bluuebunny/crossref_metadata_2025 huggingface dataset:
Global Proficiency Study of Human Papillomavirus Genotyping
Authors: Eklund Carina , Zhou Tiequn , Dillner Joakim
Published: September 2010

Internationally comparable quality assurance of Human Papillomavirus (HPV) DNA detection and typing methods is essential for evaluation of HPV vaccines and effective monitoring and implementation of HPV vaccination programs. Therefore, the World Health Organization (WHO) HPV Laboratory Network (LabNet) designed an international proficiency study. Following announcement at the WHO website, the responding laboratories performed HPV typing using one or more of their usual assays on 43 coded samples composed of titration series of purified plasmids of 16 HPV types (HPV6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68). Detection of at least 50 IU of HPV16 or HPV18 DNA and of 500 genome equivalents (GE) of the other 14 HPV types (in samples with single and multiple HPV types) was considered proficient. Fifty-four laboratories worldwide submitted a total of 84 data sets. More than 21 HPV-genotyping assays were used. Commonly used methods were Linear Array, Lineblot, InnoLiPa, Clinical Array, type-specific real-time PCR, PCR-Luminex and microarray assays. The major oncogenic HPV types (HPV16 and -18) were detected in 89.7% (70/78) and 92.2% (71/77) of the data sets, respectively. HPV types 56, 59, and 68 were the least commonly detected types (in less than 80% of the data sets). Twenty-eight data sets reported multiple false-positive results and were considered nonproficient. In conclusion, we found that international proficiency studies, traceable to international standards, allow standardized quality assurance for different HPV-typing assays and enable the comparison of data generated from different laboratories worldwide.
Specific Detection of Rinderpest Virus by Real-Time Reverse Transcription-PCR in Preclinical and Clinical Samples from Experimentally Infected Cattle
Authors: Carrillo C. , Prarat M. , Vagnozzi A. , Calahan J. D. , Smoliga G. , Nelson W. M. , Rodriguez L. L.
Published: September 2010

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID
50
) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as postmortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples.
Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence
Authors: Gryadunov Dimitry , Nicot Florence , Dubois Martine , Mikhailovich Vladimir , Zasedatelev Alexander , Izopet Jacques
Published: September 2010

The genotype of the hepatitis C virus (HCV) is essential for determining treatment duration in clinical practice and for epidemiological and clinical studies. Currently, few genotyping assays that determine the HCV subtype are available. This report describes a microarray-based molecular technique for identifying the HCV genotype and subtype. It uses low-density hydrogel-based biochips containing genotype- and subtype-specific oligonucleotides based on the sequences of the NS5B region of the HCV genome. The biochip contains 120 oligonucleotides that identify genotypes 1 to 6 and 36 (1a, 1b, 1c, 1d, 1e, 2a, 2b, 2c, 2d, 2i, 2j, 2k, 2l, 2m, 3a, 3b, 3k, 4a, 4c, 4d, 4f, 4h, 4i, 4k, 4n, 4o, 4p, 4r, 4t, 5a, 6a, 6b, 6d, 6g, 6h, and 6k) subtypes. The procedure included amplification of a 380-nucleotide (nt) fragment of NS5B and its hybridization on the biochip. Tests on 345 HCV-positive samples showed that the assay agreed with NS5B sequencing 100% for the genotype and 99.7% for the subtype. The hybridization on the microarray and the NS5B sequence were in 100% agreement for identifying the most common subtypes, 1a, 1b, 4a, 4d, and 3a. This approach is a promising tool for HCV genotyping, especially for implementing the new anti-HCV drugs that require accurate identification of clinically relevant subtypes.
In Vitro
Susceptibility of the Yeast Pathogen
Cryptococcus
to Fluconazole and Other Azoles Varies with Molecular Genotype
Authors: Chong Hin Siong , Dagg Rebecca , Malik Richard , Chen Sharon , Carter Dee
Published: September 2010

