新疆出血热病毒核蛋白基因的高效表达及其在诊断中的初步应用

目的　克隆并测定了克里米亚 刚果出血热病毒 (CCHFV)中国分离株 (新疆出血热病毒 ,XHFV)BA8816 6株核蛋白 (NP)基因的序列并实现其在细菌中的高效表达与临床诊断的应用。方法　病毒RNA经RT PCR扩增出完整的NP基因。将扩增产物进行序列分析并克隆至融合表达载体pET32a ,使重组质粒在大肠杆菌BL 2 1中高效表达。将融合蛋白经初步纯化后包被ELISA板用于抗体检测。结果　XHFVBA8816 6株NP基因序列以及推导的氨基酸序列与其它XHFV的NP基因和蛋白序列同源性较高 ,在进化树上形成独立的分支。BA8816 6株NP基因编码 4 82个氨基酸的核蛋白 ,推测的相对分子质量 (Mr)约为 5 4× 10 3。在细菌中表达的融合蛋白经印迹试验证明具有良好的抗原性。以所建立的ELISA方法检测疫区人和动物血清的结果与IFA一致 ,并与临床诊断有很好的符合率。结论　BA8816 6株与其它XHFVBA6 6 0 19、BA84 0 2的NP基因在进化上关系密切 ,综合M基因的序列分析结果 ,人源分离株BA8816 6可能是来自蜱的BA84 0 2变异株。表达于细菌中的核蛋白可作为安全的诊断性抗原用于临床检测及流行病学调查 ,所建立的方法准确、特异、简便、快速     

Genetic Characterization of the M RNA Segment of Crimean-Congo Hemorrhagic Fever Virus Strains Isolated in Russia and Tajikistan

The data on the structure of the M genome segment of CCHF virus strains from Russia and Central Asia (Tajikistan) are presented. Data obtained have been compared with other available published sequences of the middle segment of strains from China, Nigeria, and Pakistan. It has been found that all the known strains can be divided into four genetic groups, based on the nucleotide sequence of the M genome segment and an amino acid sequence of the glycoprotein precursor it encodes, whereas VLG/TI29414 and STV/HU29223 strains from Russia form a separate group. The CCHF virus strain from Tajikistan, TADJ/HU8966, was genetically related to strains 7803 and 75024 from China, and together with these and the Nigerian IbAr 10200 strain, it forms another group.     

Human defined antigenic region on the nucleoprotein of Crimean-Congo hemorrhagic fever virus identified using truncated proteins and a bioinformatics approach

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. In this study, amino acid sequence data for the CCHFV nucleoprotein (NP) was used to identify potential linear epitopic regions which were subsequently included in the design of large and small truncated recombinant NP antigens and peptide libraries. Two truncated recombinant CCHFV NP antigens were prepared based on results of prediction studies to include epitopic regions and exclude hydrophobic regions that could influence protein expression and solubility. Serum samples were collected from acute and convalescent patients. An IgG antibody response was detected in 16/16 samples tested using the large recombinant NP-based ELISA and in 2/16 using the small recombinant NP-based ELISA. A total of 60 peptides covering predicted epitopic regions of the NP were synthesized and peptide NRGGDENPRGPVSR at amino acid position 182–195, reacted with 13/16 human serum samples. In summary, functional assays are required to determine the biological activity of predicted epitopes for development of peptide based assays for antibody detection. Bacterially expressed complete NP antigens have previously been shown to be useful tools for antibody detection. Truncation of the antigen to remove the hydrophobic C terminus had no impact on the ability of the antigen to detect IgG antibody in human sera. The results indicate that the region from amino acids 123 to 396 includes a highly antigenic region of the NP with application in development of antibody detection assays.     

抗家鼠型HFRS病毒McAb13E2VH基因的克隆及序列分析

从分泌高亲和力抗家鼠型ＨＦＲＳ病毒型特异性ＭｃＡｂ的杂交瘤细胞系１３Ｅ２中提取细胞总ＲＮＡ，经逆转录合成ＣＤＮＡ，并以之为模板，用特异性引物进行ＰＣＲ，扩增出约３６０ｂｐ产物，将之克隆入ＰＵＣ１８质粒中。序列分析表明，所克隆的基因符合编码小鼠抗体ＶＨ的特征。１３Ｅ２ＶＨ基因由２６６个ｂｐ组成，编码１２２个氨基酸，隶属于小鼠重链ⅡＡ亚组。     

