bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization ofSalmonella entericaSerovar Typhimurium and MonophasicSalmonellaSerovar 1,4,[5],12:i:- Isolates in Thailand
Authors: Amavisit P. , Boonyawiwat W. , Bangtrakulnont A.
Published: June 2005

Duplex PCR was developed to screenSalmonella entericaserovar Typhimurium phage type DT104 and related strains in Thailand because a phage typing laboratory of serovar Typhimurium is not available. Of 46 isolates of serovar Typhimurium and 32 isolates ofS. entericaserovar 1,4,[5],12:i:-, 15 (33%) and 30 (94%) were duplex PCR positive, respectively. All isolates were submitted for phage typing to analyze the specificity of the PCR assay. Among serovar Typhimurium isolates that yielded positive duplex PCRs, only seven isolates were phage types DT104 or U302, and eight isolates were undefined types, whereas the negative PCR isolates were either other phage types, including DT7, DT12, DT66, DT79, DT166, DT170, DT193, and DT208 or an undefined type. The serovar Typhimurium and serovar 1,4,[5],12:i:- isolates that were duplex PCR positive were further subtyped by using XbaI PFGE to reveal their genetic relatedness. All serovar Typhimurium phage type DT104 strains had indistinguishable chromosomal patterns. The isolates of phage type U302 and most of the serovar 1,4,[5],12:i:- isolates that were duplex PCR positive yielded similar pulsed-field gel electrophoresis patterns. The patterns of PCR-negative isolates distinctly differed from the patterns of PCR-positive isolates. A total of 26% of all isolates had a dominant R-type ACSSuTG that was not found in the isolates of phage type DT104.
Central European Dobrava Hantavirus Isolate from a Striped Field Mouse (
Apodemus agrarius
)
Authors: Klempa Boris , Stanko Michal , Labuda Milan , Ulrich Rainer , Meisel Helga , Krüger Detlev H.
Published: June 2005

Dobrava virus (DOBV) is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS) in Europe. It is hosted by at least two rodent species,
Apodemus flavicollis
and
A. agrarius
. According to their natural hosts they form the distinct genetic lineages DOBV-Af and DOBV-Aa, respectively. We have now established a DOBV isolate named Slovakia (SK/Aa) from an
A. agrarius
animal captured in Slovakia. The complete S and M and partial L segment nucleotide sequences of the new isolate were determined. Phylogenetic analyses showed that the SK/Aa isolate clustered together with the other DOBV-Aa sequences amplified from
A. agrarius
before and can be taken as the representative of this genetic lineage. SK/Aa, in comparison with a DOBV-Af isolate, was used for serotyping neutralizing antibodies of HFRS patients in Central Europe. Most patients' sera exhibited a higher endpoint titer when probed with our new isolate, suggesting that DOBV-Aa strains are responsible for most of the DOBV-caused HFRS cases in this region.
Isolation and Molecular Characterization of
Staphylococcus sciuri
in the Hospital Environment
Authors: Dakić Ivana , Morrison Donald , Vuković Dragana , Savić Branislava , Shittu Adebayo , Ježek Petr , Hauschild Tomasz , Stepanović Srdjan
Published: June 2005

Staphylococcus sciuri
is a principally animal-associated bacterial species, but its clinical relevance for humans is increasing. Our study aimed to provide the first insight into the prevalence of this bacterium in a hospital environment. A 3-month surveillance was conducted in a hospital located in Belgrade, Serbia, and 1,028 samples taken from hands of medical personnel, medical devices, and various hospital surfaces were screened for
S. sciuri
presence. In total, 108 isolates were obtained, which resulted in a relatively high rate of colonization (10.5%). These isolates, along with 7
S. sciuri
strains previously isolated in the same hospital (
n
= 115), were phenotypically and genotypically characterized. Antimicrobial susceptibility testing revealed that 73% of the strains were resistant to one or more antibiotics, with 4.3% strains displaying multiresistance. Examination of 16S-23S ribosomal DNA intergenic spacer length polymorphism identified the strains at the subspecies level, and 74 (64.3%) strains of
S. sciuri
subsp.
sciuri
, 37 (32.2%) strains of
S. sciuri
subsp.
rodentium
, and 4 (3.5%) strains of
S. sciuri
subsp.
carnaticus
were established. Pulsed-field gel electrophoresis (PFGE) analysis showed 21 distinct pulsotypes, including 17 main types and 4 subtypes. One dominant cluster with 62 strains was found, while 19 (90.5%) of the PFGE types and subtypes identified had 5 or fewer strains. The predominance of small PFGE clusters suggests that the ubiquitous presence of
S. sciuri
in the outside environment presents the continuous source for colonization of the hospital environment. The presence of one dominant PFGE cluster of strains indicates that some
S. sciuri
strains may be capable for adaptation to hospital environment conditions and continuous existence in this environment.
Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6
Authors: Wakeley P. R. , Johnson N. , McElhinney L. M. , Marston D. , Sawyer J. , Fooks A. R.
Published: June 2005

Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the
Lyssavirus
genus.
Unique Endemicity of Cryptosporidiosis in Children in Kuwait
Authors: Sulaiman Irshad M. , Hira Parsotam R. , Zhou Ling , Al-Ali Faiza M. , Al-Shelahi Fatima A. , Shweiki Hussein M. , Iqbal Jamshaid , Khalid Nabila , Xiao Lihua
Published: June 2005

To understand the transmission of
Cryptosporidium
infection in children, fecal specimens from