bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection and Multigenic Characterization of a Herpesvirus Associated with Malignant Catarrhal Fever in White-Tailed Deer (
Odocoileus virginianus
) from Missouri
Authors: Kleiboeker Steven B. , Miller Margaret A. , Schommer Susan K. , Ramos-Vara Jose A. , Boucher Magalie , Turnquist Susan E.
Published: July 2002

Between 1998 and 2001, tissues from four captive white-tailed deer were observed to have histologic lesions of systemic lymphocytic vasculitis. These lesions suggested malignant catarrhal fever, although epizootic hemorrhagic disease and bluetongue were included in the differential diagnosis. Initial diagnostic efforts, including virus isolation and reverse transcription-PCR for epizootic hemorrhagic disease virus and bluetongue virus, failed to identify an etiologic agent. However, consensus primer PCR targeted to the herpesvirus DNA polymerase gene detected viral genomic DNA in each of these four cases. Nucleotide sequence analysis of the amplified product demonstrated that the detected virus was identical over the compared region to the recently described malignant catarrhal fever virus of white-tailed deer (H. Li, N. Dyer, J. Keller, and T. B. Crawford, J. Clin. Microbiol. 38:1313-1318, 2000). Additional nucleotide sequencing of both the DNA polymerase gene and DNA packaging gene followed by phylogenetic analysis solidified this newly recognized herpesvirus as a member of the
Gammaherpesvirinae
and suggests that this virus, along with ovine herpesvirus 2, alcelaphine herpesvirus 1, alcelaphine herpesvirus 2 and caprine herpesvirus 2, may be part of a separate clade within this subfamily.
Application of the C
18
-Carboxypropylbetaine Specimen Processing Method to Recovery of
Mycobacterium avium
subsp.
paratuberculosis
from Ruminant Tissue Specimens
Authors: Thornton Charles G. , MacLellan Kerry M. , Stabel Judith R. , Carothers Christine , Whitlock Robert H. , Passen Selvin
Published: July 2002

The causative agent of Johne's disease is
Mycobacterium avium
subsp.
paratuberculosis
. This is a chronic, debilitating gastrointestinal disorder that affects ruminants and is responsible for significant economic loss. The specimen processing method that combines C
18
-carboxypropylbetaine (CB-18) treatment and lytic enzyme decontamination has been shown to improve the diagnosis of mycobacterioses. This processing method was applied to the isolation of
M. avium
subsp.
paratuberculosis
from ruminant tissue samples. The BACTEC 12B liquid culture system was used but was supplemented with 1% egg yolk emulsion, 4 μg of mycobactin J, and 0.5% pyruvate (12B/EMP) for use in conjunction with this method. The final concentration of antibiotics used was 10 μg of vancomycin, 30 μg of amphotericin B, and 20 μg of nalidixic acid (VAN) per ml. A 7H10-based solid medium was also used that included mycobactin J, pyruvate, and VAN but excluded the egg yolk emulsion (7H10/MPV). Several
M. avium
subsp.
paratuberculosis
isolates were examined during the evaluation of this processing method. It was observed that treatment with lytic enzymes stimulated the growth of
M. avium
subsp.
paratuberculosis
; however, the growth of one isolate was markedly inhibited due to the presence of vancomycin. Subsequently, the vancomycin concentration in the VAN formulation was reduced to 2 μg/ml. A blinded panel of 60 previously characterized tissue samples from bovine and bison were then processed and analyzed by smear and culture. Historically, 31 and 37 specimens were classified as positive by histology and culture, respectively. The overall sensitivity and specificity of smear relative to culture following CB-18 processing were 97.6 and 89.5%, respectively. The 12B/EMP/VAN liquid culture system recovered
M. avium
subsp.
paratuberculosis
from 39 specimens, whereas 7H10/MPV and Herrold's egg yolk media recovered
M. avium
subsp.
paratuberculosis
from 26 and 16 specimens, respectively. The average times to positive were 7.4 ± 8.3, 29.9 ± 2.6, and 24 ± 0 days, respectively. The contamination rates were 4.8, 22.6, and 20.0%, respectively.
Evidence of the Presence of IS
1245
and IS
1311
or Closely Related Insertion Elements in Nontuberculous Mycobacteria outside of the
Mycobacterium avium
Complex
Authors: Keller Andreas P. , Beggs Marjorie L. , Amthor Beate , Bruns Fabian , Meissner Peter , Haas Walter H.
Published: July 2002

