bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity and Clinical Impact of
Acinetobacter baumannii
Colonization and Infection at a Military Medical Center
Authors: Petersen Kyle , Cannegieter Suzanne C. , van der Reijden Tanny J. , van Strijen Beppie , You David M. , Babel Britta S. , Philip Andrew I. , Dijkshoorn Lenie
Published: November 2010

The epidemiology of
Acinetobacter baumannii
emerging in combat casualties is poorly understood. We analyzed 65 (54 nonreplicate)
Acinetobacter
isolates from 48 patients (46 hospitalized and 2 outpatient trainees entering the military) from October 2004 to October 2005 for genotypic similarities, time-space relatedness, and antibiotic susceptibility. Clinical and surveillance cultures were compared by amplified fragment length polymorphism (AFLP) genomic fingerprinting to each other and to strains of a reference database. Antibiotic susceptibility was determined, and multiplex PCR was performed for OXA-23-like, -24-like, -51-like, and -58-like carbapenemases. Records were reviewed for overlapping hospital stays of the most frequent genotypes, and risk ratios were calculated for any association of genotype with severity of Acute Physiology and Chronic Health Evaluation II (APACHE II) score or injury severity score (ISS) and previous antibiotic use. Nineteen genotypes were identified; two predominated, one consistent with an emerging novel international clone and the other unique to our database. Both predominant genotypes were carbapenem resistant, were present at another hospital before patients' admission to our facility, and were associated with higher APACHE II scores, higher ISSs, and previous carbapenem antibiotics in comparison with other genotypes. One predominated in wound and respiratory isolates, and the other predominated in wound and skin surveillance samples. Several other genotypes were identified as European clones I to III.
Acinetobacter
genotypes from recruits upon entry to the military, unlike those in hospitalized patients, did not include carbapenem-resistant genotypes.
Acinetobacter
species isolated from battlefield casualties are diverse, including genotypes belonging to European clones I to III. Two carbapenem-resistant genotypes were epidemic, one of which appeared to belong to a novel international clone.
“
Mycobacterium avium
subsp.
hominissuis
” in Neck Lymph Nodes of Children and their Environment Examined by Culture and Triplex Quantitative Real-Time PCR
Authors: Kaevska Marija , Slana Iva , Kralik Petr , Reischl Udo , Orosova Jaroslava , Holcikova Alena , Pavlik Ivo
Published: November 2010

“
Mycobacterium avium
subsp.
hominissuis
” often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting
M. avium
subsp.
hominissuis
using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of
M. avium
subsp.
hominissuis
in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to
M. avium
subsp.
avium
, which was present due to the breeding of infected domestic hens. The high infectious dose of
M. avium
subsp.
hominissuis
or the increased susceptibility of humans to
M. avium
subsp.
hominissuis
compared to
M. avium
subsp.
avium
could be the reason why the children were infected with
M. avium
subsp.
hominissuis.
Human Papillomavirus (HPV) DNA Triage of Women with Atypical Squamous Cells of Undetermined Significance with Amplicor HPV and Hybrid Capture 2 Assays for Detection of High-Grade Lesions of the Uterine Cervix
Authors: Dufresne Simon , Sauthier Philippe , Mayrand Marie-Hélène , Petignat Patrick , Provencher Diane , Drouin Pierre , Gauthier Philippe , Dupuis Marie-Josée , Michon Bertrand , Ouellet Stéphan , Hadjeres Rachid , Ferenczy Alex , Franco Eduardo L. , Coutlée François
Published: November 2010

Up to 20% of women having a cytology smear showing atypical squamous cells of undetermined significance (ASC-US) and infected with high-risk human papillomavirus (HR HPV) have high-grade cervical intraepithelial neoplasia (CIN 2/3). Results obtained with the Amplicor HPV and Hybrid Capture 2 (HC-2) assays for HR HPV DNA detection in women referred to colposcopy for an ASC-US smear were compared. Cervical samples in PreservCyt were tested for the presence of 13 HR HPV types with HC-2, with Amplicor at three cutoffs for positivity (0.2, 1.0, and 1.5 optical density units), and for 36 genotypes with the Linear Array (LA). Of 396 eligible women, 316 did not have CIN, 47 had CIN 1, 29 had CIN 2/3, and 4 had CIN of unknown grade. HR HPV was detected in 129 (32.6%) and 164 (41.4%) samples with HC-2 and Amplicor HPV (cutoff, 0.2), respectively (
P
= 0.01). Overall, 112 specimens were positive and 215 were negative with the HC-2 and Amplicor HPV assays (agreement of 82.6%; 95% confidence interval [CI], 78.5 to 86.0). The clinical sensitivity and specificity of Amplicor HPV at cutoffs of 0.2, 1.0 and 1.5 and of HC-2 for detection of CIN 2/3 were 89.7% (95% CI, 72.8 to 97.2) and 62.5% (95% CI, 57.5 to 52.4), 89.7% (95% CI, 72.8 to 97.2) and 64.5% (95% CI, 59.4 to 69.2), 89.7% (95% CI, 72.8 to 97.2) and 64.7% (95% CI, 59.7 to 69.5), and 93.1% (95% CI, 77.0 to 99.2) and 72.2% (95% CI, 67.4 to 76.5), respectively. Both HR HPV detection tests identified women with ASC-US who would benefit the most from colposcopy. Women with persistent HR HPV infection need further investigation despite a first normal colposcopy.
Distinguishing Acute from Chronic Hepatitis C Virus (HCV) Infection Based on Antibody Reactivities to Specific HCV Structural and Nonstructural Proteins
Authors: Araujo Aufra C. , Astrakhantseva Irina V. , Fields Howard A. , Kamili Saleem
Published: November 2010

