bluuebunny/crossref_metadata_2025 huggingface dataset:
Geoacoustic inversion of short range source data using a plane wave reflection coefficient approach
Authors: Stotts S. A. , Knobles D. P. , Keller J. A. , Piper J. N. , Thompson L. A.
Published: December 2006

Acoustic time series data were collected in a shallow, hard bottom lake environment located in central Texas using both short range (2m) implosive data, obtained with the source and a single hydrophone located near mid-depth in the waveguide, along with longer range implosive and explosive data from a near surface source to a bottom mounted hydrophone. Matched field inversions using simulated annealing were performed with a ray trace plus complex plane wave reflection coefficient forward propagation model that was validated in previous work. Isolating bottom interacting paths to perform the inversions is shown to be essential to reduce parameter uncertainties in the hard bottom environment and enables a systematic approach to the inversions which establishes the number of layers needed to represent the lake environment. Measured transmission loss data from a towed source were compared through a RMS error analysis to modeled transmission loss, constructed with the parameters from inversions of data from several source types, to further establish the validity of the inversion approach for this environment. Geoacoustic parameters obtained by inversions of short range, low frequency impulsive data are used to predict transmission loss at longer ranges and higher frequencies. The range dependence of the global minimum is discussed.
A test of the Binaural Equal-Loudness-Ratio hypothesis for tones
Authors: Marozeau Jeremy , Epstein Michael , Florentine Mary , Daley Becky
Published: December 2006

It is well known that a tone presented binaurally is louder than the same tone presented monaurally. It is less clear how this loudness ratio changes as a function of level. The present experiment was designed to directly test the Binaural Equal-Loudness-Ratio hypothesis (BELRH), which states that the loudness ratio between equal-SPL monaural and binaural tones is independent of SPL. If true, the BELRH implies that monaural and binaural loudness functions are parallel when plotted on a log scale. Cross-modality matches between string length and loudness were used to directly measure binaural and monaural loudness functions for nine normal listeners. Stimuli were 1-kHz 200-ms tones ranging in level from 5 dB SL to 100dB SPL. A two-way ANOVA showed significant effects of level and mode (binaural or monaural) on loudness, but no interaction between the level and mode. Consequently, no significant variations were found in the binaural-to-monaural loudness ratio across the range of levels tested. This finding supports the BELRH. In addition, the present data were found to closely match loudness functions derived from binaural level differences for equal loudness using the model proposed by Whilby et al. [J. Acoust. Soc. Am. 119, 3931–3939 (2006)].
Precursor arrivals in the Yellow Sea, their distinction from first-order head waves, and their geoacoustic inversion
Authors: Dahl Peter H. , Choi Jee Woong
Published: December 2006

Measurements made as part of the 1996 Yellow Sea experiment at location 37° N, 124° E, undertaken by China and the U.S. are analyzed. Signals generated by explosive sources were received by a 60-m-length vertical line array deployed in waters 75m deep. Evidence is presented that precursor arrivals measured at ranges less than 1km are refracted waves that are zeroth order in their ray series classification, and this directly points to the existence of a gradient in sediment sound speed. In contrast, first-order head waves, which are much weaker in amplitude, would exist only if this gradient were absent. It is found that the energy spectrum of precursor arrivals agrees well with a zeroth-order model, i.e., it is proportional to the source amplitude spectrum, S(f), where f is frequency, rather than a first-order model, which would have it proportional to S(f)∕f. From travel time analysis the sediment sound speed just below the water-sediment interface is estimated to be 1573m∕s with a gradient of 1.1s−1, and from analysis of the energy spectrum of the precursor arrivals the sediment attenuation is estimated to be 0.08dB∕m∕kHz over the frequency range 150–420Hz. The results apply to a nominal sediment depth of 100m.
Simulations of large acoustic scintillations in the Straits of Florida
Authors: Tang Xin , Tappert F. D. , Creamer Dennis B.
Published: December 2006

Using a full-wave acoustic model, Monte Carlo numerical studies of intensity fluctuations in a realistic shallow water environment that simulates the Straits of Florida, including internal wave fluctuations and bottom roughness, have been performed. Results show that the sound intensity at distant receivers scintillates dramatically. The acoustic scintillation index SI increases rapidly with propagation range and is significantly greater than unity at ranges beyond about 10km. This result supports a theoretical prediction by one of the authors. Statistical analyses show that the distribution of intensity of the random wave field saturates to the expected Rayleigh distribution with SI=1 at short range due to multipath interference effects, and then SI continues to increase to large values. This effect, which is denoted supersaturation, is universal at long ranges in waveguides having lossy boundaries (where there is differential mode attenuation). The intensity distribution approaches a log-normal distribution to an excellent approximation; it may not be a universal distribution and comparison is also made to a K distribution. The long tails of the log-normal distribution cause “acoustic intermittency” in which very high, but rare, intensities occur.
Antibiotic Administration Early in Life Impairs Specific Humoral Responses to an Oral Antigen and Increases Intestinal Mast Cell Numbers and Mediator Concentrations
Authors: Nutten S. , Schumann A. , Donnicola D. , Mercenier A. , Rami S. , Garcia-Rodenas C. L.
Published: December 2006

