bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Evolution of Swine Influenza A (H3N2) Viruses in China from 1970 to 2006
Authors: Yu Hai , Hua Rong-Hong , Zhang Qiang , Liu Tian-Qiang , Liu Hui-Li , Li Guo-Xin , Tong Guang-Zhi
Published: January 2008

Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.
Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections
Authors: Soetens Oriane , Vauloup-Fellous Christelle , Foulon Ina , Dubreuil Pascal , De Saeger Ben , Grangeot-Keros Liliane , Naessens Anne
Published: January 2008

Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (
P
= 0.036).
Outbreak Caused by a Multidrug-Resistant
Klebsiella pneumoniae
Clone Carrying
bla
VIM-12
in a University Hospital
Authors: Tokatlidou Despina , Tsivitanidou Maria , Pournaras Spyros , Ikonomidis Alexandros , Tsakris Athanassios , Sofianou Danae
Published: January 2008

From November 2006 to April 2007, nine nonrepetitive isolates of
Klebsiella pneumoniae
with reduced susceptibility or resistance to carbapenems were recovered from clinical specimens from separate patients hospitalized in a tertiary care hospital. The imipenem-EDTA synergy test was positive for all isolates. PCR, sequencing, and transferability experiments revealed the novel
bla
VIM-12
metallo-β-lactamase gene, which was plasmid mediated and located in a class 1 integron. Pulsed-field gel electrophoresis demonstrated a single macrorestriction pattern, indicating the clonal spread of VIM-12-producing
K. pneumoniae
.
Evaluation of Eight Different Bioinformatics Tools To Predict Viral Tropism in Different Human Immunodeficiency Virus Type 1 Subtypes
Authors: Garrido Carolina , Roulet Vanessa , Chueca Natalia , Poveda Eva , Aguilera Antonio , Skrabal Katharina , Zahonero Natalia , Carlos Silvia , García Federico , Faudon Jean Louis , Soriano Vincent , de Mendoza Carmen
Published: January 2008

Human immunodeficiency virus type 1 (HIV-1) tropism can be assessed using phenotypic assays, but this is quite laborious, expensive, and time-consuming and can be made only in sophisticated laboratories. More accessible albeit reliable tools for testing of HIV-1 tropism are needed in view of the prompt introduction of CCR5 antagonists in clinical practice. Bioinformatics tools based on V3 sequences might help to predict HIV-1 tropism; however, most of these methods have been designed by taking only genetic information derived from HIV-1 subtype B into consideration. The aim of this study was to evaluate the performances of several genotypic tools to predict HIV-1 tropism in non-B subtypes, as data on this issue are scarce. Plasma samples were tested using a new phenotypic tropism assay (Phenoscript-tropism; Eurofins), and results were compared with estimates of coreceptor usage using eight different genotypic predictor softwares (Support Vector Machine [SVM], C4.5, C4.5 with positions 8 to 12 only, PART, Charge Rule, geno2pheno coreceptor, Position-Specific Scoring Matrix X4R5 [PSSM
X4R5
], and PSSM
sinsi
). A total of 150 samples were tested, with 115 belonging to patients infected with non-B subtypes and 35 drawn from subtype B-infected patients, which were taken as controls. When non-B subtypes were tested, the concordances between the results obtained using the phenotypic assay and distinct genotypic tools were as follows: 78.8% for SVM, 77.5% for C4.5, 82.5% for C4.5 with positions 8 to 12 only, 82.5% for PART, 82.5% for Charge Rule, 82.5% for PSSM
X4R5
, 83.8% for PSSM
sinsi
, and 71.3% for geno2pheno. When clade B viruses were tested, the best concordances were seen for PSSM
X4R5
(91.4%), PSSM
sinsi
(88.6%), and geno2pheno (88.6%). The sensitivity for detecting X4 variants was lower for non-B than for B viruses, especially in the case of PSSM
sinsi
(38.4% versus 100%, respectively), SVM
wetcat
(46% versus 100%, respectively), and PART (30% versus 90%, respectively). In summary, while inferences of HIV-1 coreceptor usage using genotypic tools seem to be reliable for clade B viruses, their performances are poor for non-B subtypes, in which they particularly fail to detect X4 variants.
Major Uropathogenic
Escherichia coli
Strain Isolated in the Northwest of England Identified by Multilocus Sequence Typing
Authors: Lau Siu Ha , Reddy Suganya , Cheesbrough John , Bolton Frederick J. , Willshaw Geraldine , Cheasty Tom , Fox Andrew J. , Upton Mathew
Published: January 2008