Currently available serological assays for detection of antibodies to hepatitis C virus (HCV) cannot reliably discriminate acute from chronic HCV infection. We developed a multiplexed, flow-cytometric microsphere immunoassay to measure anti-HCV-IgG reactivities to the core, NS3, NS4, and NS5 HCV recombinant proteins and applied it to 99 serum samples from 24 anti-HCV seroconverters and 141 anti-HCV-IgG and HCV RNA-positive plasma specimens from chronically infected people. Differences in the geometric means or means of signal/cutoff ratios between the two sample sets were statistically significant for all the antigens tested. A multivariate logistic regression model correctly classified the samples in two groups, with a cross-validation accuracy of 90.8% for the acute group and 97.2% for the chronic group. The immunoassay described has the potential to distinguish acute from chronic HCV infection.
Is Infection by
Dermatophilus congolensis
Underdiagnosed?
Authors: Amor Aranzazu , Enríquez Ana , Corcuera Maria Teresa , Toro Carlos , Herrero Dolores , Baquero Margarita
Published: November 2010

Dermatophilus congolensis
, which affects animal species, is an uncommon human infection. Few cases, mainly in tropical areas, have been reported. We describe the first human infection in Spain in a traveler returning from Central America. Diagnosis of human infection may be underestimated in people in contact with animals.
Development and Characterization of a Highly Specific and Sensitive SYBR Green Reverse Transcriptase PCR Assay for Detection of the 2009 Pandemic H1N1 Influenza Virus on the Basis of Sequence Signatures
Authors: Medina Rafael A. , Rojas Mark , Tuin Astrid , Huff Stephen , Ferres Marcela , Martinez-Valdebenito Constanza , Godoy Paula , García-Sastre Adolfo , Fofanov Yuriy , SantaLucia John
Published: November 2010

The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.
Molecular Detection, Epidemiology, and Genetic Characterization of Novel European Field Isolates of Equine Infectious Anemia Virus
Authors: Cappelli Katia , Capomaccio Stefano , Cook Frank R. , Felicetti Michela , Marenzoni Maria Luisa , Coppola Giacomo , Verini-Supplizi Andrea , Coletti Mauro , Passamonti Fabrizio
Published: November 2010

The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of
gag
genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total
gag
gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of
gag
gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the “clades” described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.
Identification of Oseltamivir Resistance among Pandemic and Seasonal Influenza A (H1N1) Viruses by an His275Tyr Genotyping Assay Using the Cycling Probe Method
Authors: Suzuki Yasushi , Saito Reiko , Sato Isamu , Zaraket Hassan , Nishikawa Makoto , Tamura Tsutomu , Dapat Clyde , Caperig-Dapat Isolde , Baranovich Tatiana , Suzuki Takako , Suzuki Hiroshi
Published: November 2010

Neuraminidase inhibitors are agents used against influenza viruses; however, the emergence of drug-resistant strains is a major concern. Recently, the prevalence of oseltamivir-resistant seasonal influenza A (H1N1) virus increased globally and the emergence of oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses was reported. In this study, we developed a cycling probe real-time PCR method for the detection of oseltamivir-resistant seasonal influenza A (H1N1) and pandemic influenza A (H1N1) 2009 viruses. We designed two sets of primers and probes that were labeled with 6-carboxyfluorescein or 6-carboxy-X-rhodamine to identify single nucleotide polymorphisms (SNPs) that correspond to a histidine and a tyrosine at position 275 in the neuraminidase protein, respectively. These SNPs confer susceptibility and resistance to oseltamivir, respectively. In the 2007-2008 season, the prevalence of oseltamivir-resistant H1N1 viruses was 0% (0/72), but in the 2008-2009 season, it increased to 100% (282/282). In the 2009-2010 season, all of the pandemic influenza A (H1N1) 2009 viruses were susceptible to oseltamivir (0/73, 0%). This method is sensitive and specific for the screening of oseltamivir-resistant influenza A (H1N1) viruses. This method is applicable to routine laboratory-based monitoring of drug resistance and patient management during antiviral therapy.
Diversity of Pulsed-Field Gel Electrophoresis Patterns of
Mycobacterium abscessus
Type 2 Clinical Isolates
Authors: Matsumoto Cristianne Kayoko , Chimara Erica , Bombarda Sidney , Duarte Rafael Silva , Leão Sylvia Cardoso
Published: November 2010

