bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid and Simple Approach for Identification of
Mycobacterium tuberculosis
Complex Isolates by PCR-Based Genomic Deletion Analysis
Authors: Parsons Linda M. , Brosch Roland , Cole Stewart T. , Somoskövi Ákos , Loder Arthur , Bretzel Gisela , van Soolingen Dick , Hale Yvonne M. , Salfinger Max
Published: July 2002

Although the virulences and host ranges differ among members of the
Mycobacterium tuberculosis
complex (TBC;
M. tuberculosis
,
M. africanum
,
M. canettii
,
M. microti
,
M. bovis
, and
M. bovis
BCG), commercially available molecular assays cannot differentiate these organisms because of the genetic identities of their 16S rRNA gene sequences. Comparative genomic analyses with the complete DNA sequence of
M. tuberculosis
H37Rv has provided information on regions of difference (RD 1 to RD 16) deleted in members of the TBC other than
M. tuberculosis
. To determine whether deletion analysis could accurately differentiate members of TBC, we used PCR to assess the presence or absence of specific regions of the genome in 88 well-characterized isolates of
M. tuberculosis
,
M. africanum
,
M. microti
,
M. bovis
, and
M. bovis
BCG. The identifications obtained by use of the specific deletion profiles correlated 100% with the original identifications for all TBC members except
M. africanum
, but further characterization resulted in profiles specific for all members. Although six RD regions were used in the analyses with the original 88 isolates, it was found that the use of RD 1, RD 9, and RD 10 was sufficient for initial screenings, followed by the use of RD 3, RD 5, and RD 11 if the results for any of the first three regions were negative. When 605 sequential clinical isolates were screened, 578 (96%) were identified as
M. tuberculosis
, 6 (1%) were identified as
M. africanum
, 8 (1%) were identified as
M. bovis
, and 13 (2%) were identified as
M. bovis
BCG. Since PCR-based assays can be implemented in most clinical mycobacteriology laboratories, this approach provides a rapid and simple means for the differentiation of members of TBC, especially
M. bovis
and
M. tuberculosis
, when it is important to distinguish between zoonotic sources (i.e., cattle and unpasteurized dairy products) and human sources of tuberculosis disease.
Sensitivity of the Procleix HIV-1/HCV Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA in a High-Risk Population
Authors: Jackson J. B. , Smith K. , Knott C. , Korpela A. , Simmons A. , Piwowar-Manning E. , McDonough S. , Mimms L. , Vargo J. M.
Published: July 2002

The Procleix HIV-1/HCV Assay is a high-throughput nucleic acid test for the simultaneous detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNA during blood donor screening. This study evaluated the clinical sensitivity of the Procleix assay and assessed the assay's ability to identify HIV-1- and HCV-infected individuals undetected by standard serologic tests. Plasma samples were obtained prospectively from 539 individuals at high risk for HIV-1 and HCV infection at seven clinics affiliated with Johns Hopkins University. Samples were tested in the Procleix HIV-1/HCV Assay and, if reactive, were then tested in the Procleix HIV-1 and HCV discriminatory assays to differentiate the source of viral RNA positivity. Of these 539 subjects, 287 (53.2%) tested reactive in the Procleix HIV-1/HCV Assay. In discriminatory assay testing, 12 of 287 subjects (4.2%) were reactive for HIV-1 RNA only, 260 (90.6%) were reactive for HCV RNA only, and 11 (3.8%) were coinfected with HIV-1 and HCV. The clinical sensitivity for samples tested neat was 100% for HIV-1 and 99.3% for HCV. Three subjects with Procleix HCV reactive/seronegative results seroconverted upon follow-up and were confirmed as Procleix HCV yield cases. The Procleix HIV-1/HCV Assay is a highly sensitive test that detects ongoing and early HIV-1 and HCV infection in a significant number of subjects at high risk for these diseases. Confirmation of Procleix yield cases upon follow-up demonstrated the ability of the Procleix HIV-1/HCV Assay to detect the presence of HIV-1 and HCV in blood earlier than standard serologic tests.
Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification
Authors: Ahmad Suhail , Khan Zaiba , Mustafa Abu S. , Khan Zia U.
Published: July 2002