In this study, we assessed the effect of administering the antibiotic amoxicillin to rat pups on the immune response to orally fed ovalbumin (OVA). We first established that amoxicillin administration durably altered the gut microbiota of these animals. In parallel, we observed that the induction of the specific humoral response to ovalbumin was impaired when it occurred during antibiotic administration to the rat pups. We also examined the consequences of those observations on further allergic reactions. Amoxicillin administration had no significant impact on subsequent sensitization to OVA, as nonexacerbated systemic allergic responses were induced in antibiotic-treated animals. However, increased rat mast cell protease II levels and higher mast cell numbers were detected in their small intestines, independently of the antigen administration. Globally, our data suggest that antibiotic administration early in life negatively affects the specific immune response to a luminal antigen when it is first introduced during antibiotic administration. The increased mast cell numbers and mediator concentrations in the intestinal mucosae of the antibiotic-treated animals may testify to the early stages of an altered immune system homeostasis.
Enzyme-Linked Immunosorbent Assay with Conserved Immunoreactive Glycoproteins gp36 and gp19 Has Enhanced Sensitivity and Provides Species-Specific Immunodiagnosis ofEhrlichia canisInfection
Authors: Cárdenas Ana Maria , Doyle C. Kuyler , Zhang Xiaofeng , Nethery Kimberly , Corstvet Richard E. , Walker David H. , McBride Jere W.
Published: December 2006

Ehrlichia canisis the primary etiologic agent of canine monocytic ehrlichiosis, a globally distributed and potentially fatal disease of dogs. We previously reported on the identification of two conserved major immunoreactive antigens, gp36 and gp19, which are the first proteins to elicit anE. canis-specific antibody response, and gp200 and p28, which elicit strong antibody responses later in the acute phase of the infection. In this report, the sensitivities and specificities of five recombinantE. canisproteins for the immunodiagnosis ofE. canisinfection by an enzyme-linked immunosorbent assay (ELISA) were evaluated. Recombinant polypeptides gp36, gp19, and gp200 (N and C termini) exhibited 100% sensitivity and specificity for immunodiagnosis by the recombinant glycoprotein ELISA compared with the results obtained by an indirect fluorescent-antibody assay (IFA) for the detection of antibodies in dogs that were naturally infected withE. canis. Moreover, the enhanced sensitivities of gp36 and gp19 for immunodiagnosis by the recombinant glycoprotein ELISA compared to those obtained by IFA were demonstrated with dogs experimentally infected withE. canis, in which antibodies were detected as much as 2 weeks earlier, on day 14 postinoculation. gp36 and gp19 were not cross-reactive with antibodies in sera fromE. chaffeensis-infected dogs and thus provided species-specific serologic discrimination betweenE. canisandE. chaffeensisinfections. This is the first demonstration of the improved detection capability of the recombinant protein technology compared to the capability of the “gold standard” IFA and may eliminate the remaining obstacles associated with the immunodiagnosis ofE. canisinfections, including species-specific identification and the lack of sensitivity associated with low antibody titers early in the acute phase of the infection.
Capsular Serotype K1 or K2, Rather than
magA
and
rmpA
, Is a Major Virulence Determinant for
Klebsiella pneumoniae
Liver Abscess in Singapore and Taiwan
Authors: Yeh Kuo-Ming , Kurup A. , Siu L. K. , Koh Y. L. , Fung Chang-Phone , Lin Jung-Chung , Chen Te-Li , Chang Feng-Yee , Koh Tse-Hsien
Published: December 2006

