Construction and evaluation of a universal influenza mRNA vaccine / 陆军军医大学学报

Objective To construct a universal influenza mRNA vaccine and evaluate its immunogenicity.Methods The antigen sequence of hemagglutinin(HA),nucleoprotein(NP)and matrix protein 2 ectodomain(M2e)in influenza A/California/04/2009 was optimized.HA,NP and 3 tandem M2e(3M2e)were cloned into pcDNA3.1 vector,respectively.Then the mRNAs were synthesized by linearization,in vitro transcription,enzymatic capping and enzymatic tailing,and named as mRNA-HA,mRNA-NP and mRNA-3M2e,respectively.The protein expression of the 3 kinds of mRNAs in 293T cells was detected by immunofluorescence assay.Comb-mRNA vaccine was prepared by enveloped mRNA-HA,mRNA-NP and mRNA-3M2e with lipid nanoparticles,respectively,and the particle size and potential were identified.Twenty-eight 6-week-old female BALB/c mice(18～22 g)were randomly divided into LNP group(n=14)and Comb-mRNA group(n=14).Hemagglutination inhibition(HI)method and microneutralization(MN)test were used to evaluate the serum antibody titer induced by Comb-mRNA vaccines.The mice were infected by 5LD50 wild-type H1 N1 influenza virus to evaluate the protective efficacy.Results The mRNA-HA,mRNA-NP and mRNA-3M2e were successfully constructed,and the 3 mRNAs could be expressed in 293T cells.The average size of mRNA encapsulated by lipid nanoparticles was 119.53±6.5 nm,and the average potential was-8.23±1.3 mV.The geometric mean titer(GMT)of HI and MN in the Comb-mRNA group were 179.6 and 201.6,compared with the LNP group.The ratio of IFN-γ+CD4+/CD8+Tcells was increased.The Comb-mRNA group could provide protection against 5LD50 wild type influenza H1 N1 virus after 2 weeks of booster immunization.Conclusion Comb-mRNA,an influenza vaccine candidate,can induce immune responses and protect mice from influenza virus challenge.

Host Targets Interacting with Influenza Virus NP and Mechanism of Gardenia Jasminoides Iridoid Glycoside Against Influenza Virus / 中国实验方剂学杂志

ObjectiveTo explore host factors interacting with influenza virus nucleoprotein （NP） and study their effects on influenza virus replication， as well as the mechanism of gardenia jasminoides iridoid glycoside （IGE） in inhibiting influenza virus. MethodA yeast two-hybrid system was utilized to screen host factors that interacted with influenza virus NP. Heterogeneous nuclear ribonucleoprotein D0 （HNRNPD）， glucosamine-6-phosphate deaminase 1 （GNPDA1）， poly（rC）-binding protein 1 （PCBP1）， and protein inhibitor of activated signal transducer and activator of transcription （STAT） protein 1 （PIAS1） were validated by immunoprecipitation assay. The effects of PIAS1 and HNRNPD on influenza virus replication were compared by a dual luciferase assay， and the effects of IGE on influenza virus replication were examined in the presence of transfected ribonucleoprotein （RNP） and knockdown of PIAS1. ICR mice were randomly divided into a normal group， model group， oseltamivir phosphate group， and high， medium， and low dose IGE groups， with 10 mice in each group. In addition to the normal group， each group was infected with the influenza A virus FM1 strain by nasal drip to establish a viral pneumonia model. The high， medium， and low dose IGE groups were given drugs of 50， 25， and 12.5 mg∙kg-1 by gavage， and the oseltamivir phosphate group was given the drug of 27.5 mg∙kg-1 by gavage. Equal amounts of distilled water were instilled in the normal and model groups for four consecutive days. Later， protein expression of PIAS1， NP， phosphorylated （p）-STAT3， STAT3， p-STAT1， and STAT1 were detected in the lung tissue by Western blot. ResultIn yeast two-hybrid assays， 16 potential host targets interacting with influenza virus NP were identified. Immunoprecipitation experiments revealed that HNRNPD and PIAS1 could interact with influenza virus NP. The dual luciferase reporter assays found that both PIAS1 knockdown and overexpression significantly affected IAV RNP activity （P<0.05， P<0.01）， and the effect of HNRNPD on IAV RNP was not significant. Both high and low dose IGE groups reduced influenza virus replication （P<0.05） and reversed the increase in influenza virus replication caused by the knockdown of PIAS1（P<0.05， P<0.01）. The expressions of PIAS1， NP， p-STAT3， p-STAT1， and STAT1 in the lung tissue of infected mice were reduced to different degrees in each IGE group （P<0.05， P<0.01）. ConclusionPIAS1 interacts with influenza virus NP and is able to inhibit influenza virus replication. IGE may exert antiviral effects by inhibiting the activity of IAV RNP through the PIAS1/STAT1 pathway.

