bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating Long-Term Ecological Variability Using the Global Population Dynamics Database
Authors: Inchausti Pablo , Halley John
Published: March 2006

The Global Population Dynamics Database (GPDD) is an important new source of information for ecologists, resource managers, and environmental scientists interested in the dynamics of natural populations. It comprises more than 4500 time series of population abundance for over 1800 animal species across many taxonomic groups and geographical locations. The GPDD offers great potential for asking comparative questions about the nature of population variability. We illustrate this by characterizing some critical features of ecological variability, variance growth, and spectral reddening.
Maribavir Pharmacokinetics and the Effects of Multiple-Dose Maribavir on Cytochrome P450 (CYP) 1A2, CYP 2C9, CYP 2C19, CYP 2D6, CYP 3A,
N
-Acetyltransferase-2, and Xanthine Oxidase Activities in Healthy Adults
Authors: Ma Joseph D. , Nafziger Anne N. , Villano Stephen A. , Gaedigk Andrea , Bertino Joseph S.
Published: March 2006

Maribavir (1263W94, VP-41263) is an oral anticytomegalovirus agent under clinical development. The pharmacokinetics and safety of maribavir and the effects of maribavir on the activities of cytochrome P450 (CYP) 1A2, CYP 2C9, CYP 2C19, CYP 2D6, CYP 3A,
N
-acetyltransferase-2 (NAT-2), and xanthine oxidase (XO) were evaluated in a randomized, double-blind, placebo-controlled study. Twenty healthy subjects received a five-drug phenotyping cocktail of caffeine (CYP 1A2, NAT-2, XO), warfarin plus vitamin K (CYP 2C9), omeprazole (CYP 2C19), dextromethorphan (CYP 2D6), and midazolam (CYP 3A) 4 days before and after 7 days of treatment with maribavir at 400 mg twice daily (16 subjects) or placebo (4 subjects) for 10 days. Maribavir did not affect the CYP 1A2, CYP 2C9, CYP 3A, NAT-2, or XO activities. Bioequivalence was not demonstrated for CYP 2C19 and CYP 2D6, suggesting a decrease or inhibition of CYP 2C19 and CYP 2D6 activities. The pharmacokinetics of maribavir following a single dose and after 10 days of treatment were similar, with minimal accumulation at steady state. Maribavir was safe and well tolerated. Taste disturbance was the most frequently reported adverse event. These results will further guide evaluation of the drug interaction potential and clinical development of maribavir.
Garenoxacin Treatment of Experimental Endocarditis Caused by Viridans Group Streptococci
Authors: Anguita-Alonso Paloma , Rouse Mark S. , Piper Kerryl E. , Steckelberg James M. , Patel Robin
Published: March 2006

The activity of garenoxacin was compared to that of levofloxacin or penicillin in a rabbit model of
Streptococcus mitis
group (penicillin MIC, 0.125 μg/ml) and
Streptococcus sanguinis
group (penicillin MIC, 0.25 μg/ml) endocarditis. Garenoxacin and levofloxacin had MICs of 0.125 and 0.5 μg/ml, respectively, for both study isolates. Rabbits with catheter-induced aortic valve endocarditis were given no treatment, penicillin at 1.2 × 10
6
IU/8 h intramuscularly, garenoxacin at 20 mg/kg of body weight/12 h intravenously, or levofloxacin at 40 mg/kg/12 h intravenously. For both isolates tested, garenoxacin area under the curve (AUC)/MIC and maximum concentration of drug in serum (
C
max
)/MIC ratios were 368 and 91, respectively. Rabbits were sacrificed after 3 days of treatment; cardiac valve vegetations were aseptically removed and quantitatively cultured. For
S. mitis
group experimental endocarditis, all studied antimicrobial agents were more active than no treatment (
P
< 0.001), whereas for
S. sanguinis
group endocarditis, no studied antimicrobial agents were more active than no treatment. We conclude that AUC/MIC and
C
max
/MIC ratios may not predict activity of some quinolones in experimental viridans group endocarditis and that garenoxacin and levofloxacin may not be ideal choices for serious infections caused by some quinolone-susceptible viridans group streptococci.
Using Bacteriophages To Reduce Formation of Catheter-Associated Biofilms byStaphylococcus epidermidis
Authors: Curtin John J. , Donlan Rodney M.
Published: March 2006

