Hepatitis E Virus Seroprevalence in Free-Ranging Deer in Canada.

Hepatitis E virus infection (HEV) is an important public health concern not only in traditional endemic areas, but also in some industrialized countries where both domesticated and wild animals have been recognized as potential zoonotic reservoirs implicated in HEV transmission. While the prevalence of infection in the deer population in Europe and Asia has been thoroughly investigated, it remains largely undetermined in North America. We assessed the presence of HEV in three different species of free-range deer in Canada. The seroprevalence of HEV among deer in Canada was 8.8% in white-tailed deer, 4.5% in mule deer and 3.2% in caribou. Hepatitis E virus RNA was not detected. Overall, data indicate that HEV infection occurs in deer in Canada. The absence of viraemia and the low seroprevalence especially in barren-ground caribou which is an important part of the diet in many northern communities suggests that the risk of zoonotic transmission may be less pronounced compared to other countries.

NPY Y2 receptors in the central amygdala reduce cued but not contextual fear.

The amygdala is fundamental for associative fear and extinction learning. Recently, also the central nucleus of the amygdala (CEA) has emerged as a site of plasticity actively controlling efferent connections to downstream effector brain areas. Although synaptic transmission is primarily mediated by glutamate and GABA, neuropeptides critically influence the overall response. While neuropeptide Y (NPY) acting via postsynaptic Y1 receptors exerts an important anxiolytic and fear-reducing action, the role of the predominantly presynaptic Y2 receptors is less defined. To investigate the role of Y2 receptors in the CEA we employed viral-vector mediated over-expression of the Y2 selective agonist NPY3-36 in fear conditioning and extinction experiments. NPY3-36 over-expression in the CEA resulted in reduced fear expression during fear acquisition and recall. Interestingly, this effect was blocked by intraperitoneal injection of a brain-penetrant Y2 receptor antagonist. Furthermore, over-expression of NPY3-36 in the CEA also reduced fear expression during fear extinction of CS-induced but not context-related fear. Again, fear extinction appeared delayed by peripheral injection of a Y2 receptor antagonist JNJ-31020028. Importantly, mice with over-expression of NPY3-36 in the CEA also displayed reduced spontaneous recovery and reinstatement, suggesting that Y2 receptor activation supports a permanent suppression of fear. Local deletion of Y2 receptors in the CEA, on the other hand, increased the expression of CS-induced freezing during fear recall and fear extinction. Thus, NPY inhibits fear learning and promotes cued extinction by reducing fear expression also via activation of presynaptic Y2 receptors on CEA neurons.

Differential internalization and nuclear uncoating of self-complementary adeno-associated virus pseudotype vectors as determinants of cardiac cell transduction.

Recently it was shown that several new pseudotyped adeno-associated virus (AAV) vectors support cardioselective expression of transgenes. The molecular mechanisms underlying this propensity for cardiac cell transduction are not well understood. We comparatively analyzed AAV vector attachment, internalization, intracellular trafficking, and nuclear uncoating of recombinant self-complementary (sc) AAV2.2 versus pseudotyped scAAV2.6 vectors expressing green fluorescence protein (GFP) in cells of cardiac origin. In cardiac-derived HL-1 cells and primary neonatal rat cardiomyocytes (PNCMs), expression of GFP increased rapidly after incubation with scAAV2.6-GFP, but remained low after scAAV2.2-GFP. Internalization of scAAV2.6-GFP was more efficient than that of scAAV2.2-GFP. Nuclear translocation was similarly efficient for both, but differential nuclear uncoating rates emerged as a key additional determinant of transduction: 30% of all scAAV2.6-GFP genomes translocated to the nucleus became uncoated within 48 h, but only 16% of scAAV2.2-GFP genomes. In contrast to this situation in cells of cardiac origin, scAAV2.2-GFP displayed more efficient internalization and similar (tumor cell line HeLa) or higher (human microvascular endothelial cell (HMEC)) uncoating rates than scAAV.2.6-GFP in non-cardiac cell types. In summary, both internalization and nuclear uncoating are key determinants of cardiac transduction by scAAV2.6 vectors. Any in vitro screening for the AAV pseudotype most suitable for cardiac gene therapy - which is desirable since it may allow significant reductions in vector load in upcoming clinical trials--needs to quantitate both key steps in transduction.

