bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Interleukin 1α (IL-1α), IL-4, and IL-6 Polymorphisms on Genetic Susceptibility to Chronic Osteomyelitis
Authors: Tsezou Aspasia , Poultsides Lazaros , Kostopoulou Fotini , Zintzaras Elias , Satra Maria , Kitsiou-Tzeli Sofia , Malizos Konstantinos N.
Published: October 2008

The association between cytokine gene polymorphisms and chronic osteomyelitis was investigated in order to determine whether genetic variability in cytokine genes predisposes to osteomyelitis susceptibility. Significant genotypic and allelic associations were observed between interleukin 1α (IL-1α) −889-C/T, IL-4 −1098-G/T and −590-C/T, and IL-6 −174-G/C polymorphisms and osteomyelitis in the Greek population, pointing towards their potential involvement in osteomyelitis pathogenesis.
Role of Dendritic Cells in Enhancement of Herpes Simplex Virus Type 1 Latency and Reactivation in Vaccinated Mice
Authors: Mott Kevin R. , Ghiasi Homayon
Published: October 2008

Ocular infection with herpes simplex virus type 1 (HSV-1) frequently leads to recurrent infection, which is a major cause of corneal scarring. Thus, the prevention of the establishment of latency should be a primary goal of vaccination against HSV-1. To this end, we have examined the contribution of dendritic cells (DCs) to the efficacy of a vaccine against ocular HSV-1 infection. Transgenic mice (expressing a CD11c-diphtheria toxin receptor-green fluorescent protein construct) with a BALB/c background were immunized with a vaccine consisting of DNA that encodes five HSV-1 glycoproteins or were immunized with vector control DNA. The vaccinated mice were then depleted of their DCs through the injection of diphtheria toxin before and after ocular challenge with HSV-1. Analyses of HSV-1 replication in the eye, blepharitis, corneal scarring, and the survival of the infected mice upon primary infection indicated that DC depletion neither promoted nor compromised the efficacy of the vaccine. In contrast, DC depletion was associated with an approximately fivefold reduction in the level of latent virus in the trigeminal ganglia (TGs) of latently infected mice, as well as a significant reduction in the reactivation rate of latent virus. The possibility that DCs enhance the latency of HSV-1 in the TGs of ocularly infected mice suggests for the first time that DCs, rather than acting as “immune saviors,” can exacerbate disease and compromise vaccine efficacy by enhancing viral latency and reactivation.
Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR
Authors: Wang Wei , Ren Peijun , Mardi Sek , Hou Lili , Tsai Cheguo , Chan Kwok Hung , Cheng Peter , Sheng Jun , Buchy Philippe , Sun Bing , Toyoda Tetsuya , Lim Wilina , Peiris J. S. Malik , Zhou Paul , Deubel Vincent
Published: October 2008

Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.
Ciprofloxacin-Resistant
Salmonella enterica
Serovar Typhi from Kuwait with Novel Mutations in
gyrA
and
parC
Genes
Authors: Dimitrov Tzonyo , Dashti Ali A. , Albaksami Ossama , Udo Edet E. , Jadaon Mehrez M. , Albert M. John
Published: October 2008

Blood isolates of
Salmonella enterica
serovar Typhi from two recently returned Bangladeshi patients in Kuwait were ciprofloxacin resistant, with ciprofloxacin MICs of 12 mg/liter for both isolates. Both isolates had three novel
gyrA
mutations (55-Leu→Trp, 87-Asp→Ala, and 106-Gln→Arg) and three novel
parC
mutations (84-Glu→Lys, 106-Trp→Gly, and 128-Tyr→Asp).
Uncultivated Bacteria as Etiologic Agents of Intra-Amniotic Inflammation Leading to Preterm Birth
Authors: Han Yiping W. , Shen Tao , Chung Peter , Buhimschi Irina A. , Buhimschi Catalin S.
Published: October 2008