Use of indwelling catheters is often compromised as a result of biofilm formation. This study investigated if hydrogel-coated catheters pretreated with a coagulase-negative bacteriophage would reduceStaphylococcus epidermidisbiofilm formation. Biofilms were developed on hydrogel-coated silicone catheters installed in a modified drip flow reactor. Catheter segments were pretreated with the lyticS. epidermidisbacteriophage 456 by exposing the catheter lumen to a 10-log-PFU/ml culture of the bacteriophage for 1 h at 37°C prior to biofilm formation. The untreated mean biofilm cell count was 7.01 ± 0.47 log CFU/cm2of catheter. Bacteriophage treatment with and without supplemental divalent cations resulted in log-CFU/cm2reductions of 4.47 (P< 0.0001) and 2.34 (P= 0.001), respectively. Divalent cation supplementation without bacteriophage treatment provided a 0.67-log-CFU/cm2reduction (P= 0.053). Treatment of hydrogel-coated silicone catheters with anS. epidermidisbacteriophage in an in vitro model system significantly reduced viable biofilm formation byS. epidermidisover a 24-h exposure period, suggesting the potential of bacteriophage for mitigating biofilm formation on indwelling catheters and reducing the incidence of catheter-related infections.
Triaryl Pyrazoline Compound Inhibits Flavivirus RNA Replication
Authors: Puig-Basagoiti Francesc , Tilgner Mark , Forshey Brett M. , Philpott Sean M. , Espina Noel G. , Wentworth David E. , Goebel Scott J. , Masters Paul S. , Falgout Barry , Ren Ping , Ferguson David M. , Shi Pei-Yong
Published: March 2006

Triaryl pyrazoline {[5-(4-chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-1-yl]-phenyl-methanone} inhibits flavivirus infection in cell culture. The inhibitor was identified through high-throughput screening of a compound library using a luciferase-expressing West Nile (WN) virus infection assay. The compound inhibited an epidemic strain of WN virus without detectable cytotoxicity (a 50% effective concentration of 28 μM and a compound concentration of ≥300 μM required to reduce 50% cell viability). Besides WN virus, the compound also inhibited other flaviviruses (dengue, yellow fever, and St. Louis encephalitis viruses), an alphavirus (Western equine encephalitis virus), a coronavirus (mouse hepatitis virus), and a rhabdovirus (vesicular stomatitis virus). However, the compound did not suppress an orthomyxovirus (influenza virus) or a retrovirus (human immunodeficiency virus type 1). Mode-of-action analyses in WN virus showed that the compound did not inhibit viral entry or virion assembly but specifically suppressed viral RNA synthesis. To examine the mechanism of inhibition of dengue virus, we developed two replicon systems for dengue type 1 virus: (i) a stable cell line that harbored replicons containing a luciferase reporter and a neomycin phosphotransferase selection marker and (ii) a luciferase-expressing replicon that could differentiate between viral translation and RNA replication. Analyses of the compound in the dengue type 1 virus replicon systems showed that it weakly suppressed viral translation but significantly inhibited viral RNA synthesis. Overall, the results demonstrate that triaryl pyrazoline exerts a broad spectrum of antiflavivirus activity through potent inhibition of viral RNA replication. This novel inhibitor could be developed for potential treatment of flavivirus infection.
Activity and Mechanism of Action of
N
-Methanocarbathymidine against Herpesvirus and Orthopoxvirus Infections
Authors: Prichard Mark N. , Keith Kathy A. , Quenelle Debra C. , Kern Earl R.
Published: March 2006

N
-Methanocarbathymidine [(
N
)-MCT] is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. The antiviral activity of this molecule is dependent on the type I thymidine kinase (TK) in herpes simplex virus and also appears to be dependent on the type II TK expressed by cowpox and vaccinia viruses, suggesting that it is a substrate for both of these divergent forms of the enzyme. The drug is also a good inhibitor of viral DNA synthesis in both viruses and is consistent with inhibition of the viral DNA polymerase once it is activated by the viral TK homologs. This mechanism of action explains the rather unusual spectrum of activity, which is limited to orthopoxviruses, alphaherpesviruses, and Epstein-Barr virus, since these viruses express molecules with TK activity that can phosphorylate and thus activate the drug. The compound is also effective in vivo and reduces the mortality of mice infected with orthopoxviruses, as well as those infected with herpes simplex virus type 1 when treatment is initiated 24 h after infection. These results indicate that (
N
)-MCT is active in vitro and in vivo, and its mechanism of action suggests that the molecule may be an effective therapeutic for orthopoxvirus and herpesvirus infections, thus warranting further development.
Contribution ofrpoB2RNA Polymerase β Subunit Gene to Rifampin Resistance inNocardiaSpecies
Authors: Ishikawa Jun , Chiba Kazuhiro , Kurita Haruyo , Satoh Hiroyuki
Published: March 2006