A total of 88 uropathogenic
Escherichia coli
isolates, including 68 isolates from urine and 20 isolates from blood, were characterized by multilocus sequence typing (MLST). MLST has identified an important genetic lineage of
E. coli
, designated sequence type 131 (ST-131), represented by 52 of these isolates, 51 of which were resistant to extended-spectrum cephalosporins. ST-131 appears to be a drug-resistant uropathogenic strain of
E. coli
responsible for causing urinary tract infections and bacteremia and is widely disseminated among both community and hospital patients from different geographical areas in the northwest of England. Application of MLST has helped to define the population biology which may underpin the epidemiology of pathogenic
E. coli
strains. The portability of MLST allows the accurate monitoring of this antibiotic-resistant uropathogenic strain of
E. coli
and will enhance surveillance for this important group of organisms.
Candida nivariensis
, an Emerging Pathogenic Fungus with Multidrug Resistance to Antifungal Agents
Authors: Borman Andrew M. , Petch Rebecca , Linton Christopher J. , Palmer Michael D. , Bridge Paul D. , Johnson Elizabeth M.
Published: January 2008

In 2005,
Candida nivariensis
, a yeast species genetically related to
Candida glabrata
, was described following its isolation from three patients in a single Spanish hospital. Between 2005 and 2006, 16 fungal isolates with phenotypic similarities to
C. nivariensis
were submitted to the United Kingdom Mycology Reference Laboratory for identification. The strains originated from various clinical specimens, including deep, usually sterile sites, from patients at 12 different hospitals in the United Kingdom. PCR amplification and sequencing of the D1D2 and internal transcribed spacer 1 (ITS1) regions of the nuclear ribosomal gene cassette confirmed that these isolates from the United Kingdom are genetically identical to
C. nivariensis
. Biochemically,
C. glabrata
and
C. nivariensis
are distinguished by their differential abilities to assimilate trehalose. However, in contrast to the original published findings, we found that
C. glabrata
isolates, but not
C. nivariensis
isolates, are capable of assimilating this substrate. Antifungal susceptibility tests revealed that
C. nivariensis
isolates are less susceptible than
C. glabrata
isolates to itraconazole, fluconazole, and voriconazole and to have significantly higher flucytosine MICs than
C. glabrata
strains. Finally,
C. nivariensis
could be rapidly distinguished from the other common pathogenic fungus species by pyrosequencing of the ITS2 region. In the light of these data, we believe that
C. nivariensis
should be regarded as a clinically important emerging pathogenic fungus.
Human Papillomavirus Type 16 Variant Analysis of E6, E7, and L1 Genes and Long Control Region in Biopsy Samples from Cervical Cancer Patients in North India
Authors: Pande Shailja , Jain Neeraj , Prusty Bhupesh K. , Bhambhani Suresh , Gupta Sanjay , Sharma Rajyashri , Batra Swaraj , Das Bhudev C.
Published: January 2008

