protein expression in yeast for 120 h, the maximum amount of total secreted protein was 1.437 g/L. The percentage of recombinant cecropinXJ was estimated to be 79.45% of the total protein. After purification with Ni-NTA agarose column, recombinant cecropinXJ was noted to exert strong antimicrobial activities against a broad-spectrum of microorganisms, including Gram-negative and Gram-positive bacteria. Its minimal inhibitory concentration (MIC) against Escherichia coli ATCC25922 was 0.81 µM. In addition, transmission electron microscopy (TEM) analysis indicated that the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms. CecropinXJ had a small hemolytic effect on red blood cells even with a peptide concentration of 200 µM. Thus, cecropinXJ acts selectively on bacterial membranes. Purified recombinant antibacterial peptide, cecropinXJ, retained a high stability against E. coli ATCC25922 over a temperature range from 4 °C to 100 °C and a pH range from pH 2.0 to 12.0. Taken together, this study demonstrates that recombinant cecropinXJ can be produced in large quantities in yeast with genetic engineering methods, and that it has strong and rapid antimicrobial activities against all of microorganisms tested. Our results suggest that cecropinXJ is a potential candidate for therapy.

Purification and characterization of a novel antifungal protein with antiproliferation and anti-HIV-1 reverse transcriptase activities from Peganum harmala seeds.

A novel antifungal protein, designated as PHP, was isolated from the seeds of Peganum harmala, by cationic exchange chromatography on Resource S column and gel filtration on Sephadex 75 10/300 GL column. PHP was found to form a homodimer of about 16 kDa. Isoelectric focusing polyacrylamide gel electrophoresis analysis showed that the isoelectric point of PHP was ∼8.4. The N-terminal 20-amino acid sequence of PHP, ITCPQVTQSLAPCVPYLISG, resembles the non-specific lipid transfer proteins in certain plants. PHP exhibited lipid-binding activity. Furthermore, PHP exerted antifungal activity against Alternaria alternate, Penicillium degitatum, Rhizopus stuolonifer, and Magnaporthe grisea, and its antifungal activity was stable in the temperature range 4-60°C, and in the pH range 4-10. It inhibited the mycelial growth in A. alternate, P. degitatum, R. stuolonifer, and M. grisea with 50% inhibitory concentration (IC(50)) of 1.5, 37.5, 8.44, and 12.19 µM, respectively. PHP was also able to inhibit the proliferation of esophagus carcinoma (Eca-109), cervical carcinoma (HeLa), gastric carcinoma (MGC-7), and melanoma (B16) cells with IC(50) of 0.7, 2.74, 3.13, and 1.47 µM, respectively. Moreover, PHP significantly inhibited HIV-1 reverse transcriptase (RT) with an IC(50) of 1.26 µM. It did not have hemagglutinating and antibacterial activities. In conclusion, a novel antifungal protein with antiproliferation and anti-HIV-1 RT activities was obtained from P. harmala seeds.

Draft genome sequence of Yersinia pestis strain 2501, an isolate from the great gerbil plague focus in Xinjiang, China.

We deciphered the genome of Yersinia pestis strain 2501, isolated from the Junggar Basin, a newly discovered great gerbil plague focus in Xinjiang, China. The total length of assembly was 4,597,322 bp, and 4,265 coding sequences were predicted within the genome. It is the first Y. pestis genome from this plague focus.

Isolation and characterization of a novel lectin with antifungal and antiproliferative activities from Sophora alopecuroides seeds.