Cryptococcosis is primarily caused by
Cryptococcus neoformans
and
Cryptococcus gattii.
These two pathogenic species each divide into four distinct molecular genotypes. In this study, we examined whether genotype influenced susceptibility to antifungal drugs used to treat cryptococcosis using the broth microdilution method described by the Clinical and Laboratory Standards Institute.
C. gattii
isolates belonging to molecular genotype VGII had significantly higher MIC values for flucytosine and all azole antifungal agents tested, particularly fluconazole, than isolates of other
C. gattii
genotypes. In an extended analysis of fluconazole susceptibility, VGII isolates from the north and west of Australia required higher drug levels for inhibition than those from Vancouver Island, Canada. Within
C. neoformans
, genotype VNII had significantly lower geometric mean MICs for fluconazole than genotype VNI. These results indicate that cryptococcal species, molecular genotype, and region of origin may be important when deciding treatment options for cryptococcosis.
Changing Epidemiology of Methicillin-Resistant
Staphylococcus aureus
in Iceland from 2000 to 2008: a Challenge to Current Guidelines
Authors: Holzknecht Barbara Juliane , Hardardottir Hjördis , Haraldsson Gunnsteinn , Westh Henrik , Valsdottir Freyja , Boye Kit , Karlsson Sigfus , Kristinsson Karl Gustaf , Gudlaugsson Olafur
Published: September 2010

The epidemiology of methicillin-resistant
Staphylococcus aureus
(MRSA) is continuously changing. Iceland has a low incidence of MRSA. A “search and destroy” policy (screening patients with defined risk factors and attempting eradication in carriers) has been implemented since 1991. Clinical and microbiological data of all MRSA patients from the years 2000 to 2008 were collected prospectively. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), sequencing of the repeat region of the
Staphylococcus
protein A gene (
spa
typing), staphylococcal cassette chromosome
mec
(SCC
mec
) typing, and screening for the Panton-Valentine leukocidin (PVL) gene. Two hundred twenty-six infected (60%) or colonized (40%) individuals were detected (annual incidence 2.5 to 16/100,000). From 2000 to 2003, two health care-associated outbreaks dominated (
spa
types t037 and t2802), which were successfully controlled with extensive infection control measures. After 2004, an increasing number of community-associated (CA) cases without relation to the health care system occurred. A great variety of clones (40 PFGE types and 49
spa
types) were found, reflecting an influx of MRSA from abroad. The USA300 and Southwest Pacific (SWP) clones were common. SCC
mec
type IV was most common (72%), and 38% of the isolates were PVL positive. The incidence of MRSA in Iceland has increased since 1999 but remains low and has been stable in the last years. The search and destroy policy was effective to control MRSA in the health care setting. However, MRSA in Iceland is now shifting into the community, challenging the current Icelandic guidelines, which are tailored to the health care system.
Multilocus Microsatellite Markers for Molecular Typing of
Candida glabrata
: Application to Analysis of Genetic Relationships between Bloodstream and Digestive System Isolates
Authors: Enache-Angoulvant A. , Bourget M. , Brisse S. , Stockman-Pannier C. , Diancourt L. , François N. , Rimek D. , Fairhead C. , Poulain Daniel , Hennequin C.
Published: September 2010

Candida glabrata
has emerged as the second most common etiologic agent, after
Candida albicans
, of superficial and invasive candidiasis in adults. Strain typing is essential for epidemiological investigation, but easy-to-use and reliable typing methods are still lacking. We report the use of a multilocus microsatellite typing method with a set of eight markers on a panel of 180 strains, including 136 blood isolates from hospitalized patients and 34 digestive tract isolates from nonhospitalized patients. A total of 44 different alleles were observed, generating 87 distinct genotypes. In addition to perfect reproducibility, typing ability, and stability, the method had a discriminatory power calculated at 0.97 when all 8 markers were associated, making it suitable for tracing strains. In addition, it is shown that digestive tract isolates differed from blood culture isolates by exhibiting a higher genotypic diversity associated with different allelic frequencies and preferentially did not group in clonal complexes (CCs). The demonstration of the occurrence of microevolution in digestive strains supports the idea that
C. glabrata
can be a persistent commensal of the human gut.
Respiratory Syncytial Virus Induces Host RNA Stress Granules To Facilitate Viral Replication
Authors: Lindquist Michael E. , Lifland Aaron W. , Utley Thomas J. , Santangelo Philip J. , Crowe James E.
Published: September 2010

Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response.
Sphingomyelin Activates Hepatitis C Virus RNA Polymerase in a Genotype-Specific Manner
Authors: Weng Leiyun , Hirata Yuichi , Arai Masaaki , Kohara Michinori , Wakita Takaji , Watashi Koichi , Shimotohno Kunitada , He Ying , Zhong Jin , Toyoda Tetsuya
Published: September 2010

Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.
The HIV-1 Central Polypurine Tract Functions as a Second Line of Defense against APOBEC3G/F
Authors: Hu Chunling , Saenz Dyana T. , Fadel Hind J. , Walker William , Peretz Mary , Poeschla Eric M.
Published: September 2010

HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3′ polypurine tract. This peculiarity of reverse transcription results in a central DNA “flap” structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the central polypurine tract (cPPT) and the central termination sequence (CTS). To avoid an ambiguity of previous studies, we did so without affecting integrase coding. DNA flap formation was disrupted but single-cycle infection was unaffected in all target cells tested, regardless of cell cycle status. Spreading HIV-1 infection was also normal in most T cell lines, and flap mutant viruses replicated equivalently to the wild type in nondividing cells, including macrophages. However, spreading infection of flap mutant HIV-1 was impaired in non-
vif
-permissive cells (HuT78, H9, and primary human peripheral blood mononuclear cells [PBMCs]), suggesting APOBEC3G (A3G) restriction. Single-cycle infections confirmed that
vif
-intact flap mutant HIV-1 is restricted by producer cell A3G/F. Combining the Δ
vif
and cPPT-CTS mutations increased A3G restriction synergistically. Moreover, RNA interference knockdown of A3G in HuT78 cells released the block to flap mutant HIV-1 replication. Flap mutant HIV-1 also accrued markedly increased A3G-mediated G→A hypermutation compared to that of wild-type HIV-1 (a full log
10
in the 0.36 kb downstream of the mutant cPPT). We suggest that the triple-stranded DNA structure, the flap, is not the consequential outcome. The salient functional feature is central plus-strand initiation, which functions as a second line of defense against single-stranded DNA editing by A3 proteins that survive producer cell degradation by Vif.
The Varicella-Zoster Virus ORFS/L (ORF0) Gene Is Required for Efficient Viral Replication and Contains an Element Involved in DNA Cleavage
Authors: Kaufer Benedikt B. , Smejkal Benjamin , Osterrieder Nikolaus
Published: September 2010

The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U
L
) and unique short (U
S
), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U
L
and U
S
. VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U
L
. An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U
L
, directly adjacent to the
a
-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5′ region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5′ terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U
L
, we concluded that our results can provide an explanation for the almost exclusive orientation of the U
L
seen in packaged VZV DNA.
Extracellular 2′-5′ Oligoadenylate Synthetase Stimulates RNase L-Independent Antiviral Activity: a Novel Mechanism of Virus-Induced Innate Immunity
Authors: Kristiansen Helle , Scherer Christina A. , McVean Maralee , Iadonato Shawn P. , Vends Susanne , Thavachelvam Karthiga , Steffensen Thomas B. , Horan Kristy A. , Kuri Thomas , Weber Friedemann , Paludan Søren R. , Hartmann Rune
Published: September 2010

The 2′-5′ oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, bothin vitroandin vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.
Intracellular Distribution of NS1 Correlates with the Infectivity and Interferon Antagonism of an Avian Influenza Virus (H7N1)
Authors: Keiner Bjoern , Maenz Benjamin , Wagner Ralf , Cattoli Giovanni , Capua Ilaria , Klenk Hans-Dieter
Published: September 2010