The rapid detection and identification of
Candida
species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of
Candida
are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of
Candida
species in serum specimens. The universal outer primers amplified the 3′ end of 5.8S ribosomal DNA (rDNA) and the 5′ end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350- to 410-bp fragments from the four commonly encountered
Candida
species, viz.,
C. albicans
,
C. tropicalis
,
C. glabrata
, and
C. parapsilosis.
The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of
Candida
detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of
Candida
species with 76 clinical
Candida
isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of
Candida
species in sera from culture-proven (
n
= 12), suspected (
n
= 16), and superficially colonized (
n
= 10) patients and healthy subjects (
n
= 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two
Candida
species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for
Candida
, nine patients (56%) were positive by snPCR; two of them had dual infection with
C. albicans
and either
C. tropicalis
or
C. glabrata
. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of
Candida
species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one
Candida
species is an additional advantage.
Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing
Escherichia coli
Authors: Reischl Udo , Youssef Mohammad T. , Kilwinski Jochen , Lehn Norbert , Zhang Wen Lan , Karch Helge , Strockbine Nancy A.
Published: July 2002

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing
Escherichia coli
(STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (
stx
1
and
stx
2
) and another that simultaneously detects the genes for intimin (
eae
) and enterohemolysin (E-
hly
). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of
E. coli
negative for
stx
genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the
stx
1
,
eae
, and E-
hly
genes and 96 and 100%, respectively, for the
stx
2
gene. No
stx
2
genes were detected from 10
stx
2f
-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.
Differences between
Staphylococcus aureus
Isolates from Medical and Nonmedical Hospital Personnel
Authors: Cespedes Christian , Miller Maureen , Quagliarello Bianca , Vavagiakis Peter , Klein Robert S. , Lowy Franklin D.
Published: July 2002

It is unclear whether the levels of
Staphylococcus aureus
colonization of hospital personnel with patient exposure are increased or whether personnel become colonized with more antibiotic-resistant strains. Differences in nasal and hand carriage of
S. aureus
between medical and nonmedical hospital personnel were examined. No differences in nasal carriage between the two groups were found; however, there was a trend that suggested differences in the rates of hand carriage of
S
.
aureus
(18% of nonmedical personnel and 10% of medical personnel). Medical personnel were colonized with more antibiotic-resistant isolates than nonmedical personnel (mean, 2.8 versus 2.1 isolates [
P
< 0.03]), and the strain profiles indicated that they tended to be more clonal in origin, suggesting that exposure to hospital isolates alters the colonization profile.
Transmission of
Campylobacter hyointestinalis
from a Pig to a Human
Authors: Gorkiewicz Gregor , Feierl Gebhard , Zechner Rudolf , Zechner Ellen L.
Published: July 2002

We report on a case of human gastroenteritis caused by the pathogen
Campylobacter hyointestinalis
. Recurrent watery diarrhea and intermittent vomiting were the most significant symptoms of the previously healthy patient. Whole-cell protein electrophoresis and 16S rRNA gene sequencing were used to identify this
Campylobacter
species. Investigation of the patient's surroundings led to the recovery of a second
C. hyointestinalis
strain originating from porcine feces. Subsequent typing of the human and the porcine isolates by pulsed-field gel electrophoresis revealed similar macrorestriction profiles, indicating transmission of this pathogen.
Large-Scale Outbreak of Infection with
Mycobacterium chelonae
subsp.
abscessus
after Penicillin Injection
Authors: Zhibang Yang , BiXia Zhang , Qishan Lu , Lihao Chen , Xiangquan Liu , Huaping Li
Published: July 2002