High-risk human papillomaviruses (HPVs), particularly HPV types 16 and 18 (HPV-16 and HPV-18, respectively), play a cardinal role in the etiology of cervical cancer. The most prevalent type, HPV-16, shows intratypic sequence variants that are known to differ in oncogenic potential and geographic distribution. This study was designed to analyze sequence variations in E6, E7, and L1 genes and the LCR (for long control region) of HPV-16 in cervical cancer patients to identify the most prevalent and novel HPV-16 variants and to correlate them with the severity of the disease. Cervical biopsies from 60 HPV-16-positive cancer cases were analyzed by PCR and DNA sequencing. The most frequently observed variations were T350G (100%) in E6, T789C (87.5%) in E7, A6695C (54.5%) in L1, and G7521A (91.1%) in the LCR. In addition, only one novel variant (T527A) in E6 and four new variants each in L1 (A6667C, A6691G, C6906T, and A6924C) and in the LCR (C13T, A7636C, C7678T, and G7799A) were identified. While E7 was found to be highly conserved, the variant 350G of E6 was the most prevalent in all of the histopathological grades. The majority of LCR variants were found at the YY1 transcription factor binding sites. Interestingly, a complete absence of the Asian lineage and a high prevalence of European lineages in E6, E7, L1, and the LCR (85, 86.7, 67.7, and 63.3%, respectively) indicate a possible epidemiological linkage between Europe and India with regard to the dissemination of HPV-16 infections in India.
Homogeneous Amplification of Genital Human Alpha Papillomaviruses by PCR Using Novel Broad-Spectrum GP5+ and GP6+ Primers
Authors: Schmitt Markus , Dondog Bolormaa , Waterboer Tim , Pawlita Michael
Published: January 2008

Human papillomavirus (HPV) DNA detection and typing are important for diagnosis and management of HPV-associated diseases. One of the most commonly used PCR methods, GP5+/6+, shows weaknesses in amplifying certain types. To circumvent this limitation, we developed and validated broad-spectrum primers targeting the GP5+/6+ region. The addition of eight upstream and two downstream BSGP5+/6+ (BS) primers improved amplification of plasmids of 14 genital HPV types 10- to 1,000-fold versus GP5+/6+ PCR without altering sensitivity for the 10 others. For these 24 types, an analytic sensitivity of ≤1,000 plasmid copies in the presence of 100 ng cellular DNA was obtained. Additionally, we integrated an internal β-globin PCR into both HPV PCR systems, allowing simultaneous DNA quality control without affecting the sensitivity of HPV detection. Furthermore, we describe five additional low-risk HPV probes used in multiplex HPV genotyping (MPG) for simultaneous identification of all 15 high-risk, 3 putative high-risk, and 9 low-risk HPV genotypes. The performance of BSGP5+/6+ multiplexed with β-globin primers was compared to that of standard GP5+/6+ with DNA from 1,112 cervical scrapings. There was 79% overall agreement (kappa = 0.816). BSGP5+/6+ was significantly more sensitive than GP5+/6+ for detection of HPV 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, detecting 212 additional HPV infections and increasing the proportion of multiple infections from 17.2 to 26.9% in cancer patients. In conclusion, BSGP5+/6+ multiplexed with β-globin PCR provides an improvement in type-specific amplification sensitivity and homogeneity compared to GP5+/6+ and offers simultaneous internal control of DNA quality. BSGP5+/6+-MPG, therefore, is suitable for epidemiologic and also diagnostic applications.
Comparison of Results Obtained with Amplicor HIV-1 DNA PCR Test Version 1.5 Using 100 versus 500 Microliters of Whole Blood
Authors: Piwowar-Manning Estelle , Lugalia Lebah , Kafufu Bosco , Jackson J. Brooks
Published: January 2008

The Amplicor HIV-1 DNA PCR assay (Roche Diagnostics, Branchburg, NJ) requires 500 μl of whole blood for a diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, and this amount is often difficult to obtain from infants. A comparison was performed using 100 and 500 μl of whole blood from infants less than 18 months of age. The concordance rate for HIV DNA PCR-negative and -positive samples was 100% for the two different volumes.
Kaposi's Sarcoma-Associated Herpesvirus
ori
-
Lyt
-Dependent DNA Replication: Involvement of Host Cellular Factors
Authors: Wang Yan , Li Hong , Tang Qiyi , Maul Gerd G. , Yuan Yan
Published: January 2008