Highly pathogenic avian influenza viruses of subtype H7N1 that emerged during an outbreak in 1999 and 2000 in Italy differ from their low-pathogenicity precursor viruses by changes in several genes, including three mutations in the NS1 protein. Two of them involve amino acid exchanges located within or closely adjacent to the nuclear export signal of NS1. The third mutation resulted in a new stop codon and thereby a C-terminal truncation of the NS1 protein of the highly pathogenic viruses. To find out whether these mutations contribute to the phenotypic differences between the highly pathogenic and low pathogenic viruses, we generated recombinants of the highly pathogenic A/ostrich/Italy/984/00 strain that contained the nuclear export signal and/or the extended C terminus of NS1 of a low pathogenic virus (A/chicken/Italy/1082/99). Using these recombinants we could demonstrate that replication rate and spread of infection in chicken fibroblast cultures, as well as infectivity for chicken embryos is reduced, whereas the mean death time for chicken embryos is increased, when the highly pathogenic virus acquires the NS1 motifs of the low pathogenic virus. Analysis of beta interferon transcription in chicken fibroblasts infected with the recombinants revealed that the mutations observed in the nuclear export signal of the highly pathogenic viruses were responsible for the enhanced interferon antagonism of these viruses. Cell fractionation and immunofluorescence studies in chicken fibroblasts showed that the nuclear export signal of the highly pathogenic viruses is responsible for cytoplasmic accumulation of NS1, whereas the C-terminal truncation promotes transport into the nucleoli. Comparative analysis in human A549 cells indicated that intracellular distribution of NS1 is host specific. Taken together, these observations support the concept that compartmentalization of NS1 within the cell contributes to the pathogenicity of avian influenza viruses.
Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding
Authors: Chutinimitkul Salin , Herfst Sander , Steel John , Lowen Anice C. , Ye Jianqiang , van Riel Debby , Schrauwen Eefje J. A. , Bestebroer Theo M. , Koel Björn , Burke David F. , Sutherland-Cash Kyle H. , Whittleston Chris S. , Russell Colin A. , Wales David J. , Smith Derek J. , Jonges Marcel , Meijer Adam , Koopmans Marion , Rimmelzwaan Guus F. , Kuiken Thijs , Osterhaus Albert D. M. E. , García-Sastre Adolfo , Perez Daniel R. , Fouchier Ron A. M.
Published: September 2010

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues
in vitro
, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.
Virally Induced Cellular MicroRNA miR-155 Plays a Key Role in B-Cell Immortalization by Epstein-Barr Virus
Authors: Linnstaedt Sarah D. , Gottwein Eva , Skalsky Rebecca L. , Luftig Micah A. , Cullen Bryan R.
Published: September 2010

Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). LCL formation serves as a model for lymphomagenesis, and LCLs are phenotypically similar to EBV-positive diffuse large B-cell lymphomas (DLBCLs), which represent a common AIDS-associated malignancy. B-cell infection by EBV induces the expression of several cellular microRNAs (miRNAs), most notably miR-155, which is overexpressed in many tumors and can induce B-cell lymphomas when overexpressed in animals. Here, we demonstrate that miR-155 is the most highly expressed miRNA in LCLs and that the selective inhibition of miR-155 function specifically inhibits the growth of both LCLs and the DLBCL cell line IBL-1. Cells lacking miR-155 are inefficient in progressing through S phase and spontaneously undergo apoptosis. In contrast, three other B-cell lymphoma lines, including two EBV-positive Burkitt's lymphoma cell lines, grew normally in the absence of miR-155 function. These data identify the induction of cellular miR-155 expression by EBV as critical for the growth of both laboratory-generated LCLs and naturally occurring DLBCLs and suggest that targeted inhibition of miR-155 function could represent a novel approach to the treatment of DLBCL
in vivo
.
Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses
Authors: Liu Wen-Chun , Lin Shih-Chang , Yu Yen-Ling , Chu Ching-Liang , Wu Suh-Chin
Published: September 2010

