bluuebunny/crossref_metadata_2025 huggingface dataset:
One-Point Population Analysis and Effect of Osmolarity on Detection of Hetero-Vancomycin-Resistant
Staphylococcus aureus
Authors: Jung Sook-In , Kiem Sungmin , Lee Nam Yong , Kim Yeon-Sook , Oh Won Sup , Cho Hwang Lae , Peck Kyong Ran , Song Jae-Hoon
Published: July 2002

The original screening test and one-point population analysis (OPPA) were compared for the detection of hetero-vancomycin-resistant
Staphylococcus aureus
, and the influence of osmolarity on those tests was evaluated. The positivity rates and the reproducibilities were similar for both tests. The addition of NaCl increased the MIC as well as the positivity rates for both tests and the reproducibility of OPPA.
Rescue of Mumps Virus from cDNA
Authors: Clarke David K. , Sidhu Mohinderjit S. , Johnson J. Erik , Udem Stephen A.
Published: July 2002

A complete DNA copy of the genome of a Jeryl Lynn strain of mumps virus (15,384 nucleotides) was assembled from cDNA fragments such that an exact antigenome RNA could be generated following transcription by T7 RNA polymerase and cleavage by hepatitis delta virus ribozyme. The plasmid containing the genome sequence, together with support plasmids which express mumps virus NP, P, and L proteins under control of the T7 RNA polymerase promoter, were transfected into A549 cells previously infected with recombinant vaccinia virus (MVA-T7) that expressed T7 RNA polymerase. Rescue of infectious virus from the genome cDNA was demonstrated by amplification of mumps virus from transfected-cell cultures and by subsequent consensus sequencing of reverse transcription-PCR products generated from infected-cell RNA to verify the presence of specific nucleotide tags introduced into the genome cDNA clone. The only coding change (position 8502, A to G) in the cDNA clone relative to the consensus sequence of the Jeryl Lynn plaque isolate from which it was derived, resulting in a lysine-to-arginine substitution at amino acid 22 of the L protein, did not prevent rescue of mumps virus, even though an amino acid alignment for the L proteins of paramyxoviruses indicates that lysine is highly conserved at that position. This system may provide the basis of a safe and effective virus vector for the in vivo expression of immunologically and biologically active proteins, peptides, and RNAs.
Genetic Analysis of a Poliovirus/Hepatitis C Virus (HCV) Chimera: Interaction between the Poliovirus Cloverleaf and a Sequence in the HCV 5′ Nontranslated Region Results in a Replication Phenotype
Authors: Zhao Wei Dong , Lahser Frederick C. , Wimmer Eckard
Published: July 2002

Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5′-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5′ nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.
Functional Characterization of the Dimer Linkage Structure RNA of Moloney Murine Sarcoma Virus
Authors: Ly Hinh , Nierlich Donald P. , Olsen John C. , Kaplan Andrew H.
Published: July 2002

Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255–7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.
Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein
Authors: Poppers Jeremy , Mulvey Matthew , Khoo David , Mohr Ian
Published: July 2002

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.
Two Patches of Amino Acids on the E2 DNA Binding Domain Define the Surface for Interaction with E1
Authors: Chen Grace , Stenlund Arne
Published: July 2002

The E1 and E2 proteins from bovine papillomavirus bind cooperatively to the viral origin of DNA replication (ori), forming a complex which is essential for initiation of DNA replication. Cooperative binding has two components, in which (i) the DNA binding domains (DBDs) of the two proteins interact with each other and (ii) the E2 transactivation domain interacts with the helicase domain of E1. By generating specific point mutations in the DBD of E2, we have defined two patches of amino acids that are involved in the interaction with the E1 DBD. These same mutations, when introduced into the viral genome, result in severely reduced replication of the viral genome, as well as failure to transform mouse cells in tissue culture. Thus, the interaction between the E1 and E2 DBDs is important for the establishment of the viral genome as an episome and most likely contributes to the formation of a preinitiation complex on the viral ori.
DNA Methylation of Helper Virus Increases Genetic Instability of Retroviral Vector Producer Cells
Authors: Young Won-Bin , Lindberg Gary L. , Link Charles J.
Published: July 2002

Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 × 10
5
CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 × 10
5
CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5′ long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast, retroviral vectors did not exhibit significant methylation and maintained consistent transcription activity. Treatment with 5-azacytidine, a methylation inhibitor, partially reversed the helper virus DNA methylation and restored a portion of vector production. The preference for methylation of helper virus sequences over vector sequences may have important implications for host-virus interaction. Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production. The maintenance of increased viral envelope-receptor interference might also prevent replication-competent retrovirus formation.
Locus Control Region of the Human
CD2
Gene in a Lentivirus Vector Confers Position-Independent Transgene Expression
Authors: Kowolik Claudia M. , Hu Jun , Yee Jiing-Kuan
Published: July 2002

Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the β-globin gene locus or the
CD2
gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various
cis
-acting elements such as LCRs.
Fidelity of Leader and Trailer Sequence Usage by the Respiratory Syncytial Virus and Avian Pneumovirus Replication Complexes
Authors: Marriott Anthony C. , Smith Joanne M. , Easton Andrew J.
Published: July 2002

The specificity of usage of promoters for replication and transcription by the pneumoviruses human respiratory syncytial virus (HRSV) and avian pneumovirus (APV) was studied using minigenomes containing a reporter gene. When infectious HRSV or APV was used as helper virus, replication could occur only if both the leader and trailer regions (containing the replicative and transcriptional promoters) were derived from the helper virus. In contrast, when the HRSV replication complex was supplied from cDNA plasmids, a minigenome containing either the APV leader or trailer was recognized and substantial levels of replication and transcription occurred. These data suggest that in pneumovirus-infected cells, helper virus functions can discriminate between genomes on the basis of the terminal sequences and that there is an association between the leader and trailer required for productive replication. This association is required only in virus-infected cells, not when replication and transcription are mediated by plasmid-directed expression of the component proteins required for replication and transcription. The possible implications of this are discussed.
Biologic Studies of Chimeras of Highly and Moderately Virulent Molecular Clones of Simian Immunodeficiency Virus SIVsmPBj Suggest a Critical Role for Envelope in Acute AIDS Virus Pathogenesis
Authors: Haddrick Malcolm , Brown Charles R. , Plishka Ronald , Buckler-White Alicia , Hirsch Vanessa M. , Ginsberg Harold
Published: July 2002

Previous studies identified three molecular clones of the acutely pathogenic SIVsmPBj strain that varied in terms of relative in vivo pathogenicity. One clone, SIVsmPBj6.6, reproducibly induced a rapidly fatal disease in pigtailed macaques. In contrast, a highly related clone (SIVsmPBj6.9) was only minimally pathogenic in macaques. PBj6.6 and PBj6.9 shared a tyrosine substitution at position 17 in the Nef protein that is a major determinant of virulence but differed at one residue in Vpx (C89R), three residues within the envelope (D119G, R871G, G872R), and a single residue in Nef (F252L). SIVsmPBj6.9 was less efficient in inducing proliferation of resting macaque peripheral blood mononuclear cells in vitro than SIVsmPBj6.6 and exhibited a marked reduction in infectivity relative to SIVsmPBj6.6. Chimeric viruses for each of these variable residues were constructed, and their biologic properties were compared to those of the parental strains. Differences in Vpx and Nef did not alter the basic biologic phenotype of the chimeras. However, the D119G substitution in the envelope of SIVsmPBj6.9 was associated with a marked reduction in the infectivity of this virus relative to SIVsmPBj6.6. An associated processing defect in gp160 of SIVsmPBj6.9 and chimeras expressing the D119G substitution suggests that a reduction in virion envelope incorporation is the mechanistic basis for reduced virion infectivity. In vivo studies revealed that substitution of the PBj6.9 amino acid into PBj6.6 (D119) abrogated the pathogenicity of this previously pathogenic virus. Introduction of the PBj6.9 G119, however, did not confer full virulence to the parental PBj6.9 virus, implicating one or all of the other four substitutions in the virulence of SIVsmPBj6.6.
Role of Chimeric Murine Leukemia Virus
env
β-Turn Polyproline Spacers in Receptor Cooperation
Authors: Valsesia-Wittmann Sandrine
Published: July 2002

