bluuebunny/crossref_metadata_2025 huggingface dataset:
Dog Dealers' Days May Be Numbered
Authors: Grimm David
Published: February 2010

Legislators want to shut down the pipeline of “random source” dogs and cats to laboratories, but some researchers worry about the impact on science.
Cytokines across the Night in Chronic Fatigue Syndrome with and without Fibromyalgia
Authors: Nakamura Toru , Schwander Stephan K. , Donnelly Robert , Ortega Felix , Togo Fumiharu , Broderick Gordon , Yamamoto Yoshiharu , Cherniack Neil S. , Rapoport David , Natelson Benjamin H.
Published: February 2010

The symptoms of chronic fatigue syndrome (CFS) are consistent with cytokine dysregulation. This has led to the hypothesis of immune dysregulation as the cause of this illness. To further test this hypothesis, we did repeated blood sampling for cytokines while patients and matched healthy controls slept in the sleep lab. Because no one method for assaying cytokines is acknowledged to be better than another, we assayed for protein in serum, message in peripheral blood lymphocytes (PBLs), and function in resting and stimulated PBLs. We found no evidence of proinflammatory cytokine upregulation. Instead, in line with some of our earlier studies, we did find some evidence to support a role for an increase in interleukin-10, an anti-inflammatory cytokine. Although the changes were small, they may contribute to the common complaint in CFS patients of disrupted sleep.
Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector
Authors: Pratt William D. , Wang Danher , Nichols Donald K. , Luo Min , Woraratanadharm Jan , Dye John M. , Holman David H. , Dong John Y.
Published: February 2010

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of
Zaire ebolavirus
(ZEBOV) and
Sudan ebolavirus
(SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.
Development and Evaluation of a Magnetic Immunochromatographic Test To Detect
Taenia solium
, Which Causes Taeniasis and Neurocysticercosis in Humans
Authors: Handali Sukwan , Klarman Molly , Gaspard Amanda N. , Dong X. Fan , LaBorde Ronald , Noh John , Lee Yeuk-Mui , Rodriguez Silvia , Gonzalez Armando E. , Garcia Hector H. , Gilman Robert H. , Tsang Victor C. W. , Wilkins Patricia P.
Published: February 2010

Taeniasis/cysticercosis caused by
Taenia solium
is a frequent parasitic infection of the human brain in most of the world. Rapid and simple screening tools to identify taeniasis and cysticercosis cases are needed for control programs, mostly to identify tapeworm carriers which are the source of infection and need to be treated, or as tools for point-of-care case detection or confirmation. These screening assays should be affordable, reliable, rapid, and easy to perform. Immunochromatographic tests meet these criteria. To demonstrate proof of principle, we developed and evaluated two magnetic immunochromatographic tests (MICTs) for detection of human
Taenia solium
taeniasis antibodies (ES33-MICT) and neurocysticercosis antibodies (T24-MICT). These assays detected stage-specific antibodies by using two recombinant proteins, rES33 for detection of taeniasis antibodies and rT24H for detection of cysticercosis antibodies. The sensitivity and specificity of the ES33-MICT to detect taeniasis infections were 94.5% and 96%, respectively, and those of the T24-MICT to detect cases of human cysticercosis with two or more viable brain cysts were 93.9% and 98.9%, respectively. These data provide proof of principle that the ES33- and T24-MICTs provide rapid and suitable methods to identify individuals with taeniasis and cysticercosis.
Generation of Merkel Cell Polyomavirus (MCV)-Like Particles and Their Application to Detection of MCV Antibodies
Authors: Touzé Antoine , Gaitan Julien , Arnold Françoise , Cazal Raphaël , Fleury Maxime J. , Combelas Nicolas , Sizaret Pierre-Yves , Guyetant Serge , Maruani Annabel , Baay Marc , Tognon Mauro , Coursaget Pierre
Published: February 2010

The genome of a new human polyomavirus, known as Merkel cell polyomavirus (MCV), has recently been reported to be integrated within the cellular DNA of Merkel cell carcinoma (MCC), a rare human skin cancer. To investigate MCV seroprevalence in the general population, we expressed three different MCV VP1 in insect cells using recombinant baculoviruses. Viruslike particles (VLPs) were obtained with only one of the three VP1 genes. High-titer antibodies against VP1 VLPs were detected in mice immunized with MCV VLPs, and limited cross-reactivity was observed with BK polyomavirus (BKV) and lymphotropic polyomavirus (LPV). MCV antibodies were detected in 77% of the general population, with no variations according to age.
Laboratory and Clinical Characteristics of
Staphylococcus lugdunensis
Prosthetic Joint Infections
Authors: Shah Neel B. , Osmon Douglas R. , Fadel Hind , Patel Robin , Kohner Peggy C. , Steckelberg James M. , Mabry Tad , Berbari Elie F.
Published: February 2010

