To understand the immunogenicity of purified inactivated severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), concentration by ultrafiltration as well as molecular-sieve chromatography (MSC) were used for purification of inactivated SFTSVs. Inactivated viruses in purified samples were analyzed and identified by western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the glycoprotein (GP) and nucleoprotein (NP) antigen titers of which were detected using a double-sandwich enzyme-linked immunosorbent assay (ELISA). Purified inactivated SFTSVs were enriched and observed by electron microscopy, and the total protein concentration detected using the bicinchoninic acid assay. Purified inactivated SFTSVs were applied to New Zealand rabbits via two immunization programs to evaluate immunogenicity and to compare the immune effect. After SFTSVs were inactivated and concentrated by ultrafiltration, MSC revealed two typical elution peaks. The sample of one peak was identified as inactivated virions, in which GP and NP were detected by SDS-PAGE, western blotting and ELISA. Main corponent of the other peak was NP. After concentration by ultrafiltration, purified inactivated SFTSVs with purity >90% and total protein concentration of 1. 1 mg/mL were obtained, and the typical electron microscopy of bunyavirus was observed. In the sera of animals immunized with purified inactivated SFTSVs, SFTSV-specific IgG antibody and neutralizing antibody were detected at high titers. However, antibody titers were affected by the immunization program. Effect of immunization on days 0, 14 and 28 was significantly better than that on days 0, 7 and 28. Our work revealed that cultivation of SFTSVs contained intact virus particles and large amounts of free NP. Using MSC, purified inactivated SFTSVs of high purity could be obtained. Purified inactivated SFTSVs induced high titers of neutralizing antibody and virus-specific IgG antibody showing satisfactory immunogenicity, which provides important clues for further study on a vaccine for the inactivated virus.
Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Bunyaviridae Infections ; immunology ; virology ; Humans ; Neutralization Tests ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Bunyaviridae Infections ; immunology ; virology ; Female ; Humans ; Hybridomas ; immunology ; Mice ; Mice, Inbred BALB C ; Phlebovirus ; immunology ; Viral Structural Proteins ; immunology

Effects of dihydroartemisinin (DHA) on the expression level of Alpha-7 .3 giardin mRNA in C2 Giardia lam-blia was investigated in this study to explore the damage to skeleton protein of C 2 Giardia lamblia .Giardia lamblia was culti-vated respectively for 2 ,4 ,8 ,and 12 hours with modified TYI-S-33 medium containing 100 μg/mL and 200 μg/mL DHA , while the control group performed in the same experimental conditions without DHA .The expressive quantity of Alpha-7 .3 gi-ardin mRNA was determined by using real-time reverse transcription PCR ,and then we found that the expressive quantities of Alpha-7 .3 giardin mRNA with DHA were significantly lower than those in the control group .It’s suggested that dihydroarte-misinin has obvious inhibitory effect on the expression level of Alpha-7 .3 giardin mRNA in C2 Giardia lamblia .The actions of dihydroartemisinin on skeleton protein of C2 Giardia lamblia are effective .

Objective: To obtain the optimum of lentiviral library packaging based on CRISPR/cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9). Methods: Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence antibody (IFA) and enzyme linked immunosorbent assay (ELISA) were used to detect the lentivirus titers in condition of different ratio of packaging plasmids, different addition of lipofectamine 3000 reagent and different time points post-transfection. Then, high-throughput sequencing was performed to evaluate the representation and distribution of single guide (sg)RNAs in the library. Results: The lentivirus titer was the highest when the molar ratio of psPAX2∶pMD2.0G∶Lentivirus library was 2∶1∶1, and the optimum addition of Lipofectamine 3000 reagent was 10 μl, while the result of ELISA were correspondent to that of RT-PCR. The IFA result showed that the lentivirus titer was the highest at 60 h post-transfecion. The coverage of sgRNAs in the lentivirus library packaged with the optimum we obtained was 99.3%, and the read counts of sgRNAs was observed in a normal distribution. Conclusions The optimal lentivirus library packaging was obtained, and this can provide basis for CRISPR/cas9-based screening.