Values of sperm nucleoprotein transformation in predicting recurrent abortion / 临床检验杂志

Objective To investigate the values of nucleoprotein transformation in sperm for predicting recurrent abortion.Methods A total of 521 infertile couples with complete test indicators on fertility were selected from the reproductive medical clinic of our hospital from 2019 to 2022,among which the ages of the male were from 23 to 56 years old.The following factors causing recurrent abortion were excluded,including the age of woman,body mass index,metabolic disease,antiphospholipid syndrome,uterine and accessory abnormalities,history of caesarean section and intrauterine myoma/cervical conectomy,peripheral blood chromosome abnormalities of both the couple,and adverse life history,such as smoking/alcohol abuse.According to the abortion situation,they were divided into the recurrent abortion group(≥two spontaneous abortions),one spontaneous abortion group and no abortion group.Tukey's multiple comparison was performed to compare the differences of nucleoprotein transformation of sperm in each group by using GraphPad6.0 sta-tistical software.The Pearson method was used to analyze the correlation between nucleoprotein transformation and recurrent abortion.The predictive values of nucleoprotein transformation in recurrent abortion were analyzed by the parameters of sensitivity,specificity,positive predictive value,negative predictive value,Youden index and odd ratio.Results The percentages of abnormal nucleoprotein transformation in recurrent abortion group[(33.31±13.83)％]were significantly higher than those in non-abortion group[(26.85± 15.38)％]and the one abortion group[(28.20±12.50)％,P＜0.05].Pearson correlation analysis showed that there was a significant positive correlation between abnormal nucleoprotein transformation and recurrent abortion.The sensitivity and specificity of nucleoprote-in transformation for predicting recurrent abortion were 45.24％and 73.64％,respectively.All of the data of positive predictive value(15.70％),negative predictive value(92.53％),Youden index(18.88％)and odd ratio(2.31)of nucleoprotein transformation in predicting recurrent abortion were higher than those of high DNA stainability(10.64％,90.31％,1.05％and 1.11).Conclusion In the spouses of patients with recurrent abortion,the immaturity of sperm nuclear protein is significantly increased and significantly posi-tively correlated with recurrent abortion.The abnormal nucleoprotein transformation of sperm may be the important factor of recurrent a-bortion in males,and it has high predictive value for recurrent spontaneous abortion in clinical practice.

The Structure and Function of the Bunyavirus Nucleoprotein / 中国生物化学与分子生物学报

Bunyavirus is widely distributed, highly contagious, and has a high fatality rate. It is a negative- strand RNA virus that has a major impact on public health around the world. The development of vaccines and the search for drugs are the key to prevent bunyavirus infection. The nucleoprotein (NP) of viruses is necessary for the synthesis of viral RNA, which combines with viral RNA to form the nucleocapsid, participates in viral assembly and RNA transcription, and plays an important role in viral proliferation. In addition, NP also has B cell and T cell epitopes, which can induce cellular and humoral immunity, so NP is an ideal target for vaccine design and drug development. Given its abundance and specificity, NP is also commonly used in the detection of viral diseases. More and more bunyavirus NP structures and structures of NP-RNA complexes have been resolved. Researchers have discovered two important antiviral targets through these structures, the terminal arm and the RNA binding cleft. This paper reviews the function and three-dimensional structure of the bunyavirus NP and the research progress of NP as an antiviral target, in order to provide a theoretical basis for the prevention and treatment of the bunyavirus disease.