Staphylococcus lugdunensis
is a coagulase-negative staphylococcus that has several similarities to
Staphylococcus aureus
.
S. lugdunensis
is increasingly being recognized as a cause of prosthetic joint infection (PJI). The goal of the present retrospective cohort study was to determine the laboratory and clinical characteristics of
S. lugdunensis
PJIs seen at the Mayo Clinic in Rochester, MN, between 1 January 1998 and 31 December 2007. Kaplan-Meier survival methods and Wilcoxon sum-rank analysis were used to determine the cumulative incidence of treatment success and assess subset comparisons. There were 28 episodes of
S
.
lugdunensis
PJIs in 22 patients; half of those patients were females. Twenty-five episodes (89%) involved the prosthetic knee, while 3 (11%) involved the hip. Nine patients (32%) had an underlying urogenital abnormality. Among the 28 isolates in this study tested by agar dilution, 24 of 28 (86%) were oxacillin susceptible. Twenty of the 21 tested isolates (95%) lacked
mecA
, and 6 (27%) of the 22 isolates tested produced β-lactamase. The median durations of parenteral β-lactam therapy and vancomycin therapy were 38 days (range, 23 to 42 days) and 39 days (range, 12 to 60 days), respectively. The cumulative incidences of freedom from treatment failure (standard deviations) at 2 years were 92% (±7%) and 76% (±12%) for episodes treated with a parenteral β-lactam and vancomycin, respectively (
P
= 0.015).
S. lugdunensis
is increasingly being recognized as a cause of PJIs. The majority of the isolates lacked
mecA
. Episodes treated with a parenteral β-lactam antibiotic appear to have a more favorable outcome than those treated with parenteral vancomycin.
Pathogenic Strains ofYersinia enterocoliticaIsolated from Domestic Dogs (Canis familiaris) Belonging to Farmers Are of the Same Subtype as PathogenicY. enterocoliticaStrains Isolated from Humans and May Be a Source of Human Infection in Jiangsu Province, China
Authors: Wang Xin , Cui Zhigang , Wang Hua , Tang Liuying , Yang Jinchuan , Gu Ling , Jin Dong , Luo Longze , Qiu Haiyan , Xiao Yuchun , Xiong Haiping , Kan Biao , Xu Jianguo , Jing Huaiqi
Published: February 2010

We isolated 326Yersinia enterocoliticastrains from 5,919 specimens from patients with diarrhea at outpatient clinics, livestock, poultry, wild animals, insect vectors, food, and the environment in the cities of Nantong and Xuzhou in Jiangsu Province, China, from 2004 to 2008. The results showed that the 12 pathogenic strains were of the O:3 serotype. Six strains were isolated from domestic dogs (Canis familiaris) belonging to farmers and were found to be the primary carriers of pathogenicY. enterocoliticastrains, especially in Xuzhou. Pulsed-field gel electrophoresis analysis of the pathogenic strains from dogs belonging to farmers showed that they shared the same patterns as strains from diarrhea patients isolated in 1994. This indicates that the strains from domestic dogs have a close correlation with the strains causing human infections.
Molecular Characterization and Prophage DNA Contents of
Streptococcus agalactiae
Strains Isolated from Adult Skin and Osteoarticular Infections
Authors: Salloum Mazen , van der Mee-Marquet Nathalie , Domelier Anne-Sophie , Arnault Laurence , Quentin Roland
Published: February 2010

Skin and osteoarticular infections (SKI and OAI, respectively) account for almost one-third of
Streptococcus agalactiae
infections in nonpregnant adults. We evaluated the genetic diversity and phylogeny of 58
S. agalactiae
strains responsible for adult SKI or OAI and of 61
S. agalactiae
strains from cases of adult human colonization (HCol) by serotyping and multilocus sequence typing (MLST). We also assessed the prophage DNA content of the genomes of these strains by a PCR-based method. We found that 63% of SKI and 56% of OAI occurred in people aged 55 years and over. Overall, 71% of SKI strains were of serotype Ia or V, and 91% of OAI strains were of serotype Ia, III, or V. Strains of clonal complexes 1 and 23 (CC1 and CC23) were associated with 79% of SKI cases and 62% of OAI cases. Seven groups of strains, groups A, B, C, D, E, F, and G, were obtained by performing a hierarchical analysis on the basis of prophage DNA-PCR data. We found that 85% of CC1 strains clustered in DNA prophage group D, the group with the highest prophage DNA content (average, 4.4; average of absolute deviations [AVEDEV], 0.9). The CC23 strains displayed the greatest diversity in prophage DNA fragment content, but 47% of CC23 strains clustered in group B, which also had a high average prophage DNA content per strain (average, 2.3; AVEDEV, 0.6). Many (65%) of the OAI strains were in prophage DNA group D, whereas 83% of the SKI strains were in prophage DNA groups B and D. These data suggest that
S. agalactiae
strains from CC1 and CC23 may be subject to particular transduction mechanisms in gene recombination, rendering them particularly capable of invading the skin, bone, or joints in adults.
Detection of HIV-1 Drug Resistance in Women Following Administration of a Single Dose of Nevirapine: Comparison of Plasma RNA to Cellular DNA by Consensus Sequencing and by Oligonucleotide Ligation Assay
Authors: Wagner Thor A. , Kress Catherine M. , Beck Ingrid , Techapornroong Malee , Wittayapraparat Pakorn , Tansuphasawasdikul Somboon , Jourdain Gonzague , Ngo-Giang-Huong Nicole , Lallemant Marc , Frenkel Lisa M.
Published: February 2010