We have previously reported a set of Moloney murine leukemia virus derived envelopes retargeted to the Pit-2 phosphate transporter molecule, by insertion of the Pit-2 binding domain (BD) at the N terminus of the ecotropic retroviral envelope glycoproteins (S. Valsesia-Wittmann et al., J. Virol. 70:2059-2064, 1996). The resulting chimeric envelopes share two BDs: an additional N-terminal BD (Pit-2 BD) and the BD of the ecotropic envelope (mCAT-1 BD). By inserting a variety of different amino acid spacers between the two binding domains, we showed that retroviruses can potentially use the targeted cell surface receptor Pit-2, the ecotropic retroviral receptor mCAT-1, or both receptors cooperatively for entry into target cell (S. Valsesia-Wittmann et al., EMBO J 6:1214–1223, 1997). An extreme example of receptor cooperativity was encountered when envelopes with specific proline-rich interdomain spacers (PRO spacers) were tested: both receptors had to be coexpressed at the surface of the targeted cells to cooperatively allow infection. Here, we characterized the role of PRO spacer in the cooperation of receptors. We have shown that the particular organization of the PRO spacer—a β-turn polyproline—was responsible for the cooperative effect. In the native configuration of the viruses, the structure masked the regions located downstream of the PRO spacer, thus the mCAT-1 BD. After interaction with the targeted Pit-2 receptor, the BD of the backbone envelope became accessible, and we demonstrated that interaction between the mCAT-1 BD and the mCAT-1 receptor is absolutely necessary. This interaction leads to natural fusion triggering and entry of viruses into targeted cells.
Virion Association of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Requires Expression of the VZV Open Reading Frame 66 Protein Kinase
Authors: Kinchington Paul R. , Fite Karen , Seman Amy , Turse Stephanie E.
Published: July 2002

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is associated with the tegument of gradient-purified virions. Here, we show that most, if not all, of the association requires the expression of open reading frame 66 (ORF66), a protein kinase. The association of IE62 with wild-type VZV virions was confirmed using immunoelectron microscopy with IE62-specific antibodies, which reacted with virions in ultrathin sections of VZV-infected cells. Fractionated purified virions from cells infected with recombinant VZV ROka contained substantial levels of the 175-kDa virion IE62 protein and also contained the ORF66 protein. However, virions from cells infected with recombinant VZV ROka66S, in which ORF66 is disrupted, lacked not only the ORF66 protein but also most of the virion 175-kDa IE62 polypeptide. The virion-associated protein kinase activity was still present in ROka66S virions, although the 175-kDa protein substrate for the virion kinase was absent, implying that the virion protein kinase is encoded by genes other than ORF66. The very low levels of IE62 in ROka66S virions indicate that ORF66 protein mediates the redistribution of IE62 to sites of tegument assembly. IE62 was resolved into several species from VZV-infected cells which showed mobility differences between ROka and ROka66S, and a specific form of IE62 was detected in ROka virions. These results are consistent with a role for the ORF66-mediated phosphorylation of IE62 that results in cytoplasmic distribution of the regulatory protein for tegument inclusion. They support a model in which VZV tegument acquisition occurs in the cytoplasm. As such, two unusual features of VZV IE62, namely, its virion inclusion and its phosphorylation and nuclear exclusion by the ORF66 protein kinase, are functionally linked.
Characteristics of a Pathogenic Molecular Clone of an End-Stage Serum-Derived Variant of Simian Immunodeficiency Virus (SIV
F359
)
Authors: Holterman Lennart , Dubbes Rob , Mullins James , Learn Gerald , Niphuis Henk , Koornstra Wim , Koopman Gerrit , Kuhn Eva-Maria , Wade-Evans Alison , Rosenwirth Brigitte , Haaijman Joost , Heeney Jonathan
Published: July 2002

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (
Macaca mulatta
). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Δ32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.
Adenovirus Vector Designed for Expression of Toxic Proteins
Authors: Edholm Dan , Molin Magnus , Bajak Edyta , Akusjärvi Göran
Published: July 2002