Capsular serotypes,
magA
, and
rmpA
have been documented in high prevalence for
Klebsiella pneumoniae
liver abscess. To investigate the regional difference and the correlation of capsular serotype,
magA
, and
rmpA
with virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of
magA
and
rmpA
was determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of
magA
and
rmpA.
The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%).
magA
was restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried
rmpA
. Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did
rmpA
-positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of
rmpA
-positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than
rmpA
contributed to virulence. The characteristics of
K. pneumoniae
liver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than
magA
and
rmpA
in determining virulence in
K. pneumoniae
liver abscess.
Recurrent Disseminated Skin Lesions Due to
Metarrhizium anisopliae
in an Adult Patient with Acute Myelogenous Leukemia
Authors: Osorio S. , de la Cámara R. , Monteserin M. C. , Granados R. , Oña F. , Rodriguez-Tudela J. L. , Cuenca-Estrella M.
Published: December 2006

Metarrhizium anisopliae
is a common insect pathogen that rarely causes infection in animals and humans. We report the first case of a disseminated skin infection in an immunocompromised adult patient. To date, only five cases of the disease in humans have been reported. There is no standard treatment for this infection.
Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Phage Typing for Subtype Analysis of
Salmonella enterica
Serotype Enteritidis
Authors: Boxrud D. , Pederson-Gulrud K. , Wotton J. , Medus C. , Lyszkowicz E. , Besser J. , Bartkus J. M.
Published: December 2006

Strain subtyping is an important tool for detection of outbreaks caused by
Salmonella enterica
serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping
Salmonella
serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of
Salmonella
serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by
Salmonella
serotype Enteritidis.
Structural Shifts of Mucosa-Associated Lactobacilli and
Clostridium leptum
Subgroup in Patients with Ulcerative Colitis
Authors: Zhang Meiling , Liu Bianying , Zhang Yan , Wei Hua , Lei Yufeng , Zhao Liping
Published: December 2006

To understand the role of mucosa-associated microbiota in the pathogenicity of ulcerative colitis (UC), paired biopsies were obtained during colonoscopy from the ulcerated and nonulcerated gut mucosa of 24 patients with UC. Denaturing gradient gel electrophoresis analysis was employed to profile the composition of the dominant bacteria (16S rRNA gene V3 region) and three important groups: lactobacilli, the
Clostridium leptum
subgroup, and
Bacteroides
spp. The Pearson coefficient was used to estimate similarities between the bacterial communities of the paired biopsies for each patient. The average similarity values of bacterial composition between the paired samples were 94.8 ± 3.8% for dominant bacteria, 59.9 ± 26.1% for lactobacilli, 79.2 ± 22.6% for the
Clostridium leptum
subgroup, and 88.7 ± 16.4% for
Bacteroides
spp. The data revealed that lactobacilli and the
Clostridium leptum
subgroup were significantly different between the ulcerated and the nonulcerated regions. It also was noted that for lactobacilli, the composition varied significantly between biopsy sites irrespective of the location of UC in the gut but that the composition of the
Clostridium leptum
subgroup showed significant differences between paired samples from UC in the rectum and not in the left colon. Localized dysbiosis of the mucosa-associated intestinal microflora, especially for lactobacilli and the
Clostridium leptum
subgroup, may be closely related to UC.
Multitarget PCR for Diagnosis of Pertussis and Its Clinical Implications
Authors: Qin Xuan , Galanakis Emmanouil , Martin Emily T. , Englund Janet A.
Published: December 2006

PCR has greatly facilitated pertussis diagnosis due to the speed, sensitivity, and specificity of this assay compared to other detection methods. Various single-target PCR assays are currently utilized, but none is universally considered to be the “gold standard.” Our aim was to assess the use of multitarget versus single-target PCR for the diagnosis of pertussis in clinical samples. PCR assays targeting insertion sequence IS
481
(IS), pertussis toxin
ptxA
promoter region (PT), and outer membrane porin (PO), or
recA
(RA) were evaluated in respiratory specimens collected from 4,442 patients with suspected pertussis. The diagnosis of pertussis was confirmed in 309 (6.96%) patients by the 3-target IS-PT-PO/RA PCR versus 247 (5.56%) by the conventional single-target IS (
P
= 0.007). Compared to single-target IS, the three-target combination increased the proportion of positive specimens by 1.25-fold, and two-target combinations increased the proportion of positive specimens by 1.10- to 1.24-fold. In addition, nine cases of
B. parapertussis
infection were also confirmed by using the discriminative features of this multitarget PCR. Of the 89 culture-proven pertussis cases, 17 (19.1%) and 5 of the 16 patients (31.3%) admitted to intensive care unit would have been missed had only the single-target IS PCR been applied. Patients with mild disease (
P
= 0.004) and shorter hospitalization (
P
= 0.006) were less likely to have positive cultures. This consensus generating real-time PCR approach permits a sensitive detection, as well as an accurate species identification of the causative
Bordetella
pathogens for the timely management of patients.
The VP35 Protein of Ebola Virus Inhibits the Antiviral Effect Mediated by Double-Stranded RNA-Dependent Protein Kinase PKR
Authors: Feng Zongdi , Cerveny Melissa , Yan Zhipeng , He Bin
Published: December 2006