An outbreak of infection with
Mycobacterium chelonae
subsp.
abscessus
after the injection of penicillin in 86 patients attending a factory hospital is reported. The bacterium was isolated both from lids and from the soil where the drug was stored. Molecular analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and plasmids revealed a pattern identical to that of the strains isolated from the wounds. The source of the infections was soil contamination of the vial lids and was caused by improper use and sterilization of penicillin vials.
Septicemia Due to
Acinetobacter junii
Authors: Linde Hans-Jörg , Hahn Joachim , Holler Ernst , Reischl Udo , Lehn Norbert
Published: July 2002

Acinetobacter
spp. are considered to be emerging nosocomial pathogens.
Acinetobacter junii
is a rare cause of disease in humans and was associated mainly with bacteremia in preterm infants and pediatric oncologic patients. In this report we describe a case of catheter-related infection by
A. junii
in an adult oncologic patient. Application of molecular methods for precise species identification of
Acinetobacter
spp. will help to further clarify their role as human pathogens.
Human Deep Tissue Infection with an EntomopathogenicBeauveriaSpecies
Authors: Henke Markus Oliver , de Hoog G. Sybren , Gross Uwe , Zimmermann Gisbert , Kraemer Doris , Weig Michael
Published: July 2002

Beauveriaspp. are ubiquitous fungal entomopathogens that are commercially distributed as biological insecticides worldwide. In this paper we describe the clinical manifestation, diagnosis, and therapy of the first documented human deep tissue infection with an entomopathogenicBeauveriaspecies in a patient receiving immunosuppressive therapy and describe the morphological and molecular characterization of the mold.
AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Failure To Protect and Possible Enhancement of Challenge Infection by Four Cell-Based Vaccines Prepared with Autologous Lymphoblasts
Authors: Giannecchini Simone , Isola Patrizia , Sichi Olimpia , Matteucci Donatella , Pistello Mauro , Zaccaro Lucia , Del Mauro Daniela , Bendinelli Mauro
Published: July 2002

Immunogenicity and protective activity of four cell-based feline immunodeficiency virus (FIV) vaccines prepared with autologous lymphoblasts were investigated. One vaccine was composed of FIV-infected cells that were paraformaldehyde fixed at the peak of viral expression. The other vaccines were attempts to maximize the expression of protective epitopes that might become exposed as a result of virion binding to cells and essentially consisted of cells mildly fixed after saturation of their surface with adsorbed, internally inactivated FIV particles. The levels of FIV-specific lymphoproliferation exhibited by the vaccinees were comparable to the ones previously observed in vaccine-protected cats, but antibodies were largely directed to cell-derived constituents rather than to truly viral epitopes and had very poor FIV-neutralizing activity. Moreover, under one condition of testing, some vaccine sera enhanced FIV replication in vitro. As a further limit, the vaccines proved inefficient at priming animals for anamnestic immune responses. Two months after completion of primary immunization, the animals were challenged with a low dose of homologous ex vivo FIV. Collectively, 8 of 20 vaccinees developed infection versus one of nine animals mock immunized with fixed uninfected autologous lymphoblasts. After a boosting and rechallenge with a higher virus dose, all remaining animals became infected, thus confirming their lack of protection.
Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX
Authors: Dmitriev Igor P. , Kashentseva Elena A. , Curiel David T.
Published: July 2002

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.
Heparin Binds to Murine Leukemia Virus and Inhibits Env-Independent Attachment and Infection
Authors: Walker Simon J. , Pizzato Massimo , Takeuchi Yasuhiro , Devereux Stephen
Published: July 2002