To construct recombinant adenoviruses expressing biologically active proteins may be impossible, or result in a significant reduction in virus yield, if the protein expressed has an inhibitory effect on virus replication or cellular growth. To overcome this problem, we previously designed adenovirus vectors expressing foreign proteins from inducible promoters. However, during our work with a replication-deficient virus expressing the ASF/SF2 splicing factor from a progesterone antagonist-inducible gene cassette, we discovered that ASF/SF2 was expressed at a significant level in the 293 producer cell line, even in the absence of inducer. 293 cells code for adenovirus E1A and E1B proteins and thus support the growth of E1-deficient adenoviruses. Here we show that this background ASF/SF2 expression results from a low level of E1A-mediated transactivation of the basal promoter driving transgene expression. To overcome the problem of leaky expression, we reconstructed a novel gene cassette that combines an inducible promoter and a Lac repressor protein-based block to reduce transcriptional elongation. We show that this novel vector system dramatically reduced background transgene expression and therefore should be useful for the rescue and propagation of high-titer stocks of recombinant adenoviruses expressing toxic proteins.
An Intact U5-Leader Stem Is Important for Efficient Replication of Simian Immunodeficiency Virus
Authors: Guan Yongjun , Diallo Karidia , Whitney James B. , Liang Chen , Wainberg Mark A.
Published: July 2002

Previous work has shown that four deletions in simian immunodeficiency virus (SIV), termed SD1a, SD1b, SD1c, and SD6, which eliminated sequences at nucleotide positions 322 to 362, 322 to 370, 322 to 379, and 371 to 379, respectively, located downstream of the primer binding site, impaired viral replication capacity to different extents. Long-term culturing of viruses containing the SD1a, SD1b, and SD6 deletions led to revertants that possessed wild-type replication kinetics. We now show that these revertants retained the original deletions in each case but that novel additional mutations were also present. These included a large deletion termed D1 (nt +216 to +237) within the U5 region that was shown to be biologically relevant to reversion of both the SD1a and SD1b constructs. In the case of SD6, two compensatory point mutations, i.e., A+369G, termed M1, located immediately upstream of the SD6 deletion, and C+201T, termed M2, within U5, were identified and could act either singly or in combination to restore viral replication. Secondary structure suggests that an intact U5-leader stem is important in SIV for infectiousness and that the additional mutants described played important roles in restoration of this motif.
Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein
Authors: West Michael , Flanery David , Woytek Kelly , Rangasamy Dhandapani , Wilson Van G.
Published: July 2002

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1
179–191
) and HR3 (E1
241–252
), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 α-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).
A Unique Heparin-Binding Domain in the Envelope Protein of the Neuropathogenic PVC-211 Murine Leukemia Virus May Contribute to Its Brain Capillary Endothelial Cell Tropism
Authors: Jinno-Oue Atsushi , Oue Miho , Ruscetti Sandra K.
Published: July 2002

Previous studies from our laboratory demonstrated that PVC-211 murine leukemia virus (MuLV), a neuropathogenic variant of Friend MuLV (F-MuLV), had undergone genetic changes which allowed it to efficiently infect rat brain capillary endothelial cells (BCEC) in vivo and in vitro. Two amino acid changes from F-MuLV in the putative receptor binding domain (RBD) of the envelope surface protein of PVC-211 MuLV (Glu-116 to Gly and Glu-129 to Lys) were shown to be sufficient for conferring BCEC tropism on PVC-211 MuLV. Recent examination of the unique RBD of PVC-211 MuLV revealed that the substitution of Lys for Glu at position 129 created a new heparin-binding domain that overlapped a heparin-binding domain common to ecotropic MuLVs. In this study we used heparin-Sepharose columns to demonstrate that PVC-211 MuLV, but not F-MuLV, can bind efficiently to heparin and that one or both of the amino acids in the RBD of PVC-211 MuLV that are associated with BCEC tropism are responsible. We further showed that heparin can enhance or inhibit MuLV infection and that the mode of action is dependent on heparin concentration, sulfation of heparin, and the affinity of the virus for heparin. Our results suggest that the amino acid changes that occurred in the envelope surface protein of PVC-211 MuLV may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor and efficiently infect these cells.
Vaccine-Induced Serum Immunoglobin Contributes to Protection from Herpes Simplex Virus Type 2 Genital Infection in the Presence of Immune T Cells
Authors: Morrison Lynda A. , Zhu Li , Thebeau Lydia G.
Published: July 2002