An epidemic of infections by rapidly growing mycobacteria related to surgical procedures between 2004 and 2008 in Brazil was caused by a unique strain showing the
Mycobacterium abscessus
type 2 pattern when it was analyzed by the molecular method of PCR-restriction enzyme analysis of the
hsp65
gene (PRA-
hsp65
). In order to investigate the diversity of
M. abscessus
type 2 clinical isolates and to assess whether this epidemic strain was present in specimens from nonsurgical patients, we studied 52 isolates from 38 patients showing this characteristic PRA-
hsp65
pattern obtained between 2005 and 2009. All isolates were identified by sequencing of region V of the
rpoB
gene and typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. Seven isolates obtained from sputum, bronchoalveolar lavage fluid, and urine in three different Brazilian states showed
rpoB
sequences 100% similar to the
rpoB
sequence of epidemic strain INCQS 594 and PFGE patterns highly related to the patterns of isolates, evidencing the presence of the epidemic strain in isolates from patients not associated with the surgical epidemic. The remaining isolates showed diverse
rpoB
sequences, with the highest similarities being to the corresponding sequences of
M. massiliense
T
CIP 108297 (21 isolates),
M. bolletii
T
CIP 108541 (19 isolates), or
M. abscessus
T
ATCC 19977 (5 isolates). Two additional clusters could be detected by PFGE. PFGE showed 100% typeability and reproducibility and discriminatory powers, calculated by Simpson's index of diversity, of 0.978 (DraI) and 0.986 (AseI), confirming its suitability for the discrimination of
M. abscessus
type 2 isolates.
Multiplexed Typing of
Mycobacterium avium
subsp.
paratuberculosis
Types I, II, and III by Luminex xMAP Suspension Array
Authors: Gastaldelli Michele , Stefani Elisabetta , Lettini Antonia Anna , Pozzato Nicola
Published: November 2010

Differentiation among types I, II, and III is the primary step in typing
Mycobacterium avium
subsp.
paratuberculosis.
We propose an innovative approach based on detection of gyrase B (
gyrB
) gene polymorphisms by suspension array technology, with high discriminatory power and high-throughput potential.
Factors Associated with Nasal Colonization of Methicillin-Resistant
Staphylococcus aureus
among Healthy Children in Taiwan
Authors: Chen Chih-Jung , Hsu Kuang-Hung , Lin Tzou-Yien , Hwang Kao-Pin , Chen Po-Yen , Huang Yhu-Chering
Published: November 2010

Methicillin-resistant
Staphylococcus aureus
(MRSA) has been identified as a major cause of community-associated (CA)
S. aureus
infections in the past decade. The main reservoir in the community for MRSA and the factors contributing to its worldwide spread remain poorly defined. Between July 2005 and June 2008, a total of 6,057 healthy children 2 to 60 months of age were screened for carriage of
S. aureus
and
Streptococcus pneumoniae
in Taiwan. The prevalence and epidemiological factors influencing MRSA carriage were determined. MRSA strains were tested for antimicrobial susceptibility and underwent molecular characterization. The overall prevalences of MRSA and
S. aureus
carriage were 7.8% and 23.2%, respectively. A majority (88%) of MRSA isolates belonged to a common Asian-Pacific CA-MRSA lineage, multilocus sequence type 59, and were resistant to multiple non-beta-lactam antibiotics. The carriage rate of MRSA was higher among subjects 2 to 6 months old (
P
< 0.0001), residing in northern Taiwan (
P
= 0.0003), and enrolled later in the study (
P
< 0.0001). MRSA colonization was associated with the number of children in the family (adjusted odds ratio [aOR], 1.114; 95% confidence interval [CI], 1.002 to 1.240;
P
= 0.0463) and day care attendance (aOR, 1.530; 95% CI, 1.201 to 1.949;
P
= 0.0006). Breast feeding (
P
< 0.0001) and colonization with
S. pneumoniae
(
P
= 0.0170) were protective against MRSA colonization. We concluded that epidemic CA-MRSA strains increasingly colonized Taiwanese children between 2005 and 2008. The carriage rate varied significantly across different demographical features. Crowding was an independent environmental risk factor that might accelerate CA-MRSA transmission in the community.
Polymorphisms in the Intermediate Region of VacA Impact
Helicobacter pylori
-Induced Disease Development
Authors: Jones Kathleen R. , Jang Sungil , Chang Jennifer Y. , Kim Jinmoon , Chung In-Sik , Olsen Cara H. , Merrell D. Scott , Cha Jeong-Heon
Published: November 2010

