bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Relationships Deduced from
emm
and Multilocus Sequence Typing of Invasive
Streptococcus dysgalactiae
subsp.
equisimilis
and
S. canis
Recovered from Isolates Collected in the United States
Authors: Ahmad Yusra , Gertz Robert E. , Li Zhongya , Sakota Varja , Broyles Laura N. , Van Beneden Chris , Facklam Richard , Shewmaker P. Lynn , Reingold Arthur , Farley Monica M. , Beall Bernard W.
Published: April 2009

Beta-hemolytic group C and G streptococci cause a considerable invasive disease burden and sometimes cause disease outbreaks. Little is known about the critical epidemiologic parameter of genetic relatedness between isolates. We determined the
emm
types of 334
Streptococcus dysgalactiae
subsp.
equisimilis
isolates, and attempted
emm
typing of 5
Streptococcus canis
isolates from a recent population-based surveillance for invasive isolates. Thirty-four
emm
types were observed, including one from
S. canis
. We formulated multilocus sequence typing (MLST) primers with six of the seven loci corresponding to the
Streptococcus pyogenes
MLST scheme. We performed MLST with 65 of the 334 surveillance isolates (61
S. dysgalactiae
subsp.
equisimilis
isolates, 4
S. canis
isolates) to represent each
emm
type identified, including 2 to 3 isolates for each of the 25 redundantly represented
emm
types. Forty-one MLST sequence types (STs) were observed. Isolates within 16 redundantly represented
S. dysgalactiae
subsp.
equisimilis emm
types shared identical or nearly identical STs, demonstrating concordance between the
emm
type and genetic relatedness. However, seven STs were each represented by two to four different
emm
types, and 7 of the 10
S. dysgalactiae
subsp.
equisimilis
eBURST groups represented up to six different
emm
types. Thus,
S. dysgalactiae
subsp.
equisimilis
isolates were similar to
S. pyogenes
isolates, in that strains of the same
emm
type were often highly related, but they differed from
S. pyogenes
, in that
S. dysgalactiae
subsp.
equisimilis
strains with identical or closely similar STs often exhibited multiple unrelated
emm
types. The phylogenetic relationships between
S. dysgalactiae
subsp.
equisimilis
and
S. pyogenes
alleles revealed a history of interspecies recombination, with either species often serving as genetic donors. The four
S. canis
isolates shared highly homologous alleles but were unrelated clones without evidence of past recombination with
S. dysgalactiae
subsp.
equisimilis
or
S. pyogenes
.
Multilocus Variable-Number Tandem-Repeat Method for Typing
Salmonella enterica
Serovar Newport
Authors: Davis Margaret A. , Baker Katherine N. K. , Call Douglas R. , Warnick Lorin D. , Soyer Yesim , Wiedmann Martin , Gröhn Yrjö , McDonough Patrick L. , Hancock Dale D. , Besser Thomas E.
Published: April 2009

In recent years, the proportion of
Salmonella enterica
infections represented by
S. enterica
serovar Newport has increased markedly among humans and animals. Multilocus variable-number tandem-repeat analysis (MLVA) has proven to be useful in discriminating other highly clonal
Salmonella
serovars. Here, we report on the development of a highly discriminatory MLVA for
Salmonella
serovar Newport.
Cost-Effective Frozen Master Mix Modification of a Commercial Methicillin-Resistant
Staphylococcus aureus
PCR Assay
Authors: Munson Erik , Block Timothy , Voegeli Janet T. , Hryciuk Jeanne E. , Schell Ronald F.
Published: April 2009

