bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence of Genetic Drift and Founder Effect during Quasispecies Evolution of the VP2 and NS3/NS3A Genes of Bluetongue Virus upon Passage between Sheep, Cattle, and
Culicoides sonorensis
Authors: Bonneau K. R. , Mullens B. A. , MacLachlan N. J.
Published: July 2002

Bluetongue virus (BTV) is the cause of an insect-transmitted virus infection of ruminants that occurs throughout much of the world. Individual gene segments differ between field strains of BTV; thus, we hypothesized that key viral genes undergo genetic drift during alternating passage of BTV in its ruminant and insect hosts. To test this hypothesis, variation in the consensus sequence and quasispecies heterogeneity of the VP2 and NS3/NS3A genes of a plaque-purified strain of BTV serotype 10 was determined during alternating infection of vector
Culicoides sonorensis
and a sheep and calf. Consensus sequences were determined after reverse transcriptase-nested PCR amplification of viral RNA directly from ruminant blood and homogenized insects, and quasispecies heterogeneity was determined by the sequencing of clones derived from directly amplified viral RNA. Comparison of these sequences to those of the original BTV inoculum used to initiate the cycle of BTV infection demonstrated, for the first time, that individual BTV gene segments evolve independently of one another by genetic drift in a host-specific fashion, generating quasispecies populations in both ruminant and insect hosts. Furthermore, a unique viral variant was randomly ingested by
C. sonorensis
insects that fed on a sheep with low-titer viremia, thereby fixing a novel genotype by founder effect. Thus, we conclude that genetic drift and founder effect contribute to diversification of individual gene segments of field strains of BTV.
Peptides Corresponding to the Heptad Repeat Motifs in the Transmembrane Protein (gp41) of Human Immunodeficiency Virus Type 1 Elicit Antibodies to Receptor-Activated Conformations of the Envelope Glycoprotein
Authors: de Rosny Eve , Vassell Russell , Wingfield Paul T. , Wild Carl T. , Weiss Carol D.
Published: July 2002

Two heptad repeat regions in the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane subunit (gp41) self-assemble into a six-helix bundle structure that is critical for virus entry. Immunizations with peptides corresponding to these regions generated antibodies specific to the receptor-activated conformations of gp41.
Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants That Exhibit Aberrant Core Morphology and Are Blocked in Initiation of Reverse Transcription in Infected Cells
Authors: Tang Shixing , Murakami Tsutomu , Agresta Beth E. , Campbell Stephen , Freed Eric O. , Levin Judith G.
Published: July 2002

A group of conserved hydrophobic residues faces the interior of the coiled-coil-like structure within the N-terminal domain of the human immunodeficiency virus type 1 (HIV-1) capsid protein (CA). It has been suggested that these residues are important for maintaining stable structure and functional activity. To investigate this possibility, we constructed two HIV-1 clones, in which Trp23 or Phe40 was changed to Ala. We also constructed a third mutant, D51A, which has a mutation that destroys a salt bridge between Pro1 and Asp51. All three mutants are replication defective but produce virus particles. Mutant virions contain all of the viral proteins, although the amount and stability of CA are decreased and levels of virion-associated integrase are reduced. The mutations do not affect endogenous reverse transcriptase activity; however, the mutants are blocked in their ability to initiate reverse transcription in infected cells and no minus-strand strong-stop DNA is detected. The defect in reverse transcription is associated with striking defects in the morphology of mutant virus cores, as determined by transmission electron microscopy. Our data indicate that the mutations made in this study disrupt CA structure and prevent proper maturation of virus cores. We propose that this results in a defect in core stability or in an early postentry event preceding reverse transcription.
Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus
Authors: Goodin Michael M. , Austin Jennifer , Tobias Renée , Fujita Miki , Morales Christina , Jackson Andrew O.
Published: July 2002

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione
S
-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization.
Replication-Defective Vector Based on a Chimpanzee Adenovirus
Authors: Farina Steven F. , Gao Guang-ping , Xiang Z. Q. , Rux John J. , Burnett Roger M. , Alvira Mauricio R. , Marsh Jonathan , Ertl Hildegund C. J. , Wilson James M.
Published: July 2002

