bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection of Polymorphic Peptides from
GRA6
and
GRA7
Sequences of
Toxoplasma gondii
Strains To Be Used in Serotyping
Authors: Sousa Susana , Ajzenberg Daniel , Marle Maggy , Aubert Dominique , Villena Isabelle , da Costa José Correia , Dardé Marie-Laure
Published: June 2009

The evaluation of
Toxoplasma gondii
isolates obtained from geographical environments other than Europe and North America revealed the existence of atypical strains that are not included in the three archetypal clonal lineages (lineages I, II, and III).
GRA6
and
GRA7
are polymorphic genes that have been used for the genotyping of
Toxoplasma
. The coding regions of
GRA6
and
GRA7
from 49 nonarchetypal strains were sequenced and compared with the sequences of type I, II, and III reference strains. Eighteen and 10 different amino acid sequences were found for
GRA6
and
GRA7
, respectively. The polymorphisms found between the different sequences were analyzed, with the objective of defining peptides to be used for the serotyping of
Toxoplasma
infections. Two peptides specific for clonal lineages I and III (peptides GRA7I and GRA7III, respectively) were selected from the
GRA7
locus. Three peptides specific for some atypical strains (peptides Am6, Af6, and Am7) were selected from both the
GRA6
and the
GRA7
loci. Serum samples from humans infected with
Toxoplasma
strains of known genotypes were serotyped with the selected peptides. Peptide GRA7III seems to be a good candidate for the serotyping of infections caused by type III strains. Peptide GRA7I had a very low sensitivity. Peptides Am6 and Af6 had low specificities, since they reacted with serum samples from patients infected with strains belonging to the three archetypal lineages. Although peptide Am7 was specific, it had low sensitivity.
Development of an Artificial-Antigen-Presenting-Cell-Based Assay for the Detection of Low-Frequency Virus-Specific CD8+T Cells in Whole Blood, with Application for Measles Virus
Authors: Ndhlovu Zaza M. , Angenendt Monika , Heckel Diana , Schneck Jonathan P. , Griffin Diane E. , Oelke Mathias
Published: June 2009

Evaluation of the immune responses induced by childhood vaccines requires measurement of T-cell, as well as antibody, responses. However, cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. We developed a novel assay for quantifying virus-specific CD8+T cells that combines the use of HLA-A2 immunoglobulin-based artificial antigen-presenting cells (aAPCs) for stimulation of antigen-specific CD8+T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-γ) mRNA. This assay was optimized using a well-established cytomegalovirus (CMV) CD8+T-cell system. The aAPC-qRT-PCR assay had comparable sensitivity to intracellular cytokine staining (ICS) in detecting CMV-specific CD8+T cells with a detection limit of less than 0.004%. The assay was applied to the detection of low-frequency measles virus (MV)-specific CD8+T cells by stimulating blood from five MV-immune HLA-A*0201 donors with four different MV-specific peptides (MV peptide aAPCs). Stimulation with three of the MV peptide aAPCs resulted in significant increases in IFN-γ mRNA ranging from 3.3- to 13.5-fold. Our results show that the aAPC-qRT-PCR assay is highly sensitive and specific and can be standardized for screening MV-specific CD8+T cells in vaccine trials. The technology should be transferable to analysis of CD8+T-cell responses to other antigens.
Nosocomial Outbreak of a Non-Cefepime-Susceptible Ceftazidime-Susceptible
Pseudomonas aeruginosa
Strain Overexpressing MexXY-OprM and Producing an Integron-Borne PSE-1 ß-Lactamase
Authors: Peña C. , Suarez C. , Tubau F. , Juan C. , Moya B. , Dominguez M. A. , Oliver A. , Pujol M. , Ariza J.
Published: June 2009

Cefepime (FEP) and ceftazidime (CAZ) are broad-spectrum cephalosporins that display similar MICs for wild-type
Pseudomonas aeruginosa
strains. Recently,
P. aeruginosa
isolates showing a discordance in susceptibility to CAZ and FEP have been noted at the Hospital de Bellvitge in Barcelona, Spain, and a clustering was suspected. During the study period (March to December 2007), 51 patients, particularly those in an intensive care units (ICUs) (
n
= 29 [57%]), infected or colonized with at least one
P. aeruginosa
non-FEP-susceptible and CAZ-susceptible (Fep
ns
Caz
s
) phenotype strain were detected. Twenty-three (45%) patients were infected, and the respiratory tract was the most frequent site of infection. Changes in the consumption of antimicrobials in the ICUs were observed over time: a progressive reduction in the levels of consumption of carbapenems (247 defined daily doses [DDD]/1,000 patient days to 66 DDD/1,000 patient days;
P
= 0.008), after restriction of its use in 2006, and an expected increase in the rate of piperacillin-tazobactam use (42 DDD/1,000 patient days in 2004 to 200 DDD/1,000 patient days in 2007;
P
< 0.001). Throughout the whole study period, only a single clone of a
P. aeruginosa
Fep
ns
Caz
s
phenotype strain was identified by pulsed-field gel electrophoresis analysis to be associated with the hyperexpression of MexXY-OprM and the production of an integron-borne PSE-1 ß-lactamase. In conclusion, we identified an epidemic
P. aeruginosa
clone of an Fep
ns
Caz
s
phenotype strain involving 51 patients, in particular, ICU patients. The combination of the overexpression of an efflux pump and PSE-1 ß-lactamase production is associated with the multidrug-resistant phenotype. The dominant use of a single class of antibiotics could have provided the selective pressure required for the emergence and spread of this
P. aeruginosa
strain.
Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1
Authors: Zhan Sien , Li Jinming , Xu Ruihuan , Wang Lunan , Zhang Kuo , Zhang Rui
Published: June 2009

