bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of PACS-1 in Trafficking of Human Cytomegalovirus Glycoprotein B and Virus Production
Authors: Crump Colin M. , Hung Chien-Hui , Thomas Laurel , Wan Lei , Thomas Gary
Published: September 2003

The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.
Intermolecular Interactions in a Two-Layered Viral Capsid That Requires a Complex Symmetry Mismatch
Authors: Limn Chang-Kwang , Roy Polly
Published: September 2003

The surface of the bluetongue virus core forms a T=13 quasiequivalent icosahedral protein shell with 260 trimers of a single gene product: VP7 protein. Underneath is a smooth layer, made up of VP3 protein, which appears to guide and nucleate the assembly of VP7 trimers. The contacts between the two shells are extensive but nonspecific, and construction of the T=13 icosahedral shell requires polymorphism in the association of the VP7 subunits, each of which has two domains that contribute to trimer formation. We used structural and relative sequence information to guide an investigation of how such a complex structure is achieved during virus assembly and what residues are required to form a stable capsid. Fifteen single or multiple site-specific substitution mutations were introduced into the helical domain of VP7, which is closely associated with the VP3 layer, and the effects on capsid assembly were analyzed. Our data show that both the position and the nature of single residues are critical for the attachment of VP7 to VP3 and that formation of a stable VP7 lattice is not the automatic consequence of trimer formation.
Microarray Profiling of Antibody Responses against Simian-Human Immunodeficiency Virus: Postchallenge Convergence of Reactivities Independent of Host Histocompatibility Type and Vaccine Regimen
Authors: Neuman de Vegvar Henry E. , Amara Rama Rao , Steinman Lawrence , Utz Paul J. , Robinson Harriet L. , Robinson William H.
Published: September 2003

We developed antigen microarrays to profile the breadth, strength, and kinetics of epitope-specific antiviral antibody responses in vaccine trials with a simian-human immunodeficiency virus (SHIV) model for human immunodeficiency virus (HIV) infection. These arrays contained 430 distinct proteins and overlapping peptides spanning the SHIV proteome. In macaques vaccinated with three different DNA and/or recombinant modified vaccinia virus Ankara (rMVA) vaccines encoding Gag-Pol or Gag-Pol-Env, these arrays distinguished vaccinated from challenged macaques, identified three novel viral epitopes, and predicted survival. Following viral challenge, anti-SHIV antibody responses ultimately converged to target eight immunodominant B-cell regions in Env regardless of vaccine regimen, host histocompatibility type, and divergent T-cell specificities. After challenge, responses to nonimmunodominant epitopes were transient, while responses to dominant epitopes were gained. These data suggest that the functional diversity of anti-SHIV B-cell responses is highly limited in the presence of persisting antigen.
Herpes Simplex Virus Type 2 Induces Rapid Cell Death and Functional Impairment of Murine Dendritic Cells In Vitro
Authors: Jones C. A. , Fernandez M. , Herc K. , Bosnjak L. , Miranda-Saksena M. , Boadle R. A. , Cunningham A.
Published: September 2003

Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h postinfection, wild-type (wt) HSV-2 (186) abortively infected murine bone marrow-derived DC and induced early cell death compared to UV-inactivated HSV-2 or mock-infected DC. HSV-2-induced loss of DC viability was more rapid than that induced by HSV-1 and was due, in part, to apoptosis, as shown by TEM, caspase-3 activation, and terminal deoxynucleotidyl transferase-mediated dCTP biotin nick end labeling. HSV induced type-specific changes in the murine DC immunophenotype. At 12 h postinfection, wt HSV-2 upregulated DC major histocompatibility complex (MHC) class II expression, and in contrast to UV-inactivated HSV-2, downregulated expression of MHC class I, but it had no effect on surface CD40, CD80, or CD86. Wt HSV-1 (MC-1) induced only CD40 upregulation. More-profound effects on the DC immunophenotype were observed in HSV-2-infected neonatal DC. Wt HSV of either serotype impaired murine DC-induced T-cell alloproliferation and lipopolysaccharide-induced DC interleukin-12 secretion. Thus, there are marked differences in the levels of HSV-induced cytolysis in DC according to the HSV serotype, although HSV-2 displays immunomodulatory effects on the DC immunophenotype and function similar to those of HSV-1.
Mutations in the 5′ End of the Human Immunodeficiency Virus Type 1 Polypurine Tract Affect RNase H Cleavage Specificity and Virus Titer
Authors: McWilliams Mary Jane , Julias John G. , Sarafianos Stefan G. , Alvord W. Gregory , Arnold Edward , Hughes Stephen H.
Published: September 2003

The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5′ end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5′ end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5′ end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.
Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo
Authors: Lundberg Patric , Welander Paula , Han Xiao , Cantin Edouard
Published: September 2003