The VP35 protein of Ebola virus is a viral antagonist of interferon. It acts to block virus or double-stranded RNA-mediated activation of interferon regulatory factor 3, a transcription factor that facilitates the expression of interferon and interferon-stimulated genes. In this report, we show that the VP35 protein is also able to inhibit the antiviral response induced by alpha interferon. This depends on the VP35 function that interferes with the pathway regulated by double-stranded RNA-dependent protein kinase PKR. When expressed in a heterologous system, the VP35 protein enhanced viral polypeptide synthesis and growth in Vero cells pretreated with alpha/beta interferon, displaying an interferon-resistant phenotype. In correlation, phosphorylation of PKR and eIF-2α was suppressed in cells expressing the VP35 protein. This activity of the VP35 protein was required for efficient viral replication in PKR
+/+
but not PKR
−/−
mouse embryo fibroblasts. Furthermore, VP35 appears to be a RNA binding protein. Notably, a deletion of amino acids 1 to 200, but not R312A substitution in the RNA binding motif, abolished the ability of the VP35 protein to confer viral resistance to interferon. However, the R312A substitution rendered the VP35 protein unable to inhibit the induction of the beta interferon promoter mediated by virus infection. Together, these results show that the VP35 protein targets multiple pathways of the interferon system.
Cell-Free Human Immunodeficiency Virus Type 1 Transcytosis through Primary Genital Epithelial Cells
Authors: Bobardt Michael D. , Chatterji Udayan , Selvarajah Suganya , Van der Schueren Bernadette , David Guido , Kahn Bruce , Gallay Philippe A.
Published: December 2006

Although the transport of human immunodeficiency virus type 1 (HIV-1) through the epithelium is critical for HIV-1 colonization, the mechanisms controlling this process remain obscure. In the present study, we investigated the transcellular migration of HIV-1 as a cell-free virus through primary genital epithelial cells (PGECs). The absence of CD4 on PGECs implicates an unusual entry pathway for HIV-1. We found that syndecans are abundantly expressed on PGECs and promote the initial attachment and subsequent entry of HIV-1 through PGECs. Although CXCR4 and CCR5 do not contribute to HIV-1 attachment, they enhance viral entry and transcytosis through PGECs. Importantly, HIV-1 exploits both syndecans and chemokine receptors to ensure successful cell-free transport through the genital epithelium. HIV-1-syndecan interactions rely on specific residues in the V3 of gp120 and specific sulfations within syndecans. We found no obvious correlation between coreceptor usage and the capacity of the virus to transcytose. Since viruses isolated after sexual transmission are mainly R5 viruses, this suggests that the properties conferring virus replication after transmission are distinct from those conferring cell-free virus transcytosis through the genital epithelium. Although we found that cell-free HIV-1 crosses PGECs as infectious particles, the efficiency of transcytosis is extremely poor (less than 0.02% of the initial inoculum). This demonstrates that the genital epithelium serves as a major barrier against HIV-1. Although one cannot exclude the possibility that limited passage of cell-free HIV-1 transcytosis through an intact genital epithelium occurs in vivo, it is likely that the establishment of infection via cell-free HIV-1 transmigration is a rare event.
M-T5, the Ankyrin Repeat, Host Range Protein of Myxoma Virus, Activates Akt and Can Be Functionally Replaced by Cellular PIKE-A
Authors: Werden Steven J. , Barrett John W. , Wang Gen , Stanford Marianne M. , McFadden Grant
Published: December 2006

