Objective:To understand the epidemiological characteristics and incidence trend of severe fever with thrombocytopenia syndrome (SFTS) in China.Methods:The incidence data of SFTS in China from 2018 to 2021 were collected from Chinese Disease Prevention and Control Information System for a statistical and descriptive epidemiological analysis by using software such as Excel 2016, Joinpoint 5.0.2, SPSS 26.0, and GraphPad Prism 8.0, especially, the SFTS cases reported monthly by key provinces were analyzed.Results:From 2018 to 2021, a total of 8 835 SFTS cases were reported in 25 provinces and the annual incidence showed an upward trend. The distribution of SFTS cases showed clustering, but the cases were mainly sporadic ones. The cases began to increase in March, mainly occurred during April to October (96.79%,8 551/8 835), and peaked during May to July. The cases were mainly distributed in middle-aged and old farmers, and slight more cases were women. The average case fatality rate was 5.38%, which varied greatly with areas. The case fatality rate tended to increase with age.Conclusion:From 2018 to 2021, the epidemiological characteristics of SFTS in China remained stable, but the number of reported cases gradually increased and the distribution showed an expanding trend, to which close attention should be paid.

Objective:To investigate the prevalence of lymphocytic choriomeningitis virus (LCMV) and hantavirus (HV) specific antibodies among cross-border migrant workers for assessment of the risk of rodents-borne virus infection.Methods:From 2019 to 2020, a survey was conducted on cross border migrant workers engaged in outdoor activities, and serum samples were collected, LCMV specific IgG antibody was detected by an indirect ELISA and Western blot based on recombinant nucleoprotein, and indirect immunofluorescence assay (IFA) based on recombinant expressed glycoprotein. HV IgG antibody in serum was detected by a commercial indirect IgG ELISA kit and IFA based on hantavirus infected Vero cells.Results:A total of 139 cross-border workers, aged 25~57, were surveyed; 64% (89/139) had working experience in multiple countries, involving 26 countries, including 14 countries in Asia and 12 countries in Africa; 11.51% (16/139) of serum samples were tested positive for LCMV antibodies, and the positive samples were verified by Western blot and IFA. The antibody detection rate was slightly higher than the published infection rate from other similar studies. And, HV antibodies were detected from one serum sample (0.72%, 1/139) by ELISA and IFA. However, it was still uncertain when and where the viral infections were acquired.Conclusions:Through this serological cross-sectional preliminary analysis, the infection status and existing risks of LCMV and HV viruses among cross border migrant workers were revealed, which suggested the necessity of strengthening the prevention and control of rodents borne diseases in outdoor engineering sites.

Objective:To establish a simple, rapid and low-cost 2019 novel coronavirus (2019-nCoV) neutralizing antibody detection method.Methods:The 2019-nCoV RBD specific immunoglobulin G (RBD-IgG) detection method was established based on the principle of quantum dot immunochromatography(QDs), and the detection was evaluated by using of sera from coronavirus disease 2019 (COVID-19) convalescent patients ( N = 97), vaccinated donors ( N = 82) and healthy donors ( N = 299). The suitability of fingertip blood was evaluated by matching blood samples with peripheral blood ( N=54). Results:The 2019-nCoV RBD-IgG detection method based on QDs was successfully established. The detection result of QDSs had strong correlation ( Spearman r > 0.73, P < 0.000 1) and good consistency ( Kappa=0.93, P < 0.01) with the result of micro-neutralization test(MNT). The sensitivity and specificity were 92% and 99%, respectively. There was high correlation ( Spearman r =0.932 6, P < 0.000 1) and no significant difference ( P=0.102 6) between result of fingertip blood and peripheral blood. Fingertip blood can be used as a surrogate sample for testing. Conclusions:The 2019-nCoV neutralizing antibody detection method established in this study can provide an immediate, efficient and low-cost method selection for the assessment of herd immunity status, and provide technical support for the herd immunity monitoring of 2019-nCoV vaccinated population and the prevention and control of the epidemic.

Objective:To establish a quick on-site emergency detection method for severe fever with thrombocytopenia syndrome virus (SFTSV), dengue virus (DENV), and hantaan virus (HTNV).Methods:This research was based on the traditional TaqMan fluorescent probe technology, using the domestic rapid one-step quantitative RT-PCR kit, combined with the Magnetic induction cycler (Mic) qPCR instrument. The detection limit, specificity and repeatability of this method were evaluated by simulated samples, other virus infected samples and normal human blood samples.Results:Compared with the traditional RT-PCR assay, the required time of this method was greatly shortened, and the detection can be completed within 35 minutes. The limit of quantitation for SFTSV, DENV and HTNV are less than 100copies/PCR. No nonspecific amplification was found in the simulated negative samples and other virus infected samples. All the simulated positive sample for verification could be detected, and coefficient of variation Ct value of each group was less than 4%. Conclusions:The rapid fluorescence quantitative RT-PCR assays have certain application prospects for on-site emergency detection, and provide important technical supports and new directions for the prevention and control of common hemorrhagic fever viruses.