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.
Complete Genome Sequence of the Shrimp White Spot Bacilliform Virus
Authors: Yang Feng , He Jun , Lin Xionghui , Li Qin , Pan Deng , Zhang Xiaobo , Xu Xun
Published: July 2002

We report the first complete genome sequence of a marine invertebrate virus. White spot bacilliform virus (WSBV; or white spot syndrome virus) is a major shrimp pathogen with a high mortality rate and a wide host range. Its double-stranded circular DNA genome of 305,107 bp contains 181 open reading frames (ORFs). Nine homologous regions containing 47 repeated minifragments that include direct repeats, atypical inverted repeat sequences, and imperfect palindromes were identified. This is the largest animal virus that has been completely sequenced. Although WSBV is morphologically similar to insect baculovirus, the two viruses are not detectably related at the amino acid level. Rather, some WSBV genes are more homologous to eukaryotic genes than viral genes. In fact, sequence analysis indicates that WSBV differs from all known viruses, although a few genes display a weak homology to herpesvirus genes. Most of the ORFs encode proteins that bear no homology to any known proteins, either suggesting that WSBV represents a novel class of viruses or perhaps implying a significant evolutionary distance between marine and terrestrial viruses. The most unique feature of WSBV is the presence of an intact collagen gene, a gene encoding an extracellular matrix protein of animal cells that has never been found in any viruses. Determination of the genome of WSBV will facilitate a better understanding of the molecular mechanism underlying the pathogenesis of the WSBV virus and will also provide useful information concerning the evolution and divergence of marine and terrestrial animal viruses at the molecular level.
Dynamics of Persistent TT Virus Infection, as Determined in Patients Treated with Alpha Interferon for Concomitant Hepatitis C Virus Infection
Authors: Maggi Fabrizio , Pistello Mauro , Vatteroni Marialinda , Presciuttini Silvano , Marchi Santino , Isola Patrizia , Fornai Claudia , Fagnani Sabina , Andreoli Elisabetta , Antonelli Guido , Bendinelli Mauro
Published: July 2002

TT virus (TTV) is a recently identified widespread DNA virus of humans that produces persistent viremia in the absence of overt clinical manifestations. In an attempt to shed light on the dynamics of chronic infection, we measured the levels of TTV in the plasma of 25 persistently infected patients during the first 3 months of alpha interferon (IFN-α) treatment for concomitant hepatitis C virus (HCV) infection. The first significant decline of TTV loads was observed at day 3 versus day 1 for HCV. Subsequently, the loads of TTV became progressively lower in most patients, but some initial responders relapsed before the end of the follow-up, suggesting that at least in some subjects the effects of IFN on TTV can be very short-lived. No correlation between the responses of TTV and HCV to therapy was found. Fitting the viremia data obtained during the first week of treatment into previously developed mathematical models showed that TTV sustains very active chronic infections, with over 90% of the virions in plasma cleared and replenished daily and a minimum of approximately 3.8 × 1010virions generated per day. Low levels of TTV were occasionally detected in the peripheral blood mononuclear cells of patients who had cleared plasma viremia, thus corroborating previous results showing that these cells may support TTV replication and/or persistence.
Double-Stranded RNA-Mediated Interference with Plant Virus Infection
Authors: Tenllado F. , Dı́az-Ruı́z J. R.
Published: July 2002

Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms. Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via anAgrobacterium-mediated transient-expression assay. We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach. We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals. The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not single-stranded RNA. Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation.
Porcine Teschoviruses Comprise at Least Eleven Distinct Serotypes: Molecular and Evolutionary Aspects
Authors: Zell Roland , Dauber Malte , Krumbholz Andi , Henke Andreas , Birch-Hirschfeld Eckhard , Stelzner Axel , Prager Dieter , Wurm Rudiger
Published: July 2002

Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus
Teschovirus
. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.
In Vitro and In Vivo Infectivity and Pathogenicity of the Lymphoid Cell-Derived Woodchuck Hepatitis Virus
Authors: Lew Yuan-Yee , Michalak Tomasz I.
Published: July 2002