Sophora alopecuroides lectin (SAL), a novel lectin from the seeds of Sophora alopecuroides, was purified by ion-exchange chromatography on diethylaminoethyl (DEAE)- and carboxymethyl (CM)-Sepharose columns, followed by gel filtration on a Sephadex 75 10/300 GL column. SAL was found to be a monomer of 39916.3 Da, as determined by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC). The N-terminal 10-amino acid sequence of SAL, KPWALSFSFG, resembles those of other legume lectins. SAL exhibits hemagglutinating activity against rabbit erythrocytes at 11.9 µg/ml. Its hemagglutinating activity is stable in the pH range 7-11 and in the temperature range 30-90°C, and is stimulated by Mn(2+). The hemagglutinating activity of SAL is most potently inhibited by 50-mM d-galactose. SAL suppresses mycelial growth in Penicillium digitatum and Alternaria alternata; the IC(50) of the antifungal activity toward P. digitatum and A. alternata were found to be 3.125 and 3.338 µM, respectively. SAL suppresses the proliferation of human cervical cancer cells (HeLa) at an IC(50) of 6.25 µM (P< 0.05). But it has no inhibiting effect on bacteria. This is the first report of a lectin from seeds of S. alopecuroides.

Fine mapping of the antigenic epitopes of the Gc protein of Guertu virus.

Guertu virus (GTV), a newly discovered member of the genus Banyangvirus in the family Phenuiviridae, poses a potential health threat to humans and animals. The viral glycoprotein (GP) binds to host cell receptors to induce a neutralizing immune response in the host. Therefore, identification of the B-cell epitopes (BCEs) in the immunodominant region of the GTV Gc protein is important for the elucidation of the virus-host cell interactions and the development of GTV epitope assays and vaccines. In this study, an improved overlapping biosynthetic peptide method and rabbit anti-GTV Gc polyclonal antibodies were used for fine mapping of the minimal motifs of linear BCEs of the GTV Gc protein. Thirteen BCE motifs were identified from eleven positive 16mer-peptides, namely EGc1 (19KVCATTGRA27), EGc2 (58KKINLKCKK66), EGc3 (68SSYYVPDA75), EGc4 (75ARSRCTSVRR84), EGc5 (79CTSVRRCRWA88), EGc6 (90DCQSGCPS97), EGc7 (96PSHFTSNS103), EGc8 (115AGLGFSG121), EGc9 (148ENPHGVI154), EGc10 (179KVFHPMS185), EGc11 (230QAGMGVVG237), EGc12 (303RSHDSQGKIS312), and EGc13 (430DIPRFV435). Of these, 7 could be recognized by GTV IgG-positive sheep sera. Three-dimensional structural analysis revealed that all 13 BCEs were present on the surface of the Gc protein. Sequence alignment of the 13 BCEs against homologous proteins from 10 closely related strains of severe fever with thrombocytopenia syndrome virus from different geographical regions revealed that the amino acid sequences of EGc4, EGc5, EGc8, EGc11, and EGc12 were highly conserved, with 100% similarity. The remaining 8 epitopes (EGc1, EGc2, EGc3, EGc6, EGc7, EGc9, EGc10, and EGc13) showed high sequence similarity in the range of 71.43%-87.50%. These 13 BCEs of the GTV Gc protein provide a molecular foundation for future studies of the immunological properties of GTV glycoproteins and the development of GTV multi-epitope assays and vaccines.

Discovery of Tick-Borne Karshi Virus Implies Misinterpretation of the Tick-Borne Encephalitis Virus Seroprevalence in Northwest China.

Despite few human cases of tick-borne encephalitis virus (TBEV), high rates of TBEV seroprevalence were reported among humans and animals in Xinjiang Uygur Autonomous Region in Northwestern China. In this study, the Karshi virus (KSIV) was identified and isolated from Hyalomma asiaticum ticks in Xinjiang. It belongs to the genus Flavivirus of the family Flaviviridae and is closely related to TBEV. KSIV infects cell lines from humans, other mammals and ticks, and causes encephalitis in suckling mice. High minimum infection rates (4.96%) with KSIV were detected among tick groups. KSIV infections have occurred in sheep and marmots, resulting in antibody-positive rates of 2.43 and 2.56%, respectively. We further found that, of the KSIV antibody-positive serum samples from animals, 13.9% had TBEV exposure showing cross-reaction to KSIV, and 11.1% had KSIV infection resulting in cross-reaction to TBEV; 8.3% were likely to have co-exposure to both viruses (or may be infected with one of them and present cross-reactivity with the other). The results revealed a substantial KSIV prevalence among ticks in Xinjiang, indicating exposure of animals to KSIV and TBEV. The findings implied misinterpretation of the high rates of TBEV seroprevalence among humans and animals in previous studies. There is a need to develop detection methods to distinguish KSIV from TBEV and to perform an in-depth investigation of KSIV and TBEV prevalence and incidence in Northwestern China, which would enhance our preparation to provide medical treatment of emerging diseases caused by tick-borne viral pathogens such as KSIV.