Nocardiaspecies are gram-positive environmental saprophytes, but some cause the infectious disease nocardiosis. The complete genomic sequence ofNocardia farcinicaIFM 10152 has been determined, and analyses indicated the presence of two different RNA polymerase β subunit genes,rpoBandrpoB2, in the genome (J. Ishikawa, A. Yamashita, Y. Mikami, Y. Hoshino, H. Kurita, K. Hotta, T. Shiba, and M. Hattori, Proc. Natl. Acad. Sci. USA 101:14925-14930, 2004). These genes share 88.8% identity at the nucleotide level. Moreover, comparison of their amino acid sequences with those of other bacterial RpoB proteins suggested that the nocardial RpoB protein is likely to be rifampin (RIF) sensitive, whereas RpoB2 protein contains substitutions at the RIF-binding region that are likely to confer RIF resistance. Southern analysis indicated thatrpoBduplication is widespread inNocardiaspecies and is correlated with the RIF-resistant phenotype. The introduction ofrpoB2by using a newly developedNocardia-Escherichia colishuttle plasmid vector and transformation system conferred RIF resistance toNocardia asteroidesIFM 0319T, which has neither RIF resistance norrpoBduplication. Furthermore, unmarkedrpoB2deletion mutants ofN. farcinicaIFM 10152 showed no significant resistance to RIF. These results indicated the contribution ofrpoB2to RIF resistance inNocardiaspecies. Since this is the first example of genetic engineering of theNocardiagenome, we believe that this study, as well as our determination of theN. farcinicagenome sequence, will be a landmark inNocardiagenetics.
Interaction of Pleuromutilin Derivatives with the Ribosomal Peptidyl Transferase Center
Authors: Long Katherine S. , Hansen Lykke H. , Jakobsen Lene , Vester Birte
Published: March 2006

Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. AnEscherichia coliL3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.
Effect of Farnesol on
Staphylococcus aureus
Biofilm Formation and Antimicrobial Susceptibility
Authors: Jabra-Rizk M. A. , Meiller T. F. , James C. E. , Shirtliff M. E.
Published: March 2006

Staphylococcus aureus
is among the leading pathogens causing bloodstream infections able to form biofilms on host tissue and indwelling medical devices and to persist and cause disease. Infections caused by
S. aureus
are becoming more difficult to treat because of increasing resistance to antibiotics. In a biofilm environment particularly, microbes exhibit enhanced resistance to antimicrobial agents. Recently, farnesol was described as a quorum-sensing molecule with possible antimicrobial properties. In this study, the effect of farnesol on methicillin-resistant and -susceptible strains of
S. aureus
was investigated. With viability assays, biofilm formation assessment, and ethidium bromide uptake testing, farnesol was shown to inhibit biofilm formation and compromise cell membrane integrity. The ability of farnesol to sensitize
S. aureus
to antimicrobials was assessed by agar disk diffusion and broth microdilution methods. For both strains of staphylococci, farnesol was only able to reverse resistance at a high concentration (150 μM). However, it was very successful at enhancing the antimicrobial efficacy of all of the antibiotics to which the strains were somewhat susceptible. Therefore, synergy testing of farnesol and gentamicin was performed with static biofilms exposed to various concentrations of both agents. Plate counts of harvested biofilm cells at 0, 4, and 24 h posttreatment indicated that the combined effect of gentamicin at 2.5 times the MIC and farnesol at 100 μM (22 μg/ml) was able to reduce bacterial populations by more than 2 log units, demonstrating synergy between the two antimicrobial agents. This observed sensitization of resistant strains to antimicrobials and the observed synergistic effect with gentamicin indicate a potential application for farnesol as an adjuvant therapeutic agent for the prevention of biofilm-related infections and promotion of drug resistance reversal.
Binding of Neomycin-Class Aminoglycoside Antibiotics to Mutant Ribosomes with Alterations in the A Site of 16S rRNA
Authors: Hobbie Sven N. , Pfister Peter , Bruell Christian , Sander Peter , François Boris , Westhof Eric , Böttger Erik C.
Published: March 2006