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (∼103virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.
Characterization of Interactions between RTA and the Promoter of Polyadenylated Nuclear RNA in Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8
Authors: Song Moon Jung , Li Xudong , Brown Helen J. , Sun Ren
Published: July 2002

RTA (replication and transcription activator; also referred to as ORF50, Lyta, and ART), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, disrupts latency and drives lytic replication. RTA activates the expression of polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) of KSHV. This novel noncoding PAN RNA is the most abundant lytic transcript of KSHV; therefore, studying PAN RNA expression serves as a model system for understanding how RTA transactivates target genes during lytic replication. The RTA-responsive element of the PAN promoter (pPAN RRE) was previously identified, and our data suggested direct binding of full-length RTA to the pPAN RRE. Here, we present a detailed analysis of specific interactions between RTA and the PAN promoter. We expressed and purified the DNA-binding domain of RTA (Rdbd) to near homogeneity and measured its affinity for the pPAN RRE. In electrophoretic mobility shift assays (EMSAs), the dissociation constant (Kd) of Rdbd on the pPAN RRE was determined to be approximately 8 × 10−9M, suggesting a strong interaction between RTA and DNA. The specificity of RTA binding to the PAN promoter was confirmed with supershift assays. The Rdbd binding sequences on the PAN promoter were mapped within a 16-bp region of the pPAN RRE by methylation interference assays. However, the minimal DNA sequence for Rdbd binding requires an additional 7 bp on both sides of the area mapped by interference assays, suggesting that non-sequence-specific as well as sequence-specific interactions between RTA and DNA contribute to high-affinity binding. To better understand the molecular interactions between RTA and the PAN promoter, an extensive mutagenesis study on the pPAN RRE was carried out by using EMSAs and reporter assays. These analyses revealed base pairs critical for both Rdbd binding in vitro and RTA transactivation in vivo of the PAN promoter. The results from methylation interference, deletion analysis, and mutagenesis using EMSAs and reporter assays were closely correlated and support the hypothesis that RTA activates PAN RNA expression through direct binding to DNA.
Disulfide Bond Configuration of Human Cytomegalovirus Glycoprotein B
Authors: Lopper Matthew , Compton Teresa
Published: July 2002

Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically determining structures even for highly conserved proteins.
Molecular Evolution of H6 Influenza Viruses from Poultry in Southeastern China: Prevalence of H6N1 Influenza Viruses Possessing Seven A/Hong Kong/156/97 (H5N1)-Like Genes in Poultry
Authors: Chin P. S. , Hoffmann E. , Webby R. , Webster R. G. , Guan Y. , Peiris M. , Shortridge K. F.
Published: July 2002

The A/teal/Hong Kong/W312/97 (H6N1) influenza virus and the human H5N1 and H9N2 influenza viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza viruses were characterized by antigenic and genetic analyses of 29 H6 influenza viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-like H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?
A Soluble Factor(s) Secreted from CD8+T Lymphocytes Inhibits Human Immunodeficiency Virus Type 1 Replication through STAT1 Activation
Authors: Chang Theresa Li-Yun , Mosoian Arevik , Pine Richard , Klotman Mary E. , Moore John P.
Published: July 2002

CD8+T lymphocytes can suppress human immunodeficiency virus type 1 (HIV-1) replication by secreting a soluble factor(s) known as CD8+T-lymphocyte antiviral factor (CAF). One site of CAF action is inhibition of HIV-1 RNA transcription, particularly at the step of long terminal repeat (LTR)-driven gene expression. However, the mechanism by which CAF inhibits LTR activation is not understood. Here, we show that conditioned media from several herpesvirus saimari-transformed CD8+T lymphocytes inhibit, in a time- and dose-dependent manner, the replication of HIV-1 pseudotype viruses that express the envelope glycoproteins of vesicular stomatitis virus (HIV-1VSV). The same conditioned media also inhibit phorbol myristate acetate-induced activation of the HIV-1 LTR and activate the signal transducer and activator of transcription 1 (STAT1) protein. We have obtained direct evidence that STAT1 is necessary for CAF-mediated inhibition of LTR activation and HIV-1 replication. Thus, the inhibitory effect of CAF on HIV-1VSVreplication was abolished in STAT1-deficient cells. Moreover, CAF inhibition of LTR activation was diminished both in STAT1-deficient cells and in cells expressing a STAT1 dominant negative mutant but was restored when STAT1 was reintroduced into the STAT1-deficient cells. We also observed that CAF induced the expression of interferon regulatory factor 1 (IRF-1), and that IRF-1 gene induction was STAT-1 dependent. Taken together, our results suggest that CAF activates STAT1, leading to IRF-1 induction and inhibition of gene expression regulated by the HIV-1 LTR. This study therefore helps clarify one molecular mechanism of host defense against HIV-1.
Internalization of Echovirus 1 in Caveolae
Authors: Marjomäki Varpu , Pietiäinen Vilja , Matilainen Heli , Upla Paula , Ivaska Johanna , Nissinen Liisa , Reunanen Hilkka , Huttunen Pasi , Hyypiä Timo , Heino Jyrki
Published: July 2002