Herpesvirus lytic DNA replication requires both the
cis
-acting element, the origin, and
trans
-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (
ori
-
Lyt
) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with
ori
-
Lyt
by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV
ori
-
Lyt
. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to
ori
-
Lyt
through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on
ori
-
Lyt
. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked
ori
-
Lyt
-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased
ori
-
Lyt
-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.
Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6
Authors: Dugan Gillian E. , Hewitt Eric W.
Published: January 2008

The human cytomegalovirus (HCMV) protein US6 inhibits the transporter associated with antigen processing (TAP). Since TAP transports antigenic peptides into the endoplasmic reticulum for binding to major histocompatibility class I molecules, inhibition of the transporter by HCMV US6 impairs the presentation of viral antigens to cytotoxic T lymphocytes. HCMV US6 inhibits ATP binding by TAP, hence depriving TAP of the energy source it requires for peptide translocation, yet the molecular basis for the interaction between US6 and TAP is poorly understood. In this study we demonstrate that residues 89 to 108 of the HCMV US6 luminal domain are required for TAP inhibition, whereas sequences that flank this region stabilize the binding of the viral protein to TAP. In parallel, we demonstrate that chimpanzee cytomegalovirus (CCMV) US6 binds, but does not inhibit, human TAP. The sequence of CCMV US6 differs from that of HCMV US6 in the region corresponding to residues 89 to 108 of the HCMV protein. The substitution of this region of CCMV US6 with the corresponding residues from HCMV US6 generates a chimeric protein that inhibits human TAP and provides further evidence for the pivotal role of residues 89 to 108 of HCMV US6 in the inhibition of TAP. On the basis of these observations, we propose that there is a hierarchy of interactions between HCMV US6 and TAP, in which residues 89 to 108 of HCMV US6 interact with and inhibit TAP, whereas other parts of the viral protein also bind to TAP and stabilize this inhibitory interaction.
Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination
Authors: Balamurugan Arumugam , Lewis Martha J. , Kitchen Christina M. R. , Robertson Michael N. , Shiver John W. , Daar Eric S. , Pitt Jacqueline , Ali Ayub , Ng Hwee L. , Currier Judith S. , Yang Otto O.
Published: January 2008

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1gagDNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences fromgag,pol, andnefconfirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while thegagDNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.
Influenza A Virus Strains Differ in Sensitivity to the Antiviral Action of Mx-GTPase
Authors: Dittmann Jan , Stertz Silke , Grimm Daniel , Steel John , García-Sastre Adolfo , Haller Otto , Kochs Georg
Published: January 2008

Interferon-mediated host responses are of great importance for controlling influenza A virus infections. It is well established that the interferon-induced Mx proteins possess powerful antiviral activities toward most influenza viruses. Here we analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and human MxA proteins and found remarkable differences. Virus strains of avian origin were highly sensitive to Mx1, whereas strains of human origin showed much weaker responses. Artificial reassortments of the viral components in a minireplicon system identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the recently reconstructed 1918 H1N1 “Spanish flu” virus was much less sensitive than the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a minireplicon system. Importantly, the human 1918 virus-based minireplicon system was almost insensitive to inhibition by human MxA, whereas the avian influenza A virus H5N1-derived system was well controlled by MxA. These findings suggest that Mx proteins provide a formidable hurdle that hinders influenza A viruses of avian origin from crossing the species barrier to humans. They further imply that the observed insensitivity of the 1918 virus-based replicon to the antiviral activity of human MxA is a hitherto unrecognized characteristic of the “Spanish flu” virus that may contribute to the high virulence of this unusual pandemic strain.
Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion
Authors: Howard Megan W. , Travanty Emily A. , Jeffers Scott A. , Smith M. K. , Wennier Sonia T. , Thackray Larissa B. , Holmes Kathryn V.
Published: January 2008