A PCR assay based on the simultaneous detection of IS
1245
and IS
1311
was developed and used to determine the host range of these insertion elements. Specific PCR products were observed in
Mycobacterium malmoense
,
Mycobacterium scrofulaceum,
and
Mycobacterium nonchromogenicum
, indicating that IS
1245
and IS
1311
are not limited to the
Mycobacterium avium
complex.
Tetrazolium Microplate Assay as a Rapid and Inexpensive Colorimetric Method for Determination of Antibiotic Susceptibility of
Mycobacterium tuberculosis
Authors: Caviedes Luz , Delgado Jose , Gilman Robert H.
Published: July 2002

The emergence of multidrug-resistant tuberculosis underscores the need for low-cost, rapid methods to determine the susceptibility of
Mycobacterium tuberculosis
to antibiotics. A new, rapid, easily read, and inexpensive colorimetric method with a tetrazolium indicator performs this determination as quickly and accurately as the more expensive Alamar Blue technique.
Field Evaluation of a Rapid Immunochromatographic Test for Tuberculosis
Authors: Gounder Celine , de Queiroz Mello Fernanda Carvalho , Conde Marcus B. , Bishai William R. , Kritski Afrânio L. , Chaisson Richard E. , Dorman Susan E.
Published: July 2002

Rapid diagnostic tests for tuberculosis (TB) are needed to facilitate early treatment of TB and prevention of
Mycobacterium tuberculosis
transmission. The ICT Tuberculosis test is a rapid, card-based immunochromatographic test for detection of antibodies directed against
M. tuberculosis
antigens. The objective of the study was to evaluate the performance of the ICT Tuberculosis test for the diagnosis of active pulmonary TB (PTB) with whole blood, plasma, and serum from patients suspected of having PTB and from asymptomatic controls in a setting with a high prevalence of PTB. Seventy patients suspected of having PTB (and who were later confirmed to have or not to have PTB by use of
M. tuberculosis
culture as the “gold standard”) and 42 controls were studied. Twenty-one controls were neither vaccinated with
Mycobacterium bovis
bacillus Calmette-Guérin (BCG) nor tuberculin skin test (TST) positive (group A controls), and 21 controls were TST positive and/or had previously been vaccinated with BCG (group B controls). Study subjects were drawn from one hospital and one primary health care unit in Rio de Janeiro City, Brazil. One version of the test (ICT-1) was evaluated by using whole blood, plasma, and serum samples. Sera obtained for this study were frozen and later tested with a manufacturer-modified version of the test (ICT-2). Among the patients suspected of having PTB, the sensitivities of the ICT-1 with whole blood, serum, and plasma were 83, 65, and 70%, respectively, and the specificities were 46, 67, and 56%, respectively. Among the group A controls, the specificities of ICT-1 with the three specimen types were 95, 100, and 95%, respectively. Among the group B controls, the specificities of ICT-1 with the three specimen types were 71, 86, and 86%, respectively. Among the patients suspected of having PTB, the sensitivity of ICT-2 was 70% and the specificity was 65%. Among the group A controls, the specificity of ICT-2 was 95%, and among the group B controls, the specificity of ICT-2 was 81%. With a 29% observed prevalence of PTB among patients suspected of having PTB, the positive predictive values of the ICT tests ranged from 39 to 50% and the negative predictive values ranged from 82 to 87%. The ICT Tuberculosis tests were not sufficiently predictive to warrant their widespread use as routine diagnostic tests for PTB in this setting. However, further evaluation of these tests in specific epidemiologic settings may be warranted.
Pulmonary Infiltrates in Immunosuppressed Patients: Analysis of a Diagnostic Protocol
Authors: Danés Cristina , González-Martín Julián , Pumarola Tomàs , Rañó Ana , Benito Natividad , Torres Antoni , Moreno Asunción , Rovira Montserrat , Puig de la Bellacasa Jorge
Published: July 2002