Helicobacter pylori
is the etiological agent of diseases such as gastritis, gastric and duodenal ulcers, and two types of gastric cancers. While some insight has been gained into the etiology of these diverse manifestations, by and large, the reason that some individuals develop more severe disease remains elusive. Recent studies have focused on the roles of
H. pylori
toxins CagA and VacA on the disease process and have suggested that both toxins are intimately involved. Moreover, CagA and VacA are polymorphic within different
H. pylori
strains, and particular polymorphisms seem to show a correlation with the development of particular disease states. Among VacA polymorphisms, the intermediate region has recently been proposed to play a major role in disease outcome. In this article, we describe a detailed sequence analysis of the polymorphic intermediate region of
vacA
from strains obtained from a large South Korean population. We show that polymorphisms found at amino acid position 196 are associated with more severe disease manifestations. Additionally, polymorphisms found at amino acid position 231 are linked to disease in strains that carry the non-EPIYA-ABD allele of CagA. Collectively, these data help explain the impact of the VacA intermediate region on disease and lead to the hypothesis that there are allele-driven interactions between VacA and CagA.
Novel Multiplex Oligonucleotide-Conjugated Bead Suspension Array for Rapid Identification of Enterovirus 71 Subgenogroups
Authors: Wu Y. , Tan E. L. , Yeo A. , Chan K. P. , Nishimura H. , Cardosa M. J. , Poh C. L. , Quak S. H. , Chow Vincent T.
Published: November 2010

A high-throughput multiplex bead suspension array was developed for the rapid subgenogrouping of EV71 strains, based on single nucleotide polymorphisms observed within the VP1 region with a high sensitivity as low as 1 PFU. Of 33 viral isolates and 55 clinical samples, all EV71 strains were successfully detected and correctly subgenogrouped.
Predominance of Porcine P[23] Genotype Rotaviruses in Piglets with Diarrhea in Northern Thailand
Authors: Okitsu Shoko , Khamrin Pattara , Thongprachum Aksara , Maneekarn Niwat , Mizuguchi Masashi , Ushijima Hiroshi
Published: November 2010

Of 131 stool samples collected from piglets with diarrhea in northern Thailand between July 2006 and August 2008, 14 (10.7%) were positive for group A rotavirus. Sequence analysis showed that 13 strains (92.9%) belonged to the rare P[23] genotype combination with G9 or G3 genotypes.
Identification of Novel Endogenous Betaretroviruses Which Are Transcribed in the Bovine Placenta
Authors: Baba Kenji , Nakaya Yuki , Shojima Takayuki , Muroi Yoshikage , Kizaki Keiichiro , Hashizume Kazuyoshi , Imakawa Kazuhiko , Miyazawa Takayuki
Published: November 2010

Sequences of retroviral origin occupy approximately 10% of mammalian genomes. Various infectious endogenous retroviruses (ERVs) and functional retroviral elements have been reported for several mammals but not cattle. Here, we identified two proviruses, designated bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2, containing full-length envelope (env) genes in the bovine genome. Phylogenetic analysis revealed that they belong to the genusBetaretrovirus. By reverse transcription (RT)-PCR, both BERV-K1 and -K2envmRNAs were detected in the placenta and cultured bovine trophoblast cells. Real-time RT-PCR analysis using RNAs isolated from various bovine tissues revealed that BERV-K1envmRNA was preferentially expressed in the placenta. Moreover, we also found the expression of doubly spliced transcripts, named theREBK1andREBK2genes. Both the REBK1 and REBK2 proteins have motifs for a putative nuclear localization signal and a nuclear export signal. REBK1 and REBK2 fused with green fluorescent proteins were localized mainly in the nuclei when they were expressed in bovine and porcine cells. In theenvand 3′ long terminal repeats of BERV-K1 and -K2, we found regulatory elements responsible for the splicing and transport of viral RNAs and/or translation of theenvgenes. Although we have not identified the expressed Env proteins in bovine tissues, these data suggest that both BERV-K1 and BERV-K2 express Env proteins and that these proteins may have physiological functionsin vivo.
Viral Serine/Threonine Protein Kinases
Authors: Jacob Thary , Van den Broeke Céline , Favoreel Herman W.
Published: November 2010