Since dendritic cells may play a key role in defense against influenza virus infection, we examined the effects of recombinant hemagglutinin (HA) proteins derived from mouse-adapted H1N1 (A/WSN/1933), swine-origin 2009 pandemic H1N1 (A/Texas/05/2009), and highly pathogenic avian influenza H5N1 (A/Thailand/KAN-1/2004) viruses on mouse myeloid dendritic cells (mDCs). The results reveal that tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12) p70, and major histocompatibility complex class II (MHC-II) expression was increased in mDCs after treatment with recombinant HA proteins of H1N1 and H5N1. The specificity of recombinant HA treatments for mDC activation was diminished after proteinase K digestion. HA apparently promotes mDC maturation by enhancing CD40 and CD86 expression and suppressing endocytosis. No significant differences in mDC activation were observed among recombinant proteins of H1N1 and H5N1. The stimulation of mDCs by HA proteins of H1N1 and H5N1 was completely MyD88 dependent. These findings may provide useful information for the development of more-effective influenza vaccines.
High-Throughput, Sensitive Quantification of Repopulating Hematopoietic Stem Cell Clones
Authors: Kim Sanggu , Kim Namshin , Presson Angela P. , An Dong Sung , Mao Si Hua , Bonifacino Aylin C. , Donahue Robert E. , Chow Samson A. , Chen Irvin S. Y.
Published: September 2010

Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5′ and the 3′ vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.
Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions
Authors: Helle François , Vieyres Gabrielle , Elkrief Laure , Popescu Costin-Ioan , Wychowski Czeslaw , Descamps Véronique , Castelain Sandrine , Roingeard Philippe , Duverlie Gilles , Dubuisson Jean
Published: September 2010

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.
Evolution of Functional and Sequence Variants of the Mammalian XPR1 Receptor for Mouse Xenotropic Gammaretroviruses and the Human-Derived Retrovirus XMRV
Authors: Yan Yuhe , Liu Qingping , Wollenberg Kurt , Martin Carrie , Buckler-White Alicia , Kozak Christine A.
Published: September 2010

Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the
Xpr1
receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known
Xpr1
variants in
Mus
and a novel fifth
Xpr1
gene found in
Mus molossinus
and
Mus musculus
. We describe the geographic and species distribution of the
Mus Xpr1
variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent
Xpr1
is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-
Mus
mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive
Xpr1
variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.
Reevaluating Herpes Simplex Virus Hemifusion
Authors: Jackson Julia O. , Longnecker Richard
Published: September 2010

Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.
Andes Virus Regulation of Cellular MicroRNAs Contributes to Hantavirus-Induced Endothelial Cell Permeability
Authors: Pepini Timothy , Gorbunova Elena E. , Gavrilovskaya Irina N. , Mackow Jonathan E. , Mackow Erich R.
Published: September 2010

Hantaviruses infect human endothelial cells (ECs) and cause two diseases marked by vascular permeability defects, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Vascular permeability occurs in the absence of EC lysis, suggesting that hantaviruses alter normal EC fluid barrier functions. ECs infected by pathogenic hantaviruses are hyperresponsive to vascular endothelial growth factor (VEGF), and this alters the fluid barrier function of EC adherens junctions, resulting in enhanced paracellular permeability. Vascular permeability and VEGF-directed responses are determined by EC-specific microRNAs (miRNAs), which regulate cellular mRNA transcriptional responses. miRNAs mature within cytoplasmic processing bodies (P bodies), and the hantavirus nucleocapsid (N) protein binds RNA and localizes to P bodies, suggesting that hantaviruses may modify miRNA functions within infected ECs. Here we assessed changes in EC miRNAs following infection by the HPS-causing Andes hantavirus (ANDV). We analyzed 352 human miRNAs within ANDV-infected ECs using quantitative real-time (RT)-PCR arrays. Fourteen miRNAs, including six miRNAs that are associated with regulating vascular integrity, were upregulated >4-fold following infection by ANDV. Nine miRNAs were downregulated 3- to 3,400-fold following ANDV infection; these included miR-410, involved in regulating secretion, and miR-218, which is linked to the regulation of EC migration and vascular permeability. We further analyzed changes in miR-126, an EC-specific miRNA that regulates vascular integrity by suppressing SPRED1 and PIK3R2 mRNAs. While miR-126 levels were only slightly altered, we found that SPRED1 and PIK3R2 mRNA levels were increased 10- and 7-fold, respectively, in ANDV-infected ECs but were unaltered in ECs infected by the nonpathogenic Tula hantavirus (TULV). Consistent with increased SPRED1 expression, we found that the level of phospho-cofilin was decreased within ANDV-infected ECs. Moreover, small interfering RNA (siRNA) knockdown of SPRED1 dramatically decreased the permeability of ANDV-infected ECs in response to VEGF, suggesting that increased SPRED1 contributes to EC permeability following ANDV infection. These findings suggest that interference with normal miRNA functions contributes to the enhanced paracellular permeability of ANDV-infected ECs and that hantavirus regulation of miRNA functions is an additional determinant of hantavirus pathogenesis.
Vinylferrocene as paradigm for the two-step reaction mode with ketenes. The case of tert-butylcyanoketene (Moore’s ketene)
Authors: Mihai Marina , Pârvulescu Luminitza , Marton Anca , Draghici Constantin , Varga Richard A. , Gheorghiu Mircea D.
Published: September 2010