A diagnostic protocol was started to study the etiology of pulmonary infiltrates in immunosuppressed patients. The diagnostic yields of the different techniques were analyzed, with special emphasis on the importance of the sample quality and the role of rapid techniques in the diagnostic strategy. In total, 241 patients with newly developed pulmonary infiltrates within a period of 19 months were included. Noninvasive or invasive evaluation was performed according to the characteristics of the infiltrates. Diagnosis was achieved in 202 patients (84%); 173 patients (72%) had pneumonia, and specific etiologic agents were found in 114 (66%). Bronchoaspirate and bronchoalveolar lavage showed the highest yields, either on global analysis (23 of 35 specimens [66%] and 70 of 134 specimens [52%], respectively) or on analysis of each type of pneumonia. A tendency toward better results with optimal-quality samples was observed, and a statistically significant difference was found in sputum bacterial culture. Rapid diagnostic tests yielded results in 71 of 114 (62.2%) diagnoses of etiological pneumonia.
Cell-Dependent Requirement of Human Immunodeficiency Virus Type 1 gp41 Cytoplasmic Tail for Env Incorporation into Virions
Authors: Akari Hirofumi , Fukumori Tomoharu , Adachi Akio
Published: July 2002

Growth kinetics in lymphocytic H9 and M8166 cells of two mutants of human immunodeficiency virus type 1 (HIV-1) with deleted gp41 cytoplasmic tails were examined. While the mutant viruses designated CTdel-44 and CTdel-144 were able to grow in M8166 cells, they were unable to grow in H9 cells. Transfection and single-round infectivity assays demonstrated that they are defective in the early phase of viral replication in H9 cells. Analysis of the mutant virions revealed drastically reduced incorporation of Env gp120 (compared with the incorporation of wild-type virions) in H9 cells but normal incorporation in M8166 cells. These results indicate that the HIV-1 cytoplasmic tail of gp41 determines virus infectivity in a cell-dependent manner by affecting incorporation of Env into virions and suggest the involvement of a host cell factor(s) in the Env incorporation.
Adaptation and Selection of Prion Protein Strain Conformations following Interspecies Transmission of Transmissible Mink Encephalopathy
Authors: Bartz Jason C. , Bessen Richard A. , McKenzie Debbie , Marsh Richard F. , Aiken Judd M.
Published: July 2002

Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrP
Sc
). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrP
Sc
following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrP
Sc
strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10
−5
dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrP
Sc
strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrP
Sc
conformations that underwent selection until one type of PrP
Sc
conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrP
Sc
conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrP
Sc
conformation that can determine the TSE strain phenotype.
Molecular Characterization of an Avian Astrovirus
Authors: Koci Matthew D. , Seal Bruce S. , Schultz-Cherry Stacey
Published: July 2002

Astroviruses are known to cause enteric disease in several animal species, including turkeys. However, only human astroviruses have been well characterized at the nucleotide level. Herein we report the nucleotide sequence, genomic organization, and predicted amino acid sequence of a turkey astrovirus isolated from poults with an emerging enteric disease.
CD4-Independent, CCR5-Dependent Simian Immunodeficiency Virus Infection and Chemotaxis of Human Cells
Authors: Iyengar Sujatha , Schwartz David H. , Clements Janice E. , Hildreth James E. K.
Published: July 2002