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8+cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.
T-Cell Receptor/CD28 Engagement When Combined with Prostaglandin E2Treatment Leads to Potent Activation of Human T-Cell Leukemia Virus Type 1
Authors: Dumais Nancy , Paré Marie-Ève , Mercier Simon , Bounou Salim , Marriot Susan J. , Barbeau Benoit , Tremblay Michel J.
Published: September 2003

Infection with human T-cell leukemia virus type 1 (HTLV-1) is characterized by long latency periods, indicating that viral gene expression is under tight control. There is presently little information available regarding the nature of extracellular stimuli that can transactivate the regulatory elements of HTLV-1 (i.e., long terminal repeat [LTR]). To gain insight into the biological importance of externally induced activation pathways in virus gene expression, primary and established T cells were transfected with HTLV-1-based reporter gene vectors and then were treated with agents that cross-linked the T-cell receptor (TCR) or the costimulatory CD28 molecule with prostaglandin E2(PGE2). We demonstrated that a potent induction of HTLV-1 LTR-driven reporter gene activity was seen only when the three agents were used in combination. Interestingly, similar observations were made when using C91/PL, a cell line that carries integrated HTLV-1 proviral DNA. This TCR-CD28-PGE2-mediated increase in virus transcription was dependent on protein kinase A activation and induction of the cAMP response element binding protein. Experiments with a mutated reporter construct further revealed the importance of the Tax-responsive elements in the HTLV-1 LTR in the observed up regulation of virus gene expression when TCR/CD28 engagement was combined with PGE2treatment. The protein tyrosine kinases p56lckand the transmembrane tyrosine phosphatase CD45 were all found to be involved in TCR-CD28-PGE2-directed increase in HTLV-1 LTR activity. This study presents new information on the possible mechanisms underlying reactivation of this retrovirus.
Varicella-Zoster Virus ORF47 Protein Kinase, Which Is Required for Replication in Human T Cells, and ORF66 Protein Kinase, Which Is Expressed during Latency, Are Dispensable for Establishment of Latency
Authors: Sato Hitoshi , Pesnicak Lesley , Cohen Jeffrey I.
Published: September 2003

Varicella-zoster virus (VZV) results in a lifelong latent infection in human sensory and cranial nerve ganglia after primary infection. VZV open reading frame 47 (ORF47) and ORF66 encode protein kinases that phosphorylate several viral proteins, including VZV glycoprotein gE and ORF32, ORF62, and ORF63 proteins. Here we show that the ORF47 protein kinase also phosphorylates gI. While ORF47 is essential for virus replication in human T cells and skin, we found the gene to be dispensable for establishment of latent infection in dorsal root ganglia of rodents. ORF66 protein is expressed during latency. Rodents infected with VZV unable to express ORF66 developed latent infection at a rate similar to that for the parental virus. ORF63 transcripts, a hallmark of VZV latency, were also detected in similar numbers of animals infected with the ORF47 and ORF66 mutants and with the parental virus. VZV mutants unable to express four of the six genes that do not have herpes simplex virus (HSV) homologs (ORFs 1, 13, 32, 57) were also unimpaired for establishment of latency. While a truncated HSV VP16 mutant was previously reported to be unable to establish latency in a mouse model, we found that VZV with a deletion of ORF10, the homolog of HSV VP16, was dispensable for establishment of latency. Thus, seven genes, including one expressed during latency, are dispensable for establishing latent VZV infection.
Borna Disease Virus Nucleoprotein Interacts with the Cdc2-Cyclin B1 Complex
Authors: Planz Oliver , Pleschka Stephan , Oesterle Katja , Berberich-Siebelt Friederike , Ehrhardt Christina , Stitz Lothar , Ludwig Stephan
Published: September 2003

Transition from G
2
to M phase, a cell cycle checkpoint, is regulated by the Cdc2-cyclin B1 complex. Here, we report that persistent infection with Borna disease virus (BDV), a noncytolytic RNA virus infecting the central nervous system, results in decelerated proliferation of infected host cells due to a delayed G
2
-to-M transition. Persistent BDV-infected rat fibroblast cells showed reduced proliferation compared to uninfected cells. In pull-down assays we observed an interaction of the viral nucleoprotein with the Cdc2-cyclin B1 complex. Transfection of the viral nucleoprotein but not of the phosphoprotein also results in decelerated proliferation. This phenomenon was found in BDV-susceptible primary rat fibroblast cells and also in primary mouse cells, which are not susceptible to BDV infection. This is the first evidence that the noncytolytic Borna disease virus can manipulate host cell functions via interaction of the viral nucleoprotein with mitotic entry regulators. BDV preferentially infects and persists in nondividing neurons. The present report could give an explanation for this selective choice of host cell by BDV.
High Rates of Human Immunodeficiency Virus Type 1 Recombination: Near-Random Segregation of Markers One Kilobase Apart in One Round of Viral Replication
Authors: Rhodes Terence , Wargo Heather , Hu Wei-Shau
Published: September 2003