The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

Immune responses elicited by influenza A mRNA vaccine based on lipopolyplex-encapsulated virus nucleoprotein and matrix protein 2 extracellular domain fusion in mice / 中华微生物学和免疫学杂志

Objective:To evaluate the immunogenicity of a novel influenza virus mRNA vaccine based on conserved antigens delivered by lipopolyplex (LPP) platform in a mouse model.Methods:Four copies of genes coding for extracellular domain of matrix 2 protein (M2e) and nucleoprotein (NP) of influenza A virus were synthetized after codon optimization. The fusion antigens were transcribed in vitro and delivered by LPP platform, named as LPP-4M2eNP. Expression of M2e and NP in eukaryotic cells was detected by immunofluorescence assay (IFA). BALB/c mice were inoculated intramuscularly twice with 10 μg or 30 μg LPP-4M2eNP vaccine at an interval of four weeks. Antibody response was detected by ELISA and cellular-mediated immunity (CMI) was detected by enzyme-linked immunospot assay (ELISPOT). Results:IFA showed that NP and M2e were expressed correctly in eukaryotic cells. Single dose immunization could induce significant antigen (NP, M2e)-specific CMI and antigen (NP, M2e)-specific antibody response was induced in mice with Th1 type bias after boost immunization. Moreover, NP-specific CMI was increased significantly after the second immunization, while no significant change in M2e-specific CMI was observed.Conclusions:Stronger CMI was triggered in mice by single dose of LPP-4M2eNP vaccine. Furthermore, robust humoral and cellular immune responses were induced after boost immunization. This study suggested that LPP-4M2eNP vaccine, which based on conserved antigen of influenza A and delivered by LPP platform, had great potential for development and application.

Effect of micro RNA-296-3p regulated cell on proliferation, migration and invasion of tongue squamous cell carcinoma by targeting nucleoprotein 1 / 中国医师进修杂志

Objective:To investigate the effects of micro RNA(miR)-296-3p on cell proliferation, migration and invasion of tongue squamous cell carcinoma and its underlying mechanism.Methods:Normal oral epithelial mucosal keratinocytes HOK was used as the control. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression of miR-296-3p and nucleoprotein 1(NUPR1) mRNA in tongue squamous cell carcinoma cell lines CAL27, SCC15 and SCC9, and Western blot was used to detect the expression of NUPR1 protein. CAL27 cells were divided into NC group, miR-con group, miR-296-3p group, si-con group, si-NUPR1 group, miR-296-3p+pcDNA group and miR-296-3p+pcDNA-NUPR1 group, then CCK8 method was used to detect the cell activity. Transwell was used to detect the cell migration and invasion. Western blot was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), matrixmetallo proteinase-2(MMP-2) and matrixmetallo proteinase-9(MMP-9). Dual-luciferase reporter assay system was implemented to verify the relationship between miR-296-3pand NUPR1.Results:Compared with the HOK cells, the content of miR-296-3p in the tongue squamous cell line CAL27, SCC15 and SCC9 groups was significantly reduced (0.54 ± 0.08, 0.38 ± 0.05, 0.59 ± 0.07 vs. 1.04 ± 0.12, t = 10.401, 15.231 , 9.718, P<0.05), while the expression levels of NUPR1mRNA (5.94 ± 0.40, 4.48 ± 0.45, 5.19 ± 0.48 vs.0.94 ± 0.12, t = 35.918, 22.803, 25.769) and protein (0.79 ± 0.09, 0.54 ± 0.05, 0.62 ± 0.08 vs. 0.28 ± 0.04, t = 15.535, 12.182, 11.404) were significantly increased (all P<0.05). Compared with those in the NC group or miR-con group, CAL27 cell activity, migration number, invasion number and the protein expression of PCNA, MMP-2 and MMP-9 in the miR-296-3p group were decreased (all P<0.05). Compared with those of the NC group or the si-con group, the CAL27 cell activity, migration number, number of invasions and the protein expression of PCNA, MMP-2 and MMP-9 in the si-NUPR1 group were decreased (all P<0.05). miR-296-3p negatively regulatedthe expression of NUPR1 in CAL27 cells. Compared with the miR-296-3p+pcDNA group, CAL27 cell viability (138.34 ± 5.73 vs. 98.54 ± 5.89, t = 14.530), migration number (95.28 ± 7.56 vs. 67.92 ± 5.23, t = 8.929), invasion number (184.53 ± 17.57 to 101.26 ± 10.64, t = 12.162) and the protein expression of PCNA (0.68 ± 0.07 to 0.35 ± 0.06, t = 10.738), MMP-2 (0.43 ± 0.05 to 0.29 ± 0.04, t = 6.559) and MMP-9 (0.58 ± 0.06 vs. 0.33 ± 0.08, t = 7.500) in the miR-296-3p+pcDNA-NUPR1 group were increased (all P<0.05). Conclusions:miR-296-3p may inhibitthe proliferation, migration and invasion of tongue squamous cell carcinoma cells by negatively regulating NUPR1.