Aminoglycoside antibiotics that bind to the aminoacyl-tRNA site (A site) of the ribosome are composed of a common neamine core in which a glycopyranosyl ring is attached to position 4 of a 2-deoxystreptamine moiety. The core is further substituted by one (ribostamycin), two (neomycin and paromomycin), or three (lividomycin A) additional sugars attached to position 5 of the 2-deoxystreptamine. To study the role of rings III, IV, and V in aminoglycoside binding, we used isogenic
Mycobacterium smegmatis
Δ
rrnB
mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site. MICs were determined to investigate drug-ribosome interactions, and the results were compared with that of the previously published crystal structure of paromomycin bound to the ribosomal A site. Our analysis demonstrates that the stacking interaction between ring I and G1491 is largely sequence independent, that rings III and IV each increase the strength of drug binding to the ribosome, that ring IV of the 6′-NH
3
+
aminoglycosides compensates for loss of interactions between ring II and U1495 and between ring III and G1491, that the aminoglycosides rely on pseudo-base pairing between ring I and A1408 for binding independently of the number of sugar rings attached to the neamine core, that addition of ring V to the 6′-OH 4,5-aminoglycoside paromomycin does not alter the mode of binding, and that alteration of the U1406 · U1495 wobble base pair to the Watson-Crick interaction pair 1406C-1495G yields ribosomal drug susceptibilities to 4,5-aminoglycosides comparable to those seen with the wild-type A site.
Multidimensional Volumetric Imaging of Pulmonary Infiltrates for Measuring Therapeutic Response to Antifungal Therapy in Experimental Invasive Pulmonary Aspergillosis
Authors: Petraitis Vidmantas , Petraitiene Ruta , Solomon Jeffrey , Kelaher Amy M. , Murray Heidi A. , Mya-San Christine , Bhandary Avi K. , Sein Tin , Avila Nilo A. , Basevicius Algidas , Bacher John , Walsh Thomas J.
Published: March 2006

Pulmonary infiltrates in neutropenic hosts with invasive pulmonary aspergillosis are caused by vascular invasion, hemorrhagic infarction, and tissue necrosis. Monitoring the dynamics of pulmonary infiltrates of invasive aspergillosis is an important tool for assessing response to antifungal therapy. We, therefore, introduced a multidimensional volumetric imaging (MDVI) method for analysis of the response of the volume of pulmonary infiltrates over time to antifungal therapy in experimental invasive pulmonary aspergillosis (IPA) in persistently neutropenic rabbits. We developed a semiautomatic method to measure the volume of lung lesions, which was implemented as an extension of the MEDx visualization and analysis software using ultrafast computerized tomography (UFCT). Volumetric infiltrate measures were compared with UFCT reading, histopathological resolution of lesions, microbiological clearance of
Aspergillus fumigatus
, and galactomannan index (GMI). We also studied the MDVI method for consistency and reproducibility in comparison to UFCT. Treatment groups consisted of deoxycholate amphotericin B (DAMB) at 0.5 or 1 mg/kg of body weight/day and untreated controls (UC). Therapeutic monitoring of pulmonary infiltrates using MDVI demonstrated a significant decrease in the infiltrate volume in DAMB-treated rabbits in comparison to UC (
P
≤ 0.001). Volumetric data by MDVI correlated with conventional CT pulmonary scores (
r
= 0.83,
P
≤ 0.001). These results correlated with validated biological endpoints: pulmonary infarct scores (
r
= 0.85,
P
≤ 0.001), lung weights (
r
= 0.76,
P
≤ 0.01), residual fungal burden (
r
= 0.65,
P
≤ 0.05), and GMI (
r
= 0.78,
P
≤ 0.01). MDVI correlated with key biological markers, improved the objectivity of radiological assessment of therapeutic response to antifungal therapy, and warrants evaluation for monitoring therapeutic response in immunocompromised patients with invasive aspergillosis.
Microflora Changes with Norfloxacin and Pivmecillinam in Women with Recurrent Urinary Tract Infection
Authors: Norinder Birgit Stattin , Norrby Ragnar , Palmgren Ann-Chatrin , Hollenberg Sofia , Eriksson Ulla , Nord Carl Erik
Published: March 2006

Similar changes in the periurethral and vaginal microflora were observed in 19 women with recurrent urinary tract infection following treatment with norfloxacin (NOR) or pivmecillinam (PIV).
Escherichia coli
strains were suppressed by both treatments.
Staphylococcus
spp. and enterococci colony counts increased following PIV treatment in the periurethral flora but remained stable with NOR.
Genetic Basis of Tetracycline and Minocycline Resistance in Potentially Probiotic
Lactobacillus plantarum
Strain CCUG 43738
Authors: Huys Geert , D'Haene Klaas , Swings Jean
Published: March 2006

The potentially probiotic strain
Lactobacillus plantarum
CCUG 43738, which displayed atypical phenotypic resistance to tetracycline (MIC, 512 μg/ml) and minocycline (MIC, 256 μg/ml), was found to contain a
tet
(S) gene located on a plasmid of approximately 14 kb. Plasmid curing with novobiocin eliminated this plasmid and restored the tetracycline-susceptible phenotype of the host strain.
Variability in the Region Downstream of the
bla
CMY-2
β-Lactamase Gene in
Escherichia coli
and
Salmonella enterica
Plasmids
Authors: Kang Min-Su , Besser Thomas E. , Call Douglas R.
Published: March 2006