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban.

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

Tamoxifen-regulated adenoviral E1A chimeras for the control of tumor selective oncolytic adenovirus replication in vitro and in vivo.

Pharmacological control is a desirable safety feature of oncolytic adenoviruses (oAdV). It has recently been shown that oAdV replication may be controlled by drug-dependent transcriptional regulation of E1A expression. Here, we present a novel concept that relies on tamoxifen-dependent regulation of E1A activity through functional linkage to the mutated hormone-binding domain of the murine estrogen receptor (Mer). Four different E1A-Mer chimeras (ME, EM, E(DeltaNLS)M, MEM) were constructed and inserted into the adenoviral genome under control of a lung-specific surfactant protein B promoter. The highest degree of regulation in vitro was seen for the corresponding oAdVs Ad.E(DeltaNLS)M and Ad.MEM, which exhibited an up to 100-fold higher oAdV replication in the presence as compared with the absence of 4-OH-tamoxifen. Moreover, destruction of nontarget cells was six- and 13-fold reduced for Ad.E(DeltaNLS)M and Ad.MEM, respectively, as compared with Ad.E. Further investigations supported tamoxifen-dependent regulation of Ad.E(DeltaNLS)M and Ad.MEM in vivo. Induction of Ad.E(DeltaNLS)M inhibited growth of H441 lung tumors as efficient as a control oAdV expressing E1A. E(DeltaNLS)M and the MEM chimeras can be easily inserted into a single vector genome, which extends their application to existing oAdVs and strongly facilitates in vivo application.

Correlation of the frequency of petite formation by isolates of Saccharomyces cerevisiae with virulence.

In previous studies on the colony phenotype switching of Saccharomyces cerevisiae, we observed that the least virulent isolates formed greater numbers of petite colonies when grown at body temperature, 37 degrees C. To determine if there is a link between virulence and petite formation, we examined the frequency of spontaneous petite formation for virulent clinical isolates (YJM128, YJM309), an intermediate virulent segregant of YJM128 (YJM145) and avirulent clinical (YJM308) and nonclinical S. cerevisiae (Y55, YJM237) after growth at 37 degrees C. The rank order of increasing frequency of petite formation was YJM128 = YJM145 < YJM309 < Y 55 < YJM308 = YJM237, which is similar to the rank-order of virulence in CD-1 mice. To assess the virulence of petites in vivo, two mouse models, CD-1 and DBA/ 2N, were infected i.v. with 10(7) cfu of either the parental grand or a spontaneously derived petite from one of four isolates previously classified with differing degrees of virulence: YJM128, YJM309, YJM145 and Y55. In both CD-1 and DBA/2N, the mean log10 cfu of grands recovered from the brain was significantly higher than that of the petites (P<0001). Overall, petites were significantly less virulent than the parental strains. However, death of some DBA/2N mice caused by YJM128 petite 1 showed that petites are not totally avirulent. To see if S. cerevisiae isolates form petite colonies in vivo, both mouse models were infected with parental grands of YJM128 and Y55. Recovered colonies were counted and confirmed as grand or petite, and the frequency of petite colonies in the brain, the target organ, correlated with the in vitro results. Overall, these studies show an inverse correlation between the frequency of petite-colony formation and the previously determined virulence of S. cerevisiae in CD-1 mice. Furthermore, petites were significantly less virulent than the parental grands, in most cases, and petites are spontaneously formed in vivo at a frequency inversely correlated to the virulence of the strain.

Adeno-associated virus type 2 protein interactions: formation of pre-encapsidation complexes.