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.
The Nuclear and Adherent Junction Complex Component Protein Ubinuclein Negatively Regulates the Productive Cycle of Epstein-Barr Virus in Epithelial Cells
Authors: Gruffat Henri , Lupo Julien , Morand Patrice , Boyer Véronique , Manet Evelyne
Published: November 2010

The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral
trans
-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus.
Genetic Dissection of Interferon-Antagonistic Functions of Rabies Virus Phosphoprotein: Inhibition of Interferon Regulatory Factor 3 Activation Is Important for Pathogenicity
Authors: Rieder Martina , Brzózka Krzysztof , Pfaller Christian K. , Cox James H. , Stitz Lothar , Conzelmann Karl-Klaus
Published: November 2010

The rabies virus (RV) phosphoprotein (P) is a type I interferon (IFN) antagonist preventing both transcriptional induction of IFN and IFN-mediated JAK/STAT signaling. In addition, P is an essential cofactor of the viral polymerase and is required for encapsidation of viral RNA into nucleoprotein during replication. By site-directed mutagenesis, we have identified a domain of P required for efficient inhibition of IFN induction. Phosphoproteins lacking amino acids (aa) 176 to 181, 182 to 186, or 176 to 186 were severely compromised in counteracting phosphorylation of IRF3 and IRF7 by TBK1 or IKKi while retaining the full capacity of preventing nuclear import of activated STATs and of supporting virus transcription and replication. Recombinant RV carrying the mutated phosphoproteins (the SAD ΔInd1, SAD ΔInd2, and SAD ΔInd1/2 viruses) activated IRF3 and beta IFN (IFN-β) transcription in infected cells but still blocked STAT-mediated expression of IFN-stimulated genes. Due to a somewhat higher transcription rate, the SAD ΔInd1 virus activated IRF3 more efficiently than the SAD ΔInd2 virus. After intracerebral injection into mouse brains at high doses, the SAD ΔInd1 virus was completely apathogenic for wild-type (wt) mice, while the SAD ΔInd2 virus was partially attenuated and caused a slower progression of lethal rabies than wt RV. Neurovirulence of IFN-resistant RV thus correlates with the capacity of the virus to prevent activation of IRF3 and IRF7.
Inhibition of Calmodulin-Dependent Kinase Kinase Blocks Human Cytomegalovirus-Induced Glycolytic Activation and Severely Attenuates Production of Viral Progeny
Authors: McArdle Jessica , Schafer Xenia L. , Munger Joshua
Published: November 2010

Viruses depend on the host cell to provide the energy and biomolecular subunits necessary for production of viral progeny. We have previously reported that human cytomegalovirus (HCMV) infection induces dramatic changes to central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and nucleotide biosynthesis. Here, we explore the mechanisms involved in HCMV-mediated glycolytic activation. We find that HCMV virion binding and tegument protein delivery are insufficient for HCMV-mediated activation of glycolysis. Viral DNA replication and late-gene expression, however, are not required. To narrow down the list of cellular pathways important for HCMV-medicated activation of glycolysis, we utilized pharmaceutical inhibitors to block pathways reported to be both involved in metabolic control and activated by HCMV infection. We find that inhibition of calmodulin-dependent kinase kinase (CaMKK), but not calmodulin-dependent kinase II (CaMKII) or protein kinase A (PKA), blocks HCMV-mediated activation of glycolysis. HCMV infection was also found to target calmodulin-dependent kinase kinase 1 (CaMKK1) expression, increasing the levels of CaMKK1 mRNA and protein. Our results indicate that inhibition of CaMKK has a negligible impact on immediate-early-protein accumulation yet severely attenuates production of HCMV viral progeny, reduces expression of at least one early gene, and blocks viral DNA replication. Inhibition of CaMKK did not affect the glycolytic activation induced by another herpes virus, herpes simplex virus type 1 (HSV-1). Furthermore, inhibition of CaMKK had a much smaller impact on HSV-1 replication than on that of HCMV. These data suggest that the role of CaMKK during the viral life cycle is, in this regard, HCMV specific. Taken together, our results suggest that CaMKK is an important factor for HCMV replication and HCMV-mediated glycolytic activation.
Increased Susceptibility of Human-PrP Transgenic Mice to Bovine Spongiform Encephalopathy Infection following Passage in Sheep
Authors: Plinston Chris , Hart Patricia , Chong Angela , Hunter Nora , Foster James , Piccardo Pedro , Manson Jean C. , Barron Rona M.
Published: November 2010

