Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection

Although antibody-mediated immune mechanisms have been shown to be important in immunity to ASF, it remains unclear what role virus neutralizing antibodies play in the protective response. Virus neutralizing epitopes have been identified on three viral proteins, p30, p54, and p72. To evaluate the role(s) of these proteins in protective immunity, pigs were immunized with baculovirus-expressed p30, p54, p72, and p22 from the pathogenic African swine fever virus (ASFV) isolate Pr4. ASFV specific neutralizing antibodies were detected in test group animals. Following immunization, animals were challenged with 10(4) TCID(50) of Pr4 virus. In comparison to the control group, test group animals exhibited a 2-day delay to onset of clinical disease and reduced viremia levels at 2 days postinfection (DPI); however, by 4 DPI, there was no significant difference between the two groups and all animals in both groups died between 7 and 10 DPI. These results indicate that neutralizing antibodies to these ASFV proteins are not sufficient for antibody-mediated protection.     

rhTGF-β1及转染TGF-β1基因对兔角膜内皮细胞增殖的影响

目的： 探讨不同浓度的重组人转化生长因子-β₁(rhTGF-β₁)及TGF-β₁基因转染对体外培养兔角膜内皮细胞增殖的影响。方法: 用MTT法检测不同浓度rhTGF-β₁作用下角膜内皮细胞的增殖。用脂质体介导转染方法，将TGF-β₁基因转移入培养的兔角膜内皮细胞，HE染色法观察细胞组织形态学变化；ELISA法检测转染细胞培养上清中TGF-β₁表达量；流式细胞仪检测细胞生长周期变化；DNA电泳法检测转染细胞凋亡情况。结果: MTT检测示5-20 μg/L rhTGF-β1抑制角膜内皮细胞增殖；0.5-1 μg/L组对增殖无影响；0.05~0.1 μg/L组促进细胞增殖。TGF-β₁基因转染细胞形态无明显异常，细胞培养上清中TGF-β₁的浓度约为(98±3)ng/L。流式细胞仪检测示，基因转染组S期和G₂/M期细胞比例减少、PI值降低，但加入EGF后细胞生长基本正常。DNA电泳检测示基因转染组未见凋亡带。结论: rhTGF-β₁对角膜内皮细胞增殖的影响具有剂量依赖性；TGF-β₁基因转染影响角膜内皮细胞增殖、但不诱发凋亡，其抑制作用可被外源性EGF拮抗。     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

TCRVβ7.1基因修饰T细胞对乳腺癌细胞杀伤作用的研究

目的：观察TCPVβ7．1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性的影响。方法：脂质体包裹PdWA3.1vβ7.1后转染健康人PBMC，流式细胞仪检测PdWA3.1Vβ7.1基因表达，改良MIT法检测TCRVβ7．1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性。结果：TCRVβ7．1基因转染可显著增加正常人PBMC该基因表达，转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性有显著性差异。结论：用TCR基因修饰可明显提高正常人PBMC对乳腺癌细胞杀伤作用。     

Matrix protein mediated shutdown of host cell metabolism limits vesicular stomatitis virus-induced interferon-alpha responses to plasmacytoid dendritic cells

Upon infection with many different viruses, plasmacytoid dendritic cells (pDC) produce large amounts of type I interferon (IFN-/β). To address why upon vesicular stomatitis virus (VSV) infection pDC, but not conventional myeloid DC (mDC), are induced to produce IFN-, pDC and mDC were differentiated from bone marrow cells (BM-DC). Upon VSV infection BM-pDC produced IFN-, whereas BM-mDC did not. Notably, upon infection with VSV-M2, a VSV variant expressing a M51R mutant matrix (M) protein that showed a reduced sequestration of host cell metabolism, BM-pDC and BM-mDC mounted massive IFN- responses. Both DC subsets showed comparable RNA levels of retinoic acid inducible gene-I (RIG-I) and Toll-like receptor (TLR) 7 and were able to respond upon triggering with double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) analogs. Moreover, upon VSV-M2 infection IFN- production by both DC subsets was largely dependent on viral replication. Interestingly, upon virus infection BM-pDC, but not BM-mDC, up-regulated mRNA levels of nuclear export factors Nup96/98, probably reflecting cellular mechanisms to circumvent viral escape strategies. Collectively, these results indicated that cell types induced to produce IFN- upon viral infection are not primarily defined by cellular receptor configurations but rather by complex virus/host cell interactions.     