A human cell polarity protein Lgl2 regulates influenza A virus nucleoprotein exportation from nucleus in MDCK cells

In this study, the regulatory effect of the overexpression of polarity protein Lgl2 on the nuclear export ofinfluenza A virus nucleoprotein in infected cells was investigated. A stable Tet-Off inducible MDCK cell lineexpressing a fusion protein comprising Lgl2 and an enhanced yellow fluorescent protein were used. TCID50analysis and neuraminidase activity analysis revealed that replication of influenza A virus was inhibited in Lgl2overexpressing cells. By immunofluorescence microscopical observation at different time point post virusinfection, a retention of NP in cellular nucleus was found in Lgl2 overexpressing cells. Compared with normalMDCK cells, change in claudin-1 distribution between cell contacts caused by Lgl2 overexpression impairedthe barrier function of tight junction. These results suggest that changes in cell polarity induced by Lgl2overexpressing will affect virus NP transportation.

Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein

PURPOSE: The aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (DIVA) strategy accompanying the marker vaccine lacking an immunodominant epitope (IDE) of nucleoprotein of Newcastle disease virus (NDV). MATERIALS AND METHODS: Recombinant epitope-repeat protein (rERP) gene encoding eight repeats of the IDE sequence (ETQFLDLMRAVANSMR) by tetra-glycine linker was synthesized. Recombinant baculovirus carrying the rERP gene was generated to express the rERP in insect cells. Specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) employing the rERP was evaluated. RESULTS: The rERP with molecular weight of 20 kDa was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. The rERP was antigenically functional as demonstrated by Western blotting. An indirect ELISA employing the rERP was developed and its specificity and sensitivity was determined. The ELISA test allowed discrimination of NDV infected sera from epitope deletion virus vaccinated sera. CONCLUSION: The preliminary results represent rERP ELISA as a promising DIVA diagnostic tool.

Effect of Modified Si Junzitang Drug Serum on Expression of Apoptosis-related Molecules of Gastric Cancer Cell SGC-7901 / 中国实验方剂学杂志

Objective: To explore the effect of modified Si Junzitang (MSJZT) drug serum on the expression of apoptosis-related molecules of gastric cancer cell SGC-7901 and further its anti-tumor mechanism.Method: A total of 40 SD rats were randomly divided into four groups:low-dose,middle-dose,high-dose MSJZT (0.213,0.426,0.853 g·kg-1) groups and normal group (n=10).The treatment groups were administrated through gastric perfusion,and the normal group was given the equivalent volume of normal saline for 10 days.1.5 h after the last treatment,chloral hydrate peritoneal anesthesia was performed,blood was collected from heart,and different doses of serum were separated to prepare drug-containing serum of low-dose,middle-dose,high-dose MSJZT groups,in order to incubate SGC-7901 gastric cancer cell.Early and late apoptosis rates were detected with flow cytometry.Afterwards,the tumor suppressor gene p53,c-nucleoprotein gene (c-Myc),cysteine-aspartic acid protease-3(Caspase-3),B-cell lymphoma-2(Bcl-2) mRNA expressions were confirmed by fluorescence quantitative polymerase chain reaction (Real-time PCR).The protein expressions of p53,c-Myc,Caspase-3,Bcl-2 were detected by immunofluorescence.Result: Compared with the normal group,the high-dose MSJZT group could obviously increase the apoptosis rate to 22.58%(PPPPPPConclusion: MSJZT drug serum could exert an anti-tumor effect by inhibiting the expression of the anti-apoptotic protein Bcl-2,and promoting the expressions of pro-apoptotic-related molecules p53,c-Myc,Caspase-3.