Intra-amniotic infection and inflammation are major causes of preterm birth (PTB). However, intra-amniotic inflammation is often detected in the absence of infection. This may partly be due to the culturing methods employed in hospital laboratories, which are unable to detect the uncultivated species. In this study, intra-amniotic microbial infections associated with PTB were examined by both culture and 16S rRNA-based culture-independent methods and were corroborated by the presence of intra-amniotic inflammation. Amniotic fluid (AF) specimens from 46 pregnancies complicated by PTB and 16 asymptomatic women were analyzed. No bacterial DNA was amplified in AF collected from the asymptomatic women. Among the 46 samples associated with PTB, bacterial DNA was amplified from all (16/16) of the culture-positive samples and 17% (5/30) of the culture-negative samples. In the culture-positive group, additional species were detected in more than half (9/16) of the cases by PCR and clone analysis. Altogether, approximately two- thirds of the species detected by the culture-independent methods were not isolated by culture. They included both uncultivated and difficult-to-cultivate species, such as
Fusobacterium nucleatum
,
Leptotrichia
(
Sneathia
) spp., a
Bergeyella
sp., a
Peptostreptococcus
sp.,
Bacteroides
spp., and a species of the order
Clostridiales
. To examine intra-amniotic inflammation, an AF proteomic fingerprint (mass-restricted score) was determined by surface-enhanced laser desorption ionization-time-of-flight mass spectrometry. Inflammation was detected in all five samples which were culture negative but PCR positive. Women who were PCR positive more often had elevated interleukin-6 levels in their AF, histological chorioamnionitis, and funisitis and delivered neonates with early-onset neonatal sepsis. Previously unrecognized, uncultivated, or difficult-to-cultivate species may play a key role in the initiation of PTB.
Phenotypic and Genotypic Analysis of Enterotoxigenic
Escherichia coli
in Samples Obtained from Egyptian Children Presenting to Referral Hospitals
Authors: Shaheen H. I. , Abdel Messih I. A. , Klena J. D. , Mansour A. , El-Wakkeel Z. , Wierzba T. F. , Sanders J. W. , Khalil S. B. , Rockabrand D. M. , Monteville M. R. , Rozmajzl P. J. , Svennerholm A. M. , Frenck R. W.
Published: October 2008

Hospital surveillance was established in the Nile River Delta to increase the understanding of the epidemiology of diarrheal disease among Egyptian children. Between September 2000 and August 2003, samples obtained from children less than 5 years of age who had diarrhea and who were seeking hospital care were cultured for enteric bacteria. Colonies from each culture with a morphology typical of that of
Escherichia coli
were tested for the heat-labile (LT) and heat-stable (ST) toxins by a GM-1-specific enzyme-linked immunosorbent assay and colonization factor (CF) antigens by an immunodot blot assay. Enterotoxigenic
E. coli
(ETEC) isolates were recovered from 320/1,540 (20.7%) children, and ETEC isolates expressing a known CF were identified in 151/320 (47%) samples. ST CFA/I, ST CS6, ST CS14, and LT and ST CS5 plus CS6 represented 75% of the CFs expressed by ETEC isolates expressing a detectable CF. Year-to-year variability in the proportion of ETEC isolates that expressed a detectable CF was observed (e.g., the proportion that expressed CFA/I ranged from 10% in year 1 to 21% in year 3); however, the relative proportions of ETEC isolates expressing a CF were similar over the reporting period. The proportion of CF-positive ETEC isolates was higher among isolates that expressed ST. ETEC isolates expressing CS6 were isolated significantly less often (
P
< 0.001) than isolates expressing CFA/I in children less than 1 year of age. Macrorestriction profiling of CFA/I-expressing ETEC isolates by using the restriction enzyme XbaI and pulsed-field gel electrophoresis demonstrated a wide genetic diversity among the isolates that did not directly correlate with the virulence of the pathogen. The genome plasticity demonstrated in the ETEC isolates collected in this work suggests an additional challenge to the development of a globally effective vaccine for ETEC.
Concurrent Genotyping and Quantitation of Cytomegalovirus gB Genotypes in Solid-Organ-Transplant Recipients by Use of a Real-Time PCR Assay
Authors: Pang Xiaoli , Humar Atul , Preiksaitis Jutta K.
Published: October 2008

We have developed a real-time genotyping and quantitative PCR (RT-GQ-PCR) assay to genotype cytomegalovirus (CMV) and quantify viral loads simultaneously in solid organ transplant (SOT) recipients. Special minor-groove DNA-binding probes were designed based on sequence polymorphism in the gB gene to increase genotyping specificity for gB1 to gB4. For validation, 28 samples with known genotypes determined by restriction fragment analysis (RFA) and 121 with unknown genotypes were tested. All samples were from SOT patients with CMV viremia. A 100% concordance for genotyping was achieved by using the RT-GQ-PCR with known genotypes determined by RFA. The RT-GQ-PCR identified more cases of CMV infections with mixed genotypes than RFA did. No cross-reaction between genotypes was observed. All four gB genotypes were detected in the 121 samples of unknown genotype. gB1 was the predominant single genotype (
n
= 61, 50.4%), followed by gB2 (
n
= 26, 21.0%), gB3, (
n
= 11, 9.1%), and gB4 (
n
= 3, 2.5%). Mixed-genotype infections were detected in 17% (20/121) of the samples. Patients with mixed-genotype infections had significantly higher CMV viral loads than those with single-genotype infections (
P
= 0.019). The RT-GQ-PCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.7 to 10.7 log
10
copies/ml) and very good precision (coefficient of variation, ∼1.78%). With the prominent feature of concurrent CMV gB genotyping and quantitation in a single reaction, the new assay provides a rapid and cost-effective method for monitoring CMV infection in SOT recipients.
Isopropyl Alcohol Compared with Isopropyl Alcohol plus Povidone-Iodine as Skin Preparation for Prevention of Blood Culture Contamination
Authors: Kiyoyama Tomonori , Tokuda Yasuharu , Shiiki Soichi , Hachiman Teruyuki , Shimasaki Teppei , Endo Kazuo
Published: October 2008

