PCR detection of Actinobacillus pleuropneumoniaeapxIV gene in formalin-fixed,paraffin-embedded lung tissues and comparison with in situ hybridization

AIMS: Formalin-fixed, paraffin-embedded lung tissues from pigs experimentally infected with 12 Actinobacillus pleuropneumoniae serotypes were used to develop nested PCR for the detection of apxIV gene. METHODS AND RESULTS: The PCR results from formalin-fixed, paraffin-embedded tissues were compared with in situ hybridization. The apxIV gene was detected in formalin-fixed, paraffin-embedded lung tissues from all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes by nested PCR. In situ hybridization produced a distinct positive signal in all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes. Agreement rates between nested PCR and in situ hybridization were 100% for the detection of apxIV gene in formalin-fixed paraffin-embedded lung tissues. Acceptable PCR signals were detected from lung tissues fixed for periods up to 180 days. CONCLUSIONS: The apxIV gene is species-specific rather than serotype-specific and is therefore an important diagnostic marker. The nested PCR assay would be a useful method for the detection of apxIV gene to diagnose A. pleuropneumoniae infection when formalin-fixed tissues are submitted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirmed the possibility of using formalin-fixed, paraffin-embedded tissues for the diagnosis of A. pleuropneumoniae infection in pigs.     

Prevalence of Varicella-Zoster Virus DNA in Dissociated Human Trigeminal Ganglion Neurons and Nonneuronal Cells

Previous analyses using in situ hybridization alone or together with PCR have yielded conflicting results regarding the cell type in which latent varicella-zoster virus (VZV) resides. We separated human trigeminal ganglia (TG) into neuronal and nonneuronal fractions, followed by primary and nested PCR to quantitate VZV DNA at the single cell level. Both TG from each of eight cadavers were dissociated and separated into neuronal and nonneuronal cell suspensions by differential filtration. Analysis of the neuron fraction (5,000 neurons per sample) revealed VZV DNA in 9 of 16 samples, with copy numbers ranging from 1 to 12, whereas only 2 of 16 nonneuronal cell samples were positive for VZV DNA, with 1 copy each. Further analysis of 10 samples of 100 neurons and the corresponding nonneuronal cell fractions from each TG of a single subject revealed VZV DNA in 3 of 10 samples of the left TG (range, 2 to 5 copies) and in 1 of 10 samples of the right TG (2 copies) but in none of the 20 nonneuronal cell fractions. These data indicate that latent VZV DNA is present primarily, if not exclusively, in neurons, at a frequency of two to five copies per latently infected neuron.     

Inhibition of Virion Maturation by Simultaneous Deletion of Glycoproteins E, I, and M of Pseudorabies Virus

Glycoprotein M (gM), the product of the UL10 gene of pseudorabies virus (PrV), is one of the few nonessential glycoproteins conserved throughout the Herpesviridae. In contrast to wild-type PrV strains, the UL10 gene product of the attenuated PrV vaccine strain Bartha (PrV-Ba) is not modified by N-glycans due to a mutation in the DNA sequence encoding the consensus N-glycosylation motif. To assay function of the UL10 protein in PrV-Ba, a UL10-deletion mutant (PrV-Ba-UL10(-)) was isolated. Surprisingly, in contrast to gM-deleted wild-type PrV, PrV-Ba-UL10(-) was severely impaired in plaque formation, inducing only foci of very few infected RK13, Vero, and PSEK cells and tiny plaques on MDBK cells. Since this effect was significantly more dramatic than in wild-type PrV, additional mutations known to be present in PrV-Ba were analyzed for their contribution to this phenotype. trans-complementation of the mutated PrV-Ba UL21 or gC protein by the wild-type version had no influence on the observed phenotype. In contrast, complementation of the gE/gI deletion rescued the phenotype. The synergistic effect of deletions in gE/gI and gM on plaque size was verified by construction of a gE/I/M triple mutant derived from wild-type PrV which exhibited the same phenotype. The dramatic effect of deletion of gM on plaque size in a gE/I- virus background was mainly attributable to a function of gM, and not of the gM/gN complex, as shown by analysis of a gE/I/N triple mutant. Interestingly, despite the strong effect on plaque size, penetration was not significantly impaired. In noncomplementing cells infected with the gE/I/M triple mutant, electron microscopy showed absence of secondary envelopment in the cytoplasm but occurrence of intracytoplasmic accumulations of nucleocapsids in association with electron dense material, presumably tegument proteins. These structures were not observed after infection of cells expressing either gE/I or gM. We suggest that gE/I and gM are required for late stages in virion morphogenesis prior to final envelopment in the cytoplasm.     