检测流行性出血热病毒抗体的滴金免疫测定法的建立及应用

应用自行制备的直径１５ｎｍ的胶体金标记ＳＰＡ，建立了检测血清流行性出血热病毒抗体的滴金免疫测定法。与同步建立的ＥＬＩＳＡ法对比检测临床标本，结果有良好的一致性，两法符合率为９６．４％。本法中抗原、抗体通过渗滤在膜上进行反应，数分种内即可用肉眼观察结果，操作简单，试剂稳定、安全。     

高效液相色谱纯化抗肾综合征出血热病毒血凝素单克隆抗体及特性鉴定

本文报告从BALB/c小鼠免疫腹水及该腹水经40％饱和硫酸铵盐析沉淀的γ球蛋白用高效液相色谱层析技木(HPLC)纯化、制备抗肾综合征出血热病毒血凝素单克隆抗体。HPLC一步法对IgG1的回收率为52％,纯度97.4％;HPLC二步法回收率为67％～73％,纯度98.1％;盐析法回收率为85％,纯度≥70％。间接免疫荧光法测定HPLC纯化样品(二步法)的比活性提高49～51倍,血凝抑制滴度为1:5120。     

Cloning,Expression and Sequence Analysis of the Classical Swine Fever Virus Nucleocapsid Protein

The DNA complementary to the 5′-terminal 1929 nucleotides of classical swine fever virus (CSFV; alias hog cholera virus, HCV) LPC vaccine strain RNA was cloned and sequenced. The sequence encompasses a 5′-noncoding region (NCR) of 264 nucleotides and an open reading frame (ORF) of 1665 nucleotides. The cloned sequence contains genes of four viral proteins, P23, nucleocapsid (core) protein, E0 and part of E1 proteins. Alignment of the 5′-terminal 1929 nucleotides of LPC strain with other strains of CSFV showed well conservation and a homology as high as 84–95% was found between these strains. The cDNA of CSFV-LPC core was cloned into an expression vector, and a fusion protein of 38.5 kDa was obtained which reacted strongly to CSFV antiserum. Purification of the core fusion protein was achieved by a single-step affinity chromatography and the purified product could be recognized by the sera of CSFV-infected swine in ELISA assay. Phylogenetic analysis of the 5′-terminal 1929 nucleotides between pestiviruses revealed that the 5′-end region seems to be suitable for differentiation of different strains of CSFV. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phylogenetic characterization of circulating Dengue and Alkhumra Hemorrhagic Fever viruses in western Saudi Arabia and lack of evidence of Zika virus in the region: A retrospective study, 2010‐2015

Flaviviruses represent a global public health concern. They consist of ∼70 viruses with almost half of them causing human diseases with unspecified febrile illnesses. Cities in western Saudi Arabia are endemic for viruses (DENV) with sporadic infections due to Alkhumra hemorrhagic fever virus (AHFV). They also represent a major destination for travelers coming for annual religious pilgrimages (Hajj and Umrah) from all over the world. However, whether other flaviviruses are circulating is not known because of the limited number of surveillance studies. Here, we retrospectively screened 690 samples for flaviviruses in samples from patients with unexplained febrile illnesses between 2010 and 2015 in western Saudi Arabia using a pan‐flaviviruses RT‐PCR assay. Despite Zika virus RNA was not detected, this study confirms circulation and/or sporadic spread of DENV‐2, DENV‐3, and AHFV, higher prevalence of DENV‐2, and a role for visitors from DENV endemic countries in DENV importation into the Kingdom. Further analysis also showed very low genetic diversity of AHFV confirming its slow microevolution. Accordingly, continuous and prospective surveillance for flaviviruses using such assay are warranted in Saudi Arabia which receives millions of Muslims annually to implement effective control measures in light of the global widespread and outbreaks of several flaviviruses.

     

肾综合征出血热病毒微量细胞培养法和空斑法的比较

对肾综合征出血热病毒的微量细胞的培养法和空斑法进行了比较。４株ＨＦＲＸ病毒及３株特异性ＭｃＡｂ的测定结果表明，微量细胞培养法测量的病毒滴度高于空斑法，但后者检测ＭｃＡｂ的中和效价却高于前者。     