Certain glycosaminoglycans (GAGs), including heparin, inhibit infection by murine leukemia virus (MLV). We now show that this is due to inhibition of virus attachment independent of the interaction between viral envelope proteins (Env) and their cellular receptors. Heparin blocked the binding of both Env-deficient and amphotropic MLV (MLV-A) particles to NIH 3T3 fibroblasts, CHO cells which lack the amphotropic retroviral receptor Pit-2, and CHO cells transfected with Pit-2 (CHO-Pit-2). Heparin also inhibited the transduction of NIH 3T3 cells by MLV-A over a similar concentration range. This effect was observed within 15 min of exposure to retrovirus. Preloading target cells with heparin had no effect on transduction and both MLV-A and Env-deficient retrovirus bound efficiently to heparin-coated agarose beads, suggesting that heparin interacts with the virus rather than the target cell. This requires both a strong negative charge and a specific structure since GAGs with different charge and carbohydrate composition inhibited virus infection variably. The specificity of GAG-virus interaction also depends on the producer cells, since virus packaged by murine GP+EnvAM12 cells was 1,000-fold more sensitive to inhibition by chondroitin sulfate A than was virus packaged by human FLYA13 packaging cells. No evidence for an interaction between MLV and cell surface proteoglycans was found, however, since the attachment of MLV-A and envelope-defective virus to proteoglycan-deficient CHOpgsA-745 cells was similar to that seen with both wild-type and CHO-Pit-2 cells. Although the molecular mechanism is unclear, this study presents evidence that Env receptor-independent attachment is an important step in MLV infection.
Template Requirements for De Novo RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase on the Viral X RNA
Authors: Kim Meehyein , Kim Hajeong , Cho Seong-Pil , Min Mi-Kyung
Published: July 2002

The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from
Esherichia coli
, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3′ to 5′. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3′ end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3′ end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.
Coreceptor Phenotype of Natural Human Immunodeficiency Virus with Nef Deleted Evolves In Vivo, Leading to Increased Virulence
Authors: Jekle Andreas , Schramm Birgit , Jayakumar Prerana , Trautner Verena , Schols Dominique , De Clercq Erik , Mills John , Crowe Suzanne M. , Goldsmith Mark A.
Published: July 2002

The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the
nef
long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4
+
T cells than a reference dualtropic,
nef
-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4
+
T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5
+
CD4
+
T cells, the cytotoxic effect of the 1999 isolate toward CCR5
−
CD4
+
T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5
+
and CCR5
−
CXCR4
+
cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5
+
cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this
nef
-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS.
Origin of Human Immunodeficiency Virus Type 1 Quasispecies Emerging after Antiretroviral Treatment Interruption in Patients with Therapeutic Failure
Authors: Kijak Gustavo H. , Simon Viviana , Balfe Peter , Vanderhoeven Jeroen , Pampuro Sandra E. , Zala Carlos , Ochoa Claudia , Cahn Pedro , Markowitz Martin , Salomon Horacio
Published: July 2002

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1
pol
and
env
were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of
pol
revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from
env
sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.
Activity of Polymerase Proteins of Vaccine and Wild-Type Measles Virus Strains in a Minigenome Replication Assay
Authors: Bankamp Bettina , Kearney Sean P. , Liu Xin , Bellini William J. , Rota Paul A.
Published: July 2002

The relative activities of five measles virus (MV) polymerase (L) proteins were compared in an intracellular, plasmid-based replication assay. When coexpressed with N and P proteins from an attenuated strain, L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from an MV minigenome than the three wild-type L proteins. Northern blot analysis demonstrated that the differences in reporter protein production correlated with mRNA transcription levels. Increased activity of polymerases from attenuated viruses equally affected mRNA transcription and minigenome replication. The higher level of transcription may be a consequence of increased template availability or may be an independent effect of the elevated activity of the attenuated polymerases. Coexpression of wild-type L proteins with homologous N and P proteins did not affect the activity of the wild-type polymerases, indicating that the differential activity was a function of the L proteins alone. Use of a minigenome that incorporated two nucleotide changes found in the genomic leader of the three wild-type viruses did not raise the activity of the wild-type L proteins. These data demonstrate that increased polymerase activity differentiates attenuated from wild-type viruses and suggest that functions involved in RNA synthesis contribute to the attenuated phenotype of MV vaccine strains.
The RNA Polymerase of Influenza A Virus Is Stabilized by Interaction with Its Viral RNA Promoter
Authors: Brownlee George G. , Sharps Jane L.
Published: July 2002