Despite a number of studies on the efficacies of antiseptics for the prevention of blood culture contamination, it still remains unclear which antiseptic should be used. Although the combination of povidone-iodine and isopropyl alcohol has been traditionally used in many institutions, the application of povidone-iodine needs extra time, and there is little evidence that this combination could have an additive effect in reducing contamination rates. To elucidate the additive efficacy of povidone-iodine, we compared two antiseptics, 70% isopropyl alcohol only and 70% isopropyl alcohol plus povidone-iodine, in a prospective, nonrandomized, and partially blinded study in a community hospital in Japan between 1 October 2007 and 21 March 2008. All blood samples for culture were drawn by first-year residents who received formal training on collection techniques. Skin antisepsis was performed with 70% isopropyl alcohol plus povidone-iodine on all inpatient wards and with only 70% isopropyl alcohol in the emergency department. For the group of specimens from inpatient wards cultured, 13 (0.46%) of 2,797 cultures were considered contaminated. For the group of specimens from the emergency department cultured, 12 (0.42%) of 2,856 cultures were considered contaminated. There was no significant difference in the contamination rates between the two groups (relative risk, 0.90; 95% confidence interval, 0.41 to 1.98;
P
= 0.80). In conclusion, the use of a single application of 70% isopropyl alcohol is a sufficient and a more cost- and time-effective method of obtaining blood samples for culture than the use of a combination of isopropyl alcohol and povidone-iodine. The extremely low contamination rates in both groups suggest that the type of antiseptic used may not be as important as the use of proper technique.
Occurrence, Distribution, and Origins of
Streptococcus pneumoniae
Serotype 6C, a Recently Recognized Serotype
Authors: Jacobs Michael R. , Bajaksouzian Saralee , Bonomo Robert A. , Good Caryn E. , Windau Anne R. , Hujer Andrea M. , Massire Christian , Melton Rachael , Blyn Lawrence B. , Ecker David J. , Sampath Rangarajan
Published: October 2008

The prevalence of
Streptococcus pneumoniae
serotype 6C, a recently recognized serotype that cross-reacts serologically with serotype 6A, was investigated. Isolates of serotype 6A in various collections were recovered, and serotype 6C was differentiated from 6A by multiplex PCR of DNA extracts by using appropriate primers. Antimicrobial susceptibility was performed by Clinical and Laboratory Standards Institute broth microdilution, and selected isolates were typed by pulsed-field gel electrophoresis, repetitive sequence-based PCR typing, and rapid multilocus sequence typing (MLST) by electrospray ionization mass spectrometry of PCR products. A total of 60 serotype 6C isolates were found: 30 of 122 Cleveland isolates collected from 1979 to 2007, 19 of 39 pediatric isolates collected nationwide in 2005 and 2006, and 11 pediatric isolates from Massachusetts collected in 2006 and 2007. Only four isolates were recovered prior to introduction of the conjugate pneumococcal vaccine in 2000; the earliest isolate was recovered in 1989. The sources of the isolates included blood (
n
= 5), the lower respiratory tract (
n
= 27), the sinus (
n
= 5), the ear (
n
= 2), and the nasopharynx (
n
= 18); isolates were recovered from 49 children and 11 adults. Pediatric isolates were found in all six major U.S. geographic regions. Antimicrobial susceptibility showed that 22 isolates were nonsusceptible to penicillin, macrolides, and trimethoprim-sulfamethoxazole, 8 had other resistance patterns, and 30 were fully susceptible. The three typing methods used showed similar clusters of up to eight isolates per cluster. MLST showed five clusters related to serotype 6A, two clusters related to serotype 6B, one cluster related to serotype 3, and one cluster related to serotype 34. This study documents the occurrence, nationwide distribution, diversity, likely origins, and increasing incidence after 2001 of this recently recognized serotype. Serotype 6C warrants consideration for addition to future conjugate pneumococcal vaccines.
Emergence and Characterization of Community-Associated Methicillin-Resistant
Staphyloccocus aureus
Infections in Denmark, 1999 to 2006
Authors: Larsen A. R. , Stegger M. , Böcher S. , Sørum M. , Monnet D. L. , Skov R. L.
Published: October 2008