Herpes simplex type virus 2 (HSV-2) is a sexually transmitted pathogen that causes genital lesions and spreads to the nervous system to establish acute and latent infections. Systemic but not mucosal cellular and humoral immune responses are elicited by immunization of mice with a replication-defective mutant of HSV-2, yet the mice are protected against disease caused by subsequent challenge of the genital mucosa with virulent HSV-2. In this study, we investigated the role of immune serum antibody generated by immunization with a replication-defective HSV-2 vaccine prototype strain in protection of the genital mucosa and the nervous system from HSV-2 infection. Passive transfer of replication-defective virus-immune serum at physiologic concentrations to SCID or B-cell-deficient mice had no effect on replication of challenge virus in the genital mucosa but did significantly reduce the incidence and severity of genital and neurologic disease. In contrast, B-cell-deficient mice immunized with replication-defective HSV-2 were able to control replication of challenge virus in the genital mucosa, but not until 3 days postchallenge, and were not completely protected against genital and neurologic disease. Passive transfer of physiologic amounts of immune serum to immunized, B-cell-deficient mice completely restored their capacity to limit replication of challenge virus in the genital mucosa and prevented signs of genital and systemic disease. In addition, the numbers of viral genomes in the lumbosacral dorsal root ganglia of immunized, B-cell-deficient mice were dramatically reduced by transfer of immune serum prior to challenge. These results suggest that there is an apparent synergism between immune serum antibody and immune T cells in achieving protection and that serum antibody induced by vaccination with replication-defective virus aids in reducing establishment of latent infection after genital infection with HSV-2.
Using Recombinant Coxsackievirus B3 To Evaluate the Induction and Protective Efficacy of CD8+T Cells during Picornavirus Infection
Authors: Slifka Mark K. , Pagarigan Robb , Mena Ignacio , Feuer Ralph , Whitton J. Lindsay
Published: July 2002

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDprocleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were ∼1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+T-cell response to the encoded epitope; this has implications for the proposed role of “cross-priming” during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in ∼50-fold-lower virus titers in the heart and ∼6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.
Properties of the Surface Envelope Glycoprotein Associated with Virulence of Simian-Human Immunodeficiency Virus SHIV
SF33A
Molecular Clones
Authors: Chakrabarti Lisa A. , Ivanovic Tijana , Cheng-Mayer Cecilia
Published: July 2002

In vivo adaptation of simian-human immunodeficiency virus (SHIV) clone SHIV
SF33
resulted in the emergence of pathogenic isolate SHIV
SF33A
, which caused a rapid and severe CD4
+
T-cell depletion when inoculated into rhesus macaques. Two molecular clones generated by inserting the
env
V1-to-V5 region amplified from SHIV
SF33A
-infected animals into the parental SHIV
SF33
genome retained a pathogenic phenotype. The gp120 envelope glycoproteins of pathogenic clones SHIV
SF33A2
and SHIV
SF33A5
conferred a threefold increase in viral entry and fusogenicity compared to the parental glycoprotein. Changes in gp120 were also responsible for a higher replication capacity and cytopathicity in primary CD4
+
T-cell cultures. Last, gp120 carried the determinants of SHIV
SF33A
neutralization resistance. Thus, changes in SHIV
SF33A
gp120 produced a set of properties that could account for the pathogenic phenotype observed in vivo. Measurement of antibody binding to SHIV
SF33A
viral particles revealed an increased exposure of the CD4-induced epitope recognized by the 17b monoclonal antibody in a region that was shown to contribute to coreceptor binding. Exposure of this epitope occurred in the absence of CD4 binding, suggesting that the envelope glycoprotein of pathogenic SHIV
SF33A
clones folded in a conformation that was primed for interaction with CXCR4 or for the subsequent step of fusion.
cis
-Acting Sequences 5E, M, and 3E Interact To Contribute to Primer Translocation and Circularization during Reverse Transcription of Avian Hepadnavirus DNA
Authors: Mueller-Hill Karlyn , Loeb Daniel D.
Published: July 2002