The expense inherent to molecular diagnostics may be an overriding concern for a variety of clinical laboratories in the development of PCR-based methicillin-resistant
Staphylococcus aureus
(MRSA) active surveillance programs. BD GeneOhm MRSA assay master mix was reconstituted, aliquoted into SmartCycler tubes in 25-μl volumes, and frozen at −70°C. One hundred percent of archival nasal swab lysates yielded the expected PCR results when incubated in master mix frozen for 1, 2, 3, and 4 weeks. A 98.8% concordance of the final result was observed upon prospective PCR analysis of 320 clinical lysates utilizing freshly reconstituted master mix and 2-week-frozen master mix. Initial unresolved rates generated by frozen master mix and freshly reconstituted master mix differed by 1.6% (
P
= 0.16). Of 50 MRSA-positive lysates, the titers of 32 (64%) were determined to the same value upon initial tandem frozen master mix and freshly reconstituted master mix utilization; the titers of an additional 14 were determined to the same value upon repeat testing. Frozen master mix maintains potency for at least 4 weeks, facilitating detection of MRSA from nasal swab lysates, and may decrease the amount of unused reagent up to an average of 33%.
Rapid Group-, Serotype-, and Vaccine Strain-Specific Identification of Poliovirus Isolates by Real-Time Reverse Transcription-PCR Using Degenerate Primers and Probes Containing Deoxyinosine Residues
Authors: Kilpatrick David R. , Yang Chen-Fu , Ching Karen , Vincent Annelet , Iber Jane , Campagnoli Ray , Mandelbaum Mark , De Lina , Yang Su-Ju , Nix Allan , Kew Olen M.
Published: April 2009

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.
Evaluation of the New Vitek 2 ANC Card for Identification of Medically Relevant Anaerobic Bacteria
Authors: Mory Francine , Alauzet Corentine , Matuszeswski Céline , Riegel Philippe , Lozniewski Alain
Published: April 2009

Of 261 anaerobic clinical isolates tested with the new Vitek 2 ANC card, 257 (98.5%) were correctly identified at the genus level. Among the 251 strains for which identification at the species level is possible with regard to the ANC database, 217 (86.5%) were correctly identified at the species level. Two strains (0.8%) were not identified, and eight were misidentified (3.1%). Of the 21 strains (8.1%) with low-level discrimination results, 14 were correctly identified at the species level by using the recommended additional tests. This system is a satisfactory new automated tool for the rapid identification of most anaerobic bacteria isolated in clinical laboratories.
Detection of
Streptococcus pneumoniae
Strain Cocolonization in the Nasopharynx
Authors: Brugger Silvio D. , Hathaway Lucy J. , Mühlemann Kathrin
Published: April 2009

Colonization with more than one distinct strain of the same species, also termed cocolonization, is a prerequisite for horizontal gene transfer between pneumococcal strains that may lead to change of the capsular serotype. Capsule switch has become an important issue since the introduction of conjugated pneumococcal polysaccharide vaccines. There is, however, a lack of techniques to detect multiple colonization by
S. pneumoniae
strains directly in nasopharyngeal samples. Two hundred eighty-seven nasopharyngeal swabs collected during the prevaccine era within a nationwide surveillance program were analyzed by a novel technique for the detection of cocolonization, based on PCR amplification of a noncoding region adjacent to the pneumolysin gene (
plyNCR
) and restriction fragment length polymorphism (RFLP) analysis. The numbers of strains and their relative abundance in cocolonized samples were determined by terminal RFLP. The pneumococcal carriage rate found by PCR was 51.6%, compared to 40.0% found by culture. Cocolonization was present in 9.5% (10/105) of samples, most (9/10) of which contained two strains in a ratio of between 1:1 and 17:1. Five of the 10 cocolonized samples showed combinations of vaccine types only (
n
= 2) or combinations of nonvaccine types only (
n
= 3). Carriers of multiple pneumococcal strains had received recent antibiotic treatment more often than those colonized with a single strain (33% versus 9%,
P
= 0.025). This new technique allows for the rapid and economical study of pneumococcal cocolonization in nasopharyngeal swabs. It will be valuable for the surveillance of
S. pneumoniae
epidemiology under vaccine selection pressure.
Rapid Variable-Number Tandem-Repeat Genotyping for
Mycobacterium leprae
Clinical Specimens
Authors: Kimura Miyako , Sakamuri Rama Murthy , Groathouse Nathan A. , Rivoire Becky L. , Gingrich David , Krueger-Koplin Susan , Cho Sang-Nae , Brennan Patrick J. , Vissa Varalakshmi
Published: April 2009