The epidemiology of methicillin-resistant
Staphylococcus aureus
(MRSA) infections has changed worldwide. From being strictly nosocomial, MRSA is now frequently found as a community-associated (CA) pathogen. Denmark has been a low-prevalence country for MRSA since the mid-1970s but has in recent years experienced an increasing number of CA-MRSA cases. The aim of this study was to describe the emergence of CA-MRSA infections in Denmark. All Danish MRSA specimens and corresponding clinical data from 1999 to 2006 were investigated. Isolates were analyzed by antibiotic resistance and molecular typing and were assigned to clonal complexes (CC). Clinical data were extracted from discharge summaries and general practitioners' notes, from which assessments of community association were made for all infected cases. CA-MRSA cases constituted 29.4% of all MRSA infections (
n
= 1,790) and an increasing proportion of the annual numbers of MRSA infections during the study period. CA-MRSA was associated with a young age, skin and soft tissue infections, and non-Danish origin. Transmission between household members was frequently reported. Molecular typing showed >60 circulating clones, where 89.4% of the isolates belonged to five CC (CC80, CC8, CC30, CC5, and CC22), 81.2% carried staphylococcal cassette chromosome
mec
IV, and 163/244 (69.4%) were positive for Panton-Valentine leukocidin. Clinical and microbiological characteristics indicated that import of MRSA occurs frequently. Resistance to ≥3 antibiotic classes was observed for 48.8% of the isolates. The emergence of CA-MRSA in Denmark was caused by diverse strains, both well-known and new CA-MRSA strains. The results suggest multiple introductions of MRSA as an important source for CA-MRSA infections in Denmark.
Ubiquitin-Independent Degradation of Hepatitis C Virus F Protein
Authors: Yuksek Kamile , Chen Wen-ling , Chien David , Ou Jing-hsiung James
Published: October 2008

Hepatitis C virus (HCV) F protein is encoded by the +1 reading frame of the viral genome. It overlaps with the core protein coding sequence, and multiple mechanisms for its expression have been proposed. The full-length F protein that is synthesized by translational ribosomal frameshift at codons 9 to 11 of the core protein sequence is a labile protein. By using a combination of genetic, biochemical, and cell biological approaches, we demonstrate that this HCV F protein can bind to the proteasome subunit protein α3, which reduces the F-protein level in cells in a dose-dependent manner. Deletion-mapping analysis identified amino acids 40 to 60 of the F protein as the α3-binding domain. This α3-binding domain of the F protein together with its upstream sequence could significantly destabilize the green fluorescent protein, an otherwise stable protein. Further analyses using an F-protein mutant lacking lysine and a cell line that contained a temperature-sensitive E1 ubiquitin-activating enzyme indicated that the degradation of the F protein was ubiquitin independent. Based on these observations as well as the observation that the F protein could be degraded directly by the 20S proteasome in vitro, we propose that the full-length HCV F protein as well as the F protein initiating from codon 26 is degraded by an ubiquitin-independent pathway that is mediated by the proteasome subunit α3. The ability of the F protein to bind to α3 raises the possibility that the HCV F protein may regulate protein degradation in cells.
Bromodomain Protein Brd4 Regulates Human Immunodeficiency Virus Transcription through Phosphorylation of CDK9 at Threonine 29
Authors: Zhou Meisheng , Huang Keven , Jung Kyung-Jin , Cho Won-Kyung , Klase Zach , Kashanchi Fatah , Pise-Masison Cynthia A. , Brady John N.
Published: October 2008

Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.
The Addition of Tumor Necrosis Factor plus Beta Interferon Induces a Novel Synergistic Antiviral State against Poxviruses in Primary Human Fibroblasts
Authors: Bartee Eric , Mohamed Mohamed R. , Lopez M. Cecilia , Baker Henry V. , McFadden Grant
Published: October 2008

Tumor necrosis factor (TNF) and members of the interferon (IFN) family have been shown to independently inhibit the replication of a variety of viruses. In addition, previous reports have shown that treatment with various combinations of these antiviral cytokines induces a synergistic antiviral state that can be significantly more potent than addition of any of these cytokines alone. The mechanism of this cytokine synergy and its effects on global gene expression, however, are not well characterized. Here, we use DNA microarray analysis to demonstrate that treatment of uninfected primary human fibroblasts with TNF plus IFN-β induces a distinct synergistic state characterized by significant perturbations of several hundred genes which are coinduced by the individual cytokines alone, as well as the induction of more than 850 novel host cell genes. This synergy is mediated directly by the two ligands, not by intermediate secreted factors, and is necessary and sufficient to completely block the productive replication and spread of myxoma virus in human fibroblasts. In contrast, the replication of two other poxviruses, vaccinia virus and tanapox virus, are only partially inhibited in these cells by the synergistic antiviral state, whereas the spread of both of these viruses to neighboring cells was efficiently blocked. Taken together, our data indicate that the combination of TNF and IFN-β induces a novel synergistic antiviral state that is highly distinct from that induced by either cytokine alone.
Alternatively Initiated Gene 50/RTA Transcripts Expressed during Murine and Human Gammaherpesvirus Reactivation from Latency
Authors: Gray Kathleen S. , Allen Robert D. , Farrell Michael L. , Forrest J. Craig , Speck Samuel H.
Published: October 2008