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine
PRNP
gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.
HIV-1 Replication in Langerhans and Interstitial Dendritic Cells Is Inhibited by Neutralizing and Fc-Mediated Inhibitory Antibodies
Authors: Peressin M. , Holl V. , Schmidt S. , Decoville T. , Mirisky D. , Lederle A. , Delaporte M. , Xu K. , Aubertin A. M. , Moog C.
Published: November 2010

Langerhans cells (LCs) and interstitial dendritic cells (IDCs) may be among the first human immunodeficiency virus type 1 (HIV-1) targets after sexual transmission. We generated cells of these types by differentiation of purified CD34
+
cord blood cells. After
in vitro
infection with R5-tropic strains, we obtained similar percentages of infected cells for both dendritic cell (DC) subsets. Moreover, LC infection was not increased by blockage of langerin by antilangerin. These results indicate that, under our experimental conditions, there was no evidence of any preference of HIV replication in LCs versus IDCs. The inhibitory activity of HIV-1-specific IgAs and IgGs against HIV-1 replication in LCs and IDCs was analyzed. We found that neutralizing antibodies inhibit HIV-1 infection of both DC subsets. Interestingly, HIV-1 was inhibited more efficiently by the IgGs than the corresponding IgA, due to an Fcγ receptor-dependent mechanism. Moreover, nonneutralizing inhibitory IgGs were able to inhibit infection of both LCs and IDCs. These results underline the importance of HIV-1 inhibition by the binding of the Fc part of IgGs to Fcγ receptors and suggest that the induction of neutralizing and nonneutralizing inhibitory IgGs in addition to neutralizing IgAs at mucosal sites may contribute to protection against sexual transmission of HIV-1.
RIG-I-Mediated Antiviral Signaling Is Inhibited in HIV-1 Infection by a Protease-Mediated Sequestration of RIG-I
Authors: Solis Mayra , Nakhaei Peyman , Jalalirad Mohammad , Lacoste Judith , Douville Renée , Arguello Meztli , Zhao Tiejun , Laughrea Michael , Wainberg Mark A. , Hiscott John
Published: November 2010

The rapid induction of type I interferon (IFN) is essential for establishing innate antiviral responses. During infection, cytoplasmic viral RNA is sensed by two DExD/H box RNA helicases, RIG-I and MDA5, ultimately driving IFN production. Here, we demonstrate that purified genomic RNA from HIV-1 induces a RIG-I-dependent type I IFN response. Both the dimeric and monomeric forms of HIV-1 were sensed by RIG-I, but not MDA5, with monomeric RNA, usually found in defective HIV-1 particles, acting as a better inducer of IFN than dimeric RNA. However, despite the presence of HIV-1 RNA in the
de novo
infection of monocyte-derived macrophages, HIV-1 replication did not lead to a substantial induction of IFN signaling. We demonstrate the existence of an evasion mechanism based on the inhibition of the RIG-I sensor through the action of the HIV-1 protease (PR). Indeed, the ectopic expression of PR resulted in the inhibition of IFN regulatory factor 3 (IRF-3) phosphorylation and decreased expression of IFN and interferon-stimulated genes. A downregulation of cytoplasmic RIG-I levels occurred in cells undergoing a single-cycle infection with wild-type provirus BH10 but not in cells transfected with a protease-deficient provirus, BH10-PR
−
. Cellular fractionation and confocal microscopy studies revealed that RIG-I translocated from the cytosol to an insoluble fraction during the
de novo
HIV-1 infection of monocyte-derived macrophages, in the presence of PR. The loss of cytoplasmic RIG-I was prevented by the lysosomal inhibitor E64, suggesting that PR targets RIG-I to the lysosomes. This study reveals a novel PR-dependent mechanism employed by HIV-1 to counteract the early IFN response to viral RNA in infected cells.
Genetic Diversity of Simian Immunodeficiency Virus Encoding HIV-1 Reverse Transcriptase Persists in Macaques despite Antiretroviral Therapy
Authors: Kearney Mary , Spindler Jon , Shao Wei , Maldarelli Frank , Palmer Sarah , Hu Shiu-Lok , Lifson Jeffrey D. , KewalRamani Vineet N. , Mellors John W. , Coffin John M. , Ambrose Zandrea
Published: November 2010