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5′ terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4°C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA.
Molecular Subtyping of
Treponema pallidum
subsp.
pallidum
in Lisbon, Portugal
Authors: Castro R. , Prieto E. , Águas M. J. , Manata M. J. , Botas J. , Martins Pereira F.
Published: June 2009

The objectives of this study were to evaluate the reproducibility of a molecular method for the subtyping of
Treponema pallidum
subsp.
pallidum
and to discriminate strains of this microorganism from strains from patients with syphilis. We studied 212 specimens from a total of 82 patients with different stages of syphilis (14 primary, 7 secondary and 61 latent syphilis). The specimens were distributed as follows: genital ulcers (
n
= 9), skin and mucosal lesions (
n
= 7), blood (
n
= 82), plasma (
n
= 82), and ear lobe scrapings (
n
= 32). The samples were assayed by a PCR technique to amplify a segment of the polymerase gene I (
polA
). Positive samples were typed on the basis of the analysis of two variable genes,
tpr
and
arp
. Sixty-two of the 90 samples positive for
polA
yielded typeable
Treponema pallidum
DNA. All skin lesions in which
T. pallidum
was identified (six of six [100%]) were found to contain enough DNA for typing of the organism. It was also possible to type DNA from 7/9 (77.7%) genital ulcer samples, 13/22 (59.1%) blood samples, 20/32 (62.5%) plasma samples, and 16/21 (76.2%) ear lobe scrapings. The same subtype was identified in all samples from the same patient. Five molecular subtypes (subtypes 10a, 14a, 14c, 14f, and 14g) were identified, with the most frequently found subtype being subtype 14a and the least frequently found subtype being subtype 10a. In conclusion, the subtyping technique used in this study seems to have good reproducibility. To our knowledge, subtype 10a was identified for the first time. Further studies are needed to explain the presence of this subtype in Portugal, namely, its relationship to the
Treponema pallidum
strains circulating in the African countries where Portuguese is spoken.
Emergence of KPC-Producing
Pseudomonas aeruginosa
in Trinidad and Tobago
Authors: Akpaka Patrick E. , Swanston William H. , Ihemere Hyginus N. , Correa Adriana , Torres Julian A. , Tafur Jose D. , Montealegre Maria Camila , Quinn John P. , Villegas Maria Virginia
Published: June 2009

In 2006, the first isolate of KPC-2-producing
Pseudomonas aeruginosa
in the world was identified in Colombia. Recently, similar strains have been reported in Puerto Rico. We now report KPC-2-producing
P. aeruginosa
in Trinidad and Tobago. Surveillance for similar strains is warranted, considering their wide geographic spread and known association with mobile genetic elements.
Assessing Human Immunodeficiency Virus Type 1 Tropism: Comparison of Assays Using Replication-Competent Virus versus Plasma-Derived Pseudotyped Virions
Authors: Hosoya Noriaki , Su Zhaohui , Wilkin Timothy , Gulick Roy M. , Flexner Charles , Hughes Michael D. , Skolnik Paul R. , Giguel Françoise , Greaves Wayne L. , Coakley Eoin , Kuritzkes Daniel R.
Published: June 2009

Detection of CXCR4-using human immunodeficiency virus by the Trofile assay was compared to that by assays using virus isolates or replication-competent recombinants. Concordance with the Trofile assay was good, but assays using replicating viruses did not increase substantially the ability to detect the presence of CXCR4-using virus.
Expanded Multilocus Sequence Typing for
Burkholderia
Species
Authors: Spilker Theodore , Baldwin Adam , Bumford Amy , Dowson Chris G. , Mahenthiralingam Eshwar , LiPuma John J.
Published: June 2009