The RNA polymerase of the influenza virus is responsible for the transcription and replication of the segmented RNA viral genome during infection of host cells. Polymerase function is known to be strictly dependent on interaction with its RNA promoter, but no attempts to investigate whether the virion RNA (vRNA) promoter stabilizes the polymerase have been reported previously. Here we tested whether the vRNA promoter protects the polymerase against heat inactivation. We prepared partially purified recombinant influenza A virus RNA polymerase, in the absence of influenza virus vRNA promoter sequences, by transient transfection of expression plasmids into human kidney 293T cells. The polymerase was found to be heat labile at 40°C in the absence of added vRNA. However, it was protected from heat inactivation if both the 5′ and 3′ strands of the vRNA promoter were present. By using the ability of vRNA to protect the enzyme against heat inactivation, we established a novel assay, in conjunction with a mutagenic approach, that was used to test the secondary structure requirement of the vRNA promoter for polymerase binding. Binding required a panhandle structure and the presence of local hairpin loop structures in both the 5′ and 3′ ends of vRNA, as suggested by the corkscrew model. The interaction of the vRNA promoter with the influenza virus RNA polymerase heterotrimeric complex is likely to favor a particular closed conformation of the complex, thereby ensuring the stability of the RNA polymerase within both the infected cell and the isolated virus.
Evidence for Segment-Nonspecific Packaging of the Influenza A Virus Genome
Authors: Bancroft Christa T. , Parslow Tristram G.
Published: July 2002

The influenza A virus genome is composed of eight negative-sense RNA segments (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins and studied the efficiency with which these are packaged by using a plasmid-based influenza A virus assembly system. Packaging was assessed precisely and quantitatively by scoring transduction of the fluorescent markers in a single-round infectivity assay with a flow cytometer. We found that, under conditions in which virions are limiting, pairs of alternatively tagged vRNAs compete for packaging but do so in a nonspecific manner. Reporters representing different vRNAs were not packaged additively, as would be expected under specific packaging, but instead appeared to compete for a common niche in the virion. Moreover, 3 to 5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs — a finding compatible with random, but not with specific, packaging. Probabilistic estimates suggest that in order to achieve this level of dual transduction by chance alone, each influenza A virus virion must package an average of 9 to 11 vRNAs.
Herpes Simplex Virus Type 1/Adeno-Associated Virus Hybrid Vectors Mediate Site-Specific Integration at the Adeno-Associated Virus Preintegration Site, AAVS1, on Human Chromosome 19
Authors: Heister Thomas , Heid Irma , Ackermann Mathias , Fraefel Cornel
Published: July 2002

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four
rep
genes,
rep68
, -
78
, -
40
, and -
52
. Of those,
rep68
or
rep78
are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity (∼4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV
rep68
,
rep78
, both
rep68
and -
78
, or all four
rep
genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing
rep
sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette,
rep
in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.
Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry
Authors: Yoon Miri , Spear Patricia G.
Published: July 2002

Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor.
The EBNA2 Polyproline Region Is Dispensable for Epstein-Barr Virus-Mediated Immortalization Maintenance
Authors: Gordadze Alexey V. , Poston David , Ling Paul D.
Published: July 2002