Echovirus 1
(EV1) is a human pathogen which belongs to the
Picornaviridae
family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.
The tRNA Primer Activation Signal in the Human Immunodeficiency Virus Type 1 Genome Is Important for Initiation and Processive Elongation of Reverse Transcription
Authors: Beerens Nancy , Berkhout Ben
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA
3
Lys
molecule, which binds, with its 3"-terminal 18 nucleotides (nt), to a complementary sequence in the viral genome, the primer-binding site (PBS). Besides PBS-anti-PBS pairing, additional interactions between viral RNA sequences and the tRNA primer are thought to regulate the process of reverse transcription. We previously identified a novel 8-nt sequence motif in the U5 region of the HIV-1 RNA genome that is critical for tRNA
3
Lys
-mediated initiation of reverse transcription in vitro. This motif activates initiation from the natural tRNA
3
Lys
primer but is not involved in tRNA placement and was therefore termed primer activation signal (PAS). It was proposed that the PAS interacts with the anti-PAS motif in the TΨC arm of tRNA
3
Lys
. In this study, we analyzed several PAS-mutated viruses and performed reverse transcription assays with virion-extracted RNA-tRNA complexes. Mutation of the PAS reduced the efficiency of tRNA-primed reverse transcription. In contrast, mutations in the opposing leader sequence that trigger release of the PAS from base pairing stimulated reverse transcription. These results are similar to the reverse transcription effects observed in vitro. We also selected revertant viruses that partially overcome the reverse transcription defect of the PAS deletion mutant. Remarkably, all revertants acquired a single nucleotide substitution that does not restore the PAS sequence but that stimulates elongation of reverse transcription. These combined results indicate that the additional PAS-anti-PAS interaction is needed to assemble an initiation-competent and processive reverse transcription complex.
Experimental Inoculation of Conventional Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2
Authors: Rovira A. , Balasch M. , Segalés J. , García L. , Plana-Durán J. , Rosell C. , Ellerbrok H. , Mankertz A. , Domingo M.
Published: July 2002

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (
n
= 5), PRRSV inoculated (
n
= 5), PCV2 inoculated (
n
= 7), and PRRSV and PCV2 inoculated (
n
= 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.
Altered Patterns of Cellular Gene Expression in Dermal Microvascular Endothelial Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus
Authors: Poole Lynn J. , Yu Yanxing , Kim Peter S. , Zheng Qi-Zhi , Pevsner Jonathan , Hayward Gary S.
Published: July 2002