Genetic characteristics of nucleoprotein of the epidemic measles virus in Xi’an, 2013-2017 / 中华实验和临床病毒学杂志

Objective@#To know the genetic characteristics of nucleoprotein(N)of wild type measles virus in Xi’an, 2013-2017.@*Methods@#We used the pharyngeal swab specimens of suspected measles cases which were tested positive for measles virus (MV) nucleic acid by Real-time RT-PCR to isolate MV strains with Vero-SLAM cells, extracted RNA of the strains, amplified the N genes of MV strains and the pharyngeal swab specimens which had lower Ct values by One-Step RT-PCR, and analyzed the result after sequencing.@*Results@#Thirty carboxyl terminal sequences of the N genes of MV endemic strains were obtained and identified as genotype H1a. The nucleotide and amino acid homology among the epidemic strains were 97.3%-100% and 96.7%-100%, among the epidemic strains and S191 were 91.3%-92.2% and 87.3%-90.0%, while among the epidemic strains and C47 were 92.2%-92.7% and 88.0%-90.7%. The nucleotide homology among the epidemic strains and wild strains of same period in Anhui, Gansu, Shandong, Henan, Jilin were higher than that in Shanxi, Sichuan, Hunan and Guangdong. The most different amino acid sites among the epidemic strains and the two vaccine strains which had high level of entropy were 422, 457 and 497, secondly 467, thirdly 500. The mutations at the 467 site only existed in 2017. dN/dS=0.199 and no positive selection site was found.@*Conclusions@#The dominant genotype of the epidemic MV strains in Xi’an was H1a in 2013-2017, the genetic diversity of viruses decreased in 2016-2017. We should strengthen the surveillance of etiology of MV to provide evidence for elimination of measles.

Sequencing analysis of N and G gene of rabies viruses from Sichuan province of China in 2011- 2017 / 中华实验和临床病毒学杂志

Objective@#To analyze molecular feature of rabies virus (RABV) epidemic strains in Sichuan province during 2011 to 2017, and explore differences at nucleotide, amino acid and protein modification between these street strains and vaccine strains.@*Methods@#Nucleoprotein(N) and glycoprotein(G) genes were amplified by RT-PCR using specific primers for 23 antigen-positive canine brain specimens collected from 2011 to 2017. The evolutionary relationship and immune antigenicity of N and G genes was analyzed. Bioinformatics software was used to analyze and organize data.@*Results@#We obtained the N and G genes sequences of 23 RABV strains by sequencing. Genetic evolution relationship analysis showed that all the 23 RABV strains belonged to rabies virus species and could be divided into three branches, which had apparent geographically specific characteristics but some Sichuan strains co-circulated with the epidemic strains in the eastern and northern regions of China.The N genes of Sichuan strains had nucleotide and amino acid homology of 97.4% to 100% and 99.6%-100%. The nucleotide and amino acid homology between Sichuan strains and reference strains were 72.1%-99.8% and 81.6%-100%, respectively. There were some differences in antigenic sites, cell epitopes and signal peptide sequences between vaccine strain and Sichuan strains but no significant change was found in antigenicity, organizational preference and virulence.@*Conclusions@#The 23 strains of RABV of Sichuan belonged to rabies virus species and had no obvious differences. There were few differences between Sichuan strain and vaccine strain in amino acid sequences of G, but the virulence did not change.

Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

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.

Investigation of the dog-biting events and molecular evolution characteristics of rabies virus strains in Shuangbai county of Yunnan province / 中华实验和临床病毒学杂志

Objective@#To illustrate the epidemical characteristics of the dog-biting events and molecular evolution of rabies virus (RV) strains prevalent in Shuangbai county of Yunnan province, China.@*Methods@#Epidemical investigation on the dog-biting events and human cases were conducted and the brain tissues of the biting dogs and human cases were sampled post-mortem. Nucleoprotein (N) genes of the RVs were sequenced. Homology and phylogenetic analysis were performed using the relevant bioinformatics software.@*Results@#A total of 12 dog-biting events took place between 2011-2017 in Shuangbai county and 35 persons were bitten. Of the 12 events, 11 were investigated in time and 32 bitten persons received proper wound management and a full post-exposure vaccination course. Rabies has not developed in these wounded cases until now. However, due to failure to receive medical intervention and post-exposure treatment in time, 1 of 3 bitten persons in a single event died of rabies. RV N genes from 5 dogs and 1 person were sequenced. Phylogenetic tree showed that RV strains prevalent in Shuangbai county were closely related with the ones found in neighboring counties/cities such as Chuxiong, Lufeng, Jingdong and Xiangyun. All these strains were related to the ones denoted as clade China-I and prevalent in Sichuan province. Homology analysis showed 99.6%-100% homology in nucleotide and amino acid among the 6 RVs prevalent in Shuangbai county and those prevalent in Chuxiong, Lufeng, Xiangyun and clade China-I of Sichuan province. Compared with the China-I strains prevalent in Chuxiong, Zhaotong and Qujing prefectures between 2006-2007, the homology of nucleotide and amino acid were 97.1%-99.3% and 99.1%-99.6%, respectively.@*Conclusions@#Surveillance on the dog-biting events can prevent rabies in humans effectively. RV strains prevalent in Shuangbai county belong to clade China-I and have a close relationship with those of neighboring prefectures, cities, counties and the ones prevalent in Sichuan province.