Fine mapping epitope on Glycoprotein-Gn from Severe Fever with Thrombocytopenia Syndrome Virus.

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) was recently identified as a tick-borne pathogen that threat to human health. Since 2010, many countries including China, South Korea, and Japan have reported Human SFTS caused by SFTSV infection. The glycoprotein encoded by the SFTSV M gene is the major antigenic component on the viral surface, and responsible for the viral entry, which makes it an important viral antigen and a clinical diagnostic target. The present study aimed to map linear B cell epitopes (BCEs) on the N-terminal glycoprotein (Gn) from SFTSV strain WCH/97/HN/China/2011 using the modified biosynthetic peptide method. Five fine epitopes (E1, 196FSQSEFPD203; E2, 232GHSHKII238; E3, 256VCYKEGTGPC265; E4, 285FCKVAG290, and E5, 316SYGGM320) were identified using the rabbit antisera. Western blot analysis showed that all the five epitopes interacted with the positive serum of sheep that had been naturally infected with SFTSV. Three-dimensional structural modeling analysis showed that all identified BCEs were located on the surface of the SFTSV-Gn and contained flexible loops. The sequence alignment revealed high conservation of the identified BCEs among 13 SFTSV strains from different lineage. These mapped epitopes will escalate the understanding of the epitope distribution and pathogenic mechanism of SFTSV, and could provide a basis for the development of a SFTSV multi-epitope detection antigen.

Molecular identification of common hard ticks (Acari: Ixodidae) in Xinjiang, China.

We provide data on the cytochrome c oxidase subunit I (COI) and 16S rDNA genes for eight species of common hard ticks in Xinjiang: Dermacentor montanus, D. niveus, Haemaphysalis sulcate, Hyalomma asiaticum asiaticum, Hya. detritum, Hya. scupense, Rhipicephalus sanguineus and R. pumilio. Genetic distances, calculated based on the Kimura two-parameter (K2P) distance model, found the same trend of intraspecies level≤interspecies levelintragenus level. Phylogenetic trees, constructed with the neighbor-joining (NJ) and minimum-evolution (ME) methods, demonstrated that each species clustered into separate clades, thus confirming the usefulness of CO1 and 16S rDNA genes for tick species identification. The genera Dermacentor, Haemaphysalis and Rhipicephalus were all recovered in the phylogenetic analysis, as was the subfamily Rhipicephalinae, but a monophyletic Hyalomma was not.

Antitumor effect of a pyrazolone-based-complex [Cu(PMPP-SAL)(EtOH)] against murine melanoma B16 cell in vitro and in vivo.

Pyrazolone-based derivative metal complexes were reported to have cytotoxicity in some tumor cells. In this study, the antitumor effect of [Cu(PMPP-SAL)(EtOH)] (PMPP-SAL = N-(1-phenyl-3-methyl-4-propenylidene-5-pyrazolone)- salicylidene hydrazide anion) in murine melanoma B16 cells in vitro and in vivo was investigated. The results showed that [Cu(PMPP-SAL)(EtOH)] inhibited the survival of B16 cells in vitro, and the IC50 value was superior to cisplatin (DDP) (p < 0.001). B16 cell apoptosis was significantly higher in comparison to the control group (DMSO) (p < 0.01), and cell cycle arrest occurred at the G0/G1 phase. When challenged C57 BL/6J mice were treated with [Cu(PMPPSAL)(EtOH)], a smaller volume of B16 solid tumors were reported than the control group (p < 0.01), with lower positive expression indices of CD 34, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) (p < 0.01). Moreover, the tumor growth was suppressed in mice due to the induction of apoptosis, as detected by the TUNEL assay (p < 0.001). In summary, [Cu(PMPP-SAL)(EtOH)] effectively inhibited the growth of B16 cells in vitro and in vivo due to the induction of apoptosis and the inhibition of intra-tumoral angiogenesis, demonstrating its therapeutic potential in melanoma treatment.