Mycobacterium leprae
is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of
M. leprae
has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of
M. leprae
isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (
n
= 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the
M. leprae
genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (
n
= 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of
M. leprae
in different countries where leprosy is endemic.
Detection of Plasmid-Mediated Quinolone Resistance Genes in Clinical Isolates of
Enterobacter
spp. in Spain
Authors: Cano M. E. , Rodríguez-Martínez J. M. , Agüero J. , Pascual A. , Calvo J. , García-Lobo J. M. , Velasco C. , Francia M. V. , Martínez-Martínez L.
Published: April 2009

We have studied by PCR and DNA sequencing the presence of the
qnrA, qnrB, qnrS, aac
(
6′
)
-Ib-cr, qepA, intI1
, and IS
CR1
genes in 200 clinical isolates of
Enterobacter cloacae
(
n
= 153) and
E. aerogenes
(
n
= 47) consecutively collected between January 2004 and October 2005 in two hospitals located in Santander (northern Spain) and Seville (southern Spain). Mutations in the quinolone resistance-determining region of
gyrA
and
parC
also were investigated in organisms containing plasmid-mediated quinolone resistance genes. The isolates had different resistant phenotypes, including AmpC hyperproduction, extended-spectrum β-lactamase production, resistance or decreased susceptibility to quinolones, and/or resistance to aminoglycosides. Among the 116
E. cloacae
isolates from Santander,
qnrS1, qnrB5, qnrB2
, and
aac
(
6′
)
-Ib-cr
were detected in 22 (19%), 1 (0.9%), 1 (0.9%), and 3 (2.6%) isolates, respectively. Twenty-one, 17, and 2
qnrS1
-positive isolates also contained
bla
LAP-1
,
intI1
, and IS
CR1
, respectively. A
qnrB7
-like gene was detected in one
E. aerogenes
isolate from Santander. No plasmid-mediated quinolone resistance gene was detected in the isolates from Seville. The
qnrS1
-containing isolates corresponded to four pulsed-field gel electrophoresis patterns and showed various levels of resistance to quinolones. Six isolates were susceptible to nalidixic acid and presented reduced susceptibility to ciprofloxacin. The
qnrS1
gene was contained in a conjugative plasmid of ca. 110 kb, and when the plasmid was transferred to recipient strains that did not have a specific mechanism of quinolone resistance, the ciprofloxacin MICs ranged from 0.047 to 0.125 μg/ml.
Cutaneous Infection Caused by
Macrophomina phaseolina
in a Child with Acute Myeloid Leukemia
Authors: Srinivasan Ashok , Wickes Brian L. , Romanelli Anna M. , Debelenko Larisa , Rubnitz Jeffrey E. , Sutton Deanna A. , Thompson Elizabeth H. , Fothergill Annette W. , Rinaldi Michael G. , Hayden Randall T. , Shenep Jerry L.
Published: April 2009

We report a case of
Macrophomina phaseolina
skin infection in an immunocompromised child with acute myeloid leukemia, which was treated successfully with posaconazole without recurrence after a hematopoietic stem cell transplant. The fungus was identified by DNA sequencing using both the internal transcribed spacer and D1/D2 region of the 28S ribosomal DNA gene.
No Correlation between Giant Cell Arteritis andChlamydia pneumoniaeInfection: Investigation of 189 Patients by Standard and Improved PCR Methods
Authors: Njau Florence , Ness Thomas , Wittkop Ulrike , Pancratz Thorsten , Eickhoff Meike , Hudson Alan P. , Haller Hermann , Wagner Annette D.
Published: April 2009