Epstein-Barr virus nuclear antigen 2 (EBNA2) is required for EBV-mediated immortalization of primary human B cells and is a direct transcriptional activator of viral and cellular genes. The prototype EBNA2 protein contains a unique motif in which 43 out of 45 amino acids are prolines (polyproline region [PPR]). Previous genetic analysis has shown that deletion of the PPR resulted in viruses unable to immortalize B cells, although the protein did appear transcriptionally functional (R. Yalamanchili, S. Harada, and E. Kieff, J. Virol. 70:2468-2473, 1996). The PPR's uniqueness and requirement for immortalization make it an attractive therapeutic target. However, the role of this highly unusual motif for immortalization remains enigmatic. We have recently developed a transcomplementation assay that allows both genetic and functional analyses of EBNA2 in EBV-mediated immortalization maintenance (A. V. Gordadze, R. Peng, J. Tan, G. Liu, R. Sutton, B. Kempkes, G. W. Bornkamm, and P. D. Ling, J. Virol. 75:5899-5912, 2001). Surprisingly, we found that ΔPPR-EBNA2 was able to support B-cell proliferation similar to that of wild-type EBNA2 in this assay, indicating that deletion of the PPR from EBNA2 does not result in a loss of function required for immortalization maintenance. Further analysis of this mutant EBNA2 revealed that it consistently activated the viral LMP1 and LMP2A promoters severalfold better than wild-type EBNA2 in transient cotransfection assays. In addition, one striking difference between lymphoblastoid cell lines expressing wild-type EBNA2 from those expressing ΔPPR-EBNA2 is that the latter cells have significantly reduced EBV genomic levels. The data are consistent with a model in which lower EBNA2 target gene dosage may be selected for in ΔPPR-EBNA2-dependent cell lines to compensate for hyperactive stimulation of viral genes, such as LMP-1, which is cytostatic for B cells when overexpressed. It is conceivable that the hyperactivity rather than the loss of function, as hypothesized previously, could be responsible for the inability of recombinant ΔPPR-EBNA2 EBVs to immortalize B cells.
Changes in Human Immunodeficiency Virus Type 1 Gag at Positions L449 and P453 Are Linked to I50V Protease Mutants In Vivo and Cause Reduction of Sensitivity to Amprenavir and Improved Viral Fitness In Vitro
Authors: Maguire Michael F. , Guinea Rosario , Griffin Philip , Macmanus Sarah , Elston Robert C. , Wolfram Josie , Richards Naomi , Hanlon Mary H. , Porter David J. T. , Wrin Terri , Parkin Neil , Tisdale Margaret , Furfine Eric , Petropoulos Chris , Snowden B. Wendy , Kleim Jörg-Peter
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (
P
< 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1′F) and P453L (p1/p6 PP5′L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity ≈ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.
Analysis of CD4
+
T-Cell Responses to Human Papillomavirus (HPV) Type 11 L1 in Healthy Adults Reveals a High Degree of Responsiveness and Cross-Reactivity with Other HPV Types
Authors: Williams O. Martin , Hart Keith W. , Wang Eddie C. Y. , Gelder Colin M.
Published: July 2002

Human papillomavirus type 11 (HPV-11) infection causes genital warts and recurrent respiratory papillomatosis. While there is compelling evidence that CD4
+
T cells play an important role in immune surveillance of HPV-associated diseases, little is known about human CD4
+
T-cell recognition of HPV-11. We have investigated the CD4
+
T-cell responses of 25 unrelated healthy donors to HPV-11 L1 virus-like particles (VLP). CD4
+
T-cell lines from 21 of 25 donors were established. Cell sorting experiments carried out on cells from six donors demonstrated that the response was located in the CD45RA
low
CD45RO
high
memory T-cell population. To determine the peptide specificity of these responses, epitope selection was analyzed by using 95 15-mer peptides spanning the entire HPV-11 L1 protein. No single region of L1 was immunodominant; responders recognized between 1 and 10 peptides, located throughout the protein, and peptide responses fell into clear HLA class II restricted patterns. Panels of L1 peptides specific for skin and genital HPV were used to show that the L1 CD4
+
T-cell responses were cross-reactive. The degree of cross-reactivity was inversely related to the degree of L1 sequence diversity between these viruses. Finally, responses to HPV-11 L1 peptides were elicited from ex vivo CD45RO
+
peripheral blood mononuclear cells, demonstrating that recognition of HPV-11 was a specific memory response and not due to in vitro selection during tissue culture. This is the first study of CD4
+
T-cell responses to HPV-11 in healthy subjects and demonstrates marked cross-reactivity with other skin and genital HPV types. This cross-reactivity may be of significance for vaccine strategies against HPV-associated clinical diseases.
Selective Loss of Innate CD4+Vα24 Natural Killer T Cells in Human Immunodeficiency Virus Infection
Authors: Sandberg Johan K. , Fast Noam M. , Palacios Emil H. , Fennelly Glenn , Dobroszycki Joanna , Palumbo Paul , Wiznia Andrew , Grant Robert M. , Bhardwaj Nina , Rosenberg Michael G. , Nixon Douglas F.
Published: July 2002

