bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid and Accurate Identification of Human-Associated Staphylococci by Use of Multiplex PCR
Authors: Hirotaki Shintaro , Sasaki Takashi , Kuwahara-Arai Kyoko , Hiramatsu Keiichi 
Published: August 2011

ABSTRACT

Although staphylococci are identified by phenotypic analysis in many clinical laboratories, these results are often incorrect because of phenotypic variation. Genetic analysis is necessary for definitive species identification. In the present study, we developed a simple multiplex-PCR (M-PCR) for species identification of human-associated staphylococci, which were as follows:
Staphylococcus aureus
,
S. capitis
,
S. caprae
,
S. epidermidis
,
S. haemolyticus
,
S. hominis
,
S. lugdunensis
,
S. saprophyticus
, and
S. warneri
. This method was designed on the basis of nucleotide sequences of the thermonuclease (
nuc
) genes that were universally conserved in staphylococci except the
S. sciuri
group and showed moderate sequence diversity. In order to validate this assay, 361 staphylococcal strains were studied, which had been identified at the species levels by sequence analysis of the
hsp60
genes. In consequence, M-PCR demonstrated a sensitivity of 100% and a specificity of 100%. By virtue of simplicity and accuracy, this method will be useful in clinical research.
Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid
Authors: Kim Dong Wook , Kilgore Paul Evan , Kim Eun Jin , Kim Soon Ae , Anh Dang Duc , Seki Mitsuko 
Published: August 2011

ABSTRACTHaemophilus influenzaetype b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection ofbexAand a nested PCR for Hib (Hib PCR). NoH. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.
Evaluation of Pooling Strategies for Acute HIV-1 Infection Screening Using Nucleic Acid Amplification Testing
Authors: Sullivan Timothy J. , Patel Pragna , Hutchinson Angela , Ethridge Steven F. , Parker Monica M. 
Published: August 2011

ABSTRACT
A 128-member specimen pooling scheme for acute HIV infection (AHI) detection was evaluated using 21 AHI specimens (range, 1,520 to 500,000 copies/ml) previously identified by RNA testing of 16-member plasma pools. HIV-1 RNA was detectable in 128-member pools for all 21 specimens; however, one pool created from a specimen with 1,827 copies/ml was nonreactive in one of three replicates.
Validation of a Low-Cost Human Papillomavirus Genotyping Assay Based on PGMY PCR and Reverse Blotting Hybridization with Reusable Membranes
Authors: Estrade C. , Menoud P.-A. , Nardelli-Haefliger D. , Sahli R. 
Published: August 2011

ABSTRACTThe genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n= 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n= 182) and negative samples (n= 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P< 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P= 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n= 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n= 47) were significantly associated with the weakest genotypes in samples with multiple infections (P< 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.
Characterization of Structural Features and Diversity of Variable-Region Determinants of Related Quaternary Epitopes Recognized by Human and Rhesus Macaque Monoclonal Antibodies Possessing Unusually Potent Neutralizing Activities
Authors: Krachmarov Chavdar , Lai Zhong , Honnen William J. , Salomon Aidy , Gorny Miroslaw K. , Zolla-Pazner Susan , Robinson James , Pinter Abraham 
Published: August 2011

ABSTRACT
A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909. These studies showed that determinants for the epitopes defined by the RhMAbs differed in both the V2 (positions 160, 167, and 169) and V3 (positions 313 and 315) regions from 2909, and in a number of cases, from each other. Attempts to reconstitute expression of these epitopes on the cell surface by cotransfecting Envs containing either the V2 or the V3 determinant of the epitope were not successful, suggesting that these epitopes were expressed on individual protomers in a trimer-dependent manner. Several of the V2 positions found to be critical for expression of these quaternary epitopes also significantly affected exposure and neutralization sensitivity of targets in the V3 and CD4-binding domains. These results demonstrated a considerable diversity in the fine structure of this class of epitopes and further suggested a potentially important relationship between the expression of such quaternary epitopes and V1/V2-mediated masking of immunodominant epitopes.
Fitness Costs and Diversity of the Cytotoxic T Lymphocyte (CTL) Response Determine the Rate of CTL Escape during Acute and Chronic Phases of HIV Infection
Authors: Ganusov Vitaly V. , Goonetilleke Nilu , Liu Michael K. P. , Ferrari Guido , Shaw George M. , McMichael Andrew J. , Borrow Persephone , Korber Bette T. , Perelson Alan S. 
Published: August 2011