We used sequencing and PCR mapping to investigate the regions flanking the
bla
CMY-2
β-lactamase gene carried by plasmids from
Escherichia coli
and
Salmonella enterica
. Results revealed genetic heterogeneity in the region downstream from
bla
CMY-2
and identified a putative transposable element bearing
bla
CMY-2
.
Large Conjugative Plasmids from Clinical Strains of
Salmonella enterica
Serovar Virchow Contain a Class 2 Integron in Addition to Class 1 Integrons and Several Non-Integron-Associated Drug Resistance Determinants
Authors: Rodríguez Irene , Rodicio M. Rosario , Mendoza M. Carmen , Cruz Martín M.
Published: March 2006

Two large conjugative resistance (R) plasmids from clinical strains of
Salmonella enterica
serovar Virchow carried a class 2 integron with the 5′ conserved sequence (5′CS)-
dfrA1-sat1-aadA1
-3′CS gene array, which is associated with defective Tn
7
transposons. In addition, each contained a different class 1 integron (with 5′CS-
aadA1
-3′CS or 5′CS-
sat-smr-aadA1
-3′CS gene arrays) linked to Tn
21
-Tn
9
sequences, and several non-integron-associated R determinants. An intact copy of Tn
7
(including the class 2 integron) was present in the chromosome of each strain.
Role of Residues 121 to 124 of Vesicular Stomatitis Virus Matrix Protein in Virus Assembly and Virus-Host Interaction
Authors: Connor John H. , McKenzie Margie O. , Lyles Douglas S.
Published: March 2006

The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Analysis of the membrane association of M protein in the context of infection with isogenic recombinant viruses showed that both wild-type and mutant M proteins associated with membranes to the same extent. Virus expressing the mutant M protein did show an approximately threefold-lower binding affinity of M protein for nucleocapsid-M complexes. In contrast to the relatively minor effects of the M protein mutation on virus assembly, the mutant virus exhibited growth restriction in MDBK but not BHK cells, a slower induction of apoptosis, and lower viral-protein synthesis. Despite translating less viral protein, the mutant virus produced more viral mRNA, showing that the mutant virus could not effectively promote viral translation. These results demonstrate that the 121-to-124 region of the VSV M protein plays a minor role in virus assembly but is involved in virus-host interactions and VSV replication by augmenting viral-mRNA translation.
Evidence for a Functional Link between Uncoating of the Human Immunodeficiency Virus Type 1 Core and Nuclear Import of the Viral Preintegration Complex
Authors: Dismuke David J. , Aiken Christopher
Published: March 2006

Human immunodeficiency virus type 1 (HIV-1) particles begin their replication upon fusion with the plasma membrane of target cells and release of the viral core into the host cell cytoplasm. Soon thereafter, the viral capsid, which is composed of a polymer of the CA protein, disassociates from the internal ribonucleoprotein complex. While this disassembly process remains poorly understood, the available evidence indicates that proper uncoating of the core is a key step in infection. Defects in uncoating most often lead to a failure of the virus to undergo reverse transcription, resulting in an inability to form a functional viral preintegration complex (PIC). In a previous study, we reported that an HIV-1 mutant containing two substitutions in CA (Q63A/67A) was unusual in that it was poorly infectious yet synthesized normal levels of viral DNA. Here we report that this mutant is impaired for nuclear entry. Quantitative analysis of viral DNA synthesis from infected cells by Southern blotting and real-time PCR revealed that the Q63A/Q67A mutant is impaired in the synthesis of one-long terminal repeat (1-LTR) and 2-LTR circles. Isolation of PICs from acutely infected cells revealed that the Q63A/Q67A mutant produces protein-DNA complexes similar to wild-type in yield and overall composition, but these PICs contained elevated levels of CA and were impaired for integration in vitro. These results demonstrate that mutations in CA can have deleterious effects on both nuclear targeting and integration, suggesting that these steps in the HIV-1 life cycle are dependent on proper uncoating of the viral core.
Genetic Analysis of Simian Immunodeficiency Virus Expressed in Milk and Selectively Transmitted through Breastfeeding
Authors: Rychert Jenna , Lacour Nedra , Amedee Angela Martin
Published: March 2006