Apoptotic effect of novel pyrazolone-based derivative [Cu(PMPP-SAL)(EtOH)] on HeLa cells and its mechanism.

Pyrazolone complexes have strong anti-tumor and antibacterial properties, but the anti-tumor mechanism of pyrazolone-based copper complexes has not been fully understood. In this study, the possible mechanism and the inhibitory effect of a novel pyrazolone-based derivative compound [Cu(PMPP-SAL)(EtOH)] on human cervical cancer cells (HeLa cells) was investigated. [Cu(PMPP-SAL)(EtOH)] effectively inhibited proliferation of HeLa cells in vitro with an IC50 value of 2.082 after treatment for 72 h. Cell cycle analysis showed apoptosis was induced by blocking the cell cycle in the S phase. [Cu(PMPP-SAL)(EtOH)] promoted the loss of mitochondrial membrane potential, release of cytochrome c, PARP cleavage, and activation of caspase-3/9 in HeLa cells. Additionally, [Cu(PMPP-SAL)(EtOH)] inhibited the PI3K/AKT pathway and activated the P38/MAPK, and JNK/MAPK pathways. [Cu(PMPP-SAL)(EtOH)] also inhibited the phosphorylation of Iκ-Bα in the NF-κB pathway activated by TNF-α, thus restricting the proliferation of HeLa cells which were activated by TNF-α. In conclusion, [Cu(PMPP-SAL)(EtOH)] inhibited the growth of HeLa cells and induced apoptosis possibly via the caspase-dependent mitochondria-mediated pathway. These results suggest that [Cu(PMPP-SAL)(EtOH)] can be a potential candidate for the treatment of cervical cancer.

Epidemiology and phylogenetic analysis of crimean-congo hemorrhagic fever viruses in xinjiang, china.

In 2004 and 2005, an epidemiological survey of Crimean-Congo hemorrhagic fever virus (CCHFV) was conducted in Xinjiang, China. A total of 5,629 serum samples of human and livestock were collected and tested for the CCHFV antibody, and 17,319 ticks were collected for viral identification. Reverse passive hemagglutination inhibition assays showed that the average prevalence of CCHFV antibody was 1.7% for the humans and 12.7% for the livestock. A relatively high antibody prevalence, ranging from 19.1% to 23.4%, was found in the livestock of the northwest, southwest, and northeast parts of the Tarim Basin. When the ticks were pooled to inoculate suckling mice, followed by reverse transcription-PCR (RT-PCR) to detect CCHFV RNA, the average RT-PCR-positive rates for Hyalomma asiaticum kozlovi and H. asiaticum asiaticum were 12.9% and 2.6%, respectively. A significant correlation was found between the antibody prevalence in the livestock and the CCHFV prevalence in H. asiaticum of the same geographic region. No CCHFV RNA was detected in Dermacentor nivenus, Rhipicephalus turanius, or Rhipicephalus sanguineus. A total of 27 partial S segments of CCHFVs were sequenced and used for phylogeny analysis. All but one Chinese isolate grouped into the Asia 1 clade, which contains the strains from Xinjiang and Uzbekistan, while the other strain, Fub90009, grouped with strains from the Middle East.

Cervical cancer cell lines are sensitive to sub-erythemal UV exposure.