Most simian immunodeficiency virus (SIV), human immunodeficiency virus type 2 (HIV-2), and HIV-1 infection of host peripheral blood mononuclear cells (PBMCs) is CD4 dependent. In some cases, X4 HIV-1 chemotaxis is CD4 independent, and cross-species transmission might be facilitated by CD4-independent entry, which has been demonstrated for some SIV strains in CD4−non-T cells. As expected for CCR5-dependent virus, SIV required CD4 on rhesus and pigtail macaque PBMCs for infection and chemotaxis. However, SIV induced the chemotaxis of human PBMCs in a CD4-independent manner. Furthermore, in contrast to the results of studies using transfected human cell lines, SIV did not require CD4 binding to productively infect primary human PBMCs. CD4-independent lymphocyte and macrophage infection may facilitate cross-species transmission, while reacquisition of CD4 dependence may confer a selective advantage for the virus within new host species.
Evaluation of Hepatitis C Virus Glycoprotein E2 for Vaccine Design: an Endoplasmic Reticulum-Retained Recombinant Protein Is Superior to Secreted Recombinant Protein and DNA-Based Vaccine Candidates
Authors: Heile Jens M. , Fong Yiu-Lian , Rosa Domenico , Berger Kim , Saletti Giulietta , Campagnoli Susanna , Bensi Giuliano , Capo Sabrina , Coates Steve , Crawford Kevin , Dong Christine , Wininger Mark , Baker Gary , Cousens Larry , Chien David , Ng Philip , Archangel Phillip , Grandi Guido , Houghton Michael , Abrignani Sergio
Published: July 2002

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.
Two Putative α-Helical Domains of Human Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by at Least Two Mechanisms
Authors: Kamata Masakazu , Aida Yoko
Published: July 2002

To identify the domains of Vpr that are involved nuclear localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within αH1 or αH2 resulted, by contrast, in diffuse expression over the entire cell. In addition, double mutations within both of these α-helical domains led to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the αH1 and αH2 domains fused individually with green fluorescent protein (GFP) and a Flag tag and extracted them with digitonin and Triton X-100 prior to fixation. Flag-αH1-GFP was detected in the nucleus but not in the cytoplasm, while Flag-αH2-GFP was retained predominantly in the nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two α-helical domains might be involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative α-helical domains mediate the nuclear localization of Vpr by at least two mechanisms.
The Mouse Mammary Tumor Virus Transcription Enhancers for Hematopoietic Progenitor and Mammary Gland Cells Share Functional Elements
Authors: Reuss Frank U. , Coffin John M.
Published: July 2002

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. We have previously established a critical role of an enhancer element within the long terminal repeat (LTR) for MMTV
sag
gene expression in B-lymphoid progenitor cells. We now demonstrate enhancer activity of this element in a promyelocytic progenitor cell line. We also map the position of the enhancer within the U3 region of the MMTV LTR and show that the progenitor cell enhancer shares functional elements with a previously described mammary gland-specific enhancer.
Interaction of Cellular Proteins with the 5′ End of Norwalk Virus Genomic RNA
Authors: Gutiérrez-Escolano Ana Lorena , Brito Zamirath Uribe , del Angel Rosa M. , Jiang Xi
Published: July 2002

The lack of a susceptible cell line and an animal model for Norwalk virus (NV) infection has prompted the development of alternative strategies to generate in vitro RNAs that approximate the authentic viral genome. This approach has allowed the study of viral RNA replication and gene expression. In this study, using mobility shift and cross-linking assays, we detected several cellular proteins from HeLa and CaCo-2 cell extracts that bind to, and form stable complexes with, the first 110 nucleotides of the 5′ end of NV genomic RNA, a region previously predicted to form a double stem-loop structure. These proteins had molecular weights similar to those of the HeLa cellular proteins that bind to the internal ribosomal entry site of poliovirus RNA. HeLa proteins La, PCBP-2, and PTB, which are important for poliovirus translation, and hnRNP L, which is possibly implicated in hepatitis C virus translation, interact with NV RNA. These protein-RNA interactions are likely to play a role in NV translation and/or replication.
Putative Immunodominant Human Immunodeficiency Virus-Specific CD8+T-Cell Responses Cannot Be Predicted by Major Histocompatibility Complex Class I Haplotype
Authors: Betts Michael R. , Casazza Joseph P. , Patterson Brent A. , Waldrop Shar , Trigona Wendy , Fu Tong-Ming , Kern Florian , Picker Louis J. , Koup Richard A.
Published: July 2002