Effect of mild stress in mice latently infected pseudorabies virus.

Stress is one of the important factors that induces reactivation of pseudorabies virus (PrV) in latently infected pigs. We established a murine model of latent PrV infection and examined the effects of mild stress treatment in order to demonstrate that this model simulates natural infection in the pig. Latently infected mice excreted PrV from the nasal cavity under stress treatments consisting of restraint, exposure to cold or transport. Similar reactions have been observed upon treatment with acetylcholine and dexamethasone. The present findings demonstrate that these kinds of mild stress reactivate the virus in murine latent infection models in a manner similar to the induction of latent infection in pigs in the field.     

Role of the cytoplasmic tail of pseudorabies virus glycoprotein E in virion formation

Glycoproteins M (gM), E (gE), and I (gI) of pseudorabies virus (PrV) are required for efficient formation of mature virions. The simultaneous absence of gM and the gE/gI complex results in severe deficiencies in virion morphogenesis and cell-to-cell spread, leading to drastically decreased virus titers and a small-plaque phenotype (A. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Serial passaging in noncomplementing cells of a virus mutant unable to express gM, gE, and gI resulted in a reversion of the small-plaque phenotype and restoration of infectious virus formation to the level of a gM(-) mutant. Genetic analyses showed that reversion of the phenotype was accompanied by a genomic rearrangement which led to the fusion of a portion of the gE gene encoding the cytoplasmic domain to the 3' end of the glycoprotein D gene, resulting in expression of a chimeric gD-gE protein. Since this indicated that the intracytoplasmic domain of gE was responsible for the observed phenotypic alterations, the UL10 (gM) gene was deleted in a PrV mutant, PrV-107, which specifically lacked the cytoplasmic tail of gE. Regarding one-step growth, plaque size, and virion formation as observed under the electron microscope, the mutant lacking gM and the gE cytoplasmic tail proved to be very similar to the gE/I/M triple mutant. Thus, our data indicate that it is the cytoplasmic tail of gE which is responsible for the observed phenotypic effects in conjunction with deletion of gM. We hypothesize that the cytoplasmic domain of gE specifically interacts with components of the capsid and/or tegument, leading to efficient secondary envelopment of intracytoplasmic capsids.     

Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49

Envelope glycoprotein M (gM) and the complex formed by glycoproteins E (gE) and I (gI) are involved in the secondary envelopment of pseudorabies virus (PrV) particles in the cytoplasm of infected cells. In the absence of the gE-gI complex and gM, envelopment is blocked and capsids surrounded by tegument proteins accumulate in the cytoplasm (A. R. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Here we demonstrate by yeast two-hybrid analyses that the cytoplasmic domains of gE and gM specifically interact with the C-terminal part of the UL49 gene product of PrV, which represents a major tegument protein and which is homologous to VP22 of herpes simplex virus type 1. However, deletion of the UL49 gene from PrV had only minor effects on viral replication, and ultrastructural analyses of infected cells confirmed that virus maturation and egress, including secondary envelopment in the cytoplasm, were not detectably affected by the absence of UL49. Moreover, the UL49 gene product was shown to be dispensable for virion localization of gE and gM, and mutants lacking either gE or gM incorporated the UL49 protein efficiently into virus particles. In contrast, a PrV mutant with deletions of gE-gI and gM failed to incorporate the UL49 protein despite apparently unaltered intracytoplasmic UL49 expression. In summary, we describe specific interactions between herpesvirus envelope and tegument proteins which may play a role in secondary envelopment during herpesvirus virion maturation.     