A total of 189 temporal artery biopsy samples from giant cell arteritis (GCA) patients were investigated using sensitive PCR targetingChlamydia pneumoniae. Chlamydial DNA was detected in 17 samples, 11 of which were positive for chlamydial antigens. Our data did not reveal strong evidence thatC. pneumoniaeplays an important role in the pathogenesis of GCA.
Pulmonary Disease Due to
Mycobacterium arosiense
, an Easily Misidentified Pathogenic Novel Mycobacterium
Authors: Tortoli Enrico , Adriani Beatrice , Baruzzo Serena , Degl'Innocenti Roberto , Galanti Irene , Lauria Stefania , Mariottini Alessandro , Pascarella Michela
Published: April 2009

Mycobacterium arosiense
is a newly described species. After noticing it was misidentified as
Mycobacterium intracellulare
by the commercial identification system GenoType CM (Hein, Nehren, Germany), we detected 4 such strains among 33 that were previously misidentified as
M. intracellulare
. Three more strains were found among unidentified mycobacteria not tested previously with GenoType. The first case of pulmonary disease due to
M. arosiense
is reported here, and the novel species, of which so far only one strain had been investigated, is further characterized.
Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication
Authors: Soares Jamária A. P. , Leite Flávia G. G. , Andrade Luciana G. , Torres Alice A. , De Sousa Lirlândia P. , Barcelos Lucíola S. , Teixeira Mauro M. , Ferreira Paulo C. P. , Kroon Erna G. , Souto-Padrón Thaís , Bonjardim Cláudio A.
Published: April 2009

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to ≥90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.
Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches
Authors: Baliji Surendranath , Cammer Stephen A. , Sobral Bruno , Baker Susan C.
Published: April 2009

Coronaviruses encode large replicase polyproteins which are proteolytically processed by viral proteases to generate mature nonstructural proteins (nsps) that form the viral replication complex. Mouse hepatitis virus (MHV) replicase products nsp3, nsp4, and nsp6 are predicted to act as membrane anchors during assembly of the viral replication complexes. We report the first antibody-mediated Western blot detection of nsp6 from MHV-infected cells. The nsp6-specific peptide antiserum detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6 products of approximately 23 and 25 kDa. Analysis of nsp6 transmembrane topology revealed six membrane-spanning segments and a conserved hydrophobic domain in the C-terminal cytosolic tail.
Effective Downregulation of HLA-A*2 and HLA-B*57 by Primary Human Immunodeficiency Virus Type 1 Isolates Cultured from Elite Suppressors
Authors: Nou Eric , Zhou Yan , Nou Damaris D. , Blankson Joel N.
Published: April 2009

Elite controllers or suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who control viral replication to <50 copies/ml without antiretroviral therapy. Downregulation of HLA class I molecules is an important mechanism used by HIV-1 to evade the immune system. In this study, we showed that primary isolates from ES are as effective as isolates obtained from patients with progressive HIV-1 disease at downregulating HLA-A*2 and HLA-B*57 molecules on primary CD4
+
T cells. Thus, a diminished ability of viral isolates from ES to evade HIV-specific immune responses probably does not contribute to the control of viral replication in these patients.
Cassava Brown Streak Virus
(
Potyviridae
) Encodes a Putative Maf/HAM1 Pyrophosphatase Implicated in Reduction of Mutations and a P1 Proteinase That Suppresses RNA Silencing but Contains No HC-Pro
Authors: Mbanzibwa Deusdedith R. , Tian Yanping , Mukasa Settumba B. , Valkonen Jari P. T.
Published: April 2009