One of the genetic consequences of packaging two copies of full-length viral RNA into a single retroviral virion is frequent recombination during reverse transcription. Many of the currently circulating strains of human immunodeficiency virus type 1 (HIV-1) are recombinants. Recombination can also accelerate the generation of multidrug-resistant HIV-1 and therefore presents challenges to effective antiviral therapy. In this study, we determined that HIV-1 recombination rates with markers 1.0, 1.3, and 1.9 kb apart were 42.4, 50.4, and 47.4% in one round of viral replication. Because the predicted recombination rate of two unlinked markers is 50%, we conclude that markers 1 kb apart segregated in a manner similar to that for two unlinked markers in one round of retroviral replication. These recombination rates are exceedingly high even among retroviruses. Recombination rates of markers separated by 1 kb are 4 and 4.7% in one round of spleen necrosis virus and murine leukemia virus replication, respectively. Therefore, HIV-1 recombination can be 10-fold higher than that of other retroviruses. Recombination can be observed only in the proviruses derived from heterozygous virions that contain two genotypically different RNAs. The high rates of HIV-1 recombination observed in our studies also indicate that heterozygous virions are formed efficiently during HIV-1 replication and most HIV-1 virions are capable of undergoing recombination. Our results demonstrate that recombination is an effective mechanism to break the genetic linkage between neighboring sequences, thereby reassorting the HIV-1 genome and increasing the diversity in the viral population.
Genetic Recombination during Coinfection of Two Mutants of Human Respiratory Syncytial Virus
Authors: Spann Kirsten M. , Collins Peter L. , Teng Michael N.
Published: September 2003

Recombination between coinfecting viruses had not been documented previously for a nonsegmented negative-strand RNA virus (mononegavirus). We investigated the potential of intermolecular recombination by respiratory syncytial virus (RSV) by coinfecting HEp-2 cells with two recombinant RSV (rRSV) mutants lacking either the G gene (ΔG/HEK) or the NS1 and NS2 genes (ΔNS1/2). These viruses replicate inefficiently and form pinpoint plaques in HEp-2 cells. Therefore, potential recombined viruses with a growth and/or plaque formation advantage should easily be identified and differentiated from the two parental viruses. Further identification of potential recombinants was aided by the inclusion of point mutation markers in the F and L genes of ΔG/HEK and the design of reverse transcription-PCR (RT-PCR) primers capable of detecting these markers. Independent coinfections and control single infections by these two rRSV mutants were performed. In one of six coinfections, an RSV variant was identified that produced plaques slightly larger than those of wild-type RSV in HEp-2 cells. RT-PCR and sequencing provided evidence that this variant was a recombined RSV (rec-RSV). The rec-RSV appeared to have been generated by a polymerase jump from the ΔG/HEK genome to that of ΔNS1/2 and back again in the vicinity of the SH-G-F genes. This apparently involved nonhomologous and homologous recombination events, respectively. The recombined genome was identical to that of the ΔG/HEK mutant except that all but the first 12 nucleotides of the SH gene were deleted and replaced by an insert consisting of the last 91 nucleotides of the G gene and its downstream intergenic region. This insert could have come only from the coinfecting ΔNS1/2 virus. This resulted in the formation of a short chimeric SH:G gene. Northern and Western blot analysis confirmed that the rec-RSV did not express the normal SH and G mRNAs and proteins but did express the aberrant SH:G mRNA. This provides an experimental demonstration of intermolecular recombination yielding a viable, helper-independent mononegavirus. However, the isolation of only a single rec-RSV under these optimized conditions supports the idea that RSV recombination is rare indeed.
Productive Infection Maintains a Dynamic Steady State of Residual Viremia in Human Immunodeficiency Virus Type 1-Infected Persons Treated with Suppressive Antiretroviral Therapy for Five Years
Authors: Havlir Diane V. , Strain Matthew C. , Clerici Mario , Ignacio Caroline , Trabattoni Daria , Ferrante Pasquale , Wong Joseph K.
Published: September 2003