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV; also called human herpesvirus 8) is believed to be the etiologic agent of Kaposi's sarcoma, multicentric Castleman's disease, and AIDS-associated primary effusion lymphoma. KSHV infection of human dermal microvascular endothelial cells (DMVEC) in culture results in the conversion of cobblestone-shaped cells to spindle-shaped cells, a characteristic morphological feature of cells in KS lesions. All spindle-shaped cells in KSHV-infected DMVEC cultures express the latency-associated nuclear protein LANA1, and a subfraction of these cells undergo spontaneous lytic cycle induction that can be enhanced by tetradecanoyl phorbol acetate (TPA) treatment. To study the cellular response to infection by KSHV, we used two different gene array screening systems to examine the expression profile of either 2,350 or 9,180 human genes in infected compared to uninfected DMVEC cultures in both the presence and absence of TPA. In both cases, between 1.4 and 2.5% of the genes tested were found to be significantly upregulated or downregulated. Further analysis by both standard and real-time reverse transcription-PCR procedures directly confirmed these results for 14 of the most highly upregulated and 13 of the most highly downregulated genes out of a total of 37 that were selected for testing. These included strong upregulation of interferon-responsive genes such as interferon response factor 7 (IRF7) and myxovirus resistance protein R1, plus upregulation of exodus 2 β-chemokine, RDC1 α-chemokine receptor, and transforming growth factor β3, together with strong downregulation of cell adhesion factors α
4
-integrin and fibronectin plus downregulation of bone morphogenesis protein 4, matrix metalloproteinase 2, endothelial plasminogen activator inhibitor 1, connective tissue growth factor, and interleukin-8. Significant dysregulation of several other cytokine-related genes or receptors, as well as endothelial cell and macrophage markers, and various other genes associated with angiogenesis or transformation was also detected. Western immunoblot and immunohistochemical analyses confirmed that the cellular IRF7 protein levels were strongly upregulated during the early lytic cycle both in KSHV-infected DMVEC and in the body cavity-based lymphoma BCBL1 PEL cell line.
Spiroheterocyclic Systems: New N-Substituted Spiroheterocyclic Naphthenes
Authors: Awad Ibrahim M. A. , Abdel-Hafez Ali A. , El-Zohry Maher F.
Published: July 2002

Spiro and sulfonamide derivatives have been found to be biologically active. In view of this importance, we report the synthesis of some new spiroheterocyclic naphthenes IV - VI.
Synthesis of Some 2'-C-Alkyl Derivatives of 9-(2-Phosphonomethoxyethyl)adenine and Related Compounds
Authors: Dvořáková Hana , Holý Antonín , Rosenberg Ivan
Published: July 2002

To study the effect of β-substitution in 2'-alkyl derivatives of 9-(2-phosphonomethoxyethyl)adenine (Ia) on the antiviral activity or group specificity, these derivatives were synthesized. 9-(2-Hydroxyalkyl)adenines VIII were prepared by alkylation of adenine with suitably substituted oxiranes XIII or 2-hydroxyalkyl p-toluenesulfonates IV and VI. After protection of the adenine amino group by benzoylation (compounds IX) or amidine formation (compounds X), the intermediates were alkylated with diisopropyl p-toluenesulfonyloxymethanephosphonate (XI) in the presence of sodium hydride. After deprotection, the obtained phosphonate diesters XII were converted into phosphonic acids I by transsilylation and hydrolysis. This synthetic scheme was used for the preparation of ethyl (Ie), propyl (If), 2-propyl (Ig), 2-methylpropyl (Ih), cyclopropyl (Ii), cyclohexyl (Ij), benzyl (Ik) and phenyl (Il) derivatives. The 2'-trifluoromethyl derivative XXIIa was prepared analogously from 9-(2-hydroxy-3,3,3-trifluoropropyl)adenine (XXa), obtained by alkylation of adenine sodium salt with 2-hydroxy-3,3,3-trifluoropropyl bromide. 2'-Trimethylsilyl derivative XIXa was obtained by alkylation of adenine with 2-diisopropylphosphonomethoxy-3-(4-toluenesulfonyloxy)propyltrimethylsilane (XVII) followed by transsilylation and hydrolysis of diester XVIIIa. 2,6-Diaminopurine derivatives XVIIId and XXIIb were obtained analogously. 9-(3-Phosphonomethoxybutyl)adenine (XXVIII) and 9-(2-methyl-2-phosphonomethoxypropyl)adenine (XXXV) were prepared from the corresponding hydroxy derivatives XXVIb and XXXII, respectively, by the same reaction pathway as derivatives I.
AM1 Calculations of Spin Distribution in Hydroxyanthrasemiquinones
Authors: Breza Martin , Brezová Vlasta
Published: July 2002