To develop effective intervention strategies that prevent breast milk transmission of human immunodeficiency virus (HIV), we must understand the specific viral properties and mechanisms responsible for infant infection. We have used lactating rhesus macaques infected with a pathogenic simian immunodeficiency virus (SIV) stock to analyze the viral genotypes expressed in plasma and milk throughout the disease course and to identify those variants ultimately transmitted to infants through breastfeeding. In these studies we observed mother-to-infant transmission of SIV/Delta
B670
by eight females during the chronic phase of disease, and we analyzed by heteroduplex tracking assays and sequence analysis the distribution and fluctuations in viral genotypes expressed. Each female expressed multiple V1 envelope genotypes in milk near the time of transmission, while a single genotype was found in each of the infants. Variants transmitted to infants were not expressed throughout the maternal disease course but were only detected near the time of transmission. The emergence of the transmitted genotype in the dam typically occurred in plasma before milk and was coincident with increased milk viral loads. Transmitted genotypes tended to be longer and more glycosylated and had a less negative charge over the V1 region compared to viral genotypes expressed in milk but not transmitted. These observations demonstrate that specific viral genotypes are selectively transmitted to infants through breastfeeding and support the hypothesis that transmission occurs as genotypes adapt for efficient expression in milk.
U
S
3 and U
S
3.5 Protein Kinases of Herpes Simplex Virus 1 Differ with Respect to Their Functions in Blocking Apoptosis and in Virion Maturation and Egress
Authors: Poon Alice P. W. , Benetti Luca , Roizman Bernard
Published: March 2006

Previously, we reported that the U
S
3 protein kinase blocks apoptosis, that it activates protein kinase A (PKA), that activation of PKA blocks apoptosis in cells infected with a U
S
3 deletion mutant, and that an overlapping transcriptional unit encodes a truncated kinase designated U
S
3.5. Here, we report the properties of the kinases based on comparisons of herpes simplex virus and baculoviruses expressing U
S
3 or U
S
3.5 kinase. Specifically, we report the following. (i) Both kinases mediate the phosphorylation of HDAC1, HDAC2, and the PKA regulatory IIα subunit in the absence of other viral proteins. (ii) Both enzymes mediate the phosphorylation of largely identical sets of proteins carrying the phosphorylation consensus site of PKA, but only U
S
3 blocks apoptosis, suggesting that it is U
S
3 and not PKA that is responsible for the phosphorylation of the proteins bearing the shared consensus phosphorylation site and the antiapoptotic activity. (iii) Both kinases cofractionate with mitochondria. Immune depletion of the U
S
3 and U
S
3.5 kinases from the cytoplasm removed the kinases from the supernatant fraction, but not from the mitochondrial fraction, and therefore, if the antiapoptotic activity of the U
S
3 kinase is expressed in mitochondria, the localization signal and the antiapoptotic functions are located on different parts of the protein. (iv) The U
S
3 protein kinase is required for the translocation of virus particles from the nucleus. Although the U
L
31 protein is phosphorylated in cells infected with the mutant expressing U
S
3.5 kinase, the release of virus particles from nuclei was impeded in some cells, suggesting that the U
S
3 kinase affects the modification of the nuclear membrane more efficiently than the U
S
3.5 kinase.
Full Genomic Analysis of Human Rotavirus Strain B4106 and Lapine Rotavirus Strain 30/96 Provides Evidence for Interspecies Transmission
Authors: Matthijnssens Jelle , Rahman Mustafizur , Martella Vito , Xuelei Yang , De Vos Sofie , De Leener Karolien , Ciarlet Max , Buonavoglia Canio , Van Ranst Marc
Published: March 2006

The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.
The Ribosomal Shunt Translation Strategy of
Cauliflower Mosaic Virus
Has Evolved from Ancient Long Terminal Repeats
Authors: Shababi Monir , Bourque June , Palanichelvam Karuppaiah , Cole Anthony , Xu Dong , Wan Xiu-Feng , Schoelz James
Published: March 2006

We have screened portions of the large intergenic region of the
Cauliflower mosaic virus
(CaMV) genome for promoter activity in baker's yeast (
Saccharomyces cerevisiae
) and have identified an element that contributes to promoter activity in yeast but has negligible activity in plant cells when expressed in an agroinfiltration assay. A search of the yeast genome sequence revealed that the CaMV element had sequence similarity with the R region of the long terminal repeat (LTR) of the yeast Ty1 retrotransposon, with significant statistical confidence. In plants, the same CaMV sequence has been shown to have an essential role in the ribosomal shunt mechanism of translation, as it forms the base of the right arm of the stem-loop structure that is required for the ribosomal shunt. Since the left arm of the stem-loop structure must represent an imperfect reverse copy of the right arm, we propose that the ribosomal shunt has evolved from a pair of LTRs that have become incorporated end to end into the CaMV genome.
Intrarectal Immunization with Rotavirus 2/6 Virus-Like Particles Induces an Antirotavirus Immune Response Localized in the Intestinal Mucosa and Protects against Rotavirus Infection in Mice
Authors: Agnello Davide , Hervé Christine A. , Lavaux Amandine , Darniot Magali , Guillon Patrice , Charpilienne Annie , Pothier Pierre
Published: March 2006