To provide insight into the dynamics and source of residual viremia in human immunodeficiency virus (HIV) patients successfully treated with antiretroviral therapy, 14 intensely monitored patients treated with indinavir and efavirenz sustaining HIV RNA at <50 copies/ml for >5 years were studied. Abacavir was added to the regimen of eight patients at year 5. After the first 9 months of therapy, HIV RNA levels had reached a plateau (“residual viremia”) that persisted for over 5 years. Levels of residual viremia differed among patients and ranged from 3.2 to 23 HIV RNA copies/ml. Baseline HIV DNA was the only significant pretreatment predictor of residual viremia in regression models including baseline HIV RNA, CD4 count, and patient age. In the four of five patients with detectable viremia who added abacavir to their regimen after 5 years, HIV RNA levels declined rapidly. The estimated half-life of infected cells was 6.7 days. Decrease in activated memory cells and a reduction in gamma interferon production to HIV Gag and p24 antigen in ELISpot assays were observed, consistent with a decrease in HIV replication. Thus, in patients treated with efavirenz plus indinavir, levels of residual viremia were established by 9 months, were predicted by baseline proviral DNA, and remained constant for 5 years. Even after years of highly suppressive therapy, HIV RNA levels declined rapidly after the addition of abacavir, suggesting that productive infection contributes to residual ongoing viremia and can be inhibited with therapy intensification.
Long-Term Specific Immune Responses Induced in Humans by a Human Immunodeficiency Virus Type 1 Lipopeptide Vaccine: Characterization of CD8+-T-Cell Epitopes Recognized
Authors: Gahéry-Ségard Hanne , Pialoux Gilles , Figueiredo Suzanne , Igéa Céline , Surenaud Mathieu , Gaston Jessintha , Gras-Masse Helene , Lévy Jean-Paul , Guillet Jean-Gérard
Published: September 2003

We studied the effect of booster injections and the long-term immune response after injections of an anti-human immunodeficiency virus type 1 (HIV-1) lipopeptide vaccine. This vaccine was injected alone or with QS21 adjuvant to 28 HIV-uninfected volunteers. One month later, after a fourth injection of the vaccine, B- and T-cell anti-HIV responses were detected in >85% of the vaccinated volunteers. One year after this injection, a long-term immune response was observed in >50% of the volunteers. At this point, a positive QS21 effect was observed only in the sustained B-cell and CD4+-T-cell responses. To better characterize the CD8+-T-cell response, we used a gamma interferon enzyme-linked immunospot method and a bank of 59 HIV-1 epitopes. For the six most common HLA molecules (HLA-A2, -A3, -A11, -A24, -B7 superfamily, and -B8), an average of 10 (range, 3 to 15) HIV-1 epitopes were tested. CD8+-T-cell responses were evaluated according to the HLA class I molecules of the volunteers. Each assessment was based on 18 HIV-1 epitopes in average. We showed that 31 HIV-1 epitopes elicited specific CD8+-T-cell responses after vaccination. The most frequently recognized peptides were Nef 68-76 (-B7), Nef 71-79 (-B7), Nef 84-92 (-A11), Nef 135-143 (-B7), Nef 136-145 (-A2), Nef 137-145 (-A2), Gag 259-267 (-B8), Gag 260-268 (-A2), Gag 267-274 (-A2), Gag 267-277 (-B7), and Gag 276-283 (A24). We found that CD8+-T-cell epitopes were induced at a higher number after a fourth injection (P< 0.05 compared to three injections), which indicates an increase in the breadth of HIV CD8+-T-cell epitope recognition after the boost.
Immunogenicity and Pathogenicity of Chimeric Infectious DNA Clones of Pathogenic Porcine Circovirus Type 2 (PCV2) and Nonpathogenic PCV1 in Weanling Pigs
Authors: Fenaux M. , Opriessnig T. , Halbur P. G. , Meng X. J.
Published: September 2003

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 μg of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 μg of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 μg of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 μg of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.
Molecular Phylogeny of the Psittacid Herpesviruses Causing Pacheco's Disease: Correlation of Genotype with Phenotypic Expression
Authors: Tomaszewski Elizabeth K. , Kaleta Erhard F. , Phalen David N.
Published: September 2003

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.
Characterization of Endogenous Retroviruses in Sheep
Authors: Klymiuk Nikolai , Müller Mathias , Brem Gottfried , Aigner Bernhard
Published: September 2003

Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (
Ovis aries
) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral
pro
-
pol
sequences. Three OERV β families and nine OERV γ families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV β family and two OERV γ families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.
Dissemination of Rat Cytomegalovirus through Infected Granulocytes and Monocytes In Vitro and In Vivo
Authors: van der Strate B. W. A. , Hillebrands J. L. , Lycklama à Nijeholt S. S. , Beljaars L. , Bruggeman C. A. , van Luyn M. J. A. , Rozing J. , The T. H. , Meijer D. K. F. , Molema G. , Harmsen M. C.
Published: September 2003