Vα24 natural killer T (NKT) cells are innate immune cells involved in regulation of immune tolerance, autoimmunity, and tumor immunity. However, the effect of human immunodeficiency virus type 1 (HIV-1) infection on these cells is unknown. Here, we report that the Vα24 NKT cells can be subdivided into CD4+or CD4−subsets that differ in their expression of the homing receptors CD62L and CD11a. Furthermore, both CD4+and CD4−NKT cells frequently express both CXCR4 and CCR5 HIV coreceptors. We find that the numbers of NKT cells are reduced in HIV-infected subjects with uncontrolled viremia and marked CD4+T-cell depletion. The number of CD4+NKT cells is inversely correlated with HIV load, indicating depletion of this subset. In contrast, CD4−NKT-cell numbers are unaffected in subjects with high viral loads. HIV infection experiments in vitro show preferential depletion of CD4+NKT cells relative to regular CD4+T cells, in particular with virus that uses the CCR5 coreceptor. Thus, HIV infection causes a selective loss of CD4+lymph node homing (CD62L+) NKT cells, with consequent skewing of the NKT-cell compartment to a predominantly CD4−CD62L−phenotype. These data indicate that the key immunoregulatory NKT-cell compartment is compromised in HIV-1-infected patients.
Different Effects of Nef-Mediated HLA Class I Down-Regulation on Human Immunodeficiency Virus Type 1-Specific CD8+T-Cell Cytolytic Activity and Cytokine Production
Authors: Tomiyama Hiroko , Akari Hirofumi , Adachi Akio , Takiguchi Masafumi
Published: July 2002

A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4+T cells. We confirmed this effect by using anef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4+T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4+T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4+T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4+T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4+T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8+T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8+T cells are able to partially but not completely control HIV-1 replication in vivo.
Replication of Avian Sarcoma Virus In Vivo Requires an Interaction between the Viral RNA and the TψC Loop of the tRNA
Trp
Primer
Authors: Morris Shannon , Johnson Michael , Stavnezer Ed , Leis Jonathan
Published: July 2002

Reverse transcription in avian sarcoma virus (ASV) initiates from the 3′ end of a tRNA
Trp
primer, which anneals near the 5′ end of the RNA genome. The region around the primer-binding site (PBS) forms an elaborate stem structure composed of the U5-inverted repeat (U5-IR) stem, the U5-leader stem, and the association of the tRNA primer with the PBS. There is evidence for an additional interaction between the viral U5 RNA and the TψC loop of the tRNA
Trp
(U5-TψC). We now demonstrate that this U5-TψC interaction is necessary for efficient replication of ASV in culture. By randomizing specific biologically relevant regions of the viral RNA, thereby producing a library of mutant viruses, we are able to select, through multiple rounds of infection, those sequences imparting survival fitness to the virus. Randomizing the U5-TψC interaction region of the viral RNA results in selection of largely wild-type sequences after five rounds of infection. Also recovered are mutant viruses that maintain their ability to base pair with the TψC loop of the tRNA
Trp
. To prove this interaction is specific to the tRNA primer, we constructed a second library, in which we altered the PBS to anneal to tRNA
Pro
, while simultaneously randomizing the viral RNA U5-TψC region. After five rounds of infection, the consensus sequence 5′-GPuPuCPy-3′ emerged, which is complementary to the 5′-GGTTC-3′ sequence found in the TψC loop of tRNA
Pro
. These observations confirm the importance of the U5-TψC interaction in vivo.
Shedding and Intracage Transmission of Sin Nombre Hantavirus in the Deer Mouse (
Peromyscus maniculatus
) Model
Authors: Botten Jason , Mirowsky Katy , Ye Chunyan , Gottlieb Keith , Saavedra Melissa , Ponce Liana , Hjelle Brian
Published: July 2002