The nonstructural adeno-associated virus type 2 Rep proteins are known to control viral replication and thus provide the single-stranded DNA genomes required for packaging into preformed capsids. In addition, complexes between Rep proteins and capsids have previously been observed in the course of productive infections. Such complexes have been interpreted as genome-linked Rep molecules associated with the capsid upon successful DNA encapsidation. Here we demonstrate via coimmunoprecipitation, cosedimentation, and yeast two-hybrid analyses that the Rep-VP association also occurs in the absence of packageable genomes, suggesting that such complexes could be involved in the preparation of empty capsids for subsequent encapsidation steps. The Rep domain responsible for the observed Rep-VP interactions is situated within amino acids 322 to 482. In the presence of all Rep proteins, Rep52 and, to a lesser extent, Rep78 are most abundantly recovered with capsids, whereas Rep68 and Rep40 vary in association depending on their expression levels. Rep78 and Rep52 are bound to capsids to roughly the same extent as the minor capsid protein VP2. Complexes of Rep78 and Rep52 with capsids differ in their respective detergent stabilities, indicating that they result from different types of interactions. Rep-VP interaction studies suggest that Rep proteins become stably associated with the capsid during the assembly process. Rep-capsid complexes can reach even higher complexity through additional Rep-Rep interactions, which are particularly detergent labile. Coimmunoprecipitation and yeast two-hybrid data demonstrate the interaction of Rep78 with Rep68, of Rep68 with Rep52, and weak interactions of Rep40 with Rep52 and Rep78. We propose that the large complexes arising from these interactions represent intermediates in the DNA packaging pathway.

The adeno-associated virus type 2 regulatory proteins rep78 and rep68 interact with the transcriptional coactivator PC4.

The adeno-associated virus type 2 (AAV-2) Rep78/Rep68 regulatory proteins are pleiotropic effectors of viral and cellular DNA replication, of cellular transformation by viral and cellular oncogenes, and of homologous and heterologous gene expression. To search for cellular proteins involved in mediating these functions, we used Rep68 as bait in the yeast two-hybrid system and identified the transcriptional coactivator PC4 as a Rep interaction partner. PC4 has been shown to mediate transcriptional activation by a variety of sequence-specific transcription factors in vitro. Rep amino acids 172 to 530 were sufficient and amino acids 172 to 224 were absolutely necessary for the interaction with PC4. The PC4 domains required for interaction were mapped to the C-terminal single-stranded DNA-binding domain of PC4. In glutathione S-transferase (GST) pull-down assays, in vitro-transcribed and -translated Rep78 or Rep68 proteins were bound specifically by GST-PC4 fusion proteins. Similarly, PC4 expressed in Escherichia coli was bound by GST-Rep fusion proteins, confirming the direct interaction between Rep and PC4 in vitro. Rep was found to have a higher affinity for the nonphosphorylated, transcriptionally active form of PC4 than for the phosphorylated, transcriptionally inactive form. The latter is predominant in nuclear extracts of HeLa or 293 cells. In the yeast system, but not in vitro, Rep-PC4 interaction was disrupted by a point mutation in the putative nucleotide-binding site of Rep68, suggesting that a stable interaction between Rep and PC4 in vivo is ATP dependent. This mutation has also been shown to impair Rep function in AAV-2 DNA replication and in inhibition of gene expression and inducible DNA amplification. Cytomegalovirus promoter-driven overexpression of PC4 led to transient accumulation of nonphosphorylated PC4 with concomitant downregulation of all three AAV-2 promoters in the absence of helper virus. In the presence of adenovirus, this effect was relieved. These results imply an involvement of the transcriptional coactivator PC4 in the regulation of AAV-2 gene expression in the absence of helper virus.

Control of adeno-associated virus type 2 cap gene expression: relative influence of helper virus, terminal repeats, and Rep proteins.