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Δ19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Δ19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Δ19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.
Alteration of B-Cell Subsets Enhances Neuroinvasion in Mouse Scrapie Infection
Authors: von Poser-Klein Christine , Flechsig Eckhard , Hoffmann Tanja , Schwarz Petra , Harms Harry , Bujdoso Raymond , Aguzzi Adriano , Klein Michael A.
Published: January 2008

Acquired forms of prion diseases or transmissible spongiform encephalopathies are believed to occur following peripheral exposure. Prions initially accumulate in the lymphoid system before spreading to the nervous system, but the underlying mechanisms for prion transfer between the two systems are still elusive. Here we show that ablation of the B-cell-specific transmembrane protein CD19, a coreceptor of the complement system, results in an acceleration of prion neuroinvasion. This appears to be due to an alteration of the follicular dendritic cell (FDC) network within the lymphoid tissue, thereby reducing the distance between FDCs and adjacent nerve fibers that mediate prion neuroinvasion.
Selective Downregulation of Rhesus Macaque and Sooty Mangabey Major Histocompatibility Complex Class I Molecules by Nef Alleles of Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 2
Authors: DeGottardi M. Quinn , Specht Anke , Metcalf Benjamin , Kaur Amitinder , Kirchhoff Frank , Evans David T.
Published: January 2008

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates HLA-A and -B molecules, but not HLA-C or -E molecules, based on amino acid differences in their cytoplasmic domains to simultaneously evade cytotoxic T lymphocyte (CTL) and natural killer cell surveillance. Rhesus macaques and sooty mangabeys express orthologues of HLA-A, -B, and -E, but not HLA-C, and many of these molecules have unique amino acid differences in their cytoplasmic tails. We found that these differences also resulted in differential downregulation by primary simian immunodeficiency virus (SIV) SIVsmm/macand HIV-2 Nef alleles. Thus, selective major histocompatibility complex class I downregulation is a conserved mechanism of immune evasion for pathogenic SIV infection of rhesus macaques and nonpathogenic SIV infection of sooty mangabeys.
Targets of Small Interfering RNA Restriction during Human Immunodeficiency Virus Type 1 Replication
Authors: Gao Yong , Lobritz Michael A. , Roth Justin , Abreha Measho , Nelson Kenneth N. , Nankya Immaculate , Moore-Dudley Dawn M. , Abraha Awet , Gerson Stanton L. , Arts Eric J.
Published: January 2008

Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via
env
and
gag
gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5α did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5′ of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3′-to-5′ siRNA amplification and spreading. In contrast, HIV-1 RNA 3′ of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively “naked” cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5α, or other host restriction factors.
Class VIII Myosins Are Required for Plasmodesmatal Localization of a Closterovirus Hsp70 Homolog
Authors: Avisar Dror , Prokhnevsky Alexey I. , Dolja Valerian V.
Published: January 2008

The Hsp70 homolog (Hsp70h) of
Beet yellows virus
(BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant
Nicotiana benthamiana
were sequenced and shown to encode apparent orthologs of the
Arabidopsis thaliana
myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the
Tobacco mosaic virus
movement protein and
Arabidopsis
protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.
Characterization of Hypovirus-Derived Small RNAs Generated in the Chestnut Blight Fungus by an Inducible DCL-2-Dependent Pathway
Authors: Zhang Xuemin , Segers Gert C. , Sun Qihong , Deng Fuyou , Nuss Donald L.
Published: January 2008