A single dose of nevirapine (sdNVP) to prevent mother-to-child transmission of HIV-1 increases the risk of failure of subsequent NVP-containing antiretroviral therapy (ART), especially when initiated within 6 months of sdNVP administration, emphasizing the importance of understanding the decay of nevirapine-resistant mutants. Nevirapine-resistant HIV-1 genotypes (with the mutations K103N, Y181C, and/or G190A) from 21 women were evaluated 10 days and 6 weeks after sdNVP administration and at the initiation of ART. Resistance was assayed by consensus sequencing and by a more sensitive assay (oligonucleotide ligation assay [OLA]) using plasma-derived HIV-1 RNA and cell-associated HIV-1 DNA. OLA detected nevirapine resistance in more specimens than consensus sequencing did (63% versus 33%,
P
< 0.01). When resistance was detected only by OLA (
n
= 45), the median mutant concentration was 18%, compared to 61% when detected by both sequencing and OLA (
n
= 51) (
P
< 0.0001). The proportion of women whose nevirapine resistance was detected by OLA 10 days after sdNVP administration was higher when we tested their HIV-1 RNA (95%) than when we tested their HIV-1 DNA (88%), whereas at 6 weeks after sdNVP therapy, the proportion was greater with DNA (85%) than with RNA (67%) and remained higher with DNA (33%) than with RNA (11%) at the initiation of antiretroviral treatment (median, 45 weeks after sdNVP therapy). Fourteen women started NVP-ART more than 6 months after sdNVP therapy; resistance was detected by OLA in 14% of the women but only in their DNA. HIV-1 resistance to NVP following sdNVP therapy persists longer in cellular DNA than in plasma RNA, as determined by a sensitive assay using sufficient copies of virus, suggesting that DNA may be superior to RNA for detecting resistance at the initiation of ART.
Rapid and Reliable Single Nucleotide Polymorphism-Based Differentiation ofBrucellaLive Vaccine Strains from Field Strains
Authors: Gopaul Krishna K. , Sells Jessica , Bricker Betsy J. , Crasta Oswald R. , Whatmore Adrian M.
Published: February 2010

The reliable differentiation of liveBrucellavaccine strains from field isolates is an important element in brucellosis control programs. We describe the design, validation, and implementation of a novel single nucleotide polymorphism (SNP)-based typing platform that offers a rapid, reliable, and robust tool to achieve this with improved diagnostic accuracy compared to existing molecular tests. Furthermore, the assays described are designed such that they supplement, and can be run as an intrinsic part of, a previously described assay identifyingBrucellaisolates to the species level (K. K. Gopaul, C. J. Smith, M. S. Koylass, and A. M. Whatmore, BMC Microbiol. 8:86), giving a comprehensive molecular typing platform.
Human Papillomavirus Genotyping Using Archival Vulval Dysplastic or Neoplastic Biopsy Tissues: Comparison between the INNO-LiPA and Linear Array Assays
Authors: Tan Sarah E. , Garland Suzanne M. , Rumbold Alice R. , Tabrizi Sepehr N.
Published: February 2010

The Roche Linear Array (LA) and Innogenetics INNO-LiPA human papillomavirus (HPV) genotyping assays were compared for paraffin-embedded vulval tissues. The LA detected amplifiable DNA in 28 (57%) out of 49 biopsy specimens, 20 (40%) being HPV genotyped, compared to 49 (100%) and 41 (83%), respectively, detected by the INNO-LiPA. The INNO-LiPA provides greater sensitivity for HPV genotyping in archival tissue.
Direct Culture-Independent Strain Typing of
Burkholderia cepacia
Complex in Sputum Samples from Patients with Cystic Fibrosis
Authors: Drevinek Pavel , Vosahlikova Sarka , Dedeckova Klara , Cinek Ondrej , Mahenthiralingam Eshwar
Published: February 2010

We examined if multilocus sequence typing (MLST), a method for genotyping and species identification of
Burkholderia cepacia
complex bacteria, could be applied directly to cystic fibrosis sputum. The redesigned nested-PCR MLST format was successfully used to accurately identify strains in 23 sputum samples, of which 8 were culture negative.
Detection of HIV-1 at between 20 and 49 Copies per Milliliter by the Cobas TaqMan HIV-1 v2.0 Assay Is Associated with Higher Pretherapy Viral Load and Less Time on Antiretroviral Therapy
Authors: Pascual-Pareja José F. , Martínez-Prats Lorena , Luczkowiak Joanna , Fiorante Silvana , Rubio Rafael , Pulido Federico , Otero Joaquín R. , Delgado Rafael
Published: February 2010