Recent studies of human immunodeficiency virus (HIV)-specific CD8+T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8+T-cell response complexity using cytokine flow cytometry to examine CD8+T-cell responses in 11 HLA-A2+HIV+individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8+T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8+T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8+T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8+T-cell responses will require assessment of the response to all possible epitopes.
Combination of CCR5 and CXCR4 Inhibitors in Therapy of Human Immunodeficiency Virus Type 1 Infection: In Vitro Studies of Mixed Virus Infections
Authors: Rusconi Stefano , La Seta Catamancio Simona , Citterio Paola , Bulgheroni Elisabetta , Croce Francesco , Herrmann Steven H. , Offord Robin E. , Galli Massimo , Hirsch Martin S.
Published: July 2002

We studied the combined anti-human immunodeficiency virus type 1 (HIV-1) effects of a derivative of stroma-derived factor 1β (SDF-1β), Met-SDF-1β, and a modified form of RANTES, aminooxypentane (AOP)-RANTES. The antiviral agents were tested singly or in combination at 95 and 99% virus inhibitory concentrations. Clinical R5 and X4 HIV-1 isolates were used. AOP-RANTES inhibited R5 but not X4 viruses, whereas Met-SDF-1β had the opposite effect. Combinations of these compounds inhibited mixed infections with R5 and X4 viruses (95 to 99%), whereas single drugs were less inhibitory (32 to 61%). Combinations of R5 and X4 inhibitors are promising and deserve further evaluation.
Second-Site Changes Affect Viability of Amphotropic/Ecotropic Chimeric Enveloped Murine Leukemia Viruses
Authors: O'Reilly Lucille , Roth Monica J.
Published: July 2002

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the
env
gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.
A TVA–Single-Chain Antibody Fusion Protein Mediates Specific Targeting of a Subgroup A Avian Leukosis Virus Vector to Cells Expressing a Tumor-Specific Form of Epidermal Growth Factor Receptor
Authors: Snitkovsky Sophie , Niederman Thomas M. J. , Carter Bob S. , Mulligan Richard C. , Young John A. T.
Published: July 2002

We have previously described an approach that employs retroviral receptor-ligand bridge proteins to target retroviral vectors to specific cell types. To determine whether targeted retroviral entry can also be achieved using a retroviral receptor–single-chain antibody bridge protein, the TVA-MR1 fusion protein was generated. TVA-MR1 is comprised of the extracellular domain of the TVA receptor for subgroup A avian leukosis viruses (ALV-A), fused to the MR1 single-chain antibody that binds specifically to EGFRvIII, a tumor-specific form of the epidermal growth factor receptor. We show that TVA-MR1 binds specifically to a murine version of EGFRvIII and promotes ALV-A entry selectively into cells that express this cell surface marker. These studies demonstrate that it is possible to target retroviral vectors to specific cell types through the use of a retroviral receptor–single-chain antibody fusion protein.
Cytopathicity of Human Immunodeficiency Virus Type 2 (HIV-2) in Human Lymphoid Tissue Is Coreceptor Dependent and Comparable to That of HIV-1
Authors: Schramm Birgit , Penn Michael L. , Palacios Emil H. , Grant Robert M. , Kirchhoff Frank , Goldsmith Mark A.
Published: July 2002