The disruption of one of two dicer genes,
dcl-2
, of the chestnut blight fungus
Cryphonectria parasitica
was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene
dcl-1
mutant
C. parasitica
strains but not in hypovirus-infected
dcl-2
mutant and
dcl-1 dcl-2
double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that
C. parasitica
responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in
dcl-2
expression while the expression of
dcl-1
is increased only modestly (2-fold). The expression of
dcl-2
is further increased (∼35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene,
dcl-2
. They also reveal that
dcl-2
expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.
Inhibition of IRF-3 Activation by VP35 Is Critical for the High Level of Virulence of Ebola Virus
Authors: Hartman Amy L. , Bird Brian H. , Towner Jonathan S. , Antoniadou Zoi-Anna , Zaki Sherif R. , Nichol Stuart T.
Published: January 2008

Zaire ebolavirus
causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.
Structural Basis for Potent Cross-Neutralizing Human Monoclonal Antibody Protection against Lethal Human and Zoonotic Severe Acute Respiratory Syndrome Coronavirus Challenge
Authors: Rockx Barry , Corti Davide , Donaldson Eric , Sheahan Timothy , Stadler Konrad , Lanzavecchia Antonio , Baric Ralph
Published: January 2008

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, and detailed phylogenetic and epidemiological analyses have suggested that it originated from animals. The spike (S) glycoprotein has been identified as a major component of protective immunity, and 23 different amino acid changes were noted during the expanding epidemic. Using a panel of SARS-CoV recombinants bearing the S glycoproteins from isolates representing the zoonotic and human early, middle, and late phases of the epidemic, we identified 23 monoclonal antibodies (MAbs) with neutralizing activity against one or multiple SARS-CoV spike variants and determined the presence of at least six distinct neutralizing profiles in the SARS-CoV S glycoprotein. Four of these MAbs showed cross-neutralizing activity against all human and zoonotic S variants in vitro, and at least three of these were mapped in distinct epitopes using escape mutants, structure analyses, and competition assays. These three MAbs (S109.8, S227.14, and S230.15) were tested for use in passive vaccination studies using lethal SARS-CoV challenge models for young and senescent mice with four different homologous and heterologous SARS-CoV S variants. Both S227.14 and S230.15 completely protected young and old mice from weight loss and virus replication in the lungs for all viruses tested, while S109.8 completely protected mice from weight loss and clinical signs in the presence of viral titers. We conclude that a single human MAb can confer broad protection against lethal challenge with multiple zoonotic and human SARS-CoV isolates, and we identify a robust cocktail formulation that targets distinct epitopes and minimizes the likely generation of escape mutants.
Emergence of Polyfunctional CD8+T Cells after Prolonged Suppression of Human Immunodeficiency Virus Replication by Antiretroviral Therapy
Authors: Rehr Manuela , Cahenzli Julia , Haas Anna , Price David A. , Gostick Emma , Huber Milo , Karrer Urs , Oxenius Annette
Published: January 2008

Progressive human immunodeficiency virus type 1 (HIV-1) infection is often associated with high plasma virus load (pVL) and impaired CD8+T-cell function; in contrast, CD8+T cells remain polyfunctional in long-term nonprogressors. However, it is still unclear whether CD8+T-cell dysfunction is the cause or the consequence of high pVLs. Here, we conducted a longitudinal functional and phenotypic analysis of virus-specific CD8+T cells in a cohort of patients with chronic HIV-1 infection. During the initiation and maintenance of successful antiretroviral therapy (ART), we assessed whether the level of pVL was associated with the degree of CD8+T-cell dysfunction. Under viremic conditions, HIV-specific CD8+T cells were dysfunctional with respect to cytokine secretion (gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha), and their phenotype suggested limited potential for proliferation. During ART, cytokine secretion by HIV-specific CD8+T cells was gradually restored, IL-7Rα and CD28 expression increased dramatically, and PD-1 levels declined. Thus, prolonged ART-induced reduction of viral replication and, hence, presumably antigen exposure in vivo, allows a significant functional restoration of CD8+T cells with the appearance of polyfunctional cells. These findings indicate that the level of pVL as a surrogate for antigen load has a dominant influence on the phenotypic and functional profile of virus-specific CD8+T cells.
Mitochondrial and Secretory Human Cytomegalovirus UL37 Proteins Traffic into Mitochondrion-Associated Membranes of Human Cells
Authors: Bozidis Petros , Williamson Chad D. , Colberg-Poley Anamaris M.
Published: January 2008