In the process of characterizing the requirements for expression of the essential immediate-early transcriptional activator (RTA) encoded by gene 50 of murine gammaherpesvirus 68 (MHV68), a recombinant virus was generated in which the known gene 50 promoter was deleted (G50pKO). Surprisingly, the G50pKO mutant retained the ability to replicate in permissive murine fibroblasts, albeit with slower kinetics than wild-type MHV68. 5′-rapid amplification of cDNA ends analyses of RNA prepared from G50pKO-infected fibroblasts revealed a novel upstream transcription initiation site, which was also utilized during wild-type MHV68 infection of permissive cells. Furthermore, the region upstream of the distal gene 50/RTA transcription initiation site exhibited promoter activity in both permissive NIH 3T12 fibroblasts as well as in the murine macrophage cell line RAW 264.7. In addition, in RAW 264.7 cells the activity of the distal gene 50/RTA promoter was strongly upregulated (>20-fold) by treatment of the cells with lipopolysaccharide. Reverse transcriptase PCR analyses of RNA prepared from Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-infected B-cell lines, following induction of virus reactivation, also revealed the presence of gene 50/RTA transcripts initiating upstream of the known transcription initiation site. The latter argues that alternative initiation of gene 50/RTA transcription is a strategy conserved among murine and human gammaherpesviruses. Infection of mice with the MHV68 G50pKO demonstrated the ability of this mutant virus to establish latency in the spleen and peritoneal exudate cells (PECs). However, the G50pKO mutant was unable to reactivate from latently infected splenocytes and also exhibited a significant reactivation defect from latently infected PECs, arguing in favor of a model where the proximal gene 50/RTA promoter plays a critical role in virus reactivation from latency, particularly from B cells. Finally, analyses of viral genome methylation in the regions upstream of the proximal and distal gene 50/RTA transcription initiation sites revealed that the distal promoter is partially methylated in vivo and heavily methylated in MHV68 latently infected B-cell lines, suggesting that DNA methylation may serve to silence the activity of this promoter during virus latency.
HLA-Associated Alterations in Replication Capacity of Chimeric NL4-3 Viruses Carrying
gag-protease
from Elite Controllers of Human Immunodeficiency Virus Type 1
Authors: Miura Toshiyuki , Brockman Mark A. , Brumme Zabrina L. , Brumme Chanson J. , Pereyra Florencia , Trocha Alicja , Block Brian L. , Schneidewind Arne , Allen Todd M. , Heckerman David , Walker Bruce D.
Published: October 2008

Human immunodeficiency virus type 1 (HIV-1)-infected persons who maintain plasma viral loads of <50 copies RNA/ml without treatment have been termed elite controllers (EC). Factors contributing to durable control of HIV in EC are unknown, but an HLA-dependent mechanism is suggested by overrepresentation of “protective” class I alleles, such as B*27, B*51, and B*57. Here we investigated the relative replication capacity of viruses (VRC) obtained from EC (
n
= 54) compared to those from chronic progressors (CP;
n
= 41) by constructing chimeric viruses using patient-derived
gag-protease
sequences amplified from plasma HIV RNA and inserted into an NL4-3 backbone. The chimeric viruses generated from EC displayed lower VRC than did viruses from CP (
P
< 0.0001). HLA-B*57 was associated with lower VRC (
P
= 0.0002) than were other alleles in both EC and CP groups. Chimeric viruses from B*57
+
EC (
n
= 18) demonstrated lower VRC than did viruses from B*57
+
CP (
n
= 8,
P
= 0.0245). Differences in VRC between EC and CP were also observed for viruses obtained from individuals expressing no described “protective” alleles (
P
= 0.0065). Intriguingly, two common HLA alleles, A*02 and B*07, were associated with higher VRC (
P
= 0.0140 and 0.0097, respectively), and there was no difference in VRC between EC and CP sharing these common HLA alleles. These findings indicate that cytotoxic T-lymphocyte (CTL) selection pressure on
gag-protease
alters VRC, and HIV-specific CTLs inducing escape mutations with fitness costs in this region may be important for strict viremia control in EC of HIV.
Homodimerization of Marek's Disease Virus-Encoded Meq Protein Is Not Sufficient for Transformation of Lymphocytes in Chickens
Authors: Suchodolski Paulette F. , Izumiya Yoshihiro , Lupiani Blanca , Ajithdoss Dharani K. , Gilad Oren , Lee Lucy F. , Kung Hsing-Jien , Reddy Sanjay M.
Published: October 2008

