bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of
Anaplasma marginale
and
A. phagocytophilum
in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay
Authors: Reinbold James B. , Coetzee Johann F. , Sirigireddy Kamesh R. , Ganta Roman R. 
Published: May 2010

ABSTRACT

Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity among closely related
Anaplasma
species has made the accurate determination of infection status problematic. A method for the development of simplex and duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) assays for the detection of
A. marginale
and
A. phagocytophilum
16S rRNA in plasma-free bovine peripheral blood samples is described. The duplex assay was able to detect as few as 100 copies of 16S rRNA of both
A. marginale
and
A. phagocytophilum
in the same reaction. The ratio of 16S rRNA to 16S DNA copies for
A.
marginale was determined to be 117.9:1 (95% confidence interval [95% CI], 100.7:1, 135.2:1). Therefore, the detection limit is the minimum infective unit of one
A. marginale
bacterium. The duplex assay detected nonequivalent molar ratios as high as 100-fold. Additionally, the duplex assay and a competitive enzyme-linked immunosorbent assay (cELISA) were used to screen 237 samples collected from herds in which anaplasmosis was endemic. When the cELISA was evaluated by the results of the qRT-PCR, its sensitivity and specificity for the detection of
A. marginale
infection were found to be 65.2% (95% CI, 55.3%, 75.1%) and 97.3% (95% CI, 94.7%, 99.9%), respectively.
A. phagocytophilum
infection was not detected in the samples analyzed. One- and two-way receiver operator characteristic curves were constructed in order to recommend the optimum negative cutoff value for the cELISA. Percentages of inhibition of 20 and 15.3% were recommended for the one- and two-way curves, respectively. In conclusion, the duplex real-time qRT-PCR assay is a highly sensitive and specific diagnostic tool for the accurate and precise detection of
A. marginale
and
A. phagocytophilum
infections in cattle.
Prevalence of
Haemophilus influenzae
Type b Genetic Islands among Clinical and Commensal
H. influenzae
and
H. haemolyticus
Isolates
Authors: Sandstedt Sara A. , Marrs Carl F. , Patel Mayuri , Hirasawa Hitomi , Zhang Lixin , Davis Gregg S. , Gilsdorf Janet R. 
Published: May 2010

ABSTRACT

Five genetic islands (HiGI) found in
Haemophilus influenzae
type b strain Eagan were used as hybridization probes on type b,
Haemophilus haemolyticus
, and nontypeable
H. influenzae
(NTHi) isolates. HiGI2 and HiGI7 were significantly more prevalent in NTHi isolates from children with otitis media than in those from the throats of healthy children.
Disseminated Infection with a New Genovar of
Encephalitozoon cuniculi
in a Renal Transplant Recipient
Authors: Talabani Hana , Sarfati Claudine , Pillebout Evangeline , van Gool Tom , Derouin Francis , Menotti Jean 
Published: May 2010

ABSTRACT

Disseminated microsporidiosis is a life-threatening opportunistic infection. Here, we report about a previously undescribed genovar of
Encephalitozoon cuniculi
causing disseminated infection in a non-HIV-infected renal transplant recipient. Disseminated microsporidiosis must be considered in the differential diagnosis of chronic fever in renal allograft recipients, even those without urinary symptoms.
Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase
Authors: Wolfe Leslie S. , Stanley Bradford J. , Liu Chang , Eliason William K. , Xiong Yong 
Published: May 2010

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly
in vitro
using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.
APOBEC3G Contributes to HIV-1 Variation through Sublethal Mutagenesis
Authors: Sadler Holly A. , Stenglein Mark D. , Harris Reuben S. , Mansky Louis M. 
Published: May 2010