肾综合征出血热病毒特异性双价纯化免疫血清F（ab）2的制备和临床治疗应用

１９８８年至１９９１年对收治的发病５日以内的肾综合征出血热（ＨＦＲＳ）病人应用姬鼠型ＨＦＲＳＶ陈株及家鼠型ＨＦＲＳＶＲ２２株，免疫猪所制备的特异性双价纯化免疫血清Ｆ（ａｂ）２（称Ｆ（ａｂ）２血清），治疗ＨＦＲＳ病人６５例作为研究组，以４４例作为对照组。治疗结果表明：①球结膜水肿渗出减轻，２４小时出血减轻；②白细胞病毒抗原消失迅速；③研究组出院平均早９．１天；④在洽疗后２、４日，对照组的特异性免疫荧光ＩｇＭ抗体明显高于研究组的。⑤其他实验室检测指标都以研究组为优。提纯后的免疫血清Ｆ（ａｂ）２无抗体－介导反应，无副作用及过敏反应。它含有特异性中和抗体及其他免疫因子，可中和清除体内的病毒抗原，减轻病毒血症及毛细血管壁的损伤，阻断病情发展，促进病情恢复。     

Expression of the Nucleoprotein of the Puumala Virus from the Recombinant Semliki Forest Virus Replicon: Characterization and Use as a Potential Diagnostic Tool

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), μ-capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.     

间接ELISA法的改进并用于肾综合征出血热病人血清的初步分型

采用超声、差速离心法制备肾综合征出血热病毒（ＨＦＲＳＶ）抗原。通过与免疫荧光法（ＩＦＡ）和捕获ＩｇＭＥＬＩＳＡ法（ＭａｃＥＬＩＳＡ）比较，分析了病人临床四个期血清抗体阳性率，对３组不同地区的正常人群ＨＦＲＳＶ抗体阳性血清作抑制试验，以及比较ＩＦＡ和ＥＬＩＳＡ血清抗体几何平均滴度证实本方法是特异和灵敏的。用这一方法对野鼠型和家鼠型ＨＦＲＳ病人血清，与两型病毒作交叉ＥＬＩＳＡ试验，结果呈单向反应；采用胞膜荧光反应为主的感染细胞制备抗原，可提高其分型效果。     

乙型肝炎病毒核心基因的酶切图谱及核苷酸序列分析

对慢性乙型肝炎患者的２２份肝穿刺活检组织及其中配对的６份血清用ＰＣＲ扩增出乙型肝炎病毒（ＨＢＶ）的ＰｒｅＣ／Ｃ基因，选用ＡｖａⅡ、Ｓａｕ３ＡⅠ、ＸｍｎＩ，ＢｓｔＮＩ及ＴａｑＩ５种限制性内切酶消化后，对Ｃ基因进行酶谱分析。发现有６份肝组织及１份血清出现异常酶谱。对Ｓａｕ３ＡⅠ酶解异常标本进行分子克隆及核苷酸序列分析，发现２例患者因２１３７位核苷酸点突变出现了Ｓａｕ３ＡⅠ的新酶切点。类似变化在肝癌组织的ＨＢＶＣ基因中也曾发现，其意义待进一步研究。     

Characterization of Monoclonal Antibodies to Junin Virus Nucleocapsid Protein and Application to the Diagnosis of Hemorrhagic Fever Caused by South American Arenaviruses