The AM1 method was used to obtain the optimum geometries of hydroxyanthrasemiquinones. The relation between the spin distribution data obtained by analysis of EPR spectra and by AM1 calculations depends on the position of the C atom within the aromatic skeleton, on its spin density sign and on its position relative to OH substituents. Complete annihilation of higher spin state contaminations is insufficient to eliminate the significant discrepancies between the theoretical and experimental data.
FEM and FIM Study of Reaction Zones on Platinum
Authors: Knor Zlatko , Plšek Jan , Dvořák Luboš
Published: July 2002

The initial shape of the reaction zones for the interaction of hydrogen and oxygen on the surfaces of the single crystals of platinum in FEM and FIM devices is discussed in connection with the effect of temperature and surface anisotropy. Interaction of the (H2 + O2) mixture has been studied at room temperature and the results were confronted with the interaction of hydrogen and preadsorbed oxygen at low temperature and with the anisotropy of the low-temperature field-corrosion of platinum.
Isoperichoric Focusing Phenomena Generated by Coupled Electric and Gravitational Field Forces in Bidisperse Mixtures of Colloidal Particles
Authors: Janča Josef , Gospodinova Natalia
Published: July 2002

The first successful isopycnic focusing generated by the coupled electric and gravitational fields applied to the model bidisperse mixtures of the colloidal silica and polyaniline particles of low size ratio is described. The theoretical analysis and the experimental results show that the density gradient forming liquid should not necessarily behave as a continuum regarding the focused species. This finding confirms the original prediction which was evidenced previously by the focusing under the action of high intensity centrifugal field forces only.
Cholinesterase Reactivators Derived from Pyridine-2-carbaldoxime
Authors: Bielavský Jiří , Kassa Jiří , Elsnerová Iva , Dejmek Luboš
Published: July 2002

Monoquaternary reactivators of cholinesterase 3a-3c were prepared by quaternization of substituted pyridine-2-carbaldoximes with an ester (1a, 1b) or carbamoyl (2) function in position 4. A new bisquaternary reactivator, 4-carbamoyl-2'-[(hydroxyimino)methyl]-1,1'-(but-2-ene-1,4-diyl)bispyridinium salt (5), was derived from the unsubstituted pyridine-2-carbaldoxime. The compounds were characterized by UV spectra and ionization constants. The substances exhibit antidote effects in intoxications with organophosphate cholinesterase inhibitors.
Diels-Alder Reactions of [1]Benzothieno[3,2-b]furan
Authors: Pihera Pavel , Paleček Jaroslav , Svoboda Jiří
Published: July 2002

[1]Benzothieno[3,2-b]furan reacts with substituted dienes 2 as a dienophile under formation of substituted tetrahydro[1]benzothieno[3,2-b][1]benzofuran derivatives 3-14. The cycloaddition is endo-stereoselective. Aromatization of the products leads to a series of new fused [1]benzothieno[3,2-b][1]benzofurans 17-24. The 1H NMR spectra of the new compounds were fully assigned.
Transport of Gases and Organic Vapours Through Membranes Made of Poly(amide-imide)s Crosslinked with Poly(ethylene adipate)
Authors: Šindelář Vladimír , Sysel Petr , Hynek Vladimír , Friess Karel , Šípek Milan , Castaneda Nadia
Published: July 2002

Transport of permanent gases (N2, O2, CO2) and of saturated organic vapours of benzene, toluene and methanol through membranes based on poly(ethylene adipate)-crosslinked poly(amide-imide)s was studied. The membranes containing more than 50 wt.% of non-polyimides showed higher permeability coefficients (by 2-3 orders) for the organic vapours than for the gases.
Theoretical Study of the A2Σ+-X2Π Transition of CCO-
Authors: Léonard Céline , Panten Dietmar , Rosmus Pavel , Wyss Muriel , Maier John P.
Published: July 2002

Three-dimensional potential energy functions have been generated for the A2Σ+ electronic state of C2O- using highly correlated electronic multi-reference configuration interaction wavefunctions. These were employed in variational calculations of the rovibrational states. The rotational constant has been calculated as Be = 0.3960 cm-1, with ReCO = 1.240 Å and ReCC = 1.244 Å. Rovibrational levels for J = 0 and 1 are reported for energies up to about 4 800 cm-1. Only weak anharmonic resonances are found in this energy region. The theoretically predicted A2Σ+←X2Π absorption spectrum is found to be in excellent agreement with the observed one in a neon matrix.
Preparation of [closo-CB11H12]- by Dichlorocarbene Insertion Into [nido-B11H14]-
Authors: Franken Andreas , King Benjamin T. , Rudolph Jens , Rao Photon , Noll Bruce C. , Michl Josef
Published: July 2002