The impact of antiretroviral therapy (ART) on the genetics of simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) populations has been incompletely characterized. We analyzed SIV genetic variation before, during, and after ART in a macaque model. Six pigtail macaques were infected with an SIV/HIV chimeric virus, RT-SHIV
mne
, in which SIV reverse transcriptase (RT) was replaced by HIV-1 RT. Three animals received a short course of efavirenz (EFV) monotherapy before combination ART was started. All macaques received 20 weeks of tenofovir, emtricitabine, and EFV. Plasma virus populations were analyzed by single-genome sequencing. Population diversity was measured by average pairwise difference, and changes in viral genetics were assessed by phylogenetic and panmixia analyses. After 20 weeks of ART, viral diversity was not different from pretherapy viral diversity despite more than 10,000-fold declines in viremia, indicating that, within this range, there is no relationship between diversity and plasma viremia. In two animals with consistent SIV RNA suppression to <15 copies/ml during ART, there was no evidence of viral evolution. In contrast, in the four macaques with viremias >15 copies/ml during therapy, there was divergence between pre- and during-ART virus populations. Drug resistance mutations emerged in two of these four animals, resulting in virologic failure in the animal with the highest level of pretherapy viremia. Taken together, these findings indicate that viral diversity does not decrease with suppressive ART, that ongoing replication occurs with viremias >15 copies/ml, and that in this macaque model of ART drug resistance likely emerges as a result of incomplete suppression and preexisting drug resistance mutations.
Pandemic Swine-Origin H1N1 Influenza A Virus Isolates Show Heterogeneous Virulence in Macaques
Authors: Safronetz David , Rockx Barry , Feldmann Friederike , Belisle Sarah E. , Palermo Robert E. , Brining Douglas , Gardner Don , Proll Sean C. , Marzi Andrea , Tsuda Yoshimi , LaCasse Rachel A. , Kercher Lisa , York Anthony , Korth Marcus J. , Long Dan , Rosenke Rebecca , Shupert W. Lesley , Aranda Celia Alpuche , Mattoon John S. , Kobasa Darwyn , Kobinger Gary , Li Yan , Taubenberger Jeffery K. , Richt Jürgen A. , Parnell Michael , Ebihara Hideki , Kawaoka Yoshihiro , Katze Michael G. , Feldmann Heinz
Published: November 2010

The first influenza pandemic of the new millennium was caused by a newly emerged swine-origin influenza virus (SOIV) (H1N1). This new virus is characterized by a previously unknown constellation of gene segments derived from North American and Eurasian swine lineages and the absence of common markers predictive of human adaptation. Overall, human infections appeared to be mild, but an alarming number of young individuals presented with symptoms atypical for seasonal influenza. The new SOIV also showed a sustained human-to-human transmissibility and higher reproduction ratio than common seasonal viruses, altogether indicating a higher pathogenic potential for this newly emerged virus. To study the virulence of the SOIV, we used a recently established cynomolgus macaque model and compared parameters of clinical disease, virology, host responses, and pathology/histopathology with a current seasonal H1N1 virus. We here show that infection of macaques with two genetically similar but clinically distinct SOIV isolates from the early stage of the pandemic (A/Mexico/4108/2009 and A/Mexico/InDRE4487/2009) resulted in upper and lower respiratory tract infections and clinical disease ranging from mild to severe pneumonia that was clearly advanced over the mild infection caused by A/Kawasaki/UTK-4/2009, a current seasonal strain. Unexpectedly, we observed heterogeneity among the two SOIV isolates in virus replication, host transcriptional and cytokine responses, and disease progression, demonstrating a higher pathogenic potential for A/Mexico/InDRE4487/2009. Differences in virulence may explain more severe disease, as was seen with certain individuals infected with the emerged pandemic influenza virus. Thus, the nonhuman primate model closely mimics influenza in humans.
Viral Exploitation of Host SOCS Protein Functions
Authors: Akhtar Lisa Nowoslawski , Benveniste Etty N.
Published: November 2010

Over the past decade, a family of host proteins known assuppressorsofcytokinesignaling (SOCS) have emerged as frequent targets of viral exploitation. Under physiologic circumstances, SOCS proteins negatively regulate inflammatory signaling pathways by facilitating ubiquitination and proteosomal degradation of pathway machinery. Their expression is tightly regulated to prevent excessive inflammation while maintaining protective antipathogenic responses. Numerous viruses, however, have developed mechanisms to induce robust host SOCS protein expression following infection, essentially “hijacking” SOCS function to promote virus survival. To date, SOCS proteins have been shown to inhibit protective antiviral signaling pathways, allowing viruses to evade the host immune response, and to ubiquitinate viral proteins, facilitating intracellular viral trafficking and progeny virus assembly. Importantly, manipulation of SOCS proteins not only facilitates progression of the viral life cycle but also powerfully shapes the presentation of viral disease. SOCS proteins can define host susceptibility to infection, contribute to peripheral disease manifestations such as immune dysfunction and cancer, and even modify the efficacy of therapeutic interventions. Looking toward the future, it is clear that a better understanding of the role of SOCS proteins in viral diseases will be essential in our struggle to modulate and even eliminate the pathogenic effects of viruses on the host.
The Arginine Clusters of the Carboxy-Terminal Domain of the Core Protein of Hepatitis B Virus Make Pleiotropic Contributions to Genome Replication
Authors: Lewellyn Eric B. , Loeb Daniel D.
Published: November 2010