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric
meq
gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (
meqGCN
). A recombinant virus (rMd5-MeqGCN) containing the chimeric
meqGCN
gene in place of parental
meq
was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation.
Follicular Dendritic Cells and Human Immunodeficiency Virus Type 1 Transcription in CD4+T Cells
Authors: Thacker Tyler C. , Zhou Xueyuan , Estes Jacob D. , Jiang Yongjun , Keele Brandon F. , Elton Terry S. , Burton Gregory F.
Published: October 2008

HIV replication occurs throughout the natural course of infection in secondary lymphoid tissues and in particular within the germinal centers (GCs), where follicular dendritic cells (FDCs) are adjacent to CD4+T cells. Because FDCs provide signaling that increases lymphocyte activation, we postulated that FDCs could increase human immunodeficiency virus (HIV) replication. We cultured HIV-infected CD4+T cells alone or with FDCs and measured subsequent virus expression using HIV-p24 production and reverse transcription-PCR analyses. When cultured with FDCs, infected CD4+T cells produced almost fourfold more HIV than when cultured alone, and the rate of virus transcription was doubled. Both FDCs and their supernatant increased HIV transcription and resulted in nuclear translocation of NF-κB and phosphorylated c-Jun in infected cells. FDCs produced soluble tumor necrosis factor alpha (TNF-α) ex vivo, and the addition of a blocking soluble TNF receptor ablated FDC-mediated HIV transcription. Furthermore, TNF-α was found highly expressed within GCs, and ex vivo GC CD4+T cells supported greater levels of HIV-1 replication than other CD4+T cells. These data indicated that FDCs increase HIV transcription and production by a soluble TNF-α-mediated mechanism. This FDC-mediated effect may account, at least in part, for the presence of persistent HIV replication in GCs. Therefore, in addition to providing an important reservoir of infectious virus, FDCs increase HIV production, contributing to a tissue microenvironment that is highly conducive to HIV transmission and expression.
Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry
Authors: Johannsdottir Hrefna Kristin , Mancini Roberta , Kartenbeck Jurgen , Amato Lea , Helenius Ari
Published: October 2008

Vesicular stomatitis virus (VSV) is an animal virus that based on electron microscopy and its dependence on acidic cellular compartments for infection is thought to enter its host cells in a clathrin-dependent manner. The exact cellular mechanism, however, is largely unknown. In this study, we characterized the entry kinetics of VSV and elucidated viral requirements for host cell factors during infection in HeLa cells. We found that endocytosis of VSV was a fast process with a half time of 2.5 to 3 min and that acid activation occurred within 1 to 2 min after internalization in early endosomes. The majority of viral particles were endocytosed in a clathrin-based, dynamin-2-dependent manner. Although associated with some of the surface-bound viruses, the classical adaptor protein complex AP-2 was not required for infection. Time-lapse microscopy revealed that the virus either entered preformed clathrin-coated pits or induced de novo formation of pits. Dynamin-2 was recruited to plasma membrane-confined virus particles. Thus, VSV can induce productive internalization by exploiting a specific combination of the clathrin-associated proteins and cellular functions.
Protease-Mediated Entry via the Endosome of Human Coronavirus 229E
Authors: Kawase Miyuki , Shirato Kazuya , Matsuyama Shutoku , Taguchi Fumihiro
Published: October 2008

Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.
Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein
Authors: Nash Kevin , Chen Weijun , Salganik Max , Muzyczka Nicholas
Published: October 2008

Adeno-associated virus (AAV) codes for four related nonstructural Rep proteins. AAV both replicates and assembles in the nucleus and requires coinfection with a helper virus, either adenovirus (Ad) or herpesvirus, for a productive infection. Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins, which are known to be directly involved in DNA replication, control of viral and cellular transcription, splicing, and protein translation. In this study, we used affinity-tagged Rep protein to purify cellular protein complexes that were associated with Rep in cells that had been infected with Ad and AAV. In all, we identified 188 cellular proteins from 16 functional categories, including 14 transcription factors, 6 translation factors, 15 potential splicing proteins, 5 proteins involved in protein degradation, and 13 proteins involved in DNA replication or repair. This dramatically increases the number of potential interactions over the current number of approximately 26. Twelve of the novel proteins found were further tested by coimmunoprecipitation or colocalization using confocal immunomicroscopy. Of these, 10 were confirmed as proteins that formed complexes with Rep, including proteins of the MCM complex (DNA replication), RCN1 (membrane transport), SMC2 (chromatin dynamics), EDD1 (ubiquitin ligase), IRS4 (signal transduction), and FUS (splicing). Computer analysis suggested that 45 and 28 of the 188 proteins could be placed in a pathway of interacting proteins involved in DNA replication and protein synthesis, respectively. Of the proteins involved in DNA replication, all of the previously identified proteins involved in AAV DNA replication were found, except Ad DBP. The only Ad protein found to interact with Rep was the E1b55K protein. In addition, we confirmed that Rep interacts with Ku70/80 helicase. In vitro DNA synthesis assays demonstrated that although Ku helicase activity could substitute for MCM to promote strand displacement synthesis, its presence was not essential. Our study suggests that the interaction of AAV with cellular proteins is much more complex than previously suspected and provides a resource for further studies of the AAV life cycle.
Identification of Sequences in Herpes Simplex Virus Type 1 ICP22 That Influence RNA Polymerase II Modification and Viral Late Gene Expression
Authors: Bastian Thomas W. , Rice Stephen A.
Published: October 2008