The mechanism(s) by which Sin Nombre (SN) hantavirus is maintained in deer mouse populations is unclear. Field studies indicate that transmission occurs primarily if not exclusively via a horizontal mechanism. Using an experimental deer mouse infection model in an outdoor laboratory, we tested whether infected rodents shed SN virus in urine, feces, and saliva, whether infected mice transmit infection to naïve cage mates, and whether infected dams are able to vertically transmit virus or antibody to offspring. Using pooled samples of urine, feces, and saliva collected from mice infected 8 to 120 days postinoculation (p.i.), we found that a subset of saliva samples, collected between 15 and 90 days p.i., contained viral RNA. Parallel studies conducted on wild-caught, naturally infected deer mice showed a similar pattern of intermittent positivity, also only in saliva samples. Attempts to isolate virus through inoculation of cells or naïve deer mice with the secreta or excreta of infected mice were uniformly negative. Of 54 attempts to transmit infection by cohousing infected deer mice with seronegative cage mates, we observed only a single case of transmission, which occurred between 29 and 42 days p.i. Dams passively transferred antibodies to neonatal pups via milk, and those antibodies persisted for at least 2 months after weaning, but none transmitted infection to their pups. Compared to other hantavirus models, SN virus is shed less efficiently and transmits inefficiently among cage mates. Transmission of SN virus among reservoir rodents may require factors that are not required for other hantaviruses.
Identification and Characterization of Two Closely Related Unclassifiable Endogenous Retroviruses in Pythons (Python molurusandPython curtus)
Authors: Huder Jon B. , Böni Jürg , Hatt Jean-Michel , Soldati Guido , Lutz Hans , Schüpbach Jörg
Published: July 2002

Boid inclusion body disease (BIBD) is a fatal disorder of boid snakes that is suspected to be caused by a retrovirus. In order to identify this agent, leukocyte cultures (established fromPython molurusspecimens with symptoms of BIBD or kept together with such diseased animals) were assessed for reverse transcriptase (RT) activity. Virus from cultures exhibiting high RT activity was banded on sucrose density gradients, and the RT peak fraction was subjected to highly efficient procedures for the identification of unknown particle-associated retroviral RNA. A 7-kb full retroviral sequence was identified, cloned, and sequenced. This virus contained intact open reading frames (ORFs) forgag,pro,pol, andenv, as well as another ORF of unknown function withinpol. Phylogenetic analysis showed that the virus is distantly related to viruses from both the B and D types and the mammalian C type but cannot be classified. It is present as a highly expressed endogenous retrovirus in allP. molurusindividuals; a closely related, but much less expressed virus was found in all testedPython curtusindividuals. All other boid snakes tested, includingPython regius,Python reticulatus,Boa constrictor,Eunectes notaeus, andMorelia spilota, were virus negative, independent of whether they had BIBD or not. Virus isolated fromP. moluruscould not be transmitted to the peripheral blood mononuclear cells ofB. constrictororP. regius. Thus, there is no indication that this novel virus, which we propose to name python endogenous retrovirus (PyERV), is causally linked with BIBD.