The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.
A Single N66S Mutation in the PB1-F2 Protein of Influenza A Virus Increases Virulence by Inhibiting the Early Interferon Response
In Vivo
Authors: Conenello Gina M. , Tisoncik Jennifer R. , Rosenzweig Elizabeth , Varga Zsuzsanna T. , Palese Peter , Katze Michael G.
Published: November 2010

The PB1-F2 protein of influenza A virus can contribute to viral pathogenesis of influenza virus strains. Of note, an N66S amino acid mutation in PB1-F2 has been shown to increase the pathogenesis associated with H5N1 Hong Kong/1997 and H1N1 Brevig Mission/1918 influenza viruses. To identify the mechanism of enhanced immunopathology, we evaluated the host response to two isogenic viruses that differ by a single amino acid at position 66 of the PB1-F2 protein. Various components of the adaptive immune response were ruled out as factors contributing to pathogenesis through knockout mouse studies. Transcriptional profiling of lungs from PB1-F2 66S-infected mice revealed an early delay in innate immune responses. In particular, enhanced activation of type I interferon (IFN) pathway genes, including IFN-β, RIG-I, and numerous interferon-inducible genes, was not observed until day 3 postinfection. The N66S mutant virus caused increased cellularity in the lungs, as a result of monocyte and neutrophil infiltration. Furthermore, numerous cytokines and chemokines related to monocyte and neutrophil migration and maturation were upregulated. The cellular infiltration and increased cytokine expression corresponded to increased PB1-F2 66S titer. These data suggest that PB1-F2 N66S may contribute to the delay of innate immune responses, allowing for unchecked viral growth and ultimately severe immunopathology observed in the lungs.
Indoleamine 2,3-Dioxygenase Mediates the Antiviral Effect of Gamma Interferon against Hepatitis B Virus in Human Hepatocyte-Derived Cells
Authors: Mao Richeng , Zhang Jiming , Jiang Dong , Cai Dawei , Levy Jessica M. , Cuconati Andrea , Block Timothy M. , Guo Ju-Tao , Guo Haitao
Published: November 2010

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infectionin vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells.
Sheep with Scrapie and Mastitis Transmit Infectious Prions through the Milk
Authors: Ligios Ciriaco , Cancedda Maria Giovanna , Carta Antonello , Santucciu Cinzia , Maestrale Caterina , Demontis Francesca , Saba Mariangela , Patta Cristiana , DeMartini James C. , Aguzzi Adriano , Sigurdson Christina J.
Published: November 2010

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.
Human Cytomegalovirus-Regulated Paxillin in Monocytes Links Cellular Pathogenic Motility to the Process of Viral Entry
Authors: Nogalski Maciej T. , Chan Gary , Stevenson Emily V. , Gray Scarlet , Yurochko Andrew D.
Published: November 2010

We have established that human cytomegalovirus (HCMV) infection modulates the biology of target primary peripheral blood monocytes, allowing HCMV to use monocytes as “vehicles” for its systemic spread. HCMV infection of monocytes results in rapid induction of phosphatidylinositol-3-kinase [PI(3)K] and NF-κB activities. Integrins, which are upstream of the PI(3)K and NF-κB pathways, were shown to be involved in HCMV binding to and entry into fibroblasts, suggesting that receptor ligand-mediated signaling following viral binding to integrins on monocytes could trigger the functional changes seen in infected monocytes. We now show that integrin engagement and the activation of the integrin/Src signaling pathway are essential for the induction of HCMV-infected monocyte motility. To investigate how integrin engagement by HCMV triggers monocyte motility, we examined the infected-monocyte transcriptome and found that the integrin/Src signaling pathway regulates the expression of paxillin, which is an important signal transducer in the regulation of actin rearrangement during cell adhesion and movement. Functionally, we observed that paxillin is activated via the integrin/Src signaling pathway and is required for monocyte motility. Because motility is intimately connected to cellular cytoskeletal organization, a process that is also important in viral entry, we investigated the role paxillin regulation plays in the process of viral entry into monocytes. New results confirmed that HCMV entry into target monocytes was significantly reduced in cells deficient in paxillin expression or the integrin/Src/paxillin signaling pathway. From our data, HCMV-cell interactions emerge as an essential trigger for the cellular changes that allow for HCMV entry and hematogenous dissemination.