PCR primers targeting loci in the current
Burkholderia cepacia
complex multilocus sequence typing scheme were redesigned to (i) more reliably amplify these loci from
B
.
cepacia
complex species, (ii) amplify these same loci from additional
Burkholderia
species, and (iii) enable the use of a single primer set per locus for both amplification and DNA sequencing.
Community-Associated Methicillin-Resistant
Staphylococcus aureus
Isolated in Finland in 2004 to 2006
Authors: Kanerva Mari , Salmenlinna Saara , Vuopio-Varkila Jaana , Lehtinen Pirkko , Möttönen Teemu , Virtanen Mikko J. , Lyytikäinen Outi
Published: June 2009

A nationwide population-based study on community-associated methicillin-resistant
Staphylococcus aureus
(CA-MRSA) in Finland during 2004 to 2006 showed that both incidence (1.9/100,000 population) and strain variation increased in comparison to years 1997 to 1999. There were 7 community-associated epidemic and 25 sporadic MRSA strain types. Half of these had Panton-Valentine leukocidin genes.
Development of a Multiplex Real-Time PCR for Detection and Differentiation of Malignant Catarrhal Fever Viruses in Clinical Samples
Authors: Cunha Cristina W. , Otto Lisa , Taus Naomi S. , Knowles Donald P. , Li Hong
Published: June 2009

A multiplex real-time PCR was developed using a single pair of primers and fluorescent probes specific for five malignant catarrhal fever viruses and an internal positive control. The assay was able to simultaneously detect and differentiate the viruses in clinical samples with high sensitivity (97.2%) and specificity (100%).
Genetic Diversity among Food-Borne and Waterborne Norovirus Strains Causing Outbreaks in Sweden
Authors: Lysén Maria , Thorhagen Margareta , Brytting Maria , Hjertqvist Marika , Andersson Yvonne , Hedlund Kjell-Olof
Published: June 2009

A total of 101 food-borne and waterborne outbreaks that were caused by norovirus and that resulted in more than 4,100 cases of illness were reported to the Swedish Institute for Infectious Disease Control from January 2002 to December 2006. Sequence and epidemiological data for isolates from 73 outbreaks were analyzed. In contrast to health care-related outbreaks, no clear seasonality could be observed. Sequence analysis showed a high degree of genetic variation among the noroviruses detected. Genogroup II (GII) viruses were detected in 70% of the outbreaks, and of those strains, strains of GII.4 were the most prevalent and were detected in 25% of all outbreaks. The GII.4 variants detected in global outbreaks in health care settings during 2002, 2004, and 2006 were also found in the food-borne outbreaks. GI strains totally dominated as the cause of water-related (drinking and recreational water) outbreaks and were found in 12 of 13 outbreaks. In 14 outbreaks, there were discrepancies among the polymerase and capsid genotype results. In four outbreaks, the polymerase of the recombinant GII.b virus occurred together with the GII.1 or GII.3 capsids, while the GII.7 polymerase occurred together with the GII.6 and GII.7 capsids. Mixed infections were observed in six outbreaks; four of these were due to contaminated water, and two were due to imported frozen berries. Contaminated food and water serve as important reservoirs for noroviruses. The high degree of genetic diversity found among norovirus strains causing food-borne and waterborne infections stresses the importance of the use of broad reaction detection methods when such outbreaks are investigated.
Prevalence, Distribution, and Antifungal Susceptibility Profiles of
Candida parapsilosis
,
C. orthopsilosis
, and
C. metapsilosis
in a Tertiary Care Hospital
Authors: Silva Ana P. , Miranda Isabel M. , Lisboa Carmen , Pina-Vaz Cidália , Rodrigues Acácio G.
Published: June 2009

Candida parapsilosis
, an emergent agent of nosocomial infections, was previously made up of a complex of three genetically distinct groups (groups I, II, and III). Recently, the
C. parapsilosis
groups have been renamed as distinct species:
C. parapsilosis
sensu stricto,
C. orthopsilosis
, and
C. metapsilosis
. In Portugal, no data pertaining to the distribution and antifungal susceptibility of these
Candida
species are yet available. In the present report, we describe the incidence and distribution of
C. parapsilosis
sensu stricto,
C. orthopsilosis
, and
C. metapsilosis
among 175 clinical and environmental isolates previously identified by conventional methods as
C. parapsilosis
. We also evaluated the in vitro susceptibilities of the isolates to fluconazole, voriconazole, posaconazole, amphotericin B, and two echinocandins, caspofungin and anidulafungin. Of the 175 isolates tested, 160 (91.4%) were identified as
C. parapsilosis
sensu stricto, 4 (2.3%) were identified as
C. orthopsilosis
, and 5 (2.9%) were identified as
C. metapsilosis
. Six isolates corresponded to species other than the
C. parapsilosis
group. Interestingly, all isolates from blood cultures corresponded to
C. parapsilosis
sensu stricto. Evaluation of the antifungal susceptibility profile showed that only nine (5.6%)
C. parapsilosis
sensu stricto strains were susceptible-dose dependent or resistant to fluconazole, and a single strain displayed a multiazole-resistant phenotype; two (1.3%)
C. parapsilosis
sensu stricto strains were amphotericin B resistant. All
C. orthopsilosis
and
C. metapsilosis
isolates were susceptible to azoles and amphotericin B. A high number of strains were nonsusceptible to the echinocandins (caspofungin and anidulafungin).
Performance Assessment of the GenoType MTBDR
plus
Test and DNA Sequencing in Detection of Multidrug-Resistant
Mycobacterium tuberculosis
Authors: Huang Wei-Lun , Chen Huang-Yau , Kuo Yuh-Min , Jou Ruwen
Published: June 2009