New commercial techniques for determination of the viral load (VL) in plasma are able to detect as few as 20 copies of HIV-1 RNA/ml. The relevance of this new technical threshold is uncertain. Upon multivariate analysis, factors associated with detection of VLs between 20 and 49 copies/ml by the Cobas TaqMan HIV-1 v2.0 assay in an HIV clinic were the basal VL and time on antiretroviral therapy.
Novel Duplex Real-Time PCR Assay Detects
Bordetella holmesii
in Specimens from Patients with Pertussis-Like Symptoms in Ontario, Canada
Authors: Guthrie J. L. , Robertson A. V. , Tang P. , Jamieson F. , Drews S. J.
Published: February 2010

Bordetella holmesii
is a human pathogen found mainly in immunocompromised patients. A specific real-time PCR assay was developed and successfully used to identify specimens from which
B. holmesii
was misidentified as
Bordetella pertussis
and to establish the prevalence of
B. holmesii
in Ontario patients with pertussis-like symptoms.
Incidence of
Acinetobacter
Species Other than
A. baumannii
among Clinical Isolates of
Acinetobacter
: Evidence for Emerging Species
Authors: Turton Jane F. , Shah Jayesh , Ozongwu Chika , Pike Rachel
Published: February 2010

Six hundred ninety nonduplicate isolates of
Acinetobacter
species were identified using a combination of detection of
bla
OXA-51-like
and
rpoB
sequence cluster analysis. Although most isolates were identified as
A. baumannii
(78%), significant numbers of other species, particularly
A. lwoffii
/genomic species 9 (8.8%),
A. ursingii
(4%), genomic species 3 (1.7%), and
A. johnsonii
(1.7%), were received, often associated with bacteremias.
Abl Family Tyrosine Kinases Regulate Sialylated Ganglioside Receptors for Polyomavirus
Authors: Swimm Alyson I. , Bornmann William , Jiang Mengxi , Imperiale Michael J. , Lukacher Aron E. , Kalman Daniel
Published: February 2010

Sialylated lipids serve as cellular receptors for polyomaviruses. Using pharmacological inhibitors and cell lines derived from knockout mice, we demonstrate that Abl family tyrosine kinases are required for replication of mouse polyomavirus and BK virus, a human polyomavirus associated with allograft failure following kidney transplantation. We show that decreasing Abl family kinase activity results in low levels of cell surface ganglioside receptors for mouse polyomavirus and that inhibition of sialidase activity promotes virion binding in the absence of Abl family kinase activity. These data provide evidence that Abl family kinases reduce ganglioside turnover in the plasma membrane by inhibiting host cell sialidase activity. Thus, Abl family kinases regulate the susceptibility of cells to polyomavirus infection by modulating gangliosides required for viral attachment.
Cyclosporine Blocks Incorporation of HIV-1 Envelope Glycoprotein into Virions
Authors: Sokolskaja Elena , Olivari Silvia , Zufferey Madeleine , Strambio-De-Castillia Caterina , Pizzato Massimo , Luban Jeremy
Published: February 2010

Cyclosporine (CsA) decreases HIV-1 infectivity by blocking HIV-1 capsid (CA) interaction with target cell cyclophilin A (CypA). Yet, HIV-1 virions produced in the presence of CsA also exhibit decreased infectivity that was previously shown to be independent of the well-characterized HIV-1 CA-CypA interaction. Here, we demonstrate that CsA decreases gp120 and gp41 incorporation into HIV-1 virions and that the fusion of these virions with susceptible target cells is impaired. This effect was not observed with HIV-1 virions pseudotyped with the vesicular stomatitis virus glycoprotein or with the amphotropic envelope protein of murine leukemia virus. It was independent of calcineurin signaling, the endoplasmic reticulum luminal protein cyclophilin B, and the long cytoplasmic tail of gp41. Thus, cyclosporine blocks HIV-1 infectivity via two independent mechanisms, the first involving HIV-1 CA in target cells and the second involving HIV-1 Env in producer cells.
Assembly and Biological and Immunological Properties of Newcastle Disease Virus-Like Particles
Authors: McGinnes Lori W. , Pantua Homer , Laliberte Jason P. , Gravel Kathryn A. , Jain Surbhi , Morrison Trudy G.
Published: February 2010