Hepadnaviral reverse transcription requires template switches for the genesis of relaxed circular (RC) DNA, the major genomic form in virions. Two template switches, primer translocation and circularization, are required during the synthesis of the second, or plus, strand of DNA. Studies of duck hepatitis B virus (DHBV) indicate that in addition to the requirement for repeated sequences at the donor and acceptor sites, template switching requires at least three other
cis
-acting sequences, 5E, M, and 3E. In this study we analyzed a series of variant heron hepatitis B viruses (HHBV) in which the regions of the genome that would be expected to contain 5E, M, and 3E were replaced with DHBV sequence. We found that all single and double chimeras were partially defective in the synthesis of RC DNA. In contrast, the triple chimera was able to synthesize RC DNA at a level comparable to that of unchanged HHBV. These results indicate that the three
cis
-acting sequences, 5E, M, and 3E, need to be compatible to contribute to RC DNA synthesis, suggesting that these sequences interact during plus-strand synthesis. Second, we found that the defect in RC DNA synthesis for several of the single and double chimeric viruses resulted from a partial defect in primer translocation/utilization and a partial defect in circularization. These findings indicate that the processes of primer translocation and circularization share a mechanism during which 5E, M, and 3E interact.
Cell Cycle Status Affects Coxsackievirus Replication, Persistence, and Reactivation In Vitro
Authors: Feuer Ralph , Mena Ignacio , Pagarigan Robb , Slifka Mark K. , Whitton J. Lindsay
Published: July 2002

Enteroviral persistence has been implicated in the pathogenesis of several chronic human diseases, including dilated cardiomyopathy, insulin-dependent diabetes mellitus, and chronic inflammatory myopathy. However, these viruses are considered highly cytolytic, and it is unclear what mechanisms might permit their long-term survival. Here, we describe the generation of a recombinant coxsackievirus B3 (CVB3) expressing the enhanced green fluorescent protein (eGFP), which we used to mark and track infected cells in vitro. Following exposure of quiescent tissue culture cells to either wild-type CVB3 or eGFP-CVB3, virus production was very limited but increased dramatically after cells were permitted to divide. Studies with cell cycle inhibitors revealed that cells arrested at the G1or G1/S phase could express high levels of viral polyprotein and produced abundant infectious virus. In contrast, both protein expression and virus yield were markedly reduced in quiescent cells (i.e., cells in G0) and in cells blocked at the G2/M phase. Following infection with eGFP-CVB3, quiescent cells retained viral RNA for several days in the absence of infectious virus production. Furthermore, RNA extracted from nonproductive quiescent cells was infectious when transfected into dividing cells, indicating that CVB3 appears to be capable of establishing a latent infection in G0cells, at least in tissue culture. Finally, wounding of infected quiescent cells resulted in viral protein expression limited to cells in and adjacent to the lesion. We suggest that (i) cell cycle status determines the distribution of CVB3 during acute infection and (ii) the persistence of CVB3 in vivo may rely on infection of quiescent (G0) cells incapable of supporting viral replication; a subsequent change in the cell cycle status may lead to virus reactivation, triggering chronic viral and/or immune-mediated pathology in the host.
Functional Analysis of the Murine Sarcoma Virus RNA Packaging Sequence
Authors: Izmailova Elena , Aldovini Anna
Published: July 2002

We investigated the features of the Moloney murine sarcoma virus leader sequence necessary for RNA packaging function by using a deletion analysis approach. We found that sequences that extend beyond those characterized genetically in previous reports are important for optimal packaging efficiency. A fragment covering a minimum of four potential stem-loop structures is required for the shortest packaging element compatible with gene transfer. Our results reveal the extent to which each of the segments of the packaging sequence contribute to packaging efficiency.
Influence of Iodine on Low Frequency Dielectric Properties of Anthrone
Authors: Bak Grzegorz W. , Kryszewski Marian
Published: July 2002