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.
Molecular Dissection of the Vaccinia Virus I7L Core Protein Proteinase
Authors: Byrd Chelsea M. , Bolken Tove' C. , Hruby Dennis E.
Published: September 2003

The vaccinia virus I7L gene product is predicted to be a cysteine proteinase and is demonstrated in this study to be responsible for cleavage of each of the three major core protein precursors (P4a, P4b, and P25K) in vivo. Mutagenesis of the putative catalytic triad of I7L or of the cleavage sites in the core protein precursors inhibits processing. A truncated protein lost the ability to cleave the core protein precursors.
The 5′ Untranslated Region of Alfalfa Mosaic Virus RNA 1 Is Involved in Negative-Strand RNA Synthesis
Authors: Vlot A. Corina , Bol John F.
Published: September 2003

The three genomic RNAs of alfalfa mosaic virus each contain a unique 5′ untranslated region (5′ UTR). Replacement of the 5′ UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5′ stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5′ stem-loop structure is present in RNA 2 but not in RNA 3.
Recovery of Infectious Foot-and-Mouth Disease Virus from Suckling Mice after Direct Inoculation with In Vitro-Transcribed RNA
Authors: Baranowski Eric , Molina Nicolás , Núñez José Ignacio , Sobrino Francisco , Sáiz Margarita
Published: September 2003

We assayed the infectivity of naked foot-and-mouth disease virus (FMDV) RNA by direct inoculation of suckling mice. Our results demonstrate that transcripts generated from full-length cDNA clones were infectious, as was virion-extracted RNA. Interestingly, infectious virus could be recovered from a mutant transcript encoding amino acid substitution L-147→P in capsid protein VP1, known to be noninfectious for BHK-21 cells. The model described here provides a useful tool for virulence studies in vivo, bypassing possible selection of variants during viral replication in cell culture.
Patterns of Intra- and Interhost Nonsynonymous Variation Reveal Strong Purifying Selection in Dengue Virus
Authors: Holmes Edward C.
Published: September 2003

Considerable uncertainty surrounds the evolutionary rates of and selection pressures acting on arthropod-borne RNA viruses (arboviruses). In particular, it is unclear why arboviruses such as dengue virus show substantial genetic variation within individual humans and mosquitoes yet low long-term rates of amino acid substitution. To address this question, I compared patterns of nonsynonymous variation in populations of dengue virus sampled at different levels of evolutionary divergence. Although nonsynonymous variation was abundant in viral populations within individual humans, there was a marked reduction in the frequency of nonsynonymous mutations in interhost comparisons. Moreover, intrahost genetic variation corresponded to a random pattern of mutation, and most of the sites that exhibited nonsynonymous variation within hosts were invariant at deeper phylogenetic levels. This loss of long-term nonsynonymous variation is the signature of extensive purifying selection such that more than 90% of all nonsynonymous mutations are deleterious. Consequently, although arboviruses are able to successfully adapt to diverse cell types, they are characterized by a high rate of deleterious mutation.
Impedance Study of Adsorption of Chloride Ions in Ethanol on Bi(001) Single Crystal Plane
Authors: Väärtnõu Mart , Lust Enn
Published: September 2003

The impedance spectra for Bi(001) single crystal plane in 0.1, 0.02 and 0.003 M LiCl solutions in ethanol have been measured. It was found that reproducible experimental data for Bi electrode in ethanol solutions could be obtained at ac frequencies between 0.1 and 10 000 Hz and electrode potentials from -1.6 to -0.2 V (vs saturated calomel electrode). Outside this potential region, faradaic processes were detected. By fitting the experimental data to various equivalent circuits it was found that the Frumkin-Melik-Gaikazyan model yields good fit in almost the whole potential range studied. Using this model the dependences of "true" and adsorption capacitance as well as diffusion resistance on electrode potential have been obtained. The ionic charge due to the specific adsorption has been obtained using the mixed-electrolyte method for both electrode charge and electrode potential as the independent electric variables. The Gibbs energy of adsorption of Cl- ions has been calculated using a simple virial adsorption isotherm. It was found that the adsorption of Cl- anion increases in the order methanol < ethanol < propan-2-ol. It was found that on the Bi(001) plane the electrosorption valency has a constant value in the potential range studied. It was concluded that the formed effective surface dipole is significantly screened by the solvent molecules and the metal electron gas.
Rotational Spectrum of 2,3-Benzofuran
Authors: Giuliano B. Michela , Caminati Walther
Published: September 2003