The myxoma virus (MV) ankyrin repeat, host range factor M-T5 has the ability to bind and activate cellular Akt, leading to permissive MV replication in a variety of diverse human cancer cell lines (G. Wang, J. W. Barrett, M. Stanford, S. J. Werden, J. B. Johnston, X. Gao, M. Sun, J. Q. Cheng, and G. McFadden, Proc. Natl. Acad. Sci. USA
103:
4640-4645, 2006). The susceptibility of permissive human cancer cells to MV infection is directly correlated with the basal or induced levels of phosphorylated Akt. When M-T5 is deleted from MV, the knockout virus, vMyxT5KO, can no longer productively infect a subset of human cancer cells (designated type II) that exhibit little or no endogenous phosphorylated Akt. In searching for a host counterpart of M-T5, we noted sequence similarity of M-T5 to a recently identified ankyrin repeat cellular binding protein of Akt called PIKE-A. PIKE-A binds and activates the kinase activity of Akt in a GTP-dependent manner and promotes the invasiveness of human cancer cell lines. Here, we demonstrate that transfected PIKE-A is able to rescue the ability of vMyxT5KO to productively infect type II human cancer cells that were previously resistant to infection. Also, cancer cells that were completely nonpermissive for both wild-type and vMyxT5KO infection (called type III) were rendered fully permissive following ectopic expression of PIKE-A. We conclude that the MV M-T5 host range protein is functionally interchangeable with the host PIKE-A protein and that the activation of host Akt by either M-T5 or PIKE-A is critical for the permissiveness of human cancer cells for MV.
Early Resolution of Herpes Simplex Virus Type 2 Infection of the Murine Genital Tract Involves Stimulation of Genital Parenchymal Cells by Gamma Interferon
Authors: Bird Melanie D. , Chu Chin-Fun , Johnson Alison J. , Milligan Gregg N.
Published: December 2006

Early clearance of a thymidine kinase-deficient strain of herpes simplex virus type 2 from the female genital tract required T-cell-produced gamma interferon (IFN-γ). Transfer of activated CD8+T cells to irradiated C57BL/6 mice resulted in rapid virus clearance, but clearance was greatly delayed in recipients deficient in the IFN-γ receptor (IFN-γR). Early virus clearance was demonstrated in radiation chimeras in which IFN-γR expression was limited to parenchymal cells, but resolution was significantly delayed in chimeras deficient in IFN-γR expression and chimeras expressing IFN-γR only on hematopoietic cells. Together, these results suggest that early IFN-γ-mediated protection was manifested mainly by stimulation of genital parenchymal cells.
In Vivo Packaging of Brome Mosaic Virus RNA3, but Not RNAs 1 and 2, Is Dependent on a
cis
-Acting 3′ tRNA-Like Structure
Authors: Annamalai Padmanaban , Rao A. L. N.
Published: December 2006

The four encapsidated RNAs of brome mosaic virus (BMV; B1, B2, B3, and B4) contain a highly conserved 3′ 200-nucleotide (nt) region encompassing the tRNA-like structure (TLS) which is required for packaging in vitro (Y. G. Choi, T. W. Dreher, and A. L. N. Rao, Proc. Natl. Acad. Sci. USA 99:655-660, 2002). To validate these observations in vivo, we performed packaging assays using
Agrobacterium-
mediated transient expression of RNAs and coat protein (CP) (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005). Coexpression of TLS-less constructs of B1 or B2 or B3 and CP mRNAs in
Nicotiana benthamiana
leaves resulted in packaging of TLS-less B1 and B2 but not B3, suggesting that packaging of B3 requires the TLS in
cis
. This conjecture was confirmed by the efficient packaging of a B3 chimera in which the viral TLS was replaced with a cellular tRNA
Tyr
. When
N. benthamiana
leaves were infiltrated with a mixture of transformants containing wild-type B1 (wtB1) plus wtB2 plus a TLS-less B3 (wtB1+wtB2+TLS-lessB3), the 3′ end of progeny B3 was restored by heterologous recombination with that of either B1 or B2. This intrinsic
cis-
requirement of TLS in promoting B3 packaging was further confirmed when a mixture containing agrotransformants of TLS-less B1+B2+B3 was supplemented with either wtB4 or a 3′ 200-nt or 3′ 336-nt untranslated region (UTR) of B3. Northern blot analysis followed by sequencing of B3 progeny revealed that replication of TLS-less B3, but not TLS-less B1 or B2, was fully restored due to recombination with TLS from transiently expressed wtB4 or the B3 3′ UTR. Collectively, these observations suggested that the requirement of a
cis
-acting TLS is distinct for B3 compared with B1 or B2.
Structural and Mutagenic Analysis of Foot-and-Mouth Disease Virus 3C Protease Reveals the Role of the β-Ribbon in Proteolysis
Authors: Sweeney Trevor R. , Roqué-Rosell Núria , Birtley James R. , Leatherbarrow Robin J. , Curry Stephen
Published: December 2006

The 3C protease (3C
pro
) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C
pro
, a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a β-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This β-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the β-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C
pro
with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the β-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C
pro
inhibitors.
Virus-Induced Decline in Soluble Vascular Endothelial Growth Receptor 2 Is Associated with Plasma Leakage in Dengue Hemorrhagic Fever
Authors: Srikiatkhachorn Anon , Ajariyakhajorn Chuanpis , Endy Timothy P. , Kalayanarooj Siripen , Libraty Daniel H. , Green Sharone , Ennis Francis A. , Rothman Alan L.
Published: December 2006