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.
Comparative Analysis of Complete Genome Sequences of Three Avian Coronaviruses Reveals a Novel Group 3c Coronavirus
Authors: Woo Patrick C. Y. , Lau Susanna K. P. , Lam Carol S. F. , Lai Kenneth K. Y. , Huang Yi , Lee Paul , Luk Geraldine S. M. , Dyrting Kitman C. , Chan Kwok-Hung , Yuen Kwok-Yung
Published: October 2008

In this territory-wide molecular epidemiology study of coronaviruses (CoVs) in Hong Kong involving 1,541 dead wild birds, three novel CoVs were identified in three different bird families (bulbul CoV HKU11 [BuCoV HKU11], thrush CoV HKU12 [ThCoV HKU12], and munia CoV HKU13 [MuCoV HKU13]). Four complete genomes of the three novel CoVs were sequenced. Their genomes (26,396 to 26,552 bases) represent the smallest known CoV genomes. In phylogenetic trees constructed using chymotrypsin-like protease (3CL
pro
), RNA-dependent RNA polymerase (Pol), helicase, spike, and nucleocapsid proteins, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 formed a cluster distantly related to infectious bronchitis virus and turkey CoV (group 3a CoVs). For helicase, spike, and nucleocapsid, they were also clustered with a CoV recently discovered in Asian leopard cats, for which the complete genome sequence was not available. The 3CL
pro
, Pol, helicase, and nucleocapsid of the three CoVs possessed higher amino acid identities to those of group 3a CoVs than to those of group 1 and group 2 CoVs. Unique genomic features distinguishing them from other group 3 CoVs include a distinct transcription regulatory sequence and coding potential for small open reading frames. Based on these results, we propose a novel CoV subgroup, group 3c, to describe this distinct subgroup of CoVs under the group 3 CoVs. Avian CoVs are genetically more diverse than previously thought and may be closely related to some newly identified mammalian CoVs. Further studies would be important to delineate whether the Asian leopard cat CoV was a result of interspecies jumping from birds, a situation analogous to that of bat and civet severe acute respiratory syndrome CoVs.
Effect of latanoprost and timolol on the histopathology of the human conjunctiva
Authors: Terai N , Schlötzer-Schrehardt U , Lampel J , Böhm A G , Rummelt C , Schmidt E , Pillunat L E
Published: October 2008

Aim:

To investigate the effect of timolol and latanoprost on the extracellular matrix organisation, inflammatory infiltration, and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the human conjunctiva.

Methods:

Conjunctival biopsies were obtained from the inferior fornix during routine cataract surgery from 20 patients with primary open-angle glaucoma, who had received a monotherapy either with timolol or latanoprost, and from 10 non-glaucomatous patients. Specimens were investigated by light microscopy, immunohistochemistry using antibodies against MMP-1,-3, TIMP-2,-3 and CD 68 antibodies and by quantitative transmission electron microscopy.

Results:

The number of collagen fibres was significantly decreased in latanoprost-treated conjunctival specimens compared with timolol-treated eyes (p<0.01) but showed no difference to controls. Amorphous material was increased in both treated groups compared with controls (p<0.001) but was less in latanoprost-treated specimens compared with timolol-treated eyes (p<0.001). Optically clear spaces, probably containing glycosaminoglycans, were significantly reduced in both treated groups—with less of a reduction in latanoprost—compared with timolol-treated eyes (p<0.001). A marked upregulation of MMP-1 and MMP-3 and moderately increased staining for TIMP-2 and TIMP-3 was found in epithelial cells and subepithelial stromal cells of latanoprost-treated eyes. A moderate infiltration with macrophages and inflammatory cells was observed in timolol-treated eyes.

Conclusions:

Latanoprost-treated conjunctival specimens showed a decreased stromal collagen density and a less pronounced inflammatory infiltration. The upregulation of MMP-1 and MMP-3 in latanoprost-treated eyes might explain the reduced extracellular matrix accumulation in the conjunctival stroma. Therefore, latanoprost therapy might have a more favourable effect on the outcome of glaucoma filtering surgery.
Correlation of visual recovery with presence of photoreceptor inner/outer segment junction in optical coherence images after epiretinal membrane surgery
Authors: Mitamura Y , Hirano K , Baba T , Yamamoto S
Published: October 2008

Aims:

To investigate the relationship between the presence of the photoreceptor inner and outer segment (IS/OS) junction and visual acuity after epiretinal membrane (ERM) surgery.