The complete positive-sense single-stranded RNA genome of
Cassava brown streak virus
(CBSV; genus
Ipomovirus
;
Potyviridae
) was found to consist of 9,069 nucleotides and predicted to produce a polyprotein of 2,902 amino acids. It was lacking helper-component proteinase but contained a single P1 serine proteinase that strongly suppressed RNA silencing. Besides the exceptional structure of the 5′-proximal part of the genome, CBSV also contained a Maf/HAM1-like sequence (678 nucleotides, 226 amino acids) recombined between the replicase and coat protein domains in the 3′-proximal part of the genome, which is highly conserved in
Potyviridae
. HAM1 was flanked by consensus proteolytic cleavage sites for ipomovirus NIaPro cysteine proteinase. Homology of CBSV HAM1 with cellular Maf/HAM1 pyrophosphatases suggests that it may intercept noncanonical nucleoside triphosphates to reduce mutagenesis of viral RNA.
Determinants of Secretion and Intracellular Localization of Human Herpesvirus 8 Interleukin-6
Authors: Chen Daming , Choi Young Bong , Sandford Gordon , Nicholas John
Published: April 2009

Human herpesvirus 8 (HHV-8) interleukin-6 (vIL-6) is distinct from human and other cellular IL-6 proteins in that it does not require the nonsignaling α-receptor subunit for the formation of gp130-based signal transducing complexes and also is largely retained intracellularly rather than being secreted. We and others have reported that vIL-6 is retained and is active in the endoplasmic reticulum (ER) compartment, and data from our laboratory have demonstrated that intracellular vIL-6 is functional in the autocrine promotion of proliferation and survival of HHV-8 latently infected primary effusion lymphoma cells. It has also been reported that vIL-6 secretion in gp130-deficient cells can be enhanced by introduced gp130, thereby implicating the signal transducer in vIL-6 trafficking to the cell surface. We examine here the requirements for intracellular retention and localization of vIL-6. Using vIL-6-hIL-6 chimeric and point-mutated vIL-6 proteins, we identified regions and residues of vIL-6 influencing vIL-6 secretion. However, there was no correlation between vIL-6 secretion and gp130 interaction. We found that vIL-6, but not hIL-6, could associate stably with ER-resident chaperone protein calnexin. Glycosylation-dependent interaction of vIL-6 with calnexin correlated with proper protein folding, but there was no direct relationship between vIL-6-calnexin interaction and intracellular retention. While calnexin depletion had little influence on absolute amounts of secreted vIL-6, it led to markedly reduced levels of intracellular cytokine. This was reversed by gp130 transduction, which had no detectable effect on vIL-6 secretion, but redistributed vIL-6 into ER-distinct locations in calnexin-depleted cells, specifically. Our data reveal that calnexin plays a role in ER localization of vIL-6 and that gp130 promotes ER exit, but not secretion, of the viral cytokine.
Cellular Compartments of Human Immunodeficiency Virus Type 1 Replication In Vivo: Determination by Presence of Virion-Associated Host Proteins and Impact of Opportunistic Infection
Authors: Lawn Stephen D. , Roberts Beverly D. , Griffin George E. , Folks Thomas M. , Butera Salvatore T.
Published: April 2009

Antigens derived from host cells are detectable in the envelope of human immunodeficiency virus type 1 (HIV-1) and result in a distinctive viral phenotype reflecting that of the host cell. An immunomagnetic capture assay targeting discriminatory host proteins was developed to differentiate between HIV-1 derived from macrophages and lymphocytes. HIV-1 propagated in macrophages or lymphocytes in vitro was selectively captured by monoclonal antibodies directed against the virally incorporated cell-type-specific host markers CD36 (macrophages) and CD26 (lymphocytes). Furthermore, by targeting these markers, virus of defined cellular origin was selectively captured from a mixed pool of in vitro-propagated viruses. This technique was further refined in order to determine the impact of opportunistic infection on HIV-1 expression from these cellular compartments in vivo. Analysis of cell-free virus purified from plasma of patients with HIV-1 infection suggested that in those with an opportunistic infection, viral replication occurred in activated lymphocytes. Interestingly, there was also significant replication in activated macrophages in those patients with untreated pulmonary tuberculosis. Thus, in addition to lymphocytes, the macrophage cellular pool may serve as an important source of cell-free HIV-1 in patients with opportunistic infections that lead to marked macrophage activation. This novel viral capture technique may allow researchers to address a wide range of important questions regarding virus-host dynamics.
Ultrastructural and Functional Analyses of Recombinant Influenza Virus Ribonucleoproteins Suggest Dimerization of Nucleoprotein during Virus Amplification
Authors: Ortega Joaquín , Martín-Benito Jaime , Zürcher Thomas , Valpuesta José M. , Carrascosa José L. , Ortín Juan
Published: April 2009