In strongly basic media, the [nido-B11H14]- anion reacts with the haloforms. Dehydrogenation to [closo-B11H11]2- is the only reaction observed with iodoform. With chloroform and bromoform, the cage is expanded by dihalocarbene insertion. The dominant products are the [closo-CB11H12]- and the [2-Br-closo-CB11H12]- anion, respectively. The chief side product is the [closo-B11H11]2- anion, which results from dehydrogenation of the starting material. It was identified by 11B NMR spectroscopy and isolated after acidic aqueous workup in the form of the [nido-7-OH-B11H13]- anion. Since the starting [nido-B11H14]- anion is available from NaBH4 and BF3·Et2O in 50% yield, its conversion to [closo-CB11H12]- with chloroform and base in a 40% yield represents a useful laboratory route to the numerous known but previously very expensive derivatives of [closo-CB11H12]-, highly prized for their very low nucleophilicity.
Energy metabolism in relation to body composition and gender in adolescents
Authors: van Mil E G A H , Westerterp K R , Kester A D M , Saris W H M
Published: July 2002

OBJECTIVE

To study the effect of body composition on average daily metabolic rate (ADMR) and basal metabolic rate (BMR) in adolescence, and to examine current BMR prediction equations.

STUDY

Dutch adolescents were pooled with previously reported American and British subjects (n = 90 overall). BMR and ADMR were analysed by multiple regression.

RESULTS

Fat-free mass, BMR, and ADMR were higher in the obese than in the non-obese group (mean (SD): 53.2 (10.7) kg, 8.35 (1.57) MJ/d, and 13.64 (2.78) MJ/d, compared with 41.0 (8.1) kg, 6.42 (0.94) MJ/d, and 11.16 (2.21) MJ/d, respectively). BMR remained higher when adjusted for fat-free mass, age, and sex. ADMR adjusted for BMR was similar in the two groups. WHO equations overestimated BMR in obese boys and underestimated BMR in non-obese boys.

CONCLUSIONS

BMR, but not activity, is increased in obese adolescents and in male adolescents. The WHO BMR equations for adults are recommended for obese adolescents.
Photopheresis at onset of type 1 diabetes: a randomised, double blind, placebo controlled trial
Authors: Ludvigsson J , Samuelsson U , Ernerudh J , Johansson C , Stenhammar L , Berlin G
Published: July 2002

BACKGROUND

In recent years photopheresis, an extracorporeal form of photochemotherapy using psoralen and ultraviolet A irradiation of leucocytes, has been claimed to be an effective form of immunomodulation.

AIM

To evaluate its effect in type 1 diabetes we performed a double blind, controlled study using placebo tablets and sham pheresis in the control group.

METHODS

A total of 49 children, aged 10–18 years of age at diagnosis of type 1 diabetes were included; 40 fulfilled the study and were followed for three years (19 received active treatment with photopheresis and 21 placebo treatment).

RESULTS

The actively treated children secreted significantly more C peptide in urine during follow up than control children. C peptide values in serum showed corresponding differences between the two groups. The insulin dose/kg body weight needed to achieve satisfactory HbA1c values was always lower in the photopheresis group; there was no difference between the groups regarding HbA1c values during follow up. The treatment was well accepted except for nausea (n = 3) and urticaria (n = 1) in the actively treated group. There were no differences regarding weight or height, or episodes of infection between the two groups during follow up.

CONCLUSION

Photopheresis does have an effect in addition to its possible placebo effect, shown as a weak but significant effect on the disease process at the onset of type 1 diabetes, an effect still noted after three years of follow up.
Effects of one year of resistance training on the relation between muscular strength and bone density in elderly women
Authors: Rhodes E C , Martin A D , Taunton J E , Donnelly M , Warren J , Elliot J
Published: July 2002

Objectives—There is a paucity of long term studies on exercise training in elderly women. The purpose of this study was to investigate the effects of one year of progressive resistance exercise (PRE) on dynamic muscular strength and the relations to bone mineral density (BMD) in elderly women.