Methods:

Seventy eyes of 70 consecutive patients who had undergone vitrectomy for idiopathic ERM were examined by optical coherence tomography before and 3 and 6 months after surgery. The IS/OS junction was graded into three grades. The time course of recovery of the IS/OS junction, central foveal thickness (CFT) and best corrected visual acuity (BCVA) during the postoperative period was studied.

Results:

A normal IS/OS junction was detected in 47.1% of the eyes before surgery and in 65.7% at 3 months and 75.7% at 6 months after. There was a significant correlation between the IS/OS grade and BCVA before and at 3 and 6 months after the operation (p = 0.0001, p<0.0001, p<0.0001, respectively). The preoperative IS/OS junction grade correlated significantly with BCVA at 6 months (p = 0.0239). CFT did not correlate significantly with BCVA at 3 and 6 months.

Conclusions:

The presence of a normal IS/OS junction was associated with good visual acuity after ERM surgery. A normal IS/OS junction probably indicates morphological and functional recovery of the photoreceptors.
Randomised, double-blind, placebo-controlled clinical trial of a new topical glyceryl trinitrate patch for chronic lateral epicondylosis: Table 1
Authors: Paoloni J A , Murrell G A C , Burch R M , Ang R Y
Published: October 2008

Objective:

This study aimed to determine whether a new glyceryl trinitrate patch preparation is effective in treating chronic lateral epicondylosis.

Design:

Randomised double-blind controlled clinical trial.

Setting:

Private practice patients: 154 adult patients with chronic lateral epicondylosis were recruited, with 136 patients completing the trial.

Interventions:

8 weeks of glyceryl trinitrate patch application (dosages of 72 mg/24 h, 1.44 mg/24 h, and 3.6 mg/24 h), or placebo patch application.

Main outcome measures:

Subjective global assessment of change in elbow symptoms, patient-rated tennis elbow evaluation, visual analogue pain at rest, visual analogue pain with activity, visual analogue pain intensity, grip strength, and strength testing using the Orthopaedic Research Institute-Tennis Elbow Testing System.

Results:

At 8 weeks there was a significant decrease in elbow pain with activity in the glyceryl trinitrate 0.72 mg/24 h group compared with placebo (p = 0.04). There were no other significant differences.

Conclusions:

Continuous 1.25 mg/24 h topical glyceryl trinitrate treatment, when combined with daily exercise rehabilitation, has previously demonstrated efficacy in treating chronic lateral epicondylosis. There was significantly decreased elbow pain with activity at 8 weeks in the glyceryl trinitrate 0.72 mg/24 h group (p = 0.04). This short-term dose-ranging study did not demonstrate a treatment effect of a new topical glyceryl trinitrate patch in dosages of 1.44 mg/24 h or 3.6 mg/24 h, which conflicts with previous studies on topical glyceryl trinitrate treatment.

Trial registration number:

NCT00447928.
Cost-effectiveness of interventions promoting physical activity
Authors: Müller-Riemenschneider F , Reinhold T , Willich S N
Published: October 2008

Background:

Sedentary and inactive lifestyle continues to increase and is associated with a substantial economic burden in most industrialised countries. To implement effective physical activity interventions on a broad population basis, their cost-effectiveness needs to be evaluated.

Objective:

To evaluate the cost-effectiveness of physical activity interventions targeted at healthy adults and to identify cost-effective intervention components.

Design:

Systematic review.

Methods and data sources:

The search strategy in electronic databases identified relevant literature published until June 2008. We included studies investigating the cost-effectiveness of interventions aiming to promote physical activity behaviour in healthy adults. Two researchers independently assessed publications according to predefined inclusion criteria and with regard to methodological quality. Study characteristics and predefined outcome measures were extracted and costs per participant to become sufficiently active were compared between interventions.

Results:

Out of 6543 identified publications, eight studies investigating 11 intervention strategies met the inclusion criteria. There was substantial heterogeneity in study quality, intervention strategies and intervention effects. Behavioural interventions were able to promote physical activity, with participants meeting recommended levels of physical activity, for about 800 Euros per year. Environmental interventions and interventions targeted at general practitioners seemed to have the potential to be substantially more cost-effective.

Discussion:

There was evidence that current physical activity intervention strategies can be a cost-effective means of resource allocation. But, despite the growing literature on physical activity promotion, appropriate cost-effectiveness analyses are rare and the generalisability of presented findings is limited. Further research is warranted to investigate the cost-effectiveness of behavioural and environmental intervention strategies.