Objective: To establish a fluorescent bead-based multiplex assay for the simultaneous detection of seven viral diseases endemic in Africa. Methods: The genomic sequences of the viral pathogens causing Rift valley fever, Yellow fever, Marburg virus disease, Ebola virus disease, Lassa fever, Crimean-Congo hemorrhagic fever and Chikungunya fever were compared, PCR detection target fragments were selected, and amplification primers and hybrid probes were designed. The reference samples of related pathogens were prepared by chemical synthesis of DNA and in vitro transcription RNA. The sensitivity and stability of the detection method were evaluated. The specificity was evaluated by testing 30 samples of suspected dengue fever, and hantavirus diseases, and 32 healthy human blood samples. Results: The fluorescent bead-based multiplex assay could specifically detect the corresponding pathogen, the detection limit was at a range of 10²-10⁵ copies/ μl, the specificity was 100%, and the intra-assay coefficient of variation was below 12%, and the inter-assay coefficient of variation was below 15%. Conclusions A fluorescent bead-based multiplex PCR assay for the simultaneous detection of seven viral diseases endemic in Africa was established, which may provide a new choice for the screening of suspected infectious diseases.

Objective To establish a multiplex Luminex assay for nucleic acid detection of Phlebovirus.Methods Specific primers and probes of Rift Vally fever virus(RVFV),Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and Heartland virus (HLV) were designed.Target fragments of the three viruses were amplified using target enriched multiplex (Tem) PCR,and then a multiplex Luminex assay of nucleic acid detection was established.The specificity of the developed Luminex assay was evaluated withTem-PCR products of RVFV,SFTSV,HLV and the amplification products of 7 hemorrhagic fever viruses (HFVs) including Hantand virus(HTNV),and its sensitivity and reproducibility was evaluated with in vitro transcribed viral RNA standards of gradient dilution.Results Using the developed multiplex Luminex assay of nucleic acid detection,the target sequences of RVFV,SFTSV and HLV could be detected specifically,and there was no cross-reaction with 7 HFVsincluding HTNV.In vitro transcribed RNA of 103 copies/μl,102 copies/μ1 and102copies/μl could be detected for RVFV,SFTSV and HLV detection,respectively.The coefficient of variation of detection values were all less than 15％ in each concentration more than 103 copies/μl of RNA standards for both intra-assays and inter-assays.Conclusion A multiplex Luminex assay of nucleic acid detection for phlebovirus was preliminary established to effectively detect RVFV,SFTSV and HLV,laying the foundation for the laboratory diagnosis of related infectious diseases.

Objective To investigate the effect of IFITM1/2/3 on SFTS virus infection.Methods IFITM1/2/3 expression vectors were constructed by inserting corresponding genes into pCAGEN vector respectively and the expression of IFITM1/2/3 were detected by immunofluorescence assay (IFA) and Western Blot analysis.SFTS virus load at different time post infection on Vero and THP-1 cells that overexpressing IFITM1/2/3 were determined by quantitative real-time RT-PCR.Results High intracellular antibody binding activities of IFITM 1/2/3 were observed by IFA and Western Blot assay.The results of realtime RT-PCR indicated that IFITM1/2/3 could significantly inhibit the infection of SFTS virus on the above two cell lines.Furthermore,at early stage of virus infection,the inhibition effects were more obvious for low dose infection.Conclusions IFITM1/2/3 could effectively inhibit intracellular SFTS virus infection.

Objective:To compare and analyze the clinical diagnostic values of five thyroid nodule malignant risk stratification guidelines.Methods:From October 2019 to October 2021, 926 cases of patients with 1 027 thyroid nodules were recruited in the Second Affiliated Hospital of Xi ′an Jiaotong University. All nodules were categorized individually according to 2015 American Thyroid Association for Ultrasound Malignancy Risk Stratification of Thyroid Nodules in Adults Guidelines(ATA guidelines), 2016 the Korean Society of Radiology and the Korean Society of Thyroid Radiology Thyroid Imaging Reporting and Data Systems(K-TIRADS), 2017 European Thyroid Association Thyroid Imaging Reporting and Data Systems(Eu-TIRADS), 2017 American College of Radiology Thyroid Imaging Reporting and Data Systems (ACR TI-RADS), and 2020 Chinese Thyroid Imaging Reporting and Data System (C-TIRADS). The pathological results were taken as the gold standard, the malignancy ratio of nodules of different categories in each system was calculated. ROC curves were plotted to evaluate the diagnostic efficiencies of different systems, and DeLong test was used to compare the areas under ROC curves. The sensitivity and specificity of different systems were calculated when the maximum point of the Youden index was the optimal cut-off value.Results:In the same stratified system, there were statistically significant differences in the malignant proportion of nodules of different grades ( P<0.05). The malignant proportion of nodules in the high-risk group showed no significant difference among different stratified systems ( P>0.05). Except for C-TIRADS, the malignant proportion of nodules was increased with the increase of diagnostic grade at each grade of the other four stratification systems. ROC curve showed that AUCs of ATA guidelines, K-TIRADS, EU-TIRADS, ACR TI-RADS and C-TIRADS were 0.814, 0.819, 0.814, 0.820 and 0.802, respectively, there was no statistical significance in AUC of different stratification systems (all P>0.05). The optimal truncation values in differentiating benign and malignant nodules were middle-risk malignant nodules, moderately suspicious malignant nodules, middle-risk malignant nodules, class 4 and class 4B. The diagnostic of five stratification systems showed that ATA guidelines had the highest sensitivity (0.784), C-TIRADS had the highest specificity (0.854). Conclusions:The five stratified systems have similar efficacy in differentiating benign and malignant thyroid nodules, and all of them have good diagnostic value.

