慢性乙型肝炎患者肝组织中HBV抗原表达特征及其临床意义

目的探讨慢性乙型病毒性肝炎肝活检组织中检测乙肝表面抗原(HBsAg)和乙肝核心抗原(HBcAg)表达强度及表达方式的必要性。方法采用EnVision免疫组织化学法检测196例慢性乙型肝炎患者肝穿组织中HBsAg和HBcAg的表达水平,并用荧光定量PCR检测其血清中的HBV DNA的含量。对肝组织进行炎症活动度分级和纤维化分期。结果肝组织中的HBsAg表达强度和表达方式与炎症分级、纤维化分期和血清乙肝病毒载量均无相关性(P>0.05)。HBcAg表达强度与炎症分级无相关性(r=-0.02,P>0.05);与纤维化分期呈负相关(r=-0.28,P<0.01);与血清乙肝病毒载量呈正相关(r=0.53,P<0.01)。HBcAg表达方式与炎症分级为负相关(r=-0.27,P<0.01),其中浆型组炎症活动度分级高于核型组和混合型组(P<0.01),混合型组高于核型组(P<0.01)。HBcAg表达方式与纤维化分期亦呈较弱的负相关(r=-0.23,P<0.01),其中浆型组纤维化分期高于核型组和混合型组(P<0.05)。HBcAg表达方式与血清乙肝病毒载量呈正相关(r=0.22,P<0.01)。结论区分肝组织中的HBsAg表达强度和表达方式无益于了解慢性乙型肝炎患者肝损害的程度,而检测肝组织中的HBcAg则有助于临床抗病毒治疗。     

Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

Previous results indicated that the herpes simplex virus 1 (HSV-1) U(L)31 gene is necessary and sufficient for localization of the U(L)34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U(L)31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U(L)31 gene. The replication of the U(L)31 deletion virus was restored on U(L)31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U(L)34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U(L)34 protein localized at the nuclear membrane in rabbit skin cells, and U(L)31 complementing CV1 cells infected with the U(L)31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U(L)34 protein to the nuclear membrane. We speculate that this function partially complements that of U(L)31 and may explain why U(L)31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.     

Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase

Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.     

中枢神经系统感染患儿血脑屏障与脑脊液白蛋白指数的变化

目的探讨中枢神经系统感染患儿血脑屏障（blood-brain barrier,BBB）与脑脊液（cerebrospinal-fluid,CSF）白蛋白指数的变化。方法采用溴甲酚绿法和免疫比浊法检测18例化脓性脑膜炎患儿急性期、恢复期及22例病毒性脑炎患儿急性期血清和CSF中白蛋白水平,并计算出CSF白蛋白指数。与正常对照组比较。结果化脓性脑膜炎、病毒性脑炎患儿急性期CSF白蛋白、CSF白蛋白指数显著高于正常对照组（P〈0.001）,且化脑组显著高于病脑组（P〈0.001）。化脑组恢复期患儿CSF白蛋白、CSF白蛋白指数与对照组无明显差异（P〉0.05）。结论监测CSF白蛋白和CSF白蛋白指数的水平可作为化脓性脑膜炎和病毒性脑炎早期诊断、鉴别诊断,以及判断BBB损伤程度的参考指标。     

新生儿TORCH感染的血清学检测与临床分析

目的 对新生儿进行TORCH感染的血清学检查及临床表现分析。方法 用ELASA法检测血清中TORCH(弓形体、风疹病毒、巨细胞病毒、单纯疱疹病毒)IgM。结果 2000年1月至2003年1月本院新生儿重症监护室(NICU)共收治新生儿l554例，其中48例为TORCH感染，巨细胞病毒感染率最高占52．1％；风疹病毒感染占33．3％；单纯疱疹病毒感染占14．6％。未发现弓形体抗体阳性的患儿。结论 新生儿TORCH感染可造成多器官损伤，主要为听力异常、高胆红素血症和肝功能异常、神经系统损伤、心肌损伤、血小板减少、先天性心脏病。计划免疫、母亲孕期筛查、新生儿早期筛查、干预治疗非常重要。     

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.     

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.     

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.