Some individuals infected with dengue virus develop dengue hemorrhagic fever (DHF), a viral hemorrhagic disease characterized by a transient period of localized plasma leakage. To determine the importance of vascular endothelial growth factor A (VEGF-A) in this syndrome, we compared plasma levels of VEGF-A and the soluble forms of its receptors in patients with DHF to patients with dengue fever (DF), a milder form of dengue virus infection without plasma leakage. We observed a rise in the plasma levels of free, but not total VEGF-A in DHF patients at the time of plasma leakage. This was associated with a decline in the soluble form of VEGF receptor 2 (VEGFR2) and VEGF-soluble VEGFR2 complexes, but not the soluble form of VEGFR1. The severity of plasma leakage in patients inversely correlated with plasma levels of soluble VEGFR2. In vitro, dengue virus suppressed soluble VEGFR2 production by endothelial cells but up-regulated surface VEGFR2 expression and promoted response to VEGF stimulation. In vivo, plasma viral load correlated with the degree of decline in plasma soluble VEGFR2. These results suggest that VEGF regulates vascular permeability and its activity is controlled by binding to soluble VEGFR2. Dengue virus-induced changes in surface and soluble VEGFR2 expression may be an important mechanism of plasma leakage in DHF.
Interaction of Papillomavirus E2 Protein with the Brm Chromatin Remodeling Complex Leads to Enhanced Transcriptional Activation
Authors: Ajay Kumar R. , Naidu Samisubbu R. , Wang Xiaoyu , Imbalzano Anthony N. , Androphy Elliot J.
Published: December 2006

Papillomavirus E2 is a sequence-specific DNA binding protein that regulates transcription and replication of the viral genome. The transcriptional activities of E2 are typically evaluated by transient transfection of nonreplicating E2-dependent reporters. We sought to address whether E2 activates transcription in an episomal context and its potential interaction with the chromatin remodeling proteins. Using an Epstein-Barr virus-based episomal reporter, we demonstrate that E2 stimulates transcription from an E2-dependent promoter in a chromatin context. This activation is enhanced by the presence of proteins associated with SWI/SNF complexes, which are ATP-dependent chromatin remodeling enzymes. We show that exogenous expression of the Brm ATPase enhances E2 activity in SWI/SNF-deficient cell lines and that the amino-terminal transactivation domain of E2 mediates association with the Brm complex in vivo. Using chromatin immunoprecipitation assays, we demonstrate that Brm enhances promoter occupancy by E2 in an episomal context. Our results demonstrate that E2 activates transcription from an episomal reporter system and reveal a novel property of E2 in collaborating with the Brm chromatin remodeling complex in enhancing transcriptional activation.
Disseminated Simian Varicella Virus Infection in an Irradiated Rhesus Macaque (
Macaca mulatta
)
Authors: Kolappaswamy Krishnan , Mahalingam Ravi , Traina-Dorge Vicki , Shipley Steven T. , Gilden Donald H. , Kleinschmidt-Demasters Bette K. , McLeod Charles G. , Hungerford Laura L. , DeTolla Louis J.
Published: December 2006

We describe correlative clinicopathological/virological findings from a simian varicella virus (SVV)-seronegative monkey that developed disseminated varicella 105 days after gamma-irradiation. Twelve other monkeys in the colony were also irradiated, none of which developed varicella. Before irradiation, sera from the monkey that developed disseminated infection and one asymptomatic monkey were available. Analysis indicated that subclinical reactivation of latent SVV from an asymptomatic irradiated monkey likely led to disseminated varicella in the seronegative irradiated monkey. These findings parallel those from humans with disseminated varicella infection and support the usefulness of SVV infection as a model for human varicella-zoster virus infection, particularly virus reactivation after gamma-irradiation.
Antiviral Peptides Targeting the West Nile Virus Envelope Protein
Authors: Bai Fengwei , Town Terrence , Pradhan Deepti , Cox Jonathan , Ashish  , Ledizet Michel , Anderson John F. , Flavell Richard A. , Krueger Joanna K. , Koski Raymond A. , Fikrig Erol
Published: December 2006

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.
Hepatitis B Virus X Protein Induces Perinuclear Mitochondrial Clustering in Microtubule- and Dynein-Dependent Manners
Authors: Kim Sujeong , Kim Hye-Young , Lee Seungmin , Kim Sung Woo , Sohn Seonghyang , Kim Kyongmin , Cho Hyeseong
Published: December 2006