The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent antiapoptotic protein, which prevents cytochrome
c
release from mitochondria. The UL37x1 NH
2
-terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase 1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using subcellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria, and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37
NH2
and gpUL37
COOH
were both detected in the ER and MAM fraction, even though only pUL37
NH2
is preferentially imported into mitochondria but gpUL37
COOH
is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH
2
-terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation.
Rift Valley Fever Virus Lacking the NSs and NSm Genes Is Highly Attenuated, Confers Protective Immunity from Virulent Virus Challenge, and Allows for Differential Identification of Infected and Vaccinated Animals
Authors: Bird Brian H. , Albariño César G. , Hartman Amy L. , Erickson Bobbie Rae , Ksiazek Thomas G. , Nichol Stuart T.
Published: January 2008

Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping “abortion storms” and high mortality among young animals. Human infection results in self-limiting febrile disease that in ∼1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 × 104PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, ∼1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas.
Rice Yellow Mottle Virus
, an RNA Plant Virus, Evolves as Rapidly as Most RNA Animal Viruses
Authors: Fargette D. , Pinel A. , Rakotomalala M. , Sangu E. , Traoré O. , Sérémé D. , Sorho F. , Issaka S. , Hébrard E. , Séré Y. , Kanyeka Z. , Konaté G.
Published: January 2008

The rate of evolution of an RNA plant virus has never been estimated using temporally spaced sequence data, by contrast to the information available on an increasing range of animal viruses. Accordingly, the evolution rate of
Rice yellow mottle virus
(RYMV) was calculated from sequences of the coat protein gene of isolates collected from rice over a 40-year period in different parts of Africa. The evolution rate of RYMV was estimated by pairwise distance linear regression on five phylogeographically defined groups comprising a total of 135 isolates. It was further assessed from 253 isolates collected all over Africa by Bayesian coalescent methods under strict and relaxed molecular clock models and under constant size and skyline population genetic models. Consistent estimates of the evolution rate between 4 × 10
−4
and 8 × 10
−4
nucleotides (nt)/site/year were obtained whatever method and model were applied. The synonymous evolution rate was between 8 × 10
−4
and 11 × 10
−4
nt/site/year. The overall and synonymous evolution rates of RYMV were within the range of the rates of 50 RNA animal viruses, below the average but above the distribution median. Experimentally, in host change studies, substitutions accumulated at an even higher rate. The results show that an RNA plant virus such as RYMV evolves as rapidly as most RNA animal viruses. Knowledge of the molecular clock of plant viruses provides methods for testing a wide range of biological hypotheses.
Superior performance in WAIS-R block design among top-level rugby players: Table 1
Authors: Kasahara S , Mashiko H , Niwa S-I
Published: January 2008

Objective and design:

Following a report that a well-known soccer player achieved a perfect score on the Block Design subtest of WAIS-R, WAIS-R was conducted on 31 skilled rugby players.

Participants:

Twenty-seven of the 31 players were recruited for further analyses.

Results:

In 14 of 27 players, Block Design scores were the highest of the 11 subtests. In addition to Block Design scores that exceeded the scores for the other subtests, the players’ Block Design scores were also better than scores for the standard sample.

Conclusions:

The Block Design is related to spatial cognitive ability. These results showed that highly skilled players of field and ball games possess high spatial cognitive abilities.
Analysis of heart rate variability at rest and during aerobic exercise: a study in healthy people and cardiac patients
Authors: de la Cruz Torres B , López C López , Orellana J Naranjo
Published: January 2008

Objective:

To analyse HRV at rest in healthy people and in patients with acute myocardial infarction (AMI) and how it changes during aerobic exercise.