Epidemiological studies have shown that human immunodeficiency virus type 2 (HIV-2) is markedly less pathogenic than HIV-1 in vivo. Individuals infected with HIV-2 exhibit a remarkably slow rate of disease development, and these clinical properties have been attributed presumptively to an “attenuated” phenotype of HIV-2 itself. Here, we investigated the impact of coreceptor usage on the cytopathicity of HIV-2 and compared its pathogenic potential with that of HIV-1 in a unique human lymphoid histoculture model. We found that HIV-2 strains, as well as closely related simian immunodeficiency viruses (SIV), displayed mildly or highly aggressive cytopathic phenotypes depending on their abilities to use the coreceptor CCR5 or CXCR4, respectively. A side-by-side comparison of primary X4 HIV-1 and HIV-2 strains revealed similar, high degrees of cytopathicity induced by both HIV types. Furthermore, we found that HIV-2 coreceptor specificity for CCR5 and CXCR4 determined the target cell population for T-cell depletion in lymphoid tissue. Finally, utilization of the alternate coreceptors BOB and Bonzo did not significantly increase the cytopathic properties of HIV-2. These findings demonstrate that coreceptor preference is a key regulator of target cell specificity and the cytopathic potential of HIV-2, with indistinguishable rules compared with HIV-1. Moreover, HIV-2 strains are not characterized by an intrinsically lower cytopathicity than HIV-1 strains. Therefore, direct cytopathic potential per se does not explain the unique behavior of HIV-2 in people, highlighting that other unknown factors need to be elucidated as the basis for their lesser virulence in vivo.
A Human Immunodeficiency Virus Prime-Boost Immunization Regimen in Humans Induces Antibodies That Show Interclade Cross-Reactivity and Neutralize Several X4-, R5-, and Dualtropic Clade B and C Primary Isolates
Authors: Verrier Florence , Burda Sherri , Belshe Robert , Duliege Anne-Marie , Excler Jean-Louis , Klein Michel , Zolla-Pazner Susan
Published: July 2002

A human immunodeficiency virus (HIV) vaccine that will be useful in diverse geographic regions will need to induce a broad immune response characterized by cross-clade immunity. To test whether a clade B-based HIV candidate vaccine could induce interclade humoral responses, including neutralizing activity against primary HIV-1 isolates, sera were tested from recipients of a vaccine consisting of recombinant canarypox virus vCP205 and recombinant gp120SF2. Serum antibodies exhibited strong immunochemical cross-reactivity with V3 peptides from clades B, C, and F, with weaker activity for several V3 peptides from clades A, D, G, and H; essentially no reactivity could be demonstrated with V3 peptides from clades E and O. Extensive cross-clade reactivity was also documented by enzyme-linked immunosorbent assay with all nine recombinant HIV envelope glycoproteins tested from clades B, D, and E. In addition, vaccinees' sera displayed significant neutralizing activity against 5 of 14 primary isolates tested, including one X4 virus and two dualtropic viruses (from clade B) and two R5 viruses (from clades B and C). This is the first demonstration of the induction by a candidate HIV vaccine constructed from clade B laboratory strains of HIV of neutralizing activity against R5 and clade C primary isolates. The data suggest that, by virtue of their ability to induce cross-clade immune responses, appropriately formulated HIV vaccines based on a finite number of HIV isolates may ultimately be able to protect against the wide range of HIV isolates affecting the populations of many geographic regions.
Unprecedented Degree of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Genetic Diversity in the Democratic Republic of Congo Suggests that the HIV-1 Pandemic Originated in Central Africa
Authors: Vidal Nicole , Peeters Martine , Mulanga-Kabeya Claire , Nzilambi Nzila , Robertson David , Ilunga Wantabala , Sema Hurogo , Tshimanga Kazadi , Bongo Beni , Delaporte Eric
Published: July 2002