Virus-like particles (VLPs) released from avian cells expressing the Newcastle disease virus (NDV) strain AV proteins NP, M, HN (hemagglutinin-neuraminidase), and F were characterized. The VLP-associated HN and F glycoproteins directed the attachment of VLPs to cell surfaces and fusion of VLP membranes with red blood cell membranes, indicating that they were assembled into VLPs in an authentic conformation. These particles were quantitatively prepared and used as an immunogen, without adjuvant, in BALB/c mice. The resulting immune responses, detected by enzyme-linked immunosorbent assay (ELISA), virus neutralization, and intracellular cytokine staining, were comparable to the responses to equivalent amounts of inactivated NDV vaccine virus. HN and F proteins from another strain of NDV, strain B1, could be incorporated into these VLPs. Foreign peptides were incorporated into these VLPs when fused to the NP or HN protein. The ectodomain of a foreign glycoprotein, the Nipah virus G protein, fused to the NDV HN protein cytoplasmic and transmembrane domains was incorporated into ND VLPs. Thus, ND VLPs are a potential NDV vaccine candidate. They may also serve as a platform to construct vaccines for other pathogens.
The Length of and Nonhydrophobic Residues in the Transmembrane Domain of Dengue Virus Envelope Protein Are Critical for Its Retention and Assembly in the Endoplasmic Reticulum
Authors: Hsieh Szu-Chia , Tsai Wen-Yang , Wang Wei-Kung
Published: February 2010

The morphogenesis of many enveloped viruses, in which viral nucleocapsid complex interacts with envelope (E) protein, is known to take place at various sites along the secretory pathway. How viral E protein retains in a particular intracellular organelle for assembly remains incompletely understood. In this study, we investigated determinants in the E protein of dengue virus (DENV) for its retention and assembly in the endoplasmic reticulum (ER). A chimeric experiment between CD4 and DENV precursor membrane/E constructs suggested that the transmembrane domain (TMD) of E protein contains an ER retention signal. Substitutions of three nonhydrophobic residues at the N terminus of the first helix (T1) and at either the N or C terminus of the second helix of the TMD with three hydrophobic residues, as well as an increase in the length of T1, led to the release of chimeric CD4 and E protein from the ER, suggesting that short length and certain nonhydrophobic residues of the TMD are critical for ER retention. The analysis of enveloped viruses assembled at the plasma membrane and of those assembled in the Golgi complex and ER revealed a trend of decreasing length and increasing nonhydrophobic residues of the TMD of E proteins. Taken together, these findings support a TMD-dependent sorting for viral E proteins along the secretory pathway. Moreover, similar mutations introduced into the TMD of DENV E protein resulted in the increased production of virus-like particles (VLPs), suggesting that modifications of TMD facilitate VLP production and have implications for utilizing flaviviral VLPs as serodiagnostic antigens and vaccine candidates.
Identification of a Dominant Negative Inhibitor of Human Zinc Finger Antiviral Protein Reveals a Functional Endogenous Pool and Critical Homotypic Interactions
Authors: Law Lok Man J. , Albin Owen R. , Carroll John-William N. , Jones Christopher T. , Rice Charles M. , MacDonald Margaret R.
Published: February 2010

The zinc finger antiviral protein (ZAP) is a host factor with potent antiviral activity when overexpressed in cells. ZAP blocks replication of the prototype alphavirus Sindbis virus (SINV) at a step at or before translation of the incoming viral genome. The mechanism of ZAP anti-SINV activity and the determinants of its antiviral function, however, have not been defined. Here, we have identified a dominant negative inhibitor of human ZAP. Rat ZAP with a cysteine-to-arginine mutation at position 88 (rZAPC88R), previously reported as a nonfunctional form of ZAP, increases SINV growth in cells. These results led us to discover a previously undetectable pool of endogenous functional ZAP within human cells. Investigation of the mechanism of dominant negative inhibition, combined with a comprehensive mutational analysis of the antiviral factor, revealed that homotypic associations are required for ZAP function in limiting SINV propagation.
Novel Inhibitors of Influenza Virus Fusion: Structure-Activity Relationship and Interaction with the Viral Hemagglutinin
Authors: Vanderlinden Evelien , Göktaş Fusun , Cesur Zafer , Froeyen Matheus , Reed Mark L. , Russell Charles J. , Cesur Nesrin , Naesens Lieve
Published: February 2010

A new class of
N
-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 μM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.
Sumoylation of the Epstein-Barr Virus BZLF1 Protein Inhibits Its Transcriptional Activity and Is Regulated by the Virus-Encoded Protein Kinase
Authors: Hagemeier Stacy R. , Dickerson Sarah J. , Meng Qiao , Yu Xianming , Mertz Janet E. , Kenney Shannon C.
Published: February 2010