The reaction of tert-butylcyanoketene (TBCK, Moore’s ketene; 1) with ethenylferrocene (2) yields four products: the cyclobutanone 2-trans-tert-butyl-2-cyano-3-ferrocenylcyclobutanone (3), two δ-lactones as 2:1 ketene:alkene cis and trans adducts 3-cis-tert-butyl-3-cyano-6-(1-cyano-2,2-dimethylpropylidene)-4-ferrocenyltetrahydro-2-pyrone (4) and 3-trans-tert-butyl-3-cyano-6-(1-cyano-2,2-dimethylpropylidene)-4-ferrocenyl-tetrahydro-2-pyrone (5), and an α,β-unsaturated ketone 3-cyano-1E-ferrocenyl-5,5-dimethylhex-1-en-3-one (6). The configuration of the bulky groups (t-Bu and ferrocenyl) in cyclobutanone 3 is trans. The two δ lactones are stereoisomers as a result of the cis and trans configuration of t-Bu and ferrocenyl groups. The cyclobutanone 3 isomerizes by heating or catalytically into the α,β-unsaturated ketone 6. Assigned structures for the compounds were made based on IR, 1H, 13C NMR, and for 5 also by single-crystal X-ray diffraction.
Twenty-year survival of Phellinus (Poria) weirii in Douglas-fir stumps
Authors: Hansen Everett M.
Published: September 2010

Fungus survival was examined in root systems of stumps of trees that were harvested 20 years previously. Roots were excavated with dynamite and hand tools to the outer limit of living P. weirii or until the roots went deeper than 45 cm. Sixty-nine stumps infected before harvest were excavated; P. weirii was still viable in 94%. The fungus was dying back in 68% of the stumps. Some roots as small as 1.3 cm had viable P. weirii, but the margin between living and dead P. weirii occurred at an average root diameter of 12.5 cm.The margin between living and dead P. weirii was often marked by a black zone line. The fungus survived only within roots with intact bark. Ectotrophic mycelium occurred in discontinuous patches on roots of 33% of the stumps.
Pathogenicity of Polyporustomentosus and P. tomentosus var. circinatus on seedlings of 11 conifer species
Authors: Whitney Roy D. , Bohaychuk Wendy P.
Published: September 2010

Polyporustomentosus Fr. caused significantly higher disease ratings than P. tomentosus var. circinatus Fr. in seedlings of 6 of 11 conifer species under laboratory conditions. Both fungi caused highest disease ratings on seedlings of ponderosa pine (Pinusponderosa Laws.), followed by lodgepole pine (Pinuscontorta Dougl. var. latifolia Engelm.), white spruce (Piceaglauca (Moench) Voss), black spruce (Piceamariana (Mill.) B.S.P.), and tamarack (Larixlaricina (Du Roi) K. Koch).
Estimating shrub biomass from basal stem diameters
Authors: Brown James K.
Published: September 2010