Adeno-associated virus type 2 (AAV-2) gene expression is tightly controlled by functions of the helper virus as well as by the products of its own viral rep gene. Double-immunofluorescence studies of Rep and VP protein expression in cells coinfected with AAV-2 and adenovirus type 2 showed that a large proportion of these cells expressed Rep78 and Rep52 but no capsid proteins. The percentage of Rep78/Rep52- and capsid protein-positive cells was strongly influenced by the relative ratio of AAV-2 to adenovirus type 2. In contrast, nearly all cells positive for Rep68/Rep40 were also positive for capsid protein expression. Examination of p40 promoter transactivation by individual Rep proteins in the presence of adenovirus, however, showed that both Rep78 and Rep68 efficiently stimulated p40 mRNA accumulation and capsid protein expression. This strong transactivation was reliant upon the presence of terminal repeats and correlated with template amplification. In replication-deficient expression constructs, transactivation was observed only with Rep68 and was dependent on the linear Rep binding site within the left terminal repeat which was detected in the presence of high adenovirus concentrations. In the absence of any terminal repeat sequences, Rep68 expression again led to a minor transactivation of capsid protein expression which was detectable only at low adenovirus concentrations. This low level of transactivation of capsid protein expression by Rep proteins in the absence of terminal repeats resulted in a lower efficiency of capsid assembly. The data show a dominant influence of adenovirus type 2 functions on AAV-2 gene expression, a requirement for terminal repeats for strong transactivation of the p40 promoter by Rep proteins, and differential influences of Rep78 and Rep68 on AAV-2 promoters. Implications for the production of recombinant AAV-2 vectors are discussed.

Subcellular compartmentalization of adeno-associated virus type 2 assembly.

Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformation-specific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.

Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins.

The proteins encoded by the adeno-associated virus type 2 (AAV-2) rep and cap genes obtained during a productive infection of HeLa cells with AAV-2 and adenovirus type 2 were fractionated according to solubility, cellular localization, and sedimentation properties. The majority of Rep and Cap proteins accumulated in the nucleus, where they distributed into a soluble and an insoluble fraction. Analysis of the soluble nuclear fraction of capsid proteins by sucrose density gradients showed that they formed at least three steady-state pools: a monomer pool sedimenting at about 6S, a pool of oligomeric intermediates sedimenting between 10 and 15S, and a broad pool of assembly products with a peak between 60 and 110S, the known sedimentation positions of empty and full capsids. While the soluble nuclear monomer and oligomer pool contained predominantly only two capsid proteins, the 30 to 180S assembly products contained VP1, VP2, and VP3 in a stoichiometry similar to that of purified virions. They probably represent different intermediates in capsid assembly, DNA encapsidation, and capsid maturation. In contrast, the cytoplasmic fraction of capsid proteins showed a pattern of oligomers continuously increasing in size without a defined peak, suggesting that assembly of 60S particles occurs in the nucleus. Soluble nuclear Rep proteins were distributed over the whole sedimentation range, probably as a result of association with AAV DNA. Subfractions of the Rep proteins with defined sedimentation values were obtained in the soluble nuclear and cytoplasmic fractions. We were able to coimmunoprecipitate capsid proteins sedimenting between 60 and 110S with antibodies against Rep proteins, suggesting that they exist in common complexes possibly involved in AAV DNA packaging. Antibodies against the capsid proteins, however, precipitated Rep78 and Rep68 predominantly with a peak around 30S representing a second complex containing Rep and Cap proteins.

Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of heterologous and homologous promoters.

The four Rep proteins encoded by adeno-associated virus type 2 (AAV-2) inhibit transcription of their own promoters and of several heterologous promoters. To gain insight into the molecular mechanism of Rep-mediated transcription repression, we studied the effects of the four Rep proteins on the accumulation of mRNA transcribed from the human papillomavirus type 18 upstream regulatory region HPV18 URR, the human immunodeficiency virus long terminal repeat, and the AAV-2 p5 and p19 promoters by transient transfection experiments in HeLa cells. We observed a distinct contribution of the C- and N-terminal sequences in which the four Rep proteins (Rep78, Rep68, Rep52, and Rep40) differ from each other. While Rep78 showed a more than 10-fold inhibition of the four promoters studied, transcriptional repression mediated by Rep68 and Rep52 was reduced and nearly completely abolished for Rep40. The contribution of the C terminus of Rep78 was reduced with respect to the inhibition of the AAV-2 p5 and p19 promoters. Point mutations and deletions showed that a C-terminal zinc binding motif is required for zinc binding in vitro but plays no obvious role in the inhibition of homologous and heterologous promoters. Overall, inhibition of the four different promoters was dependent on the identical Rep protein domains with the exception of the AAV-2 p5 promoter. Expression of the AAV-2 p5 promoter was inhibited by a Rep78 protein with a mutation in the nucleotide binding motif, whereas expression of the AAV-2 p19 promoter, the human immunodeficiency virus long terminal repeat, and the HPV18 URR was not. Mutational analysis of the HPV18 URR showed that several, but not a single, cis regulatory elements are involved in the inhibition process. This finding suggests that transcriptional repression is mediated by protein-protein interactions of the Rep proteins either with multiple transcription factors or with target proteins of sequence-specific transcription factors of the basal transcription machinery.