Severe fever with thrombocytopenia syndrome virus (SFTSV), is a tick-borne novel Bunyavirus, which was first discovered in the area of Dabie Mountain in China. It can cause severe fever and thrombocytopenia clinically, with a fatality rate of 10% to 30%. As the pathogenesis and infection mechanism of SFTSV are still unclear, there are no specific drugs and vaccines for SFTSV. In this paper, the mechanism of entry, replication, assembly and budding of SFTSV is reviewed, and the research progress of the treatment antibody and antiviral drugs of SFTSV is summarized, which will provide reference for the development of vaccines and antiviral drugs for SFTSV in China.

Objective:To establish real-time fluorescent quantitative RT-PCR method for detection of Avalon virus (AVAV) and Hughes virus (HUGV), two Nairoviruses in the family of Nairoviridae. Methods:The genomic sequences of the two viruses published in the international public database were collected, collated, compared and analyzed to define the detection targets, and the viral specific primers and probes were designed accordingly. Real-time fluorescent quantitative RT-PCR detection method were established, and the operating detection procedures were optimized using simulated samples prepared using in vitro transcription assay, other virus infected samples, virus strains and normal human blood samples. The detection limit, specificity and repeatability of the method were evaluated.Results:The real-time fluorescent quantitative RT-PCR method could effectively amplify and detect AVAV and HUGV viral target RNA with detection limits of about 20 copies/μl and 70 copies/μl, respectively. No nonspecific amplification was found in the samples of Kyasanur forest disease virus, influenza B virus BV and BY, influenza A virus H3N2, yellow fever virus, Japanese encephalitis virus, Crimean-Congo hemorrhagic fever virus, SFTS virus, nairobi sheep disease virus and Tahyna virus. There was no cross reaction between the two nairoviruses. The coefficient of variation was within 2% by repeated comparative analysis.Conclusions:The real-time fluorescent quantitative RT-PCR method for detection of AVAV and HUGV might be used for screening of humans, vectors and host animal samples for rapid detection of related pathogens.

Objective:To explore an early, rapid and accurate detection method of severe acute respiratory syndrome coronavirus 2 (2019-nCoV) antigen and improve the detection rate of patients with 2019-nCoV.Methods:We detected the characteristics of monoclonal antibodies (mAbs) against 2019-nCoV generated previously and established a double antibody sandwich ELISA against S protein of the virus.Results:All 12 mAbs against 2019-nCoV screened in our previous study were able to recognize purified virion and S protein. They were good candidates for detecting antibody to 2019-nCoV antigen. A double antibody sandwich ELISA established in this study could rapidly and effectively detect 2019-nCoV S protein with high sensitivity. The detection limit of 2019-nCoV S protein was lower than 1 ng/ml.Conclusions:This method could specifically detect 2019-nCoV antigen, and provided a meaningful reference for early, sensitive and specific diagnosis of COVID-19 infection.

Objective:To analyze the neutralization properties of different genotypes and mutants of severe fever with thrombocytopenia syndrome virus (SFTSV).Methods:Pseudoviruses of SFTSV of different genotypes and mutants were constructed using VSVΔG-Fluc*G backbone. Neutralization assays were established based on the pseudoviruses. DNA vaccines for different SFTSV genotypes were prepared. Serum samples were collected from guinea pigs immunized with the DNA vaccines. Neutralizing antibodies in serum samples from immunized guinea pigs and naturally infected patients were detected using neutralization assays and analyzed.Results:The pseudoviruses of five genotypes and 43 mutants were successfully constructed and the neutralization assays based the pseudoviruses were successfully established after optimizing the reaction parameters. The dilution multiple corresponding to the inhibition rate of neutralizing antibody to half of the pseudovirus infection was taken as the titer of neutralizing antibody by the reduction in pseudovirus reporter gene. The neutralization antibody titers in naturally infected patients and immunized guinea pigs were respectively in the ranges of 1∶100-1∶43 000 and 1∶100-1∶2 500 when detected with the reference HB29 pseudovirus. The neutralization antibody titers ranged from 1∶100-1∶2 500 after immunization with different genotypes of DNA vaccines. No significant statistical difference in neutralization antibody titer was observed among different genotypes or mutant strains.Conclusions:The neutralization properties of different genotypes and mutants showed no significant change, which would be very useful for developing vaccines.

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.

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.