High risk human papillomavirus (HPV) infections are the causative agent in virtually every cervical cancer as well as a host of other anogenital and oropharyngeal malignancies. These viruses must activate DNA repair pathways to facilitate their replication, while avoiding the cell cycle arrest and apoptosis that can accompany DNA damage. HPV oncoproteins facilitate each of these goals, but also reduce genome stability. Our data dissect the cytotoxic and cytoprotective characteristics of HPV oncogenes in cervical cancer cells. These data show that while the transformation of keratinocytes by HPV oncogene leaves these cells more sensitive to UV, the oncogenes also protect against UV-induced apoptosis. Cisplatin and UV resistant cervical cancer cell lines were generated and probed for their sensitivity to genotoxic agents. Cervical cancer cells can acquire resistance to one DNA crosslinking agent (UV or cisplatin) without gaining broad tolerance of crosslinked DNA. Further, cisplatin resistance may or may not result in sensitivity to PARP1 inhibition.

Fine epitope mapping of glycoprotein Gn in Guertu virus.

Guertu virus (GTV) is a tick-borne phleboviruses (TBPVs) which belongs to the genus Banyangvirus in the family of Phenuiviridae. In vitro and in vivo studies of GTV demonstrated that it was able to infect animal and human cell lines and could cause pathological lesions in mice. Glycoproteins (GP, including Gn and Gc) on the surface of Guertu virus (GTV) could bind to receptors on host cells and induce protective immunity in the host, but knowledge is now lacking on the information of B cell epitopes (BCEs) present on GTV-GP protein. The aim of this study was to identify all BCEs on Gn of the GTV DXM strain using rabbit pAbs against GTV-Gn. Seven fine BCEs and two antigenic peptides (APs) from nine reactive 16mer-peptides were identified, which are EGn1 (2PIICEGLTHS11), EGn2 (135CSQDSGT141), EGn3 (165IP EDVF170), EGn4 (169VFQEL K174), EGn5 (187IDGILFN193), EGn6 (223QTKWIQ228), EGn7 (237CHKDGIGPC245), AP-8 (299GVRVRPKCYGFSRMMA314) and AP-9 (355CASH FCSSAESGKKNT370), of which six of mapped BCEs were recognized by the IgG-positive sheep serum obtained from sheep GTV-infected naturally. Multiple sequence alignments (MSA) based on each mapped BCE motif identified that the most of identified BCEs and APs are highly conserved among 10 SFTSV strains from different countries and lineages that share relatively close evolutionary relationships with GTV. The fine epitope mapping of the GTV-Gn would provide basic data with which to explore the GTV-Gn antigen structure and pathogenic mechanisms, and it could lay the foundation for the design and development of a GTV multi-epitope peptide vaccine and detection antigen.

Fine mapping epitope on glycoprotein Gc from Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis, caused by CCHF virus (CCHFV) and which there are no diagnostic or therapeutic strategies. The C-terminus of glycoprotein (Gc) encoded by the CCHFV M gene is responsible for CCHFV binding to cellular receptors and acts as a neutralizing-antibody target. In this study, a modified biosynthetic peptide technique (BSP) was used to identify fine epitopes of Gc from the CCHFV YL04057 strain using rabbit antiserum against CCHFV-Gc. Six B cell epitopes (BCEs) and one antigenic peptide (AP) were identified: E1 (88VEDASES94), E2 (117GDRQVEE123), E3 (241EIVTLH246), AP-4 (281DFQVYHVGNLLRGDKV296), E5a (370GDTP QLDL377), E5b (373PQLDLKAR380), and E6 (443HVRSSD448). Western blotting analysis showed that each epitope interacted with the positive serum of sheep that had been naturally infected with CCHFV, and the results were consistent with that of Dot-ELISA. The multiple sequence alignment (MSA) revealed high conservation of the identified epitopes among ten CCHFV strains from different areas, except for epitopes AP-4 and E6. Furthermore, three-dimensional structural modeling showed that all identified epitopes were located on the surface of the Gc "head" domain. These mapped epitopes of the CCHFV Gc would provide a basis for further increase our understanding CCHFV glycoprotein function and the development of a CCHFV epitope-based diagnostics vaccine and detection antigen.