The purpose of this study was to document the genetic diversity of human immunodeficiency virus type 1 (HIV-1) in the Democratic Republic of Congo (DRC; formerly Zaire). A total of 247 HIV-1-positive samples, collected during an epidemiologic survey conducted in 1997 in three regions (Kinshasa [the capital], Bwamanda [in the north], and Mbuyi-Maya [in the south]), were genetically characterized in the
env
V3-V5 region. All known subtypes were found to cocirculate, and for 6% of the samples the subtype could not be identified. Subtype A is predominant, with prevalences decreasing from north to south (69% in the north, 53% in the capital city, and 46% in the south). Subtype C, D, G, and H prevalences range from 7 to 9%, whereas subtype F, J, K, and CRF01-AE strains represent 2 to 4% of the samples; only one subtype B strain was identified. The highest prevalence (25%) of subtype C was in the south, and CRF01-AE was seen mainly in the north. The high intersubtype variability among the V3-V5 sequences is the most probable reason for the low (45%) efficiency of subtype A-specific PCR and HMA (heteroduplex mobility assay). Eighteen (29%) of 62 samples had discordant subtype designations between
env
and
gag
. Sequence analysis of the entire envelope from 13 samples confirmed the high degree of diversity and complexity of HIV-1 strains in the DRC; 9 had a complex recombinant structure in gp160, involving fragments of known and unknown subtypes. Interestingly, the unknown fragments from the different strains did not cluster together. Overall, the high number of HIV-1 subtypes cocirculating, the high intrasubtype diversity, and the high numbers of possible recombinant viruses as well as different unclassified strains are all in agreement with an old and mature epidemic in the DRC, suggesting that this region is the epicenter of HIV-1 group M.
The Anti-Influenza Virus Agent 4-GU-DANA (Zanamivir) Inhibits Cell Fusion Mediated by Human Parainfluenza Virus and Influenza Virus HA
Authors: Greengard Olga , Poltoratskaia Natalia , Leikina Evgenia , Zimmerberg Joshua , Moscona Anne
Published: July 2002

4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced the number (but not the area) of plaques if present only during the adsorption period and reduced plaque area (but not number) if added only after the 90-min adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that its interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque number or blocking hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.
Expression of the Surface Glycoproteins of Human Parainfluenza Virus Type 3 by Bovine Parainfluenza Virus Type 3, a Novel Attenuated Virus Vaccine Vector
Authors: Haller Aurelia A. , Miller Tessa , Mitiku Misrach , Coelingh Kathleen
Published: July 2002

Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39°C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37°C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses.
Identification of Multiple Transcription Factors, HLF, FTF, and E4BP4, Controlling Hepatitis B Virus Enhancer II
Authors: Ishida Hisashi , Ueda Keiji , Ohkawa Kazuyoshi , Kanazawa Yoshiyuki , Hosui Atsushi , Nakanishi Fumihiko , Mita Eiji , Kasahara Akinori , Sasaki Yutaka , Hori Masatsugu , Hayashi Norio
Published: July 2002

Hepatitis B virus (HBV) enhancer II (EnII) is a hepatotropic
cis
element which is responsible for the hepatocyte-specific gene expression of HBV. Multiple transcription factors have been demonstrated to interact with this region. In this study, the region from HBV nucleotides (nt) 1640 to 1663 in EnII was demonstrated to be essential for enhancer activity and to be another target sequence of putative transcription factors. To elucidate the factors which bind to this region, we used a yeast one-hybrid screening system and cloned three transcription factors, HLF, FTF, and E4BP4, from a human adult liver cDNA library. All of these factors had binding affinity to the sequence from nt 1640 to 1663. Investigation of the effects of these factors on transcriptional regulation revealed that HLF and FTF had stimulatory activity on nt 1640 to 1663, whereas E4BP4 had a suppressing effect. FTF coordinately activated both 3.5-kb RNA and 2.4/2.1-kb RNA transcription in a transient transfection assay with an HBV expression vector. HLF, however, activated only 3.5-kb RNA transcription, and in primer extension analysis, HLF strongly stimulated the synthesis of pregenome RNA compared to precore RNA. Thus, FTF stimulated the activity of the second enhancer, while HLF stimulated the activity of the core upstream regulatory sequence, which affects only the core promoter, and had a dominant effect on the pregenome RNA synthesis.
Sialylation of the Host Receptor May Modulate Entry of Demyelinating Persistent Theiler's Virus
Authors: Zhou Lan , Luo Yu , Wu Yan , Tsao Jun , Luo Ming
Published: July 2002