The hepatitis B virus (HBV) X protein (HBx) is thought to play a key role in HBV replication and the development of liver cancer. It became apparent that HBx induces mitochondrial clustering at the nuclear periphery, but the molecular basis for mitochondrial clustering is not understood. Since mitochondria move along the cytoskeleton as a cargo of motor proteins, we hypothesized that mitochondrial clustering induced by HBx occurs by an altered intracellular motility. Here, we demonstrated that the treatment of HBx-expressing cells with a microtubule-disrupting drug (nocodazole) abrogated mitochondrial clustering, while the removal of nocodazole restored clustering within 30 to 60 min, indicating that mitochondrial transport is occurring in a microtubule-dependent manner. The addition of a cytochalasin D-disrupting actin filament, however, did not measurably affect mitochondrial clustering. Mitochondrial clustering was further studied by observations of HBV-related hepatoma cells and HBV-replicating cells. Importantly, the abrogation of the dynein activity in HBx-expressing cells by microinjection of a neutralizing anti-dynein intermediate-chain antibody, dynamitin overexpression, or the addition of a dynein ATPase inhibitor significantly suppressed the mitochondrial clustering. In addition, HBx induced the activation of the p38 mitogen-activated protein kinase (MAPK) and inhibition of the p38 kinase activity by SB203580-attenuated HBx-induced mitochondrial clustering. Taken together, HBx activation of the p38 MAPK contributed to the increase in the microtubule-dependent dynein activity. The data suggest that HBx plays a novel regulatory role in subcellular transport systems, perhaps facilitating the process of maturation and/or assembly of progeny particles during HBV replication. Furthermore, mitochondrion aggregation induced by HBx may represent a cellular process that underlies disease progression during chronic viral infection.
Thiol/Disulfide Exchange Is Required for Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein
Authors: Jain Surbhi , McGinnes Lori W. , Morrison Trudy G.
Published: December 2006

Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity—5′5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody—inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.
Wild-Type Levels of Human Immunodeficiency Virus Type 1 Infectivity in the Absence of Cellular Emerin Protein
Authors: Shun Ming-Chieh , Daigle Janet E. , Vandegraaff Nick , Engelman Alan
Published: December 2006

Preintegration complexes (PICs) mediate retroviral integration, and recent results indicate an important role for the inner nuclear membrane protein emerin in orienting human immunodeficiency virus type 1 (HIV-1) PICs to chromatin for integration. Two other host cell proteins, the barrier-to-autointegration factor (BAF) and lamina-associated polypeptide 2α (LAP2α), seemed to play a similar preintegrative role for Moloney murine leukemia virus (MMLV) in addition to HIV-1. In contrast, we determined efficient HIV-1 and MMLV infection of HeLa-P4 cells following potent down-regulation of emerin, BAF, or LAP2α protein by using short interfering RNA. Mouse embryo fibroblasts ablated for emerin protein through gene knockout support the same level of HIV-1 infection as cells derived from wild-type littermate control animals. As the expression of human emerin in mouse knockout cells fails to affect the level of infectivity achieved in its absence, we conclude that HIV-1 efficiently infects cells in the absence of emerin protein and, by extension, that emerin is not a universally important regulator of HIV-1 infectivity.
Participation of both Host and Virus Factors in Induction of Severe Acute Respiratory Syndrome (SARS) in F344 Rats Infected with SARS Coronavirus
Authors: Nagata Noriyo , Iwata Naoko , Hasegawa Hideki , Fukushi Shuetsu , Yokoyama Masaru , Harashima Ayako , Sato Yuko , Saijo Masayuki , Morikawa Shigeru , Sata Tetsutaro
Published: December 2006

To understand the pathogenesis and develop an animal model of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), the Frankfurt 1 SARS-CoV isolate was passaged serially in young F344 rats. Young rats were susceptible to SARS-CoV but cleared the virus rapidly within 3 to 5 days of intranasal inoculation. After 10 serial passages, replication and virulence of SARS-CoV were increased in the respiratory tract of young rats without clinical signs. By contrast, adult rats infected with the passaged virus showed respiratory symptoms and severe pathological lesions in the lung. Levels of inflammatory cytokines in sera and lung tissues were significantly higher in adult F344 rats than in young rats. During in vivo passage of SARS-CoV, a single amino acid substitution was introduced within the binding domain of the viral spike protein recognizing angiotensin-converting enzyme 2 (ACE2), which is known as a SARS-CoV receptor. The rat-passaged virus more efficiently infected CHO cells expressing rat ACE2 than did the original isolate. These results strongly indicate that host and virus factors such as advanced age and virus adaptation are critical for the development of SARS in rats.
Mutations in the Central Domain of Potato Virus X TGBp2 Eliminate Granular Vesicles and Virus Cell-to-Cell Trafficking
Authors: Ju Ho-Jong , Brown James E. , Ye Chang-Ming , Verchot-Lubicz Jeanmarie
Published: December 2006

Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.
Adeno-Associated Virus Site-Specific Integration Is Regulated by TRP-185
Authors: Yamamoto Noriaki , Suzuki Masato , Kawano Masa-aki , Inoue Takamasa , Takahashi Ryou-u , Tsukamoto Hiroko , Enomoto Teruya , Yamaguchi Yuki , Wada Tadashi , Handa Hiroshi
Published: December 2006

Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the
R
ep
b
inding
s
ite (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration.
T
AR
R
NA loop binding
p
rotein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3′→5′ DNA helicase activity.
Murine MusD Retrotransposon: Structure and Molecular Evolution of an “Intracellularized” Retrovirus
Authors: Ribet David , Harper Francis , Dewannieux Marie , Pierron Gérard , Heidmann Thierry
Published: December 2006

We had previously identified active autonomous copies of the MusD long terminal repeat-retrotransposon family, which have retained transpositional activity. These elements are closely related to betaretroviruses but lack an envelope (
env
) gene. Here we show that these elements encode strictly intracellular virus-like particles that can unambiguously be identified by electron microscopy. We demonstrate intracellular maturation of the particles, with a significant proportion of densely packed cores for wild-type MusD but not for a protease mutant. We show that the molecular origin of this unexpected intracellular localization is solely dependent on the N-terminal part of the Gag protein, which lacks a functional sequence for myristoylation and plasma membrane targeting: replacement of the N-terminal domain of the MusD matrix protein by that of its closest relative—the Mason-Pfizer monkey virus—led to targeting of the MusD Gag to the plasma membrane, with viral particles budding and being released into the cell supernatant. These particles can further be pseudotyped with a heterologous envelope protein and become infectious, thus “reconstituting” a functional retrovirus prone to proviral insertions. Consistent with its retroviral origin, a sequence with a constitutive transport element-like activity can further be identified at the MusD 3′ untranslated region. A molecular scenario is proposed that accounts for the transition, during evolution, from an ancestral infectious betaretrovirus to the strictly intracellular MusD retrotransposon, involving not only the loss of the
env
gene but also an inability to escape the cell—via altered targeting of the Gag protein—resulting de facto in the generation of a very successful “intracellularized” insertional mutagen.
Electron Cryotomography Reveals the Portal in the Herpesvirus Capsid
Authors: Chang Juan T. , Schmid Michael F. , Rixon Frazer J. , Chiu Wah
Published: December 2006

Herpes simplex virus type 1 is a human pathogen responsible for a range of illnesses from cold sores to encephalitis. The icosahedral capsid has a portal at one fivefold vertex which, by analogy to portal-containing phages, is believed to mediate genome entry and exit. We used electron cryotomography to determine the structure of capsids lacking pentons. The portal vertex appears different from pentons, being located partially inside the capsid shell, a position equivalent to that of bacteriophage portals. Such similarity in portal organization supports the idea of the evolutionary relatedness of these viruses.
Persistent Antigen Presentation after Acute Vesicular Stomatitis Virus Infection
Authors: Turner Damian L. , Cauley Linda S. , Khanna Kamal M. , Lefrançois Leo
Published: December 2006

Long-term antigen expression is believed to play an important role in modulation of T-cell responses to chronic virus infections. However, recent studies suggest that immune responses may occur late after apparently acute infections. We have now analyzed the CD8 T-cell response to vesicular stomatitis virus (VSV), which is thought to cause to an infection characterized by rapid virus clearance by innate and adaptive immune system components. Unexpectedly, virus-encoded antigen was detectable more than 6 weeks after intranasal VSV infection in both draining and nondraining lymph nodes by adoptively transferred CD8 T cells. Infection withListeria monocytogenesexpressing the same antigen did not result in prolonged antigen presentation. Weeks after VSV infection, discrete T-cell clustering with dendritic cells within the lymph node was observed after transfer of antigen-specific CD8 T cells. Moreover, memory CD8 T cells as defined by phenotype and function were generated from naïve CD8 T cells entering the response late after infection. These findings suggested that protracted antigen presentation after an apparently acute virus infection may contribute to an ongoing antiviral immune response.