Rotavirus (RV) is the main etiological agent of severe gastroenteritis in infants, and vaccination seems the most effective way to control the disease. Recombinant rotavirus-like particles composed of the viral protein 6 (VP6) and VP2 (2/6-VLPs) have been reported to induce protective immunity in mice when administered by the intranasal (i.n.) route. In this study, we show that administration of 2/6-VLPs by the intrarectal (i.r.) route together with either cholera toxin (CT) or a CpG-containing oligodeoxynucleotide as the adjuvant protects adult mice against RV infection. Moreover, when CT is used, RV shedding in animals immunized by the i.r. route is even reduced in comparison with that in animals immunized by the i.n. route. Humoral and cellular immune responses induced by these immunization protocols were analyzed. We found that although i.r. immunization with 2/6-VLPs induces lower RV-specific immunoglobulin G (IgG) and IgA levels in serum, intestinal anti-RV IgA production is higher in mice immunized by the i.r. route. Cellular immune response has been evaluated by measuring cytokine production by spleen and Peyer's patch cells (PPs) after ex vivo restimulation with RV. Mice immunized by the i.n. and i.r. routes display higher gamma interferon production in spleen and PPs, respectively. In conclusion, we demonstrate that i.r. immunization with 2/6-VLPs protects against RV infection in mice and is more efficient than i.n. immunization in inducing an anti-RV immune response in intestinal mucosa.
White Spot Syndrome Virus Open Reading Frame 222 Encodes a Viral E3 Ligase and Mediates Degradation of a Host Tumor Suppressor via Ubiquitination
Authors: He Fang , Fenner Beau J. , Godwin Andrew K. , Kwang Jimmy
Published: March 2006

We have characterized a white spot syndrome virus (WSSV) RING-H2-type protein, WSSV222, which is involved in ubiquitination. WSSV222 exhibits RING-H2-dependent E3 ligase activity in vitro in the presence of the specific conjugating enzyme UbcH6. Mutations in the RING-H2 domain abolished WSSV222-dependent ubiquitination, revealing the importance of this domain in WSSV222 function. Yeast two-hybrid and pull-down analyses revealed that WSSV222 interacts with a shrimp tumor suppressor-like protein (TSL) sharing 60% identity with human OVCA1. To better characterize the interaction of WSSV222 and TSL in vivo, we established a stable TSL-expressing cell line derived from the human ovarian cancer cell line A2780, where we observed a TSL-dependent prolonged G
1
phase. Furthermore, we detected WSSV222-mediated ubiquitination and MG132-sensitive degradation of TSL both in shrimp primary cell culture and in the TSL-expressing cell line. Transient expression of TSL in BHK cells leads to apoptosis, which was rescued by WSSV222. Taken together, our data suggest that WSSV222 acts as an antiapoptosis protein by ubiquitin-mediated proteolysis of TSL to ensure successful WSSV replication in shrimp.
Phosphorylation Mutants of JC Virus Agnoprotein Are Unable To Sustain the Viral Infection Cycle
Authors: Sariyer Ilker K. , Akan Ilhan , Palermo Victoria , Gordon Jennifer , Khalili Kamel , Safak Mahmut
Published: March 2006

Many eukaryotic and viral regulatory proteins are known to undergo posttranslational modifications including phosphorylation, which plays a critical role in many aspects of cell function. Previous studies from our and other laboratories indicated that the JC virus (JCV) late regulatory protein, agnoprotein, plays an important role in the JCV life cycle. Agnoprotein contains several potential phosphorylation sites, including Ser7, Ser11, and Thr21, which are potential targets for the serine/threonine-specific protein kinase C (PKC). In this study, we investigated the functional significance of these phosphorylation sites for the activity of agnoprotein. In vitro and in vivo kinase assays demonstrated that agnoprotein is a target for phosphorylation by PKC. In addition, each of the PKC phosphorylation sites was mutated to Ala singly and in combination, and the effects of these mutations on the JCV life cycle were analyzed. Although the expression of each mutant agnoprotein was detectable during the infection cycle, virus containing each of these mutations failed to propagate. These results contrast with those obtained with an agnoprotein start codon point (Pt) mutant where agnoprotein expression was completely inhibited. The Pt mutant was viable but replicates less efficiently than the wild type (WT). Moreover, conservative substitutions at PKC phosphorylation sites (Ser7, Ser11, and Thr21 to Asp) resulted in a viable virus, which further demonstrate the importance of these sites on agnoprotein function. Further analysis of the mutants by viral release assay and electron microscopy studies revealed that viral particles were efficiently released from infected cells and morphologically indistinguishable from those of WT but were deficient in DNA content. This may account for the defective propagation of the mutants. These results imply that phosphorylated forms of agnoprotein may have essential functions in the viral life cycle and serve as potential targets for therapeutic interventions to limit JCV propagation and JCV-induced diseases.
Function of Bovine CD46 as a Cellular Receptor for Bovine Viral Diarrhea Virus Is Determined by Complement Control Protein 1
Authors: Krey Thomas , Himmelreich Anke , Heimann Manuela , Menge Christian , Thiel Heinz-Jürgen , Maurer Karin , Rümenapf Till
Published: March 2006