Objective:To construct an explainable artificial intelligence(AI) model of risk characteristics of papillary thyroid carcinoma(PTC), and to explore its value of it combined with clinical features in predicting cervical lymph node metastasis(CLNM) in PTC patients.Methods:From January 2021 to September 2022, 422 patients(422 nodules) with pathologically confirmed PTC underwent thyroidectomy and neck lymph node dissection in the Second Affiliated Hospital of Xi′an Jiaotong University were retrospectively collected, the patients were randomly divided into training set and test set according to the ratio of 7∶3. Ultrasonographic features highly correlated with PTC risk characteristics were extracted by traditional machine learning method, and an intelligent prediction model with optimal probability of risk characteristics was established. Then, a risk model for predicting CLNM of PTC patients was constructed in combination with clinical features. The diagnostic effectiveness of the model was evaluated by drawing a ROC curve and calculating the area under curve (AUC).Results:In the AI explaineable model of PTC risk characteristics in the test set, the intelligent diagnosis model of calcification based on logistic regression classification showed the highest diagnostic efficiency, with an AUC of 0.87 ( P<0.05). Compared with the probability model of risk characteristic of PTC alone, the comprehensive model combined with clinical characteristics showed higher diagnostic efficiency in predicting CLNM of PTC patients, with AUC of 0.97, diagnostic critical value of 0.15, corresponding accuracy, sensitivity and specificity of 92.65%, 92.76% and 92.54%, respectively (all P<0.05). Conclusions:The explaineble risk characteristics of PTC AI model combined with clinical features can effectively predict the cervical lymph node metastasis of PTC, and then provide effective information for clinical decision-making of PTC patients.

Objective:To develop a rapid nucleic acid detection method for Zika virus (ZIKV), Dengue virus (DENV), Yellow fever virus (YFV), Chikungunya virus (CHIKV) based on microfluidic fluorescence quantitative RT-PCR technologies, in order to achieve rapid diagnosis of these four viral infections.Methods:Four sets of specific primers and probes were designed targeting the NS1 gene of ZIKV, the NS5 gene of DENV, and YFV, the E1 gene of CHIKV, respectively. The sensitivity was evaluated using in vitro transcribed RNA of ZIKV, DENV, YFV and CHIKV, and the specificity were evaluated using other viral nucleic acid. ZIKV, YFV and CHIKV detection method were verified using simulated positive samples, and DENV detection method was verified using clinical patient samples, the result of which were also compared with the quantitative RT-PCR detection method . Results:The limit of detection (LOD) of ZIKV, DENV, YFV, and CHIKV microfluidic qRT-PCR method were 14.57 copies/μl, 94.27 copies/μl, 8.25 copies/μl, and 223.19 copies/μl, respectively, and the four detection method showed no cross-reactivity with other viral nucleic acids. The prepared ZIKV, YFV and CHIKV simulated positive samples were 100% detected, and the variation coefficient of Ct value measured at each concentration were all around 2%; the 20 clinical patient specimens of DENV infection were 100% detected, which is consistent with the result of fluorescent quantitative RT-PCR detection.Conclusions:The ZIKV, DENV, YFV, and CHIKV microfluidic quantitative RT-PCR detection method showed good sensitivity, specificity, and stability. The detection could be completed within 25 minutes, which could be used for laboratory detection and early diagnosis.