Promotion of retroviral entry in the absence of envelope protein by chlorpromazine

Retrovirus packaging cell lines that express the Moloney murine leukemia virus gag, pol, and env genes and a retroviral vector genome can produce virus particles that are capable of transducing cells. Normally if the packaging cell line does not produce a functional viral fusion glycoprotein, such as the retroviral envelope protein or a foreign viral glycoprotein, then the viruses will be incapable of transducing cells. We have found that incubating envelope protein-deficient virus particles bound to cells with chlorpromazine leads to transduction. Chlorpromazine (CPZ) is a membrane-active reagent that is commonly used to induce the hemifusion to fusion transition when membrane fusion is mediated by partially defective viral glycoproteins. The concentration and pH dependence of the promotion of transduction by CPZ is consistent with a role for CPZ micelle formation in viral entry. These data indicate that caution is warranted when experiments concerning membrane fusion completion promoted by CPZ are analyzed.     

Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging

To begin elucidation of the relationship between Brome mosaic virus (BMV) replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to express either individual or desired pairs of the three genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (CP) was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1 + RNA3 or RNA2 + RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNA1 and RNA2 were constructed by deleting the 3' tRNA-like structure (3' TLS). Co-expression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing TLS-less RNA1 and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging.     

临床病毒性脑炎标本的博尔纳病病毒核酸检测

目的 探讨临床病毒性脑炎(viral encephalitis,VE)患者与博尔纳病病毒(Borna disease virus,BDV)感染的关系,分析BDV感染的病毒性脑炎患者临床特征.方法 用荧光定量巢式逆转录聚合酶链反应(FQ-nRT-PCR)方法检测病毒性脑炎患者及非感染性疾病施行蛛网膜下腔阻滞麻醉的手术患者脑脊液单个核细胞(cerebrospinal fluid mononuclear cell,CSFMC)中BDV p24基因片段,同时用β-肌动蛋白(β-actin)作为内参照,脑脊液(CSF)阳性标本基因测序分析并总结出临床特征.结果 32例病毒性脑炎脑脊液标本BDV p24基因片段检出率为12.5%(4/32),拷贝数＞102/μl.对其中一份CSF阳性标本测序后,与BDV标准病毒株Ⅴ和马源的BDV病毒株H1766序列比较同源性分别为95.35%和98.84%.在4个位点出现基因突变(nt1649 T→C、nt1656 G→A、nt1670 C→T、nt1676 C→T).该目的基因片段与马源的BDV病毒株亲缘关系最近.阳性脑炎患者主要以精神行为异常为临床特征.结论 贵州省遵义市部分病毒性脑炎的发生与BDV感染有关,主要以精神行为异常为临床特征.     

重型肝炎中甲、乙型肝炎病毒合并感染的探讨

报告经血清免疫学抗HAVIgM、抗-HBcIgM、HBV-DNA、HBsAg/IgM复合物以及乙肝三种抗原抗体系统检测48例重型肝炎中甲、乙型肝炎病毒感染情况。结果:31例为HBV感染,1例为HAV感染,1例未定型,15例(31.25％)为甲、乙型合并感染(混合感染7例、重叠感染8例)。15例中亚急性10例、慢性5例;死亡或恶化10例,与同期“单纯”感染的重肝比较,包括主要临床生化指标均无显著差异。有7例患者病程中有近期出现“急黄肝”症状的历史,随后病情突然加重;1例发生在住院期间,且血清抗-HAVIgM转阳,最后死亡。认为合并感染可使部分急、慢性肝炎病情加重,甚至发展成重型。应注意发现。     

Osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β1 genein vitro

Summary To study the osteogenic potential of cultured bone marrow stromal cells (BMSCs) transfected with transforming growth factor β₁ (TGF-β₁) genein vitro, cultured BMSCs were transfected with the complexes of pcDNA₃-TGF-β₁ and Lipofectamine Reagentin vitro. The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type I propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type I expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF-β₁ gene could promote the osteogenic potential of cultured BMSCs.