For 25 northern Rocky Mountain shrub species, linear regressions are presented for total aboveground weight on basal stem diameter (average r2 of 0.91) and for leaf weight on basal stem diameter (average r2 of 0.74). The percentage of stem-wood weight in diameter classes of 0 to 0.5 cm, 0.5 to 2 cm, and 2 to 5 cm varies with basal stem diameter and species growth characteristics (as shown in figures). For these classes, diameters of stem wood averaged 0.21 cm, 0.76 cm, and 2.37 cm, respectively; however, some species differed significantly from these averages. Coefficients of variation ranged from 60 to 340 for shrub weight per unit area sampled in 12 stands. Some distributions were positively skewed. Implications for sampling are discussed.
Threshold levels for foliar injury to Populustremuloides by sulfur dioxide and ozone
Authors: Karnosky David F.
Published: September 2010

The response of five Populustremuloides Michx. clones to sulfur dioxide (SO2) and ozone (O3) was studied in controlled fumigation chambers in the greenhouse. Threshold levels for foliar injury varied from clone to clone and were in the range of 0.35 to 0.65 ppm SO2 and 0.05 to 0.20 ppm O3 for 3-h exposures. Evidence was found for more-than-additive effects of the two pollutants when they were applied in combination.The most common symptoms for both SO2 and O3 injury were large bifacial chlorotic and necrotic areas which appeared within 20 to 48 h after fumigations. Sulfur dioxide injury rapidly bleached to a light tan color, whereas the O3 injury remained dark until leaf drop. Ozone injury also frequently extented across the veins, whereas SO2 injury usually did not.
Burning for black spruce regeneration on a lowland cutover site in southeastern Manitoba
Authors: Chrosciewicz Z.
Published: September 2010

Two experimental burns were tested on a peaty, very moist, lowland site in southeastern Manitoba to improve seedbeds and black spruce (Piceamariana (Mill.) BSP.) regeneration after cutting. The operations were conducted under different degrees of desiccation in the upper peat materials so that light and moderate burns were obtained. Unmerchantable trees that were left standing at the time of cutting constituted the main seed source on each of the burns. After five growing seasons, black spruce stocking by 0.001-acre (4.047-m2) quadrats was 94% on the moderate burn, 70% on the light burn, and 35% on the control. The numbers of seedlings associated with this stocking were 16 129, 3075, and 1898/acre (39 856, 7598, and 4690/ha), respectively. Various seedbed, regeneration, and plant-succession characteristics indicated beneficial effects of burning, and on this basis, future requirements for its postcut use on the black spruce lowlands are discussed.
Effect of mechanical stress on growth and anatomical structure of red pine: compression stress
Authors: Quirk J. Thomas , Freese Frank
Published: September 2010

Under natural conditions, the tree is stressed statically by the mass of the crown and bole. Pairs of steel rods drilled through two branch whorls were connected by turnbuckles to simulate increasing static load on a given internode. Level of soil moisture was included to discern its influence on the effect of static loading.Compression and moisture stress each reduced radial growth. After computed loading exceeded the proportional limit, the next-formed fibers were greatly expanded radially and, when macerated, exhibited extreme twisting. The fibers produced before the proportional limit was exceeded exhibited compression failures when macerated.
The evolution of permafrost in the taiga and in the forest–tundra, western Quebec–Labrador Peninsula
Authors: Payette Serge , Samson Hélène , Lagarec Daniel
Published: September 2010