ABSTRACT
The mammalian APOBEC3 proteins are an important component of the cellular innate immune response to retroviral infection. APOBEC3G can extinguish HIV-1 infectivity by its incorporation into virus particles and subsequent cytosine deaminase activity that attacks the nascent viral cDNA during reverse transcription, causing lethal mutagenesis. It has been suggested, but not formally shown, that APOBEC3G can also induce sublethal mutagenesis, which would maintain virus infectivity and contribute to HIV-1 variation. To test this, we developed a novel model system utilizing an HIV-1 vector and a panel of APOBEC3G-expressing cells. We observed proviruses with single APOBEC3G-mediated mutations (in the presence or absence of Vif), occurring at distinct hot spots and which could be rescued and shown to have infectivity. These data indicate that APOBEC3G-dependent restriction of HIV-1 can result in viable viral progeny that harbor sublethal levels of G-to-A mutations. Such mutations have the potential to contribute significantly to HIV-1 evolution, pathogenesis, immune escape, and drug resistance.
Splicing Diversity of the Human
OCLN
Gene and Its Biological Significance for Hepatitis C Virus Entry
Authors: Kohaar Indu , Ploss Alexander , Korol Evgenia , Mu Kathy , Schoggins John W. , O'Brien Thomas R. , Rice Charles M. , Prokunina-Olsson Ludmila 
Published: May 2010

ABSTRACT

Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of
OCLN
in normal human liver and observed variable expression of alternative splice variants, including two known forms (
WT-OCLN
and
OCLN-ex4del
) and six novel forms (
OCLN-ex7ext
,
OCLN-ex3pdel
,
OCLN-ex3del
,
OCLN-ex3
-
4del
,
OCLN-ex3p-9pdel
, and
OCLN-ex3p-7pdel
). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in
in vitro
infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of
OCLN
splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of
OCLN
might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of
OCLN
expression and susceptibility to HCV infection remain to be elucidated.
Genetically Engineered, Biarsenically Labeled Influenza Virus Allows Visualization of Viral NS1 Protein in Living Cells
Authors: Li Yang , Lu Xinya , Li Junwei , Bérubé Nathalie , Giest Kerri-Lane , Liu Qiang , Anderson Deborah H. , Zhou Yan 
Published: May 2010

ABSTRACT
Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.
Establishment of an H6N2 Influenza Virus Lineage in Domestic Ducks in Southern China
Authors: Huang K. , Bahl J. , Fan X. H. , Vijaykrishna D. , Cheung C. L. , Webby R. J. , Webster R. G. , Chen H. , Smith Gavin J. D. , Peiris J. S. M. , Guan Y. 
Published: May 2010

ABSTRACT
Multiple reassortment events between different subtypes of endemic avian influenza viruses have increased the genomic diversity of influenza viruses circulating in poultry in southern China. Gene exchange from the natural gene pool to poultry has contributed to this increase in genetic diversity. However, the role of domestic ducks as an interface between the natural gene pool and terrestrial poultry in the influenza virus ecosystem has not been fully characterized. Here we phylogenetically and antigenically analyzed 170 H6 viruses isolated from domestic ducks from 2000 to 2005 in southern China, which contains the largest population of domestic ducks in the world. Three distinct hemagglutinin lineages were identified. Group I contained the majority of isolates with a single internal gene complex and was endemic in domestic ducks in Guangdong from the late 1990s onward. Group II was derived from reassortment events in which the surface genes of group I viruses were replaced with novel H6 and N2 genes. Group III represented H6 viruses that undergo frequent reassortment with multiple virus subtypes from the natural gene pool. Surprisingly, H6 viruses endemic in domestic ducks and terrestrial poultry seldom reassort, but gene exchanges between viruses from domestic ducks and migratory ducks occurred throughout the surveillance period. These findings suggest that domestic ducks in southern China mediate the interaction of viruses between different gene pools and facilitate the generation of novel influenza virus variants circulating in poultry.
OrfA Downregulates Feline Immunodeficiency Virus Primary Receptor CD134 on the Host Cell Surface and Is Important in Viral Infection
Authors: Hong Yang , Fink Elizabeth , Hu Qiong-Ying , Kiosses William B. , Elder John H. 
Published: May 2010