Influenza virus ribonucleoproteins (RNPs) were reconstituted in vivo from cloned cDNAs expressing the three polymerase subunits, the nucleoprotein (NP), and short template RNAs. The structure of purified RNPs was studied by electron microscopy and image processing. Circular and elliptic structures were obtained in which the NP and the polymerase complex could be defined. Comparison of the structure of RNPs of various lengths indicated that each NP monomer interacts with approximately 24 nucleotides. The analysis of the amplification of RNPs with different lengths showed that those with the highest replication efficiency contained an even number of NP monomers, suggesting that the NP is incorporated as dimers into newly synthesized RNPs.
Roles of Matrix, p2, and N-Terminal Myristoylation in Human Immunodeficiency Virus Type 1 Gag Assembly
Authors: Morikawa Yuko , Hockley David J. , Nermut Milan V. , Jones Ian M.
Published: April 2009

Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the >1,000-kDa multimer are intermediates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV particle assembly.
Immunohistochemical Analysis of Primary Sensory Neurons Latently Infected with Herpes Simplex Virus Type 1
Authors: Yang L. , Voytek C. C. , Margolis T. P.
Published: April 2009

We characterized the populations of primary sensory neurons that become latently infected with herpes simplex virus (HSV) following peripheral inoculation. Twenty-one days after ocular inoculation with HSV strain KOS, 81% of latency-associated transcript (LAT)-positive trigeminal ganglion (TG) neurons coexpressed SSEA3, 71% coexpressed Trk
A
(the high-affinity nerve growth factor receptor), and 68% coexpressed antigen recognized by monoclonal antibody (MAb) A5; less than 5% coexpressed antigen recognized by MAb KH10. The distribution of LAT-positive, latently infected TG neurons contrasted sharply with (i) the overall distribution of neuronal phenotypes in latently infected TG and (ii) the neuronal distribution of viral antigen in productively infected TG. Similar results were obtained following ocular and footpad inoculation with KOS/62, a LAT deletion mutant in which the LAT promoter is used to drive expression of the
Escherichia coli lacZ
gene. Thus, although all neuronal populations within primary sensory ganglia appear to be capable of supporting a productive infection with HSV, some neuronal phenotypes are more permissive for establishment of a latent infection with LAT expression than others. Furthermore, expression of HSV LAT does not appear to play a role in this process. These findings indicate that there are marked differences in the outcome of HSV infection among the different neuronal populations in the TG and highlight the key role that the host neuron may play in regulating the repertoire of viral gene expression during the establishment of HSV latent infection.
Integrins α2β1 and α4β1 Can Mediate SA11 Rotavirus Attachment and Entry into Cells
Authors: Hewish Marilyn J. , Takada Yoshikazu , Coulson Barbara S.
Published: April 2009

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins α2β1 and α4β1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing α2β1 or α4β1 integrins via transfection is increased over virus binding to and infection of cells transfected with α3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of α2β1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing α2β1 was elevated over binding to control cells and was specifically blocked by the anti-α2 monoclonal antibody AK7. Virus growth in α4-transfected K562 cells which had also been induced to express α2β1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, α2β1 and α4β1, are capable of acting as cellular receptors for SA11 rotavirus.
Conditional Site-Specific Integration into Human Chromosome 19 by Using a Ligand-Dependent Chimeric Adeno-Associated Virus/Rep Protein
Authors: Rinaudo Daniela , Lamartina Stefania , Roscilli Giuseppe , Ciliberto Gennaro , Toniatti Carlo
Published: April 2009