Mdr1/P-glycoprotein expression in natural killer (NK) cells enriched from peripheral or umbilical cord blood.

The sensitivity to antineoplastic agents of subpopulations of haematopoietic cells during cancer chemotherapy is an open question. The performance of natural killer (NK) cells, possibly assisting the elimination of tumour cells under drug treatment might be of particular interest. We examined the expression of the transmembrane multidrug transporter mdr1/P-glycoprotein in NK-cells (CD56+) enriched from the peripheral blood or the umbilical cord blood from healthy donors by indirect immunocytofluorescence using the monoclonal P-glycoprotein antibody C219 and a polymerase chain reaction (PCR) approach with amplimers specific for the human mdr1 cDNA. As the antibody C219 apparently cross-reacts with the human mdr3 gene product whose functions are as yet unclear we also checked expression of this gene by PCR using mdr3 specific amplimers. Distinct, but rather inhomogeneous mdr1/P-glycoprotein expression was found in NK-cells enriched from the peripheral blood. NK-cells enriched from the umbilical cord blood showed quite strong mdr1 expression levels throughout, exceeding the values found in the moderately multidrug-resistant cell line CCRF VCR 100 which is permanently cultivated in the presence of 100 ng/ml vincristine. Mdr1/P-glycoprotein expression was mirrored by lowered sensitivities of the cultivated NK-cells towards actinomycin D or adriamycin. The drug sensitivity could be modulated by treatment of the cells with the immunosuppressive drug cyclosporin A. Expression of the mdr3 gene was low or absent in all NK-cell samples examined so far.

mdr1/P-glycoprotein gene segments analyzed from various human leukemic cell lines exhibiting different multidrug resistance profiles.

Three high-level multidrug-resistant sublines of the human T-lymphoblastoid cell line CCRF-CEM were selected independently with either actinomycin D, vincristine or adriamycin. They exhibited distinct quantitative differences of cross-resistance profiles, and showed amplification and marked expression of the mdrl/P-glycoprotein gene. DNA and RNA were prepared from the cell lines, and additionally from three cell samples of patients suffering from acute lymphatic leukemia. Applying the polymerase chain reaction (PCR) for amplification, we cloned and sequenced from these sources segments of the mdrl/P-glycoprotein gene around the codon 185 which codes for an amino acid residue possibly influencing the drug binding function of the P-glycoprotein. Altogether, only 2 single nucleotide differences in an intron were found in 2 out of 40 recombinants each harboring a 209 bp genomic or a 269 bp cDNA fragment of the mdrl/P-glycoprotein gene. Our result does not support the idea of clustered point mutations in this segment of the P-glycoprotein gene as a cause of different multidrug resistance profiles. We additionally examined another segment of the P-glycoprotein gene in its second half, essentially delivering the same negative result, though.

Computer-controlled discontinuous rotating gel electrophoresis for separation of very large DNA molecules.

The newly designed equipment for alternating field gel electrophoresis which permits the separation of very large DNA molecules and the simultaneous analysis of up to 35 samples is described. The field alternation is effected by intermittently rotating the submerged agarose gel by optitional angles. The time intervals between changes of position are controlled by a computer program driving a simple switching device which was designed to suit any technique using periodic switching or inversion of the electrical field. Because the electrophoresis unit provides an absolutely homogeneous electrical field, no distortion of migration lanes occurs and the resolution is very good. Moreover, by using a switching time interval gradient an almost perfect linear relationship between migration distances and molecule sizes in the range of about 100-1250 kilobase pairs is observed. In two-dimensional separations, different switching time programs for the first and second dimension allow maximum resolution of selected size ranges. Field inversion gel electrophoresis is possible as well. The performance of the method is demonstrated by comparing the chromosome sizes of different yeast strains.