Low frequency dielectric properties of polycrystalline anthrone and anthrone/iodine charge-transfer complex are presented. Polycrystalline anthrone proves to be a typical low-loss material. Iodization of anthrone pellets leads to a remarkable change in dielectric properties. Dielectric response of anthrone/iodine CT complex shows comparatively strong low frequency dispersion. The change in dielectric properties proves to be reversible to great extend as removing of iodine by heating at 373 K for a few hours almost completely restores the dielectric values of a virgin anthrone sample.
Crystallization of Isotactic Poly(propylene) in Solution as Followed by Slow Calorimetry
Authors: Phuong-Nguyen Hong , Delmas Geneviève
Published: July 2002

Dissolution, crystallization and second dissolution traces of isotactic poly(propylene) have been obtained in a slow temperature ramp (3 K h-1) with the C80 Setaram calorimeter. Traces of phase-change, in presence of solvent, are comparable to traces without solvent. The change of enthalpy on heating or cooling, ∆Htotal, over the 40-170 °C temperature range, is the sum of two contributions, ∆HDSC and ∆Hnetwork. The change ∆HDSC is the usual heat obtained in a fast temperature ramp and ∆Hnetwork is associated with a physical network whose disordering is slow and subject to superheating due to strain. When dissolution is complete, ∆Htotal is equal to ∆H0, the heat of fusion of perfect crystals. The values of ∆Htota for nascent and recrystallized samples are compared. Dissolution is the tool to evaluate the quality of the crystals. The repartition of ∆Htotal, into the two endotherms, reflects the quality of crystals. The crystals grown more rapidly have a higher fraction of network crystals which are stable at high T in the solvents. A complete dissolution, i.e. a high temperature (170 °C or more) is necessary to obtain good crystals. The effect of concentration, polymer molecular weight and solvent quality on crystal growth is analyzed.
Synthesis and Properties of Cobaltacarboranes with Substituted Monoatomic Bridges Between Ligands of the 6,6'-μ-RnE(1,7-C2B9H10)2-2-Co Type
Authors: Plešek Jaromír , Grüner Bohumír , Holub Josef
Published: July 2002

Syntheses, properties and constitutions of eleven asymmetric cobaltacarboranes with various substituents on monoatomic bridges between both ligands of the type 6,6'-μ-RnE(1,7-C2B9H10)2-2-Co (E = O, R = methyl; E = S, R = ethyl, propyl, isopropyl, allyl, butyl, hexyl and CH3OCOCH2; E = N, R = H (H), methyl, (H), and dimethyl) are described. Constitution assignation of all compounds are based on 1H and 11B NMR spectroscopy experiments complemented by 11B-11B COSY NMR and mass spectrometry; UV-VIS, melting points and TLC parameters are also presented. Only the racemic forms were obtained, although in principle, the meso-forms might result as well.
Synthesis of C2-Symmetrical [1,1'-Binaphthalene]-2,2'-diamines with Additional Chelating Groups Attached to the Nitrogen Atoms as Potential Ligands for Asymmetric Catalysis
Authors: Vyskočil Štěpán , Meca Luděk , Kubišta Jiří , Maloň Petr , Kočovský Pavel
Published: July 2002

Reductive amination of 2-hydroxybenzaldehyde, using a combination of NaBH4 and (R)-[1,1'-binaphthalene]-2,2'-diamine, served as the key step in the synthesis of the potentially tetradentate ligands (R)-N,N'-bis(2-hydroxybenzyl)[1,1'-binaphthalene]-2,2'-diamine and (R)-N,N'-bis(2-hydroxybenzyl)-N,N'-dimethyl[1,1'-binaphthalene]-2,2'-diamine. In analogy, amination of 2-bromobenzaldehyde with (R)-[1,1'-binaphthalene]-2,2'-diamine produced (R)-N,N'-bis(2-bromobenzyl)[1,1'-binaphthalene]-2,2'-diamine, whose conversion into the novel diphosphine ligand (R)-N,N'-bis[2-(diphenylphosphino)benzyl]-N,N'-dimethyl- [1,1'-binaphthalene]-2,2'-diamine was readily attained in two steps. CD spectra of the new binaphthyl derivatives are presented.
Increase in mortality rates from intrahepatic cholangiocarcinoma in England and Wales 1968–1998
Authors: Taylor-Robinson S D , Toledano M B , Arora S , Keegan T J , Hargreaves S , Beck A , Khan S A , Elliott P , Thomas H C
Published: July 2002