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) mediates the switch between latent and lytic EBV infection. Z not only activates early lytic viral gene transcription but also plays a direct role in lytic viral genome replication. Although a small fraction of Z is known to be sumoylated, the effects of this posttranslational modification on various different Z functions have not been well defined. In this report, we show that only the lysine at amino acid residue 12 is required for the sumoylation of Z, and that Z can be sumoylated by SUMO isoforms 1, 2, and 3. We also demonstrate that the sumo-defective Z mutants ZK12A and ZK12R have enhanced transcriptional activity. The sumoylated and nonsumoylated forms of Z were found to have a similar cellular location, both being localized primarily within the nuclear matrix. The Z sumo-defective mutants were, however, partially defective for disrupting promyelocytic leukemia (PML) bodies compared to the ability of wild-type Z. In addition, we show that lytic viral genome replication does not require the sumoylation of Z, although a Z mutant altered at both amino acids 12 and 13 is replication defective. Furthermore, we show that the sumoylation of Z is greatly increased (from less than 1 to about 11%) in lytically induced 293 cells infected with an EBV mutant virus deleted for the EBV-encoded protein kinase (EBV-PK) compared to that of 293 cells infected with wild-type EBV, and that the overexpression of EBV-PK leads to the reduced sumoylation of Z in EBV-negative cells. Our results suggest that the sumoylation of Z helps to promote viral latency, and that EBV-PK inhibits Z sumoylation during viral reactivation.
NCoR1 Mediates Papillomavirus E8^E2C Transcriptional Repression
Authors: Powell Maria L. C. , Smith Jennifer A. , Sowa Mathew E. , Harper J. Wade , Iftner Thomas , Stubenrauch Frank , Howley Peter M.
Published: February 2010

The papillomavirus E2 open reading frame encodes the full-length E2 protein as well as an alternatively spliced product called E8^E2C. E8^E2C has been best studied for the high-risk human papillomaviruses, where it has been shown to regulate viral genome levels and, like the full-length E2 protein, to repress transcription from the viral promoter that directs the expression of the viral E6 and E7 oncogenes. The repression function of E8^E2C is dependent on the 12-amino-acid N-terminal sequence from the E8 open reading frame (ORF). In order to understand the mechanism by which E8^E2C mediates transcriptional repression, we performed an unbiased proteomic analysis from which we identified six

h

igh-confidence

c

andidate

i

nteracting

p

roteins (HCIPs) for E8^E2C; the top two are NCoR1 and TBLR1. We established an interaction of E8^E2C with an NCoR1/HDAC3 complex and demonstrated that this interaction requires the wild-type E8 open reading frame. Small interfering RNA (siRNA) knockdown studies demonstrated the involvement of NCoR1/HDAC3 in the E8^E2C-dependent repression of the viral long control region (LCR) promoter. Additional genetic work confirmed that the papillomavirus E2 and E8^E2C proteins repress transcription through distinct mechanisms.
Downregulation of Cdc2/CDK1 Kinase Activity Induces the Synthesis of Noninfectious Human Papillomavirus Type 31b Virions in Organotypic Tissues Exposed to Benzo[
a
]pyrene
Authors: Alam Samina , Bowser Brian S. , Conway Michael J. , Israr Mohd , Ryndock Eric J. , Xi Long Fu , Meyers Craig
Published: February 2010

Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[
a
]pyrene (B
a
P), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since B
a
P is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that B
a
P-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, B
a
P treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16
INK4
and p27
KIP1
, which normally mediate pRb hypophosphorylation. In contrast, p21
WAF1
and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to B
a
P-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. B
a
P targeting of CDK1 occurred independently of HPV status, since B
a
P treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of B
a
P were dependent on B
a
P-mediated alteration in CDK1 kinase activity for maintaining their infectivity.
The Epstein-Barr Virus (EBV)-Encoded Protein Kinase, EBV-PK, but Not the Thymidine Kinase (EBV-TK), Is Required for Ganciclovir and Acyclovir Inhibition of Lytic Viral Production
Authors: Meng Qiao , Hagemeier Stacy R. , Fingeroth Joyce D. , Gershburg Edward , Pagano Joseph S. , Kenney Shannon C.
Published: February 2010

Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC
50
] = 1.5 μM) and ACV (IC
50
= 4.1 μM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC
50
= 19.6 μM) and ACV (IC
50
= 36.4 μM). Expression of the EBV-PK protein in
trans
restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC
50
= 1.2 μM) and ACV (IC
50
= 2.8 μM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.
Antiretroviral Therapy in the Clinic
Authors: Tsibris Athe M. N. , Hirsch Martin S.
Published: February 2010

Antiretroviral therapy in the developed world has resulted in substantial reductions in HIV-associated morbidity and mortality, changing an HIV diagnosis from a likely death sentence into a manageable chronic infection (F. J. Palella, Jr., K. M. Delaney, A. C. Moorman, M. O. Loveless, J. Fuhrer, G. A. Satten, D. J. Aschman, and S. D. Holmberg, N. Engl. J. Med.
338:
853-860, 1998). Several million years of life have been saved by effective anti-HIV treatment, although these successes should not obscure the magnitude of the ongoing worldwide HIV epidemic (R. P. Walensky, A. D. Paltiel, E. Losina, L. M. Mercincavage, B. R. Schackman, P. E. Sax, M. C. Weinstein, and K. A. Freedberg, J. Infect. Dis.
194:
11-19, 2006). Readers of the
Journal of Virology
are doubtless aware of the fundamental advances in retrovirology that have made possible the development of potent inhibitors of HIV replication. In this review, we focus on the issues surrounding how these drugs and drug regimens are actually used in clinical settings. Their proper use requires detailed knowledge of the natural history of HIV infection, the pharmacology of the individual drugs, the complexities of drug-drug interactions, and the use of sophisticated molecular tests for monitoring of viral load, immunologic response, and drug resistance.
Avian Reovirus μNS Protein Forms Homo-Oligomeric Inclusions in a Microtubule-Independent Fashion, Which Involves Specific Regions of Its C-Terminal Domain
Authors: Brandariz-Nuñez Alberto , Menaya-Vargas Rebeca , Benavente Javier , Martinez-Costas Jose
Published: February 2010