To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDR
plus
test and DNA sequencing, were assessed for the rapid detection of drug-resistant
Mycobacterium tuberculosis
for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the
rpoB
,
katG
,
inhA
regulatory region,
inhA
, and
oxyR-ahpC
genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible
M. tuberculosis
isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDR
plus
test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDR
plus
test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDR
plus
test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDR
plus
test has the advantage of a short turnaround time for drug-resistant
M. tuberculosis
detection. Overall, the two assays performed equally well in detecting RIF resistance (
P
= 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (
P
< 0.001) and MDR tuberculosis (
P
< 0.001). We suggest that new alleles of INH resistance genes should be evaluated to improve the sensitivity of the GenoType MTBDR
plus
test, especially for different geographic areas with genetically diverse
M. tuberculosis
strains.
Characterization of Atypical Isolates of
Yersinia intermedia
and Definition of Two New Biotypes
Authors: Martin Liliane , Leclercq Alexandre , Savin Cyril , Carniel Elisabeth
Published: June 2009

The species
Yersinia intermedia
is a member of the genus
Yersinia
which belongs to the
Enterobacteriaceae
family. This species is divided into eight biotypes, according to Brenner's biotyping scheme. This scheme relies on five tests (utilization of Simmons citrate and acid production from
d
-melibiose,
d
-raffinose, α-methyl-
d
-glucoside [αMG], and
l
-rhamnose). The collection of the French
Yersinia
Reference Laboratory (Institut Pasteur, Paris, France) contained 44 strains that were originally identified as
Y. intermedia
but whose characteristics did not fit into the biotyping scheme. These 44 strains were separated into two biochemical groups: variant 1 (positive for acid production from
l
-rhamnose and αMG and positive for Simmons citrate utlization) and variant 2 (positive for acid production from
l
-rhamnose and αMG). These atypical strains could correspond to new biotypes of
Y. intermedia
, to
Y. frederiksenii
strains having the atypical property of fermenting αMG, or to new
Yersinia
species. These strains did not exhibit growth or phenotypic properties different from those of
Y. intermedia
and
Y. frederiksenii
and did not harbor any of the virulence traits usually found in pathogenic species. DNA-DNA hybridizations performed between one strain each of variants 1 and 2 and the
Y. intermedia
and
Y. frederiksenii
type strains demonstrated that these variants do belong to the
Y. intermedia
species. We thus propose that Brenner's biotyping scheme be updated by adding two new biotypes: 9 (for variant 1) and 10 (for variant 2) to the species
Y. intermedia
.
Investigation of the Mechanism by Which Herpes Simplex Virus Type 1 LAT Sequences Modulate Preferential Establishment of Latent Infection in Mouse Trigeminal Ganglia
Authors: Imai Yumi , Apakupakul Kathleen , Krause Philip R. , Halford William P. , Margolis Todd P.
Published: June 2009

We previously demonstrated that herpes simplex virus type 1 (HSV-1) preferentially establishes latent infection in monoclonal antibody (MAb) A5-positive ganglionic neurons and that a 2.8-kb portion of the HSV-1 genome, corresponding to the 5′ end of the LAT (latency-associated transcript) coding region, is responsible for this phenotype (38, 65). In the current study we carried out further genetic mapping of this latency phenotype and investigated some of the mechanisms that might be responsible. Studies with the chimeric virus HSV-1 17syn+/LAT2, an HSV-1 virus engineered to express HSV-2 LAT, demonstrated that this virus exhibited an HSV-2 latency phenotype, preferentially establishing latency in MAb KH10-positive neurons. This result is complementary to that previously described for the chimeric virus HSV-2 333/LAT1 and indicate that the HSV-1 latency phenotype can be changed to that of HSV-2 by substitution of a 2.8-kb piece of complementary viral DNA. Sequential studies in which we evaluated the pattern of HSV-1 latent infection of the mouse trigeminal ganglion following ocular inoculation with viruses with deletions of functional thymidine kinase, glycoprotein E, ICP0, and US9 protein demonstrate that preferential establishment of HSV-1 latent infection in A5-positive neurons is not a consequence of (i) differential access of HSV-1 to A5-positive neurons,(ii) differential cell-to-cell spread of HSV-1 to A5-positive neurons, (iii) differential “round-trip” spread of HSV-1 to A5-positive neurons, or (iv) expression of ICP0. Additional mapping studies with the HSV-1 LAT deletion viruses
d
LAT371, 17ΔSty, and 17Δ348 indicate that most of the LAT 5′ exon is not required for HSV-1 to preferentially establish latent infection in A5-positive neurons.
Intrathecal Humoral Responses Are Inversely Associated with the Frequency of Simian Immunodeficiency Virus Macrophage-Tropic Variants in the Central Nervous System
Authors: Ryzhova Elena , Aye Pyone , Harvey Tom , Cao Wei , Lackner Andrew , González-Scarano Francisco
Published: June 2009