Methods—Forty four healthy sedentary women (mean age 68.8 years) volunteered for this study and were randomly assigned to either an exercise group or a control group. The exercise group were involved in three one hour sessions a week for 52 weeks of supervised PRE to strengthen the large muscle groups of the body, while the control group were instructed to continue their normal lifestyle. The exercise circuit included three sets of eight repetitions at 75% of one repetition maximum focused on the large muscle groups. BMD was measured by dual energyxray absoptiometry (Lunar DPX) at the lumbar spine and at three sites in the proximal femur. Other selected parameters of physical fitness were also measured.

Results—Statistical analyses (analysis of covariance) showed significant strength gains (p<0.01) in bilateral bench press (>29%), bilateral leg press (>19%), and unilateral biceps curl (>20%). No significant difference between groups was evident in body weight, grip strength, flexibility, waist to hip ratio, or the sum of eight skinfolds. Significant relations (p<0.05) were recorded between dynamic leg strength and the BMD of the femoral neck, Ward's triangle, and the lumbar spine.

Conclusions—Significant strength changes, after one year of PRE, were evident in elderly women, and the muscle increases may parallel changes in BMD; however, correlation coefficients were moderate.
Red blood cell variables in highly trained pubescent athletes: a comparative analysis
Authors: Boyadjiev N , Taralov Z
Published: July 2002

Background—A suboptimal haematological status has often been recorded in athletes involved in intensive physical activity. There have even been reports of “sports anaemia” associated with intensive physical exercise. However, studies on the effect of different types of exercise practiced over a long period of time on the red blood cell variables in pubescent athletes are very few.

Aim—To assess the basic red blood cell variables in highly trained pubescent athletes from different sports and to compare the results with those for a control untrained group. Sex related differences in these variables were also assessed.

Methods—876 highly trained athletes (559 boys and 317 girls) were included in the study. Their mean (SEM) age, weight, and duration of training were: 14.01 (0.06) years, 56.24 (0.52) kg, and 3.52 (0.07) years respectively. The control group consisted of 357 untrained subjects (171 boys and 186 girls) with mean (SEM) age and weight of 14.58 (0.09) years and 57.75 (0.67) kg. The group of athletes was divided into seven subgroups according to the sport practiced: athletics (105), swimming (107), rowing (230), wrestling (225), weight lifting (47), various team sports (92), and other sports (67). Venous blood samples were drawn from the cubital vein, and the red blood cell count, packed cell volume, haemoglobin concentration, and mean corpuscular volume were measured. Statistical indices were computed for each group and for each variable, and analysis of variance factorial analysis was performed to evaluate the statistical significance of the differences detected.

Results—The highly trained group was found to have lower red blood cell count, packed cell volume, and haemoglobin concentration (p<0.001) than the control untrained group (4.61 (0.01) × 1012/1v4.75 (0.02) × 1012/l, 0.389 (0.001)v0.404 (0.002) l/l, and 133.01 (0.38)v139.9 (0.62) g/l respectively). These variables were lower for the boys of the trained group than for the boys of the control group (p<0.001), and similarly for the girls (p<0.001). The lowest red blood cell count, packed cell volume, and haemoglobin concentration were measured in blood samples from the boys of the swimming subgroup (4.54 (0.06) × 1012/l, 0.386 (0.006) l/l, and 129.38 (1.80) g/1 respectively) and the rowing subgroup (4.66 (0.03) × 1012/l, 0.400 (0.003) l/l, and 136.21 (0.94) respectively). The same distribution was found for the girls: lowest in the rowing subgroup (4.32 (0.04) × 1012/1, 0.314 (0.003) l/l, and 124.27 (0.93) g/1) and the swimming subgroup (4.40 (0.05) × 1012/l, 0.375 (0.005) l/l, and 125.90 (1.30) g/1). No differences were found in the mean corpuscular volume.

Conclusions—Continuous (more than one year) high intensity sports training (twice a day/five days a week) results in a decrease in the basic red blood cell variables in pubescent boys and girls, this being most pronounced in the submaximal sports.