Methods:

The heartbeat signal was recorded beat to beat for 15 minutes at rest and 15 minutes while pedalling in 10 healthy and active men (H group) and 10 cardiac patients (C group). The statistical parameters in the time domain were calculated as well as the spectral analysis applying the Fast Fourier Transform (FFT) and Poincare’s graphic analysis (PGA).

Results:

At rest, H group have an average SDRR (standard deviation of RR intervals) of 71.24 msec, a pRR50 (percentage of differences higher than 50 msec in RR intervals) of 9.97% and a PGA called “comet-type”. The C group have a SDRR of 36.69 msec, a pRR50 of 1.69%, and a PGA “torpedo-type”. These data show a low or moderate risk for healthy people and a high risk for patients. The FFT analysis lies in the very-low-frequency (VLF) zone in both groups.

During exercise, H group shows a significant decrease in all parameters; the PGA turns to “torpedo-type” and the FFT remains in the VLF zone. However, C group is characterised by the maintenance of pRR50, no change in PGA and a second peak in FFT in the high-frequency zone.

Conclusion:

The HRV at rest and during aerobic exercise follows a different pattern in healthy people and in patients and it provides further information about performance during exercise.
EDITORIAL
Authors: GIORGINI BRUNO , BAZZANI ARMANDO , RAMBALDI SANDRO
Published: January 2008

In this preface we propose a synthetic survey of the main ideas which are in our aim at the origin of the "Physics and the City" Symposium (Bologna–Italy, 15–17 December 2005). These ideas are developed from different point of view in the following collected papers, also with different scientific languages, from the sociological or planning to the mathematical or physical ones. Very schematically we think that the present proceedings could prefigure the emergence of a new "science of the city", really multidisciplinary and with possible spin-off on every single science concerned. Moreover with a large range of possible applications to the actual social life, in order to contribute to a better quality of life. Surely we are at the beginning and the topology of this "science of the city" it is not still well defined, but as H. Poincaré said: Pour obtenir un résultat qui ait une valeur réelle, il ne suffit pas de mettre les choses en ordre; ce n'est pas seulement l'ordre, c'est l'ordre inattendu qui vaut quelque chose.
SPECIFICATION OF THE SOCIAL FORCE PEDESTRIAN MODEL BY EVOLUTIONARY ADJUSTMENT TO VIDEO TRACKING DATA
Authors: JOHANSSON ANDERS , HELBING DIRK , SHUKLA PRADYUMN K.
Published: January 2008

Based on suitable video recordings of interactive pedestrian motion and improved tracking software, we apply an evolutionary optimization algorithm to determine optimal parameter specifications for the social force model. The calibrated model is then used for large-scale pedestrian simulations of evacuation scenarios, pilgrimage, and urban environments.
SUPPORTING URBAN PLANNING WITH CAGE: A SOFTWARE ENVIRONMENT TO SIMULATE COMPLEX SYSTEMS
Authors: CECCHINI ARNALDO , TRUNFIO GIUSEPPE A.
Published: January 2008

The planning process is a multiactor, multilevel process, and the techniques we need for it must be suitable for all the protagonists involved. Moreover, the difficulty in dealing with the complexity of urban systems and the related difficulty of analyzing and forecasting are twofold: one kind of difficulty lies in the complexity of the system itself, and the other is due to the actions of actors, which are "acts of freedom." Correspondingly, the process of urban planning requires a set of techniques and models that have proved to be of great potential for management of communication, participation, consensus-building and system' simulation. In this paper, we describe the peculiarity of the articulated set of tools that should be used to support planning, allowing the setting up and management of processes of participation and communication tailored to the needs of specific projects — a set of friendly tools that make the relationship between technicians, clients and users effective and efficacious. Following this discussion we present a very flexible software environment based on cellular automata (Cellular Automata General Environment — CAGE) that can be used to simulate planning decisions and can be coupled with other modules dealing with the "social side" of complexity.