Members of the genus
Orthoreovirus
replicate in cytoplasmic inclusions termed viral factories. Compelling evidence suggests that the nonstructural protein μNS forms the matrix of the factories and recruits specific viral proteins to these structures. In the first part of this study, we analyzed the properties of avian reovirus factories and μNS-derived inclusions and found that they are nonaggresome cytoplasmic globular structures not associated with the cytoskeleton which do not require an intact microtubule network for formation and maturation. We next investigated the capacity of avian reovirus μNS to form inclusions in transfected and baculovirus-infected cells. Our results showed that μNS is the main component of the inclusions formed by recombinant baculovirus expression. This, and the fact that μNS is able to self-associate inside the cell, suggests that μNS monomers contain all the interacting domains required for inclusion formation. Examination of the inclusion-forming capacities of truncated μNS versions allowed us to identify the region spanning residues 448 to 635 of μNS as the smallest that was inclusion competent, although residues within the region 140 to 380 seem to be involved in inclusion maturation. Finally, we investigated the roles that four different motifs present in μNS(448-635) play in inclusion formation, and the results suggest that the C-terminal tail domain is a key determinant in dictating the initial orientation of monomer-to-monomer contacts to form basal oligomers that control inclusion shape and inclusion-forming efficiency. Our results contribute to an understanding of the generation of structured protein aggregates that escape the cellular mechanisms of protein recycling.
T Cells Facilitate Recovery from Venezuelan Equine Encephalitis Virus-Induced Encephalomyelitis in the Absence of Antibody
Authors: Brooke Christopher B. , Deming Damon J. , Whitmore Alan C. , White Laura J. , Johnston Robert E.
Published: February 2010

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genusAlphavirusthat is responsible for a significant disease burden in Central and South America through sporadic outbreaks into human and equid populations. For humans, 2 to 4% of cases are associated with encephalitis, and there is an overall case mortality rate of approximately 1%. In mice, replication of the virus within neurons of the central nervous system (CNS) leads to paralyzing, invariably lethal encephalomyelitis. However, mice infected with certain attenuated mutants of the virus are able to control the infection within the CNS and recover. To better define what role T cell responses might be playing in this process, we infected B cell-deficient μMT mice with a VEEV mutant that induces mild, sublethal illness in immune competent mice. Infected μMT mice rapidly developed the clinical signs of severe paralyzing encephalomyelitis but were eventually able to control the infection and recover fully from clinical illness. Recovery in this system was T cell dependent and associated with a dramatic reduction in viral titers within the CNS, followed by viral persistence in the brain. Further comparison of the relative roles of T cell subpopulations within this system revealed that CD4+T cells were better producers of gamma interferon (IFN-γ) than CD8+T cells and were more effective at controlling VEEV within the CNS. Overall, these results suggest that T cells, especially CD4+T cells, can successfully control VEEV infection within the CNS and facilitate recovery from a severe viral encephalomyelitis.
In VivoTropism of Attenuated and Pathogenic Measles Virus Expressing Green Fluorescent Protein in Macaques
Authors: de Vries Rory D. , Lemon Ken , Ludlow Martin , McQuaid Stephen , Yüksel Selma , van Amerongen Geert , Rennick Linda J. , Rima Bert K. , Osterhaus Albert D. M. E. , de Swart Rik L. , Duprex W. Paul
Published: February 2010