Theiler's murine encephalomyelitis virus
(TMEV) is a picornavirus of the
Cardiovirus
genus. Certain strains of TMEV may cause a chronic demyelinating disease, which is very similar to multiple sclerosis in humans, associated with a persistent viral infection in the mouse central nervous system (CNS). Other strains of TMEV only cause an acute infection without persistence in the CNS. It has been shown that sialic acid is a receptor moiety only for the persistent TMEV strains and not for the nonpersistent strains. We report the effect of sialylation on cell surface on entry and the complex structure of DA virus, a persistent TMEV, and the receptor moiety mimic, sialyllactose, refined to a resolution of 3.0 Å. The ligand binds to a pocket on the viral surface, composed mainly of the amino acid residues from capsid protein VP2 puff B, in the vicinity of the VP1 loop and VP3 C terminus. The interaction of the receptor moiety with the persistent DA strain provides new understanding for the demyelinating persistent infection in the mouse CNS by TMEV.
Selective Interactions of Polyanions with Basic Surfaces on Human Immunodeficiency Virus Type 1 gp120
Authors: Moulard Maxime , Lortat-Jacob Hugues , Mondor Isabelle , Roca Guillaume , Wyatt Richard , Sodroski Joseph , Zhao Lu , Olson William , Kwong Peter D. , Sattentau Quentin J.
Published: July 2002

It is well established that the gp120 V3 loop of T-cell-line-adapted human immunodeficiency virus type 1 (HIV-1) binds both cell-associated and soluble polyanions. Virus infectivity is increased by interactions between HIV-1 and heparan sulfate proteoglycans on some cell types, and soluble polyanions such as heparin and dextran sulfate neutralize HIV-1 in vitro. However, the analysis of gp120-polyanion interactions has been limited to T-cell-line-adapted, CXCR4-using virus and virus-derived gp120, and the polyanion binding ability of gp120 regions other than the V3 loop has not been addressed. Here we demonstrate by monoclonal-antibody inhibition, labeled heparin binding, and surface plasmon resonance studies that a second site, most probably corresponding to the newly defined, highly conserved coreceptor binding region on gp120, forms part of the polyanion binding surface. Consistent with the binding of polyanions to the coreceptor binding surface, dextran sulfate interfered with the gp120-CXCR4 association while having no detectable effect on the gp120-CD4 interaction. The interaction between polyanions and X4 or R5X4 gp120 was readily detectable, whereas weak or undetectable binding was observed with R5 gp120. Analysis of mutated forms of X4 gp120 demonstrated that the V3 loop is the major determinant for polyanion binding whereas other regions, including the V1/V2 loop structure and the NH2and COOH termini, exert a more subtle influence. A molecular model of the electrostatic potential of the conserved coreceptor binding region confirmed that it is basic but that the overall charge on this surface is dominated by the V3 loop. These results demonstrate a selective interaction of gp120 with polyanions and suggest that the conserved coreceptor binding surface may present a novel and conserved target for therapeutic intervention.
Pathogenic Conversion of Live Attenuated Simian Immunodeficiency Virus Vaccines Is Associated with Expression of Truncated Nef
Authors: Sawai Earl T. , Hamza M. Sabry , Ye Michael , Shaw Karen E. S. , Luciw Paul A.
Published: July 2002

Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large
nef
deletion (SIVmac239Δ
152
nef) or interleukin-2 in place of
nef
developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.
Production of Recombinant Snakehead Rhabdovirus: the NV Protein Is Not Required for Viral Replication
Authors: Johnson Marc C. , Simon Benjamin E. , Kim Carol H. , Leong Jo-Ann C.
Published: July 2002

Snakehead rhabdovirus
(SHRV) affects warm water fish in Southeast Asia and belongs to the genus
Novirhabdovirus
by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31°C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11,550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.
Role of the Gag Matrix Domain in Targeting Human Immunodeficiency Virus Type 1 Assembly
Authors: Ono Akira , Orenstein Jan M. , Freed Eric O.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6
Gag
or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.
The M184V Mutation in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Impairs Rescue of Chain-Terminated DNA Synthesis
Authors: Götte Matthias , Arion Dominique , Parniak Michael A. , Wainberg Mark A.
Published: July 2002

Nucleoside analog chain terminators such as 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxy-3′-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.