The rotational spectra of the ground state and of one vibrational satellite of 2,3-benzofuran have been measured by millimetre-wave absorption free jet spectroscopy in the frequency range 60-78 GHz. The value of the inertial defect (-0.072 uÅ2) shows the molecule to be planar. The shifts of the rotational constants in going from the ground to the excited state indicate that the observed vibrational satellite does not belong to the two lowest energy motions, the butterfly and 1,3-ring-twisting, which undergo relaxation upon the supersonic expansion.
Selenium(IV) Electrochemistry on Silver: A Combined Electrochemical Quartz-Crystal Microbalance and Cyclic Voltammetric Investigation
Authors: Pezzatini Giovanni , Loglio Francesca , Innocenti Massimo , Foresti Maria Luisa
Published: September 2003

The electrochemical behavior of Se(IV) on silver was investigated by cyclic voltammetry and electrochemical quartz-crystal microbalance (EQCM) measurements. As already reported in the literature, Se(IV) electrochemistry is always complex, and on silver even more, due to the formation of a compound. Our results confirm that the reduction process of Se(IV) occurs through two reaction paths, Se(IV) → Se(0) and Se(IV) → Se(-II); the product Se(-II) then reacts with Se(IV) through a comproportionation reaction. The latter step leads to red Se that, according to the literature, is the only electroactive form of Se(0). The presence of the electroactive red Se is evident both in the negative range of potentials, through the reduction Se(0) → Se(-II), and in the less negative range of potentials, through the oxidation Se(0) → Se(IV). Moreover, our measurements pointed to the formation of a deposit that never redissolves. This deposit seems to be the electroinactive gray Se. The electrochemical behavior of Se(IV) was investigated in the whole potential range accessible on silver. Our results confirm the occurrence of competitive processes whose predominance depends on the scan rate, as well as on the potential limits of voltammetry. A detailed table with the processes occurring in different potential ranges was drawn up.
Insight Into Electric Potential Distribution Within Mesoporous Titanium Dioxide Films
Authors: Solarska Renata , Morand Robin , Augustynski Jan
Published: September 2003

Charge transport and potential distribution in mesoporous semiconductor films operating in an electrolyte, especially those composed of TiO2 nanoparticles, are still the subject of wide debate. Herein we describe a series of experiments, performed under band-gap energy illumination of nanostructured TiO2 films, intended to shed new light on the actual electric potential profile across such three-dimensional electrode. Our approach stems from quite a general observation that addition of various electron acceptors to a solution containing an efficient hole scavenger (e.g., methanol, formic acid) results in a marked drop of the maximum photocurrent at the mesoporous TiO2 film electrodes whatever the applied anodic bias might be. We have chosen an electron acceptor, MV2+ dication, which-due to its negative redox potential, more negative than that of the bottom of conduction band of TiO2 in acidic media - causes a drop of the photooxidation current only in alkaline but not in acidic solutions of hole scavengers. Measurements of the incident photon-to-current efficiencies as a function of wavelength show that the drop of the photocurrent after the MV2+ addition, observed in alkaline formate solution extends practically over the whole range of wavelengths. As the optical penetration depth in TiO2 for the wavelengths close to its band edge is expected to match approximately the chosen film thickness, we can conclude that major part of the electric potential drop in the TiO2 electrode occurs actually close to the back contact.
Problems of Electrochemical Controls of Oxidation-Reduction Systems in Aqueous-Organic and Nonaqueous Media. The rH Index with Establishment of the Relevant Scales and Standards
Authors: Falciola Luigi , Mussini Patrizia R. , Mussini Torquato
Published: September 2003

The state and problems of the electrochemical controls of oxidation-reduction systems in aqueous-organic and nonaqueous solvent media with particular emphasis on the rH index of the reducing power of such systems are discussed. In the frame of a generalized treatment of the rH-metric domain, the key definitions, the acquisition of primary rHS standards, the comparison of rH scales in different solvent media, and the recommended procedure of rHX determination are described. The most recent results here obtained in the studies of the behaviour of the quinhydrone electrode with respect to the hydrogen gas electrode, and of the key cell Pt|H2|H2O|HgO|Hg|Pt are reported, and the guidelines for further development are outlined.
Electron Transfer at Boron-Doped Diamond Electrodes. Comparison with Pt, Au and RuO2
Authors: Friso Francesco , Trasatti Sergio
Published: September 2003

Electron exchange reaction rates were compared for boron-doped diamond (BDD), Pt, Au and RuO2 using [Ru(NH3)6]3+/2+ as a redox couple. The study was carried out by cyclic voltammetry; calculations were performed by Nicholson's procedure. The results show that the rate constants on BDD, Pt and Au are qualitatively similar, one order of magnitude higher than on RuO2. The outcome is explained in terms of the specific structure of the interface between oxides and electrolyte solutions.
Electrochemical Study of Mannitol Oxidation at Nickel Oxide Electrode
Authors: Tonelli Domenica , Ballarin Barbara , Berrettoni Mario , Trevisani Marcello
Published: September 2003