Junin virus (JUNV), Machupo virus, Guanarito virus, Sabia virus, and Chapare virus are members of New World arenavirus clade B and are the etiological agents of viral hemorrhagic fevers that occur in South America. In this study, we produced three monoclonal antibodies (MAbs) to the recombinant nucleocapsid protein of JUNV, designated C6-9, C11-12, and E4-2. The specificity of these MAbs was examined by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, and an epitope-mapping method. Using these MAbs, we developed antigen (Ag) capture ELISA systems. We showed that by using MAb C6-9, JUNV Ag was specifically detected. On the other hand, by using MAb C11-12 or E-4-2, the Ags of all human pathogenic South American arenaviruses were detected. The combined use of these Ag capture ELISA systems in the present study may be useful for the diagnosis of acute-phase viral hemorrhagic fever due to infection by a South American arenavirus.The South American arenaviruses Junin virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), Sabia virus (SABV), and Chapare virus (CHPV) are members of New World arenavirus clade B. JUNV, MACV, GTOV, and SABV are the etiological agents of Argentine hemorrhagic fever (AHF), Bolivian hemorrhagic fever (BHF), Venezuelan hemorrhagic fever (VHF), and Brazilian hemorrhagic fever, respectively (4). CHPV was also recently shown to be associated with cases of hemorrhagic fever in Bolivia (5). AHF emerged in the 1950s, and since then, outbreaks have occurred annually without interruption (4). The mortality rate for AHF is estimated to be 15 to 30%, but early treatment with immune plasma reduces the rate to less than 1% (6). The region at risk has been progressively expanding into northern central Argentina, and almost 5 million people are currently considered to be at risk for AHF (6, 13). Phylogenetic analysis indicates that JUNV is more closely related to MACV than to SABV or CHPV, whereas SABV and CHPV are more closely related to each other than to other New World arenaviruses (5).Arenaviruses are enveloped and contain a bisegmented RNA genome. The genome consists of two ambisense single-stranded RNA molecules, one designated L, which encodes the RNA-dependent RNA polymerase and a zinc-binding matrix protein, Z, and the other designated S, which encodes the major structural components of the virion, i.e., the nucleocapsid protein (NP) and the envelope glycoprotein precursor (15). The arenavirus NP is the most abundant protein among the viral structural proteins both in infected cells and in virions (2) and is commonly used as a target for detecting viral antigens (Ags) (20). Moreover, arenavirus NPs have been known to be the most conserved among the same virus species and, to some extent, among different arenavirus species (3, 8). Therefore, it seems likely that monoclonal antibodies (MAbs) raised against the NP of an arenavirus would also be useful for detecting other arenaviruses (20). Recently, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed by using a recombinant NP (rNP) of JUNV, obtained from a recombinant baculovirus system, and was proposed to be useful for etiologic confirmation of AHF in seroepidemiological studies (20, 26). It is considered that an Ag capture ELISA using MAbs specific for viral Ags allows rapid diagnosis of the acute phase of viral hemorrhagic fever by detecting viral Ags in blood or tissue homogenates (20). In this study, we produced MAbs to the rNP of JUNV. These MAbs were characterized by ELISA, indirect immunofluorescence assay (IFA), and an epitope-mapping method. Ag capture ELISAs were developed by using these MAbs that are specific for JUNV and that are broadly applicable for the detection of human pathogenic New World arenaviruses.     

中国狂犬病毒疫苗株（5aG）糖蛋白基因在痘苗病毒中的表达及免疫效果观察

将克隆到的中国狂犬病毒疫苗株（５ａＧ）的糖蛋白基因重组到痘苗病毒ＴＫ区，并在痘苗病毒Ｐ１１启动子的控制下，构建了狂犬－痘苗重组病毒（ＶＶａＧ）。经间接免疫荧光和Ｗｅｓｔｅｒｎ免疫印染证明，重组病毒ＶＶａＧ能良好地表达狂犬病毒糖蛋白，其分子量约为６６００。用ＶＶａＧ免疫小鼠，７ｄ便可诱生较高的狂犬病毒中和抗体，２１ｄ达４１６９，并能１００％保护狂犬病毒本毒株和国际标准攻击毒（ＣＶＳ）的致死量攻击。     

Large-Scale Gene Expression Profiling of Discrete Brain Regions: Potential, Limitations, and Application in Genetics of Aggressive Behavior

Many behavioral geneticists are interested in unraveling the molecular mechanisms underlying aggressive behavior. So far, most scientists have based their search for aggression-related genes on a preliminary functional hypothesis. Large-scale gene expression profiling techniques, such as serial analysis of gene expression (SAGE) and DNA microarrays, now enable the screening of expression levels of thousands of genes simultaneously, allowing the identification of new candidate aggression-related genes expressed in brain and thus the generation of new hypotheses. However, expression profiling in the brain is challenging, as brain is a complex heterogeneous tissue where large numbers of genes are expressed and relatively small changes in gene expression occur. In this special issue, we focus on the principles of SAGE and DNA microarrays, as well as their advantages and disadvantages and application to analysis in brain tissue in order to identify aggression-related genes.     

Baculovirus Expression of the Fusion Protein Gene of Bovine Respiratory Syncytial Virus and Utility of the Recombinant Protein in a Diagnostic Enzyme Immunoassay

The fusion (F) protein of bovine respiratory syncytial virus (BRSV) was expressed by using a baculovirus vector. Antigenicity was tested by immunofluorescence analysis with F-specific monoclonal and polyclonal antibodies. Antibodies to recombinant F protein raised in a rabbit neutralized BRSV and human respiratory syncytial virus infectivity when tested in a plaque reduction assay. The recombinant F protein was evaluated as a source of antigen in an enzyme-linked immunosorbent assay (ELISA), and this ELISA was compared with the virus neutralization (VN) test for detecting BRSV antibodies in 10 consecutive serum samples from four calves vaccinated with a live modified BRSV vaccine and from two nonvaccinated control calves. The ELISA with the baculovirus-expressed F protein as an antigen compared favorably with the VN test and is a rapid, sensitive, and specific method for detecting serum antibodies to BRSV.     

High-level Expression of the ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Pichia pastoris

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.