It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.
Activation of a Cell Entry Pathway Common to Type C Mammalian Retroviruses by Soluble Envelope Fragments
Authors: Lavillette Dimitri , Ruggieri Alessia , Russell Stephen J. , Cosset François-Loïc
Published: April 2009

Mutations that negatively or positively affect the fusion properties of murine leukemia viruses (MLVs) have been found within all subdomains of their SU (surface) and TM (transmembrane) envelope units. Yet, the interrelations between these different regions of the envelope complex during the cell entry process are still elusive. Deletion of the histidine residue of the conserved PHQV motif at the amino terminus of the amphotropic or the ecotropic MLV SU resulted in the AdelH or the MOdelH fusion-defective mutant envelope, respectively. These delH mutant envelopes are incorporated on retroviral particles at normal densities and normally mediate virion binding to cells expressing the retroviral receptors. However, both their cell-cell and virus-cell fusogenicities were fully prevented at an early postbinding stage. We show here that the fusion defect of AdelH or MOdelH envelopes was also almost completely reverted by providing either soluble SU or a polypeptide encompassing the receptor-binding domain (RBD) to the target cells, provided that the integrity of the amino-terminal end of either polypeptide was preserved. Restoration of delH envelope fusogenicity was caused by activation of the target cells via specific interaction of the latter polypeptides with the retrovirus receptor rather than by their association with the delH envelope complexes. Moreover crossactivation of the target cells, leading to fusion activation of AdelH or MOdelH envelopes, was achieved by polypeptides containing various type C mammalian retrovirus RBDs, irrespective of the type of entry-defective glycoprotein that was used for infection. Our results indicate that although they recognize different receptors for binding to the cell surface, type C mammalian retroviruses use a common entry pathway which is activated by a conserved feature of their envelope glycoproteins.
Measles Virus Structural Components Are Enriched into Lipid Raft Microdomains: a Potential Cellular Location for Virus Assembly
Authors: Manié Serge N. , Debreyne Sylvain , Vincent Séverine , Gerlier Denis
Published: April 2009

The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.
Herpes Simplex Virus Type 1 Gene UL14: Phenotype of a Null Mutant and Identification of the Encoded Protein
Authors: Cunningham Charles , Davison Andrew J. , MacLean Alasdair R. , Taus Naomi S. , Baines Joel D.
Published: April 2009

Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.
Identification of Specific Molecular Structures of Human Immunodeficiency Virus Type 1 Tat Relevant for Its Biological Effects on Vascular Endothelial Cells
Authors: Mitola Stefania , Soldi Raffaella , Zanon Ilaria , Barra Luca , Gutierrez Maria Ines , Berkhout Ben , Giacca Mauro , Bussolino Federico
Published: April 2009

Human immunodeficiency virus type 1 (HIV-1) Tat transactivates viral genes and is released by infected cells, acting as a soluble mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type 2 (VEGFR-2) and integrins. A structure-activity relationship study was performed by functional analysis of Tat substitution and deletion variants to define the Tat determinants necessary for EC activation. Variants were made (i) in the basic and (ii) in the cysteine-rich domains and (iii) in the C-terminal region containing the RGD sequence required for integrin recognition. Our results led to the following conclusions. (i) Besides a high-affinity binding site corresponding to VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the cysteine-rich variants bind only to the low-affinity binding sites and do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they have a reduced ability to activate EC in vitro, and they lack angiogenic activity. (iii) Mutants with mutations in the C-terminal region are partially defective for in vitro biological activities and in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In conclusion, regions encoded by the first exon oftatare necessary and sufficient for activation of VEGFR-2. However, the C-terminal region, most probably through RGD-mediated integrin engagement, is indispensable for full activation of an in vitro and in vivo angiogenic program.
Sequence and Functional Analysis of EBNA-LP and EBNA2 Proteins from Nonhuman Primate Lymphocryptoviruses
Authors: Peng RongSheng , Gordadze Alexey V. , Fuentes Pananá Ezequiel M. , Wang Fred , Zong Jianchao , Hayward Gary S. , Tan Jie , Ling Paul D.
Published: April 2009