The global increase in measles vaccination has resulted in a significant reduction of measles mortality. The standard route of administration for the live-attenuated measles virus (MV) vaccine is subcutaneous injection, although alternative needle-free routes, including aerosol delivery, are under investigation.In vitro, attenuated MV has a much wider tropism than clinical isolates, as it can use both CD46 and CD150 as cellular receptors. To compare thein vivotropism of attenuated and pathogenic MV, we infected cynomolgus macaques with pathogenic or attenuated recombinant MV expressing enhanced green fluorescent protein (GFP) (strains IC323 and Edmonston, respectively) via the intratracheal or aerosol route. Surprisingly, viral loads and cellular tropism in the lungs were similar for the two viruses regardless of the route of administration, and CD11c-positive cells were identified as the major target population. However, only the pathogenic MV caused significant viremia, which resulted in massive virus replication in B and T lymphocytes in lymphoid tissues and viral dissemination to the skin and the submucosa of respiratory epithelia. Attenuated MV was rarely detected in lymphoid tissues, and when it was, only in isolated infected cells. Following aerosol inhalation, attenuated MV was detected at early time points in the upper respiratory tract, suggesting local virus replication. This contrasts with pathogenic MV, which invaded the upper respiratory tract only after the onset of viremia. This study shows that despitein vitrodifferences, attenuated and pathogenic MV show highly similarin vivotropism in the lungs. However, systemic spread of attenuated MV is restricted.
Potent Vesicular Stomatitis Virus-Based Avian Influenza Vaccines Provide Long-Term Sterilizing Immunity against Heterologous Challenge
Authors: Schwartz Jennifer A. , Buonocore Linda , Suguitan Amorsolo L. , Silaghi Alex , Kobasa Darwyn , Kobinger Gary , Feldmann Heinz , Subbarao Kanta , Rose John K.
Published: February 2010

The emergence in 1997 and continuance today of a highly lethal H5N1 avian influenza virus (AIV) causing human disease has raised concern about an impending pandemic and the need for a vaccine to prepare for such an occurrence. We previously generated an efficacious vesicular stomatitis virus (VSV)-based AIV vaccine expressing H5 hemagglutinin (HA) from the fifth genomic position of VSV (J. A. Schwartz et al., Virology 366:166-173, 2007). Here we have generated and characterized VSV-based vaccines that express the A/Hong Kong/156/1997 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provide complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 strains in animals even 1 year after vaccination. Postchallenge pulmonary virus loads show that these vectors provide sterilizing immunity. Therefore, VSV-based AIV vaccines are potent, broadly cross-protective pandemic vaccine candidates.
Structural Insights into the Molecular Mechanisms of
Cauliflower Mosaic Virus
Transmission by Its Insect Vector
Authors: Hoh François , Uzest Marilyne , Drucker Martin , Plisson-Chastang Célia , Bron Patrick , Blanc Stéphane , Dumas Christian
Published: February 2010

Cauliflower mosaic virus
(CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.
Induction of Interferon and Interferon Signaling Pathways by Replication of Defective Interfering Particle RNA in Cells Constitutively Expressing Vesicular Stomatitis Virus Replication Proteins
Authors: Panda Debasis , Dinh Phat X. , Beura Lalit K. , Pattnaik Asit K.
Published: February 2010

We show here that replication of defective interfering (DI) particle RNA in HEK293 cells stably expressing vesicular stomatitis virus (VSV) replication proteins potently activates interferon (IFN) and IFN signaling pathways through upregulation of IFN-β promoter, IFN-stimulated response element (ISRE) promoter, and NF-κB promoter activities. Replication of DI particle RNA, not mere expression of the viral replication proteins, was found to be critical for induction of IFN and IFN signaling. The stable cells supporting replication of DI RNA described in this report will be useful in further examining the innate immune signaling pathways and the host cell functions in viral genome replication.
PB1-F2 Expression by the 2009 Pandemic H1N1 Influenza Virus Has Minimal Impact on Virulence in Animal Models
Authors: Hai Rong , Schmolke Mirco , Varga Zsuzsanna T. , Manicassamy Balaji , Wang Taia T. , Belser Jessica A. , Pearce Melissa B. , García-Sastre Adolfo , Tumpey Terrence M. , Palese Peter
Published: February 2010

Unlike previous pandemic viruses, the 2009 H1N1 pandemic influenza virus does not code for the virulence factor PB1-F2. The genome of the 2009 H1N1 virus contains three stop codons preventing PB1-F2 expression; however, PB1-F2 production could occur following genetic mutation or reassortment. Thus, it is of great interest to understand the impact that expression of the PB1-F2 protein might have in the context of the 2009 pandemic influenza virus, A/California/04/2009 (Cal/09). We have addressed this question by generating two Cal/09 viruses with productive PB1-F2 open reading frames containing either an asparagine at position 66 of PB1-F2 (66N) or a serine at position 66 (66S): this N66S change has previously been shown to be associated with increased virulence in mice. We used these viruses to investigate the effect on virulence conferred by expression of the 66N or the 66S PB1-F2 protein in both
in vitro
and
in vivo
systems. Our results show enhanced replication of the 66S virus in A549 cells, while studies of BALB/c and DBA/2 mice and ferrets revealed no significant differences in symptoms of infection with wild-type Cal/09 versus the 66N or 66S virus variant. Also, coinfection of mice with
Streptococcus pneumoniae
and the different viruses (recombinant wild-type [rWT] Cal/09 and the 66N and 66S viruses) did not result in significant differences in mortality. Mice infected with either PB1-F2-expressing virus did demonstrate altered protein levels of proinflammatory cytokines; differences were observed to be greater in infection caused by the 66S virus. In summary, our study demonstrates that PB1-F2 expression by the Cal/09 virus modulates the immune response to infection while having a minimal effect on virus virulence in two mammalian models.