ABSTRACTHIV-1 often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. We used single-genome amplification and sequencing of complete HIV genomes to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set point at 1 year after the infection. We found that the rate of viral escape from CTL responses in a given patient decreases dramatically from acute infection to the viral set point. Using a novel mathematical model that tracks the dynamics of viral escape at multiple epitopes, we show that a number of factors could potentially contribute to a slower escape in the chronic phase of infection, such as a decreased magnitude of epitope-specific CTL responses, an increased fitness cost of escape mutations, or an increased diversity of the CTL response. In the model, an increase in the number of epitope-specific CTL responses can reduce the rate of viral escape from a given epitope-specific CTL response, particularly if CD8+T cells compete for killing of infected cells or control virus replication nonlytically. Our mathematical framework of viral escape from multiple CTL responses can be used to predict the breadth and magnitude of HIV-specific CTL responses that need to be induced by vaccination to reduce (or even prevent) viral escape following HIV infection.
Inhibition of RIG-I-Mediated Signaling by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Deubiquitinase ORF64
Authors: Inn Kyung-Soo , Lee Sun-Hwa , Rathbun Jessica Y. , Wong Lai-Yee , Toth Zsolt , Machida Keigo , Ou Jing-Hsiung James , Jung Jae U. 
Published: August 2011

ABSTRACT
Virus infection triggers interferon (IFN)-mediated innate immune defenses in part through viral nucleic acid interactions. However, the immune recognition mechanisms by which the host identifies incoming DNA viruses are still elusive. Here, we show that increased levels of Kaposi's sarcoma-associated herpesvirus (KSHV) persistency are observed in retinoic acid-inducible gene I (RIG-I)-deficient cells and that KSHV ORF64, a tegument protein with deubiqutinase (DUB) activity, suppresses RIG-I-mediated IFN signaling by reducing the ubiquitination of RIG-I, crucial for its activation. This study suggests that RIG-I plays a potential role in sensing KSHV infection and that KSHV ORF64 DUB counteracts RIG-I signaling.
Differential Processing of Nuclear Pore Complex Proteins by Rhinovirus 2A Proteases from Different Species and Serotypes
Authors: Watters Kelly , Palmenberg Ann C. 
Published: August 2011

ABSTRACT

Human rhinoviruses (HRVs) from the HRV-A, HRV-B, and HRV-C species use encoded proteases, 2A
pro
and 3C
pro
, to process their polyproteins and shut off host cell activities detrimental to virus replication. Reactions attributed to 2A
pro
include cleavage of eIF4G-I and -II to inhibit cellular mRNA translation and cleavage of select nucleoporin proteins (Nups) within nuclear pore complexes (NPCs) to disrupt karyopherin-dependent nuclear-cytoplasmic transport and signaling. Sequence diversity among 2A
pro
proteases from different HRV clades, even within species, suggested individual viruses might carry out these processes with unique mechanistic signatures. Six different recombinant 2A
pro
proteases (A16, A89, B04, B14, Cw12, and Cw24) were compared for their relative substrate preferences and cleavage kinetics using eIF4G from cellular extracts and Nups presented in native (NPC) or recombinant formats. The enzyme panel attacked these substrates with different rates or processing profiles, mimicking the preferences observed during natural infection (A16 and B14). For eIF4G, all 2A
pro
proteases cleaved at similar sites, but the comparative rates were species specific (HRV-A > HRV-C ≫ HRV-B). For Nup substrates, 5 of the 6 enzymes had unique product profiles (order of Nup selection) or reacted at different sites within Nup62, Nup98, and Nup153. Only A16 and A89 behaved similarly in most assays. Since each type of karyopherin receptor prefers particular Nups or uses a limited cohort of binding motifs within those Nups, the consequences of individual 2A
pro
avidities could profoundly affect relative viral replication levels, intracellular signaling, or extracellular signaling, all of which are underlying triggers for different host immune responses.
Novel Adenoviruses in Wild Primates: a High Level of Genetic Diversity and Evidence of Zoonotic Transmissions
Authors: Wevers Diana , Metzger Sonja , Babweteera Fred , Bieberbach Marc , Boesch Christophe , Cameron Kenneth , Couacy-Hymann Emmanuel , Cranfield Mike , Gray Maryke , Harris Laurie A. , Head Josephine , Jeffery Kathryn , Knauf Sascha , Lankester Felix , Leendertz Siv Aina J. , Lonsdorf Elizabeth , Mugisha Lawrence , Nitsche Andreas , Reed Patricia , Robbins Martha , Travis Dominic A. , Zommers Zinta , Leendertz Fabian H. , Ehlers Bernhard 
Published: August 2011

ABSTRACTAdenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly conserved adenovirus DNA polymerase gene. A plethora of novel AdV sequences were identified, representing at least 45 distinct AdVs. From the AdV-positive individuals, 29 nearly complete hexon genes were amplified and, based on phylogenetic analysis, tentatively allocated to all known human AdV species (Human adenovirus AtoHuman adenovirus G[HAdV-A to -G]) as well as to the only simian AdV species (Simian adenovirus A[SAdV-A]). Interestingly, five of the AdVs detected in great apes grouped into the HAdV-A, HAdV-D, HAdV-F, or SAdV-A clade. Furthermore, we report the first detection of AdVs in New World monkeys, clustering at the base of the primate AdV evolutionary tree. Most notably, six chimpanzee AdVs of species HAdV-A to HAdV-F revealed a remarkably close relationship to human AdVs, possibly indicating recent interspecies transmission events.
The Breadth of Antiviral Activity of Apobec3DE in Chimpanzees Has Been Driven by Positive Selection
Authors: Duggal Nisha K. , Malik Harmit S. , Emerman Michael 
Published: August 2011

ABSTRACT

The Apobec3 family of cytidine deaminases can inhibit the replication of retroviruses and retrotransposons. Human and chimpanzee genomes encode seven
Apobec3
paralogs; of these,
Apobec3DE
has the greatest sequence divergence between humans and chimpanzees. Here we show that even though human and chimpanzee Apobec3DEs are very divergent, the two orthologs similarly restrict long terminal repeat (LTR) and non-LTR retrotransposons (MusD and Alu, respectively). However, chimpanzee Apobec3DE also potently restricts two lentiviruses, human immunodeficiency virus type 1 (HIV-1) and the simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagmTAN), unlike human Apobec3DE, which has poor antiviral activity against these same viruses. This difference between human and chimpanzee Apobec3DE in the ability to restrict retroviruses is not due to different levels of Apobec3DE protein incorporation into virions but rather to the ability of Apobec3DE to deaminate the viral genome in target cells. We further show that
Apobec3DE
rapidly evolved in chimpanzee ancestors approximately 2 to 6 million years ago and that this evolution drove the increased breadth of chimpanzee Apobec3DE antiviral activity to its current high activity against some lentiviruses. Despite a difference in target specificities between human and chimpanzee Apobec3DE, Apobec3DE is likely to currently play a role in host defense against retroelements in both species.
Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility
Authors: Morrison Monique A. , Morreale Richard J. , Akunuru Shailaja , Kofron Matthew , Zheng Yi , Wells Susanne I. 
Published: August 2011

ABSTRACT
Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.
Human and Rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H Demonstrate a Conserved Capacity To Restrict Vif-Deficient HIV-1
Authors: Hultquist Judd F. , Lengyel Joy A. , Refsland Eric W. , LaRue Rebecca S. , Lackey Lela , Brown William L. , Harris Reuben S. 
Published: August 2011

ABSTRACT

Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4
+
T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4
+
T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins—APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H—in HIV-1 restriction.
Resistance of Human Immunodeficiency Virus Type 1 to a Third-Generation Fusion Inhibitor Requires Multiple Mutations in gp41 and Is Accompanied by a Dramatic Loss of gp41 Function
Authors: Eggink Dirk , Bontjer Ilja , Langedijk Johannes P. M. , Berkhout Ben , Sanders Rogier W. 
Published: August 2011

ABSTRACT

HIV-1 entry into target cells requires the fusion of viral and cellular membranes. This process is an attractive target for therapeutic intervention, and a first-generation fusion inhibitor, T20 (Enfuvirtide; Fuzeon), was approved for clinical use in 2003. Second-generation (T1249) and third-generation (T2635) fusion inhibitors with improved stability and potency were developed. Resistance to T20 and T1249 usually requires one or two amino acid changes within the binding site. We studied the
in vitro
evolution of resistance against T2635. After 6 months of culturing, a multitude of resistance mutations was identified in all gp41 subdomains, but no single mutation provided meaningful T2635 resistance. In contrast, multiple mutations within gp41 were required for resistance, and this was accompanied by a dramatic loss of viral infectivity. Because most of the escape mutations were situated outside the T2635 binding site, a decrease in drug target affinity cannot account for most of the resistance. T2635 resistance is likely to depend on altered kinetics of six-helix bundle formation, thus limiting the time window for T2635 to interfere with membrane fusion. Interestingly, the loss of virus infectivity caused by T2635 resistance mutations in gp41 was partially compensated for by a mutation at the base of the V3 domain in gp120. Thus, escape from the third-generation HIV-1 fusion inhibitor T2635 is mechanistically distinct from resistance against its predecessors T20 and T1249. It requires the accumulation of multiple mutations in gp41, is accompanied with a dramatic loss of gp41 function, and induces compensatory mutations in gp120.
Membrane Integration of Poliovirus 2B Viroporin
Authors: Martínez-Gil Luis , Bañó-Polo Manuel , Redondo Natalia , Sánchez-Martínez Silvia , Nieva José Luis , Carrasco Luis , Mingarro Ismael 
Published: August 2011

ABSTRACT

Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of
in vitro
translation-glycosylation compatible with the translocon-mediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the
in vitro
translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.