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.
Correlation between Point Mutations in NS2 and the Viability and Cytopathogenicity of Bovine Viral Diarrhea Virus Strain Oregon Analyzed with an Infectious cDNA Clone
Authors: Kümmerer Beate M. , Meyers Gregor
Published: April 2009

Cytopathogenicity ofBovine viral diarrhea virus(BVDV) is correlated with expression of the nonstructural protein NS3, which can be generated by processing of a fusion protein termed NS2-3. For the cytopathogenic (cp) BVDV strain Oregon, NS2-3 processing is based on a set of point mutations within NS2. To analyze the correlation between NS2-3 cleavage and cytopathogenicity, a full-length cDNA clone composed of cDNA from BVDV Oregon and the utmost 5′- and 3′-terminal sequences of a published infectious BVDV clone was established. After transfection of RNA transcribed from this cDNA clone, infectious virus with similar growth characteristics to wild-type BVDV Oregon could be recovered that also exhibited a cytopathic effect. Based on this cDNA construct and published cp and noncp infectious clones, chimeric full-length cDNA clones were constructed. Analysis of the recovered viruses demonstrated that the presence of the NS2 gene of BVDV Oregon in a chimeric construct is sufficient for NS2-3 processing and a cp phenotype. Since previous studies had revealed that the amino acid serine at position 1555 of BVDV Oregon plays an important role in efficient NS2-3 cleavage, mutants of BVDV Oregon with different amino acids at this position were constructed. Some of these mutants showed NS2-3 cleavage efficiencies in the range of the wild-type sequence and allowed the recovery of viruses that behaved similarly to wild-type virus with regard to growth characteristics and cytopathogenicity. In contrast, other mutants with considerably reduced NS2-3 cleavage efficiencies propagated much more slowly and reverted to viruses expressing polyproteins with sequences allowing efficient NS2-3 cleavage. These viruses apparently induced cytopathic effects only after reversion.
Upregulation of the Genes Encoding Lysosomal Hydrolases, a Perforin-Like Protein, and Peroxidases in the Brains of Mice Affected with an Experimental Prion Disease
Authors: Kopacek Juraj , Sakaguchi Suehiro , Shigematsu Kazuto , Nishida Noriyuki , Atarashi Ryuichirou , Nakaoke Ryota , Moriuchi Ryozo , Niwa Masami , Katamine Shigeru
Published: April 2009

In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of β-
N
-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrP
Sc
) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.
Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor
Authors: Arnberg Niklas , Edlund Karin , Kidd Alistair H. , Wadell Göran
Published: April 2009

Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) α2, have recently been identified. In the absence of CAR, MHC-I α2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expresing CHO cells and MHC-I α2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via α(2→3)-linked sialic acid saccharides rather than α(2→6)-linked ones, since (i) α(2→3)-specific but not α(2→6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with α(2→3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of α(2→3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I α2.
Development of Human T-Cell Leukemia Virus Type 1-Transformed Tumors in Rats following Suppression of T-Cell Immunity by CD80 and CD86 Blockade
Authors: Hanabuchi Shino , Ohashi Takashi , Koya Yoshihiro , Kato Hirotomo , Takemura Fumiyo , Hirokawa Katsuiku , Yoshiki Takashi , Yagita Hideo , Okumura Ko , Kannagi Mari
Published: April 2009

Host immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses. Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells. During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor regression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.