Sustained simian immunodeficiency virus (SIV) infection of the central nervous system (CNS) depends on macrophage-tropic (M-tropic) strains that are often easily neutralizable. The CNS is often thought of as an immunologically privileged site that fosters replication of M-tropic quasispecies. Yet, there are limited data addressing the intrathecal antibody response or the role of the humoral response, in general, to control M-tropic strains. We investigated the temporal course of the intrathecal fusion inhibitory activity against an M-tropic viral variant and found an inverse relationship between the magnitude of this neutralization and the prevalence of M-tropic populations. These studies suggest a role for the humoral response in the suppression of M-tropic viral species in the CNS in experimental SIV infection.
Analysis of the Percentage of Human Immunodeficiency Virus Type 1 Sequences That Are Hypermutated and Markers of Disease Progression in a Longitudinal Cohort, Including One Individual with a Partially Defective Vif
Authors: Piantadosi Anne , Humes Daryl , Chohan Bhavna , McClelland R. Scott , Overbaugh Julie
Published: June 2009

Hypermutation, the introduction of excessive G-to-A substitutions by host proteins in the APOBEC family, can impair replication of the human immunodeficiency virus (HIV). Because hypermutation represents a potential antiviral strategy, it is important to determine whether greater hypermutation is associated with slower disease progression in natural infection. We examined the level of HIV-1 hypermutation among 28 antiretroviral-naive Kenyan women at two times during infection. By examining single-copy
gag
sequences from proviral DNA, hypermutation was detected in 16 of 28 individuals. Among individuals with any hypermutation, a median of 15% of
gag
sequences were hypermutated (range, 5 to 43%). However, there was no association between the level of
gag
hypermutation and the viral load or CD4 count. Thus, we observed no overall relationship between hypermutation and markers of disease progression among individuals with low to moderate levels of hypermutation. In addition, one individual sustained a typical viral load despite having a high level of hypermutation. This individual had 43% of
gag
sequences hypermutated and harbored a partially defective Vif, which was found to permit hypermutation in a peripheral blood mononuclear cell culture. Overall, our results suggest that a potential antiviral therapy based on hypermutation may need to achieve a substantially higher level of hypermutation than is naturally seen in most individuals to impair virus replication and subsequent disease progression.
The Nucleoprotein of Lymphocytic Choriomeningitis Virus Facilitates Spread of Persistent Infection through Stabilization of the Keratin Network
Authors: Labudova Martina , Tomaskova Jana , Skultety Ludovit , Pastorek Jaromir , Pastorekova Silvia
Published: June 2009

Lymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus containing a bisegmented single-stranded RNA genome with an ambisense coding strategy. MX is a noncytolytic LCMV strain with an in vitro host range restricted to only few cell lines. MX LCMV spreads via cell-cell contacts and causes persistent infection with high production of viral nucleoprotein (NP). Using a proteomic approach, we identified keratin 1 (K1), an intermediate filament network component, as a binding partner of the viral NP. The functional significance of this interaction has been examined by chemical disruption of the keratin network, resulting in a reduced spread of MX LCMV in HeLa cells. However, K1 disassembly was considerably lower in MX LCMV-infected cells than in noninfected counterparts, indicating that NP can stabilize the keratin network and thereby support the integrity of cytoskeleton. The presence of NP also resulted in increased formation of desmosomes and stronger cell-cell adhesion. Similar effects were observed in HeLa cells persistently infected with LCMV strain Armstrong. Our findings suggest that the keratin network is important for the intercellular transmission of persistent LCMV infection in epithelial cells and show that the virus can actively facilitate its own intercellular spread through the interaction between the viral NP and K1 and stimulation of cell-cell contacts.
Cardif-Mediated Signaling Controls the Initial Innate Response to Dengue Virus In Vivo
Authors: Perry Stuart T. , Prestwood Tyler R. , Lada Steven M. , Benedict Chris A. , Shresta Sujan
Published: June 2009