B1 subgenotype of human metapneumovirus detected in children with acute respiratory infection / 中华实验和临床病毒学杂志

Objective@#To reveal the prevalence of human metapneumovirus (HMPV) among pediatric patients with acute respiratory infection (ARI).@*Methods@#Nasopharyngeal aspirates were collected from hospitalized young children (1-5 years) diagnosed as ARI in Department of Respiratory Disease, Shanxi Provincial Children′s Hospital, Taiyuan city. The nucleoprotein (N) gene of HMPV was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The positive products of PCR were sequenced, made homology and phylogenetic analysis.@*Results@#One sample collected in Feb. 2017 and two samples collected in Nov. 2017 were HMPV positive in RT-PCR detection. The homologies of nucleic acid and deduced amino acid sequences of the three positive PCR products with the corresponding HMPV sequences in GenBank were over 83% and 90%, respectively. Phylogenetic analysis revealed that HMPV B1 subgenotype was detected.@*Conclusions@#HMPV B1 subgenotype was the major epidemic HMPV in young children with ARI in Taiyuan city.

Effect of baicalin Combined with phillyrin on the gene expression of influenza A virus nucleoprotein / 医学研究生学报

Objective Influenza A (InfA) is an acute respiratory infectious disease persistently threatening human health and social stability. The high mutation of the InfA virus makes very significant the development of anti-influenza drugs. Traditional Chinese herbal drugs have shown distinct advantages in the prevention and management of InfA. This study aimed to investigate the effect of baicalin combined with phillyrin on the gene expression of InfA virus nucleoprotein (NP).Methods The nucleoprotein eukaryotic expression vector of InfA virus was constructed by transient transfection of InfA virus NP eukaryotic recombinant plasmid pcDNA3.1 (+)/NP into hela cells. Six groups were designed for the experiment: hela cells, liposome, recombinant plasmid (transfected pcDNA3.1(+)/NP), baicalin, phillyrin, baicalin (15.625 μg/mL-1) + phillyrin (12.5 μg/mL-1), each treated with respective agents after transfection. After 48 hours of culture, the number of the copied InfA virus NPs in the hela cells was measured by RT-qPCR and the effect of baicalin combined with phillyrin on the gene expression of InfA virus NP in the target cells evaluated by the statistical method.Results RT-qPCR showed that the gene expression of InfA virus NP was significantly lower in the baicalin + phillyrin than in the recombinant plasmid group (t=6.966, P1) and a synergistic effect of medium- and high-dose baicalin + medium- and high-dose phillyrin (CI<1).Conclusion Baicalin combined with phillyrin can decrease the gene expression of InfA virus NP and has a synergistic effect within a certain dose range.

Preparation and identification of antibodies against respiratory syncytial virus nucleoprotein / 中华微生物学和免疫学杂志

Objective To prepare and identify the monoclonal antibodies against respiratory syn-cytial virus nucleoprotein(RSV N protein). Methods A prokaryotic expression system was used to express recombinant RSV N protein in Escherichia coli (E.coli). BALB/c mice were immunized with the recombi-nant N protein after purification. Monoclonal antibodies (McAbs) against the N protein were sorted from these BALB/c mice and then were further characterized by Western blot, ELISA and immunofluorescence. Furthermore,this study used orthogonal experiment to identify McAbs pairs,which could be used for diagno-sis. Results This study succeeded in obtaining 24 hybridoma cells that stably secreted monoclonal antibod-ies against RSV N protein. These antibodies showed good reactivity in ELISA,of which eight had strong spe-cificity in Western blot and 13 could be used in immunofluorescence. This study obtained two McAbs pairs (12F2/11H8-HRP and 12F2/15A8-HRP) that could be used in RSV detection. Conclusion This study succeeded in screening and preparation of McAbs against RSV N protein and obtaining two potential McAbs pairs for rapid detection of RSV.

Semi-Functional Quantitative Flow Cytometry Assay for Lymphocytic Choriomeningitis Virus Titration

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.

Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.