The electrocatalytic oxidation of mannitol at a nickel oxide electrode was investigated. The experimental conditions for determining mannitol concentrations have been optimised taking into account the involved electrochemistry. Unlike what previously reported in the literature, our findings lead to the conclusion that both the electrochemical reaction involving the Ni(II)/Ni(III) couple and the chemical reaction between mannitol and Ni(III) are effective in determining the overall kinetics of the electrocatalytic process. The calibration line for mannitol was linear up to 20.0 mmol l-1. Mannitol determination with the nickel oxide electrode was performed in a liquid culture medium selective for Staphylococcus aureus in order to make an indirect calibration of bacterial viable cells, but the results were not satisfactory.
A Solvent-Dependent and Electrochemically Controlled Self-Assembling/Disassembling System
Authors: Amendola Valeria , Boiocchi Massimo , Diaz Fernandez Yuri , Mangano Carlo , Pallavicini Piersandro
Published: September 2003

The bis-bidentate ligand R,S-1,2-diphenyl-N,N'-bis(2-quinolinemethylidene)ethane-1,2-diamine (ligand 4), containing two (iminomethyl)quinoline moieties separated by a cis-1,2-diphenylethylene spacer, forms stable complexes with both CuI and CuII. With CuII, the monomeric 1:1 complex [CuII(4)]2+ is obtained both in CH3CN and CH2Cl2. With CuI and overall 1:1 metal/ligand molar ratio, an equilibrium mixture is obtained in CH3CN, consisting of [CuI(4)2]+, [CuI2(4)2]2+ and [CuI2(4)(CH3CN)4]2+. The preponderant species is the two-metal one-ligand "open" complex [CuI2(4)(CH3CN)4]2+, in which each Cu+ cation is coordinated in a tetrahedral fashion by one (iminomethyl)quinoline unit and by two CH3CN molecules. Precipitation from the equilibrium mixture yields only crystals of [CuI2(4)(CH3CN)4](ClO4)2·2CH3CN, whose crystal and molecular structures have been determined. On the other hand, in the poorly coordinating CH2Cl2 solvent, only the dimeric helical [CuI2(4)2]2+ complex is obtained, when the overall metal/ligand 1:1 molar ratio is chosen. Addition of large quantities of acetonitrile to solutions of [CuI2(4)2]2+ in dichlorometane results in the formation of [CuI2(4)(CH3CN)4]2+, i.e. in the solvent-driven disassembling of the CuI helicate. While electrochemistry in CH3CN is poorly defined due to the presence of more than one CuI species, cyclic voltammetry experiments carried out in CH2Cl2 revealed a well defined behavior, with irreversible oxidation of [CuI2(4)2]2+ and irreversible reduction of [CuII(4)]2+ taking place at separate potentials (∆E ≈ 700 mV). Irreversibility and separation of the redox events are due to the self-assembling and disassembling processes following the reduction and oxidation, respectively.
Electrochemical Behaviour of trans-[FeH(CN)(dppe)2] Adducts
Authors: Almeida Sílvia S. P. R. , Guedes Da Silva M. Fátima C. , Pombeiro Armando J. L.
Published: September 2003

Electrochemical behaviour of various metallo cyano adducts of trans-[FeH(CN)(dppe)2], viz. the dinuclear complexes [FeH(dppe)2(μ-CN)PdCl2(PPh3)], [FeH(dppe)2(μ-CN)NiCl2(PCy3)], and trinuclear [{FeH(dppe)2(μ-CN)}2(ReOCl3)], [{FeH(dppe)2(μ-CN)}2PtCl(Ph)] and [{FeX(dppe)2(μ-CN)}2WCl3(OH)] (X = Cl or OH, dppe = 1,2-diphenylphosphinoethane, PCy3 = P(C6H11)3), as well as the benzoylisocyanide mononuclear adduct trans-[FeH(CNCOPh)- (dppe)2], is reported. All of them exhibit FeII/FeIII-based oxidations (which are reversible, except for trans-[FeH(CNCOPh)(dppe)2]). The metallocyanide bridges C≡N-Re-N≡C and C≡N-W-N≡C in [{FeH(dppe)2(μ-CN)}2(ReOCl3)] and [{FeX(dppe)2(μ-CN)}2WCl3(OH)], respectively, allow electronic communication between the iron centres, with possible generation of mixed-valence FeII/FeIII complexes whose comproportionation constant could be estimated in the former case. From the values of the measured oxidation potentials, the electrochemical PL and EL ligand parameters have been estimated for the metallocyanide ligands that were shown to behave as stronger net electron donors than organoisocyanides, although weaker than cyanide itself. Ligand-centred reduction processes were also observed to lead, in the cases of complexes trans-[FeH(CNCOPh)(dppe)2] (CNCOPh-based reduction) and [FeH(dppe)2(μ-CN)PdCl2(PPh3)] (PdII-based reduction in the CNPdCl2(PPh3)- metallocyanide ligand), to the dissociation of the adduct, with regeneration of the parent cyano complex trans-[FeH(CN)(dppe)2], thus reflecting the reductive decrease of the electrophilic (or Lewis acidic) character of the benzoyl and {PdCl2(PPh3)} groups.
Electronic Absorption Spectrum and Reduction Behavior of a Multicomponent, Trinuclear Ru(II) Species Containing 2,3-Bis(2'-pyridyl)pyrazine Bridging Ligands and 2,2'-Biquinoline Peripheral Ligands
Authors: Loiseau Frédérique , Serroni Scolastica , Campagna Sebastiano
Published: September 2003