Major permafrost landforms of the discontinuous permafrost zone of Hudson Bay and James Bay, Quebec–Labrador Peninsula, are described and interpreted within an ecological perspective. These landforms are not fossil permafrost bodies; they are presently evolving under aggrading and degrading developmental stages. Permafrost peat complexes and permafrost mineral complexes are differentially distributed in the midtaiga, the forest–tundra, and the shrub–tundra zones of the study area. The wooded palsa complex is the most obvious permafrost complex in the midtaiga, and the palsa complex in the forest–tundra: The wooded palsa complex is related to the forest chronosequence, whereas the palsa complex, farther north, occurs where there is a suitable macroclimate for permafrost aggradation without the influence of cool forest microclimate. The wooded cryogenic mound complex in mineral landform is mainly distributed in the southern section of the forest–tundra and near the Hudson Bay shore; the cryogenic mineral mound complex is found in the northern section. The latitudinal shift of the northern complex is necessary for permafrost initiation in open sites. Degrading stages associated with thermokarst activity are similar for all these landforms and it is suggested that palsa complex is made of incipient, mature, and residual palsas whether they belong to palsa mounds or to peat plateaus. Similar conclusions are valid for cryogenic mineral mound complex.
Field evaluation of ecological differentiation of dwarf mistletoe on shore pine and western hemlock
Authors: Smith R. B. , Wass E. F.
Published: September 2010

Hemloclc dwarf mistletoe (Arcenthobiiuntsugense (Rosend.) G. N. Jones) principally infects western hemlock (Tsugaheteropliylla (Raf.) Sarg.), but it is also damaging to shore pine (Pinuscontorta Dougl. ex Loud.) growing in coastal British Columbia. Stands of varied composition were studied in the Home Lake area of Vancouver Island to compare the levels of infection in shore pine and western hemlock. Infection of shore pine occurred whether or not infected hemlock trees were present in the stand. Observations and measurements from these field studies support indications from earlier artificial inoculation trials that two ecological races of A. tsugense exist.
Growth characteristics of Phellinus (Poria) weirii in soil and on root and other surfaces
Authors: Wallis G. W.
Published: September 2010

Growth of Phellinus (Poria) weirii (Murr.) Gilbertson from alder block inoculum into the surrounding soil to infect healthy roots was less than 10 cm. Tree-to-tree spread of this root rot fungus, beyond that which would occur as a result of root contact, may be facilitated by growth of the mycelium over roots of minor vegetation and over wood buried in soil. Ectotrophic mycelium is profuse on the bark of infected roots of Douglas fir (Pseudotsugamenziesii (Mirb.) Franco) and western hemlock (Tsugaheterophylla (Raf.) Sarg.), limited on infected roots of pine (Pinusmonticola Dougl., P. contorta Dougl, P. ponderosa Laws.), and nearly absent on infected roots of western red cedar (Thujaplicata Donn). Rate of spread of P. weirii root rot through stands with a high component of western red cedar or pine, or both, should be considerably slower than in pure stands of Douglas fir.
Naphthenate stimulation of conifer growth over a 3-year period
Authors: Wort D. J. , Kozak A.
Published: September 2010

On May 1, 18, and 30, 1972, 1-year-old seedlings of Douglas fir (Pseudotsugamenziesii (Mirb.) Franco), lodgepole pine (Pinuscontorta Dougl.), Sitka spruce (Piceasitchensis (Bong) (Carr.), western hemlock (Tsugaheterophylla (Raf.) Sarg.), and also Douglas fir planted in plastic bullets were sprayed with a 2000- or 5000-ppm solution of potassium naphthenate (KNap). The growth stage of each species on each of the three dates was classified as (A) buds unopened, (B) terminal bud well open, little or no growth in stem length, or (C) terminal bud well open, lateral buds opening, growth in stem length just beginning. Eighty-six percent of the average cumulative stem-length increments of the groups of plants given the various KNap treatments, determined at the end of each of the 3 years after spray application, were greater than those of their respective control (untreated) plants. Maximum response of all species was to 5000 ppm KNap applied at growth stage C. One, 2, and 3 years after treatment the average cumulative growth in stem length of plants given this treatment exceeded that of controls by 44.5, 25.2, and 15.2%, respectively. The most effective treatment of Douglas fir in bullets was 2000 ppm KNap, also applied at growth stage C. The survival of treated seedlings at the end of the 3rd year did not differ significantly from that of controls. A second application of 5000 ppm KNap on May 18 of the 3rd year, at which date bud growth of the four species was in the early, middle, or late phases of stage B, was without significant effect.