The role of Cardif-dependent signaling in controlling dengue virus (DENV) infection and regulating type I interferon (IFN) production in vivo was examined in Cardif-deficient mice. DENV RNA levels were significantly elevated in both the serum and lymphoid tissues of Cardif
−/−
mice at early times compared to those in wild-type animals. Type I IFN production was delayed in these locales of Cardif
−/−
mice until 18 h postinfection, indicating that Cardif regulates the initial type I IFN response in lymphoid tissues. In contrast, DENV viral loads in nonlymphoid tissues were similar between Cardif
−/−
and wild-type mice. These results reveal that RNA helicase-mediated sensing acts as a first line of innate defense against DENV infection in vivo and functions in a tissue-dependent manner.
Human Papillomavirus Type 16 Infection of Human Keratinocytes Requires Clathrin and Caveolin-1 and Is Brefeldin A Sensitive
Authors: Laniosz Valerie , Dabydeen Sarah A. , Havens Mallory A. , Meneses Patricio I.
Published: June 2009

Human papillomavirus type 16 (HPV16) has been identified as being the most common etiological agent leading to cervical cancer. Despite having a clear understanding of the role of HPV16 in oncogenesis, details of how HPV16 traffics during infection are poorly understood. HPV16 has been determined to enter via clathrin-mediated endocytosis, but the subsequent steps of HPV16 infection remain unclear. There is emerging evidence that several viruses take advantage of cross talk between routes of endocytosis. Specifically, JCV and bovine papillomavirus type 1 have been shown to enter cells by clathrin-dependent endocytosis and then require caveolin-1-mediated trafficking for infection. In this paper, we show that HPV16 is dependent on caveolin-1 after clathrin-mediated endocytosis. We provide evidence for the first time that HPV16 infection is dependent on trafficking to the endoplasmic reticulum (ER). This novel trafficking may explain the requirement for the caveolar pathway in HPV16 infection because clathrin-mediated endocytosis typically does not lead to the ER. Our data indicate that the infectious route for HPV16 following clathrin-mediated entry is caveolin-1 and COPI dependent. An understanding of the steps involved in HPV16 sorting and trafficking opens up the possibility of developing novel approaches to interfere with HPV16 infection and reduce the burden of papillomavirus diseases including cervical cancer.
Suppression of Human Immunodeficiency Virus Type 1 (HIV-1) Viremia with Reverse Transcriptase and Integrase Inhibitors, CD4
+
T-Cell Recovery, and Viral Rebound upon Interruption of Therapy in a New Model for HIV Treatment in the Humanized Rag2
−/−
γ
c
−/−
Mouse
Authors: Choudhary Shailesh K. , Rezk Naser L. , Ince William L. , Cheema Manzoor , Zhang Liguo , Su Lishan , Swanstrom Ronald , Kashuba Angela D. M. , Margolis David M.
Published: June 2009

A small animal model that reproduces human immunodeficiency virus type 1 (HIV-1) pathogenesis may allow modeling of new therapeutic strategies in ways not approachable in mononuclear cell culture. We find that, as in humans, combination antiretroviral therapy (ART) in humanized (hu-) Rag2
−/−
γ
c
−/−
mice allows suppression of viremia below the limits of detection and recovery of CD4
+
cells, while interruption of ART results in viral rebound and renewed loss of CD4
+
T cells. Failure of ART in infected mice is associated with the appearance of drug resistance mutations. The hu-Rag2
−/−
γ
c
−/−
mouse may therefore facilitate testing of novel approaches to HIV replication and persistence.
The M10 Locus of Murine Gammaherpesvirus 68 Contributes to both the Lytic and the Latent Phases of Infection
Authors: Flach B. , Steer B. , Thakur N. N. , Haas J. , Adler H.
Published: June 2009

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model to study the pathogenesis of gammaherpesvirus (γHV) infections. According to the colinear organization of the γHV genomes, the M10 locus is situated at a position equivalent to the K12 locus of KSHV, which codes for proteins of the kaposin family. The M10 locus of MHV-68 has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) with unknown function. In addition, the M10 locus contains a lytic origin of replication (oriLyt). To elucidate the function of the M10 locus during lytic and latent infections, we investigated, both in vitro and in vivo, the following four recombinant viruses which were generated using MHV-68 cloned as a bacterial artificial chromosome: (i) a mutant virus with a deletion which affects both the coding region for M10a-c and the oriLyt; (ii) a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type; (iii) a virus with an ectopic insertion of the oriLyt, which restores the function of the oriLyt but not the M10a-c coding region; and (iv) a mutant virus with a deletion in the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced. In contrast, both the revertant virus and the virus with the ectopic oriLyt insertion grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype. We conclude that the oriLyt in the M10 locus plays an important role during infection of mice with MHV-68.
Paramyxovirus Ultrastructure and Genome Packaging: Cryo-Electron Tomography of Sendai Virus
Authors: Loney Colin , Mottet-Osman Geneviève , Roux Laurent , Bhella David
Published: June 2009