伪狂犬病病毒gE基因主要抗原表位区的原核表达及其在疫苗接种和自然感染鉴别诊断中的应用

根据伪狂犬病病毒（PRV）Min-A株gE基因序列,利用PCR方法扩增了PRV-gE基因不含信号肽、胞内区和跨膜区的主要抗原表位区,并克隆到原核表达载体pGEX-6p-1中,获得的重组质粒命名为pGEX-tgE。经SDSPAGE电泳分析证实克隆的部分gE基因获得了表达,融合表达产物大小约为63kD,并在终浓度为0.6mmol/L的IPTG诱导下,3.5h其表达量达到高峰。通过改变诱导条件,有效抑制了包涵体形成,提高了重组蛋白的溶解性。Western blot分析证实表达的重组gE蛋白具有抗原反应活性。将表达产物利用亲和层析法纯化后作为ELISA抗原,通过对其特异性、敏感性及工作条件的优化试验,和对48份PRV阴性血清样品的检测结果的统计学分析,建立了猪伪狂犬病tgE-ELISA鉴别诊断方法。通过对400份送检血清样品的检测结果分析,表明其与PRV全病毒ELISA试验的符合率高达95%以上,与基于抗gE蛋白单抗竞争性ELISA的符合率达94%。此方法可用于gE基因缺失PRV疫苗免疫动物和PRV自然感染动物的鉴别诊断。     

MicroRNA-146a抑制胶质瘤细胞增殖的研究

目的：检测胶质瘤中miR-146a的表达水平,并研究miR-146a对胶质瘤细胞增殖的影响。方法：应用实时定量PCR的方法检测胶质瘤组织和癌旁组织中miR-146a的表达水平,采用脂质体细胞转染miRNA模拟物的方式过表达miR-146a,MTT法检测转染后细胞的增殖率,利用在线软件targetScan预测miRNA可能的靶基因。结果：miR-146a在胶质瘤组织中表达明显降低（P〈0.01）,相对表达水平为癌旁组织的35%,细胞转染miR-146a模拟物后,miR-146a表达明显增加,癌细胞增殖率明显降低（P〈0.01）,仅为原细胞的47%。Notch1基因是miR-146a影响胶质瘤细胞增殖活力的可能靶基因。结论：miR-146a可能通过抑制Notch1基因的表达调控胶质瘤细胞的增殖。     

逆转录病毒载体介导表达伪狂犬病病毒PK基因MDBK细胞系的建立

细胞凋亡 (apoptosis)是机体在生理条件下受刺激后 ,经过多种途径的信号传导导致细胞产生一系列形态和生物化学方面的改变而引起的细胞自我消亡的过程 ,是程序性细胞死亡 (programmedcelldeath ,PCD)的主要机制 ,它对于机体的发育 ,内外环境的平衡等都具有重要的作用[1,2 ] .研究表明 ,许多病毒具有诱导或抑制细胞凋亡的功能 ,但其发生的机理仍然不清 .随着分子生物学研究的深入 ,人们发现病毒的某些基因与细胞凋亡相关 ,其编码的基因产物或因病毒感染诱导细胞表达的蛋白抑制或诱导细胞发生凋亡 .最近研究表明 ,伪狂犬病病毒 (psedorabies…     

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels.     

Estimation of association of <Emphasis Type="Italic">CNTN6</Emphasis> copy number variation with idiopathic intellectual disability

Analysis of the prevalence of copy number variations of the CNTN6 gene, recently selected as a new candidate gene for intellectual disorders, was performed. Real-time PCR did not detect any change in the number of CNTN6 gene copies in a group of 200 patients with impaired intellectual development. However, taking into account our data from the previous aCGH analysis and published data, the overall frequency of microdeletions and microduplications of CNTN6 was estimated as 1: 265 (0.4%). The common phenotypic features of 40 patients with microdeletions and microduplications of CNTN6 appeared to be the autism spectrum disorders, developmental delay, intellectual disability, seizures, cognitive impairment, cardiological defects, and behavioral problems.     

Antibody-induced and cytoskeleton-mediated redistribution and shedding of viral glycoproteins, expressed on pseudorabies virus-infected cells.

Fluorescein isothiocyanate-labeled porcine pseudorabies virus (PrV) polyclonal antibodies were added to PrV-infected swine kidney cells in vitro at 37 degrees C. In approximately 47% of the infected cells, the addition induced passive patching and subsequent energy- and microtubule-dependent capping of all viral envelope glycoproteins, expressed on the plasma membranes of the infected cells. Further contraction and extrusion of the capped viral glycoproteins occurred in approximately 30% of the capped cells 2 h after the addition of antibodies and was accompanied by a concentration of F-actin beneath the caps. At that time, about 18% of the extruded caps were shed spontaneously into the surrounding medium. Mechanical force released 85% of the extruded caps, leaving viable cells with no microscopically detectable levels of viral glycoproteins on their plasma membranes. Experiments with PrV deletion mutants showed that viral glycoproteins gE and gI are important in triggering viral glycoprotein redistribution. Since the PrV gE-gI complex exhibits Fc receptor activity which facilitates capping, the importance of gE and gI may be partially explained by antibody bipolar bridging.     

Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread

A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.     

The absence of glycoprotein gL, but not gC or gK, severely impairs pseudorabies virus neuroinvasiveness

Penetration and propagation of herpesviruses in the nervous system require the action of several glycoproteins. To assay for a function of glycoproteins gC, gK, and gL in the neuroinvasiveness of pseudorabies virus (PrV), deletion mutants lacking one of these glycoproteins and corresponding rescuants were inoculated in the nasal cavity of adult mice. We demonstrate that the lack of gL almost prevented the virus from penetrating and propagating in trigeminal, sympathetic, and parasympathetic tracks innervating the nasal cavity, while the lack of gC and gK only slowed the invasion of the nervous system. The conclusion of this and previous studies is that only gB, gD, gH, and gL are indispensable for penetration into neurons, while gB, gH, and gL (and, in some categories of neurons, also gE and gI) are necessary for transneuronal transfer in the mouse model. The deletion of other glycoprotein genes has little effect on PrV neuroinvasiveness although it may affect the dissemination of the virus.     

Prevalence of Chlamydia trachomatis in human immunodeficiency virus-infected women in Cuba

To determine the prevalence rates and serovar distribution of Chlamydia trachomatis cervical infections in Cuban women, two different groups were selected. Group I consisted of 60 human immunodeficiency virus (HIV-1) seropositive women from different regions of Cuba and group II of 60 randomly selected women HIV seronegative and apparently healthy. C. trachomatis was detected in cervical scrapes by mean of nested polymerase chain reaction (PCR) specific for major out membrane protein. The overall prevalence rate of C. trachomatis in cervical scrapes determined by nested PCR was 10% in group I and the estimated prevalence was 6.6% for group II; 83.3% of HIV seropositive women with C. trachomatis infection reported history of pelvic inflammatory disease followed by cervicitis (50%). The control group C. trachomatis-infected women referred a history of cervicitis in 75% of cases. Other reports in the latter group included infertility and pelvic inflamatory disease in 50%. The present study is the first report of C. trachomatis prevalence in Cuba. It showed that there was not significantly difference in the prevalence rate of C. trachomatis between both groups.     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Adaptability in herpesviruses: glycoprotein D-independent infectivity of pseudorabies virus.

Initial contact between herpesviruses and host cells is mediated by virion envelope glycoproteins which bind to cellular receptors. In several alphaherpesviruses, the nonessential glycoprotein gC has been found to interact with cell surface proteoglycans, whereas the essential glycoprotein gD is involved in stable secondary attachment. In addition, gD is necessary for penetration, which involves fusion between virion envelope and cellular cytoplasmic membrane. As opposed to other alphaherpesvirus gD homologs, pseudorabies virus (PrV) gD is not required for direct viral cell-to-cell spread. Therefore, gD- PrV can be passaged in noncomplementing cells by cocultivating infected and noninfected cells. Whereas infectivity was found to be strictly cell associated in early passages, repeated passaging resulted in the appearance of infectivity in the supernatant, finally reaching titers as high as 10(7) PFU/ml (PrV gD- Pass). Filtration experiments indicated that this infectivity was not due to the presence of infected cells, and the absence of gD was verified by Southern and Western blotting and by virus neutralization. Infection of bovine kidney cells constitutively expressing PrV gD interfered with the infectivity of wild-type PrV but did not inhibit that of PrV gD- Pass. Similar results were obtained after passaging of a second PrV mutant, PrV-376, which in addition to gD also lacks gG, gI, and gE. Penetration assays demonstrated that PrV gD- Pass entered cells much more slowly than wild-type PrV. In summary, our data demonstrate the existence of a gD-independent mode of initiation of infection in PrV and indicate that the essential function(s) that gD performs in wild-type PrV infection can be compensated for after passaging. Therefore, regarding the requirement for gD, PrV seems to be intermediate between herpes simplex virus type 1, in which gD is necessary for penetration and cell-to-cell spread, and varicella-zoster virus (VZV), which lacks a gD gene. Our data show that the relevance of an essential protein can change under selective pressure and thus demonstrate a way in which VZV could have evolved from a PrV-like ancestor.     

Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.