BACKGROUND

The age standardised mortality rate per 100 000 population for all causes of liver tumours (International Classification of Disease 9 (ICD-9) 155) has almost doubled in England and Wales during the period 1979–1996. We further analysed the mortality statistics to determine which anatomical subcategories were involved.

METHODS

Mortality statistics for liver tumours of ICD-9 155, 156, and subcategories, and for tumours of the pancreas (ICD-9 157), in England and Wales were investigated from the Office for National Statistics, London, from 1968 to 1996 inclusive. Data for 1997 and 1998 were also available on intrahepatic cholangiocarcinomas.

RESULTS

There has been a marked rise in age standardised mortality rates for intrahepatic cholangiocarcinoma. Since 1993, it represents the commonest recorded cause of liver tumour related death in England and Wales. This is evident in age groups older than 45 years. In contrast, mortality trends from other primary liver tumours, including hepatocellular carcinoma, were unremarkable.

CONCLUSIONS

The observed increase in mortality from intrahepatic cholangiocarcinoma may represent better case ascertainment and diagnosis due to improved diagnostic imaging, use of image guided biopsies, or increased use of ERCP. However, the trend started before ERCP was introduced nationally, mortality rates have continued to increase steadily thereafter, and there is no clear evidence that diagnostic transfers easily explains the findings. Alternatively, these observations may represent a true increase in intrahepatic bile duct tumours. Epidemiological studies are required to determine whether there is any geographical clustering of cases around the UK.
Acute diquat poisoning with intracerebral bleeding
Authors: Saeed S A M , Wilks M F , Coupe M
Published: July 2002

A case of severe diquat poisoning complicated by the development of aggressive behaviour, oliguric renal failure, and intracerebral bleeding is described. The patient was successfully managed and made a complete recovery. In this paper special attention has been given to the major clinical differences between diquat and paraquat intoxication.
Ozonation of kraft pulp mill effluents: process dynamics
Authors: El-Din M G , Smith Daniel W
Published: July 2002

Pulp mill wastewater discharged from an aerated stabilization basin (ASB) of a kraft pulp mill was ozonated in an extra-coarse-bubble diffuser contactor. The ozonation process is dynamic. Therefore, modeling the reduction efficiencies of contaminants present in the pulp mill effluent and the ozone gas absorption process became more complex than the pure water situation. The ozonation-process-mitigated reduction of colour, absorbable organic halides (AOX), chemical oxygen demand (COD), and total organic carbon (TOC) were examined. Ozone was more effective in reducing the colour and AOX than in reducing the COD and TOC. At an ozone dose (ΔΔO3) of 230 mg L1, the maximum reduction efficiencies of colour, AOX, COD, and TOC were 86, 44, 22, and 15%, respectively. Ozonation was also very effective in increasing the biochemical oxygen demand (BOD5) of the pulp mill effluent. The biochemical oxygen demand increased by about 320% at ΔΔO3 of 230 mg L1. Ozone gas absorption dynamics were investigated by continuous monitoring of the feed-gas and off-gas ozone concentrations and flowrates. Owing to the occurrence of rapid reactions between ozone and some contaminants in the liquid phase, the mass transfer process was enhanced by an enhancement factor, E. The application of the well-known two-film theory and the assumption that the ozone reactions followed pseudo-first-order irreversible kinetics, the overall mass transfer coefficient, kLa, and the enhancement factor, E, were determined as functions of ΔΔO3. The induced reductions of pulp mill effluent contaminants and ozone gas absorption dynamics were compared to those obtained in other ozonation systems to investigate the effects of contactor design, configuration, operating conditions, and scale on the performance of the ozonation process.

Key words: absorbable organic halides, chemical oxygen demand, colour, enhancement factor, mass transfer, ozonation process dynamics, total organic carbon.