Members of the
Paramyxoviridae
such as measles, mumps, and parainfluenza viruses have pleomorphic, enveloped virions that contain negative-sense unsegmented RNA genomes. This is encapsidated by multiple copies of a viral nucleocapsid protein N to form a helical ribonucleoprotein complex (termed the nucleocapsid), which acts as the template for both transcription and replication. Structure analysis of these viruses has proven challenging, owing to disordered regions in important constituent proteins, conformational flexibility in the nucleocapsid and the pleomorphic nature of virus particles. We conducted a low-resolution ultrastructural analysis of Sendai virus, a prototype paramyxovirus, using cryo-electron tomography. Virions are highly variable in size, ranging approximately from 110 to 540 nm in diameter. Envelope glycoproteins are densely packed on the virion surface, while nucleocapsids are clearly resolved in the virion interior. Subtomogram segmentation and filament tracing allowed us to define the path of many nucleocapsids and in some cases to determine the number of putative genomes within a single virus particle. Our findings indicate that these viruses may contain between one and six copies of their genome per virion and that there is no discernible order to nucleocapsid packaging.
Virion-Incorporated Glycoprotein B Mediates Transneuronal Spread of Pseudorabies Virus
Authors: Curanovic Dusica , Enquist Lynn W.
Published: June 2009

Transneuronal spread of pseudorabies virus (PRV) is a multistep process that requires several virally encoded proteins. Previous studies have shown that PRV glycoprotein B (gB), a component of the viral fusion machinery, is required for the transmission of infection to postsynaptic, second-order neurons. We sought to identify the gB-mediated step in viral transmission. We determined that gB is not required for the sorting of virions into axons of infected neurons, anterograde transport, or the release of virions from the axon.
trans
or
cis
expression of gB on the cell surface was not sufficient for transneuronal spread of the virus; instead, efficient incorporation of gB into virions was required. Additionally, neuron-to-cell spread of PRV most likely does not proceed through syncytial connections. We conclude that, upon gB-independent release of virions at the site of neuron-cell contacts, the virion-incorporated gB/gH/gL fusion complex mediates entry into the axonally contacted cell by fusion of the closely apposed membranes.
Unusual Molecular Architecture of the Machupo Virus Attachment Glycoprotein
Authors: Bowden Thomas A. , Crispin Max , Graham Stephen C. , Harvey David J. , Grimes Jonathan M. , Jones E. Yvonne , Stuart David I.
Published: June 2009

New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel α/β fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.
A Complicated Message: Identification of a Novel PB1-Related Protein Translated from Influenza A Virus Segment 2 mRNA
Authors: Wise Helen M. , Foeglein Agnes , Sun Jiechao , Dalton Rosa Maria , Patel Sheetal , Howard Wendy , Anderson Emma C. , Barclay Wendy S. , Digard Paul
Published: June 2009

Influenza A virus segment 2 is known to encode two polypeptides in overlapping open reading frames: PB1, the polymerase, and PB1-F2, a proapoptotic virulence factor. We show that a third major polypeptide is synthesized from PB1 mRNA via differential AUG codon usage. PB1 codon 40 directs translation of an N-terminally truncated version of the polypeptide (N40) that lacks transcriptase function but nevertheless interacts with PB2 and the polymerase complex in the cellular environment. Importantly, the expression of N40, PB1-F2, and PB1 are interdependent, and certain mutations previously used to ablate PB1-F2 production affected N40 accumulation. Removal of the PB1-F2 AUG upregulated N40 synthesis, while truncating PB1-F2 after codon 8 (with a concomitant M40I change in PB1) abolished N40 expression. A virus lacking both N40 and PB1-F2 replicated normally. However, viruses that did not express N40 but retained an intact PB1-F2 gene overexpressed PB1 early in infection and replicated slowly in tissue culture. Thus, the influenza A virus proteome includes a 12th primary translation product that (similarly to PB1-F2) is nonessential for virus viability but whose loss, in particular genetic backgrounds, is detrimental to virus replication.
Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms
Authors: Sherrill Joseph D. , Stropes Melissa P. , Schneider Olivia D. , Koch Diana E. , Bittencourt Fabiola M. , Miller Jeanette L. C. , Miller William E.
Published: June 2009

The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric Gq/11proteins and not through promiscuous coupling of M33 to the Gspathway. Using inhibitors of signaling molecules downstream of Gq/11, we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-β and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38α and transcription factor NF-κB, occurs in the absence of Gq/11and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-β and PKC plays a central role in MCMV pathogenesis in vivo.
Topoisomerase I and RecQL1 Function in Epstein-Barr Virus Lytic Reactivation
Authors: Wang Pu , Rennekamp Andrew J. , Yuan Yan , Lieberman Paul M.
Published: June 2009