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.
Antibody Production and In Vitro Behavior of CD27-Defined B-Cell Subsets: Persistent Hepatitis C Virus Infection Changes the Rules
Authors: Racanelli Vito , Frassanito Maria Antonia , Leone Patrizia , Galiano Maria , De Re Valli , Silvestris Franco , Dammacco Franco
Published: March 2006

There is growing interest in the tendency of B cells to change their functional program in response to overwhelming antigen loading, perhaps by regulating specific parameters, such as efficiency of activation, proliferation rate, differentiation to antibody-secreting cells (ASC), and rate of cell death in culture. We show that individuals persistently infected with hepatitis C virus (HCV) carry high levels of circulating immunoglobulin G (IgG) and IgG-secreting cells (IgG-ASC). Thus, generalized polyclonal activation of B-cell functions may be supposed. While IgGs include virus-related and unrelated antibodies, IgG-ASC do not include HCV-specific plasma cells. Despite signs of widespread activation, B cells do not accumulate and memory B cells seem to be reduced in the blood of HCV-infected individuals. This apparent discrepancy may reflect the unconventional activation kinetics and functional responsiveness of the CD27+B-cell subset in vitro. Following stimulation with T-cell-derived signals in the absence of B-cell receptor (BCR) engagement, CD27+B cells do not expand but rapidly differentiate to secrete Ig and then undergo apoptosis. We propose that their enhanced sensitivity to BCR-independent noncognate T-cell help maintains a constant level of nonspecific serum antibodies and ASC and serves as a backup mechanism of feedback inhibition to prevent exaggerated B-cell responses that could be the cause of significant immunopathology.
Dissecting Rotavirus Particle-Raft Interaction with Small Interfering RNAs: Insights into Rotavirus Transit through the Secretory Pathway
Authors: Cuadras Mariela A. , Bordier Bruno B. , Zambrano Jose L. , Ludert Juan E. , Greenberg Harry B.
Published: March 2006

Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts; in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex.
In Vivo Ablation of CD11c-Positive Dendritic Cells Increases Susceptibility to Herpes Simplex Virus Type 1 Infection and Diminishes NK and T-Cell Responses
Authors: Kassim Sadik H. , Rajasagi Naveen K. , Zhao Xiangyi , Chervenak Robert , Jennings Stephen R.
Published: March 2006

The precise role of each of the seven individual CD11c+dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+and CD8+T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.
Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies
Authors: Ansari Israrul H. , Kwon Byungjoon , Osorio Fernando A. , Pattnaik Asit K.
Published: March 2006

Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.
The Products of the Herpes Simplex Virus Type 1 Immediate-Early U
S
1/U
S
1.5 Genes Downregulate Levels of S-Phase-Specific Cyclins and Facilitate Virus Replication in S-Phase Vero Cells
Authors: Orlando Joseph S. , Astor Todd L. , Rundle Scott A. , Schaffer Priscilla A.
Published: March 2006

Herpes simplex virus type 1 ICP22
−
/U
S
1.5
−
mutants initiate viral gene expression in all cells; however, in most cell types, the replication process stalls due to an inability to express γ
2
late proteins. Although the function of ICP22/U
S
1.5 has not been established, it has been suggested that these proteins activate, induce, or repress the activity of cellular proteins during infection. In this study, we hypothesized that cell cycle-associated proteins are targets of ICP22/U
S
1.5. For this purpose, we first isolated and characterized an ICP22
−
/U
S
1.5
−
mutant virus, 22/n199. Like other ICP22
−
/U
S
1.5
−
mutants, 22/n199 replicates in a cell-type-specific manner and fails to induce efficient γ
2
late gene expression in restrictive cells. Although synchronization of restrictive human embryonic lung cells in each phase of the cell cycle did not overcome the growth restrictions of 22/n199, synchronization of permissive Vero cells in S phase rendered them less able to support 22/n199 plaque formation and replication. Consistent with this finding, expression of cellular S-phase cyclins was altered in an ICP22/U
S
1.5-dependent manner specifically when S-phase Vero cells were infected. Collectively, these observations support the notion that ICP22/U
S
1.5 deregulates the cell cycle upon infection of S-phase permissive cells by altering expression of key cell cycle regulatory proteins either directly or indirectly.