ABSTRACT
Feline immunodeficiency virus (FIV) OrfA is an accessory protein that is critical for productive viral replication and infection in T cells. Here, we show that OrfA acts to markedly reduce cell surface expression of the FIV primary binding receptor. Downregulation does not occur at the transcriptional or translational level in that the amounts of CD134 mRNA and protein in total cell lysates are not altered between parental 104-C1 T cells and the same cell line stably expressing OrfA (104-C1-OrfA). Analysis by confocal microscopy revealed significant accumulation of CD134 in the Golgi apparatus of 104-C1 cells expressing OrfA. OrfA does not cause a generalized disruption of membrane trafficking in that surface expression of CD9 is unaffected by OrfA overexpression. Consistent with the above observations, OrfA-negative FIV-34TF10 productively infects CrFK (CD134-negative) and 104-C1-OrfA (CD134 downregulated by OrfA) cells but fails to productively infect either 104-C1 (CD134-positive) cells or GFox (CrFK cells overexpressing CD134) cells. FIV-34TF10 in which the OrfA reading frame is open (OrfArep) productively infects CrFK, GFox, 104-C1, and 104-C1-OrfA cells. We hypothesize that reduced surface expression of the receptor, a hallmark of retrovirus infections, may facilitate an increase in virus release from the infected cell by minimizing receptor interactions with budding virus particles.
Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients
Authors: Song Ruijiang , Franco David , Kao Chia-Ying , Yu Faye , Huang Yaoxing , Ho David D. 
Published: May 2010

ABSTRACT

Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection
in vitro
by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.
A Limited Number of Simian Immunodeficiency Virus (SIV)
env
Variants Are Transmitted to Rhesus Macaques Vaginally Inoculated with SIVmac251
Authors: Stone Mars , Keele Brandon F. , Ma Zhong-Min , Bailes Elizabeth , Dutra Joseph , Hahn Beatrice H. , Shaw George M. , Miller Christopher J. 
Published: May 2010

ABSTRACT

Single-genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection. Use of this strategy as a means for identifying transmitted viruses suggested that intrarectal simian immunodeficiency virus (SIV) inoculation of macaques recapitulates key features of human rectal infection. However, no studies have used the SGA strategy to identify vaginally transmitted virus(es) in macaques or to determine how early SIV diversification in vaginally infected animals compares with HIV-1 in humans. We used SGA to amplify 227 partial
env
sequences from a SIVmac251 challenge stock and from seven rhesus macaques at the earliest plasma viral RNA-positive time point after low- and high-dose intravaginal inoculation. Sequences were analyzed phylogenetically to determine the relationship of transmitted/founder viruses within and between each animal and the challenge stock. In each animal, discrete low-diversity
env
sequence lineages were evident, and these coalesced phylogenetically to identical or near-identical
env
sequences in the challenge stock, thus confirming the validity of the SGA sequencing and modeling strategy for identifying vaginally transmitted SIV. Between 1 and 10 viruses were responsible for systemic infection, similar to humans infected by sexual contact, and the set of viruses transmitted to the seven animals studied represented the full genetic constellation of the challenge stock. These findings recapitulate many of the features of sexual HIV-1 transmission in women. Furthermore, the SIV rhesus macaque model can be used to understand the factors that influence the transmission of single versus multiple SIV variants.
Bat Guano Virome: Predominance of Dietary Viruses from Insects and Plants plus Novel Mammalian Viruses
Authors: Li Linlin , Victoria Joseph G. , Wang Chunlin , Jones Morris , Fellers Gary M. , Kunz Thomas H. , Delwart Eric 
Published: May 2010

ABSTRACT

Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families
Dicistroviridae
,
Iflaviridae
,
Tetraviridae
, and
Nodaviridae
and the subfamily
Densovirinae
. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families
Luteoviridae
,
Secoviridae
,
Tymoviridae
, and
Partitiviridae
and the genus
Sobemovirus
. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families
Parvoviridae
,
Circoviridae
,
Picornaviridae
,
Adenoviridae
,
Poxviridae
,
Astroviridae
, and
Coronaviridae
. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.
Neutralization Efficiency Is Greatly Enhanced by Bivalent Binding of an Antibody to Epitopes in the V4 Region and the Membrane-Proximal External Region within One Trimer of Human Immunodeficiency Virus Type 1 Glycoproteins
Authors: Wang Pengcheng , Yang Xinzhen 
Published: May 2010

ABSTRACT

Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC
50
) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer.
Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells
Authors: Gorbunova Elena , Gavrilovskaya Irina N. , Mackow Erich R. 
Published: May 2010

ABSTRACTHantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.