Cellular topoisomerases and helicases are thought to play an essential role in herpesvirus replication and gene expression and are considered to be potential targets for antiviral therapies. Topoisomerase I (Topo I) and Topo II inhibitors can selectively inhibit Epstein-Barr virus (EBV) lytic cycle DNA replication. We found that the Topo I inhibitor camptothecin and, to a lesser extent, the Topo II inhibitor etoposide are potent inhibitors of the transcription and replication function of the EBV-encoded immediate-early protein Zta (also referred to as ZEBRA, EB1, and BZLF1). Camptothecin inhibited the Zta transcription activation of endogenous and reporter-linked viral promoters. Small interfering RNA depletion of Topo I also inhibited the Zta-dependent activation of lytic cycle DNA replication. Topo I could be coimmunoprecipitated with Zta, but this interaction was restricted to EBV-positive cells, suggesting that other viral proteins stabilize the interaction between Zta and Topo I. We also found that the RecQL1 helicase, which is known to associate with Kaposi's sarcoma-associated herpesvirus (KSHV) OriLyt, interacts with EBV OriLyt. Treatment with camptothecin reduced both Zta and RecQL1 binding to OriLyt in vivo, suggesting that Topo I promotes replication protein assembly at OriLyt.
3′
cis
-Acting Elements That Contribute to the Competence and Efficiency of Japanese Encephalitis Virus Genome Replication: Functional Importance of Sequence Duplications, Deletions, and Substitutions
Authors: Yun Sang-Im , Choi Yu-Jeong , Song Byung-Hak , Lee Young-Min
Published: June 2009

The positive-strand RNA genome of Japanese encephalitis virus (JEV) terminates in a highly conserved 3′-noncoding region (3′NCR) of six domains (V, X, I, II-1, II-2, and III in the 5′-to-3′ direction). By manipulating the JEV genomic RNA, we have identified important roles for RNA elements present within the 574-nucleotide 3′NCR in viral replication. The two 3′-proximal domains (II-2 and III) were sufficient for RNA replication and virus production, whereas the remaining four (V, X, I, and II-1) were dispensable for RNA replication competence but required for maximal replication efficiency. Surprisingly, a lethal mutant lacking all of the 3′NCR except domain III regained viability through pseudoreversion by duplicating an 83-nucleotide sequence from the 3′-terminal region of the viral open reading frame. Also, two viable mutants displayed severe genetic instability; these two mutants rapidly developed 12 point mutations in domain II-2 in the mutant lacking domains V, X, I, and II-1 and showed the duplication of seven upstream sequences of various sizes at the junction between domains II-1 and II-2 in the mutant lacking domains V, X, and I. In all cases, the introduction of these spontaneous mutations led to an increase in RNA production that paralleled the level of protein accumulation and virus yield. Interestingly, the mutant lacking domains V, X, I, and II-1 was able to replicate in hamster BHK-21 and human neuroblastoma SH-SY5Y cells but not in mosquito C6/36 cells, indicating a cell type-specific restriction of its viral replication. Thus, our findings provide the basis for a detailed map of the 3′
cis
-acting elements in JEV genomic RNA, which play an essential role in viral replication. They also provide experimental evidence for the function of 3′ direct repeat sequences and suggest possible mechanisms for the emergence of these sequences in the 3′NCR of JEV and perhaps in other flaviviruses.
Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus
Authors: Arapović Jurica , Lenac Tihana , Antulov Ronald , Polić Bojan , Ruzsics Zsolt , Carayannopoulos Leonidas N. , Koszinowski Ulrich H. , Krmpotić Astrid , Jonjić Stipan
Published: June 2009

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1δ, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1δ and RAE-1γ in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1δ form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1δ compared to RAE-1γ but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1δ. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.
Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication
Authors: McLean Jeffrey E. , Datan Emmanuel , Matassov Demetrius , Zakeri Zahra F.
Published: June 2009

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.
Benthic Macrofaunal Production of Browns Bank, Northwest Atlantic
Authors: Wildish D. J. , Wilson A. J. , Frost B.
Published: June 2009

Benthic macrofauna of the coarse sediments of Browns Bank, off southwest Nova Scotia, Canada were sampled with a modified 0.5-m2 Van Veen grab; 29 stations from 1983 to 1985. Production was estimated from alcohol-stored biomass by multiplying by the annual turnover ratio, P:B, of each species. The latter was determined from an empirically derived relationship using known or estimated lifespans. Benthic macrofaunal production averaged 64 g wet weight∙m−2∙yr−1 on Browns Bank, markedly lower than the 193 g wet weight∙m−2∙yr−1 in the mixed and finer sediments of the Bay of Fundy. Other community characteristics, such as the number of polychaete and amphipod species for the two areas were similar, which we attribute to the similar geological ages of the sediments. Prey consumed by juvenile age 0 haddock (Melannogrammus aeglefinus) consist mostly of deposit-feeding macroinfauna which produce ~1.8 × 104 t wet biomass per year, representing ~8% of the total production of Browns Bank. Two linked hypotheses are proposed to account for the suitability of Banks as juvenile gadid feeding grounds: rapid rates of suspension of macrofauna by strong tidal currents; and drifting animals are of a suitable size and kind for juvenile haddock feeding.