Electronic absorption spectrum and reduction pattern of a trinuclear Ru(II) species containing 2,3-bis(2'-pyridyl)pyrazine bridging ligands and 2,2'-biquinoline peripheral ligands have been studied and compared with other multinuclear metal complexes of the same family of systems. The structural formula of the compound investigated is [RuCl2{Ru(biq)2(μ-2,3-dpp)}2]4+ (1; biq = 2,2'-biquinoline, 2,3-dpp = 2,3-bis(2'-pyridyl)pyrazine). The electronic absorption spectrum of 1 (in acetonitrile) is dominated by intense spin-allowed ligand-centred bands in the UV region and by moderately intense spin-allowed metal-to-ligand charge-transfer bands in the visible. The studied compound undergoes several quasireversible reduction processes in the potential window investigated (up to -1.20 V vs SCE in DMF): two successive one-electron reductions at -0.52 and -0.61 V, followed by two processes involving two closely-spaced one-electron reductions each. Extensive comparison with reduction patterns of closely related species allows to assign each process to specific subunits of the multicomponent array. The effect of the peripheral biq ligands on the electronic interaction between inner subunits is evidenced.
Synthesis, Spectroscopy and Spectroelectrochemistry of Chlorocarbonyl {1,2-Bis[(2,6-diisopropylphenyl)imino]acenaphthene-κ2-N,N'}rhodium(I)
Authors: Mahabiersing Taasje , Luyten Henk , Nieuwendam Ronald C. , Hartl František
Published: September 2003

Reaction of the dinuclear complex [{Rh(CO)2}2(μ-Cl)2] with an α-diimine ligand, 1,2-bis[(2,6-diisopropylphenyl)imino]acenaphthene (iPr2Ph-bian), produces square-planar [RhCl(CO)(iPr2Ph-bian)]. For the first time, 2:1 and 1:1 α-diimine/dimer reactions yielded the same product. The rigidity of iPr2Ph-bian together with its flexible electronic properties and steric requirements of the 2,6-diisopropyl substituents on the benzene rings allow rapid closure of a chelate bond and replacement of a CO ligand instead of chloride. A resonance Raman study of [RhCl(CO)(iPr2Ph-bian)] has revealed a predominant Rh-to-bian charge transfer (MLCT) character of electronic transitions in the visible spectral region. The stabilisation of [RhCl(CO)(iPr2Ph-bian)] in lower oxidation states by the π-acceptor iPr2Ph-bian ligand was investigated in situ by UV-VIS, IR and EPR spectroelectrochemistry at variable temperatures. The construction of the novel UV-VIS-NIR-IR low-temperature OTTLE cell used in these studies is described in the last part of the paper.
Electrochromic Behaviour of Polynuclear Ruthenium Complexes on Nanocrystalline SnO2
Authors: Biancardo Matteo , Schwab Peter F. H. , Bignozzi Carlo A.
Published: September 2003

The trinuclear [{Ru(NH3)4(py-COOH)}(μ-CN){Ru(bpy)2}(μ-CN){Ru(NH3)4(py-COOH)}]6+ (py-COOH = isonicotinic acid, bpy = 2,2'-bipyridine) (1) and dinuclear [{Ru(dcb)2}2L]7- (dcb = 2,2'-bipyridine-4,4'-dicarboxylic acid, H3L = 9-phenyl-2,3,7-trihydroxy-6-fluorone) (2) complexes were prepared and their spectroelectrochemical behaviour characterised in solution and on conductive glass electrodes layered with Sb-doped nanocrystalline SnO2. The systems display a reversible redox response in a narrow potential range (-0.5 to +0.5 V vs SCE) and offer the possibility of controlling colour changes of electrochromic devices through variations of the coordinated and bridging ligands at the metal centres. SnO2(Sb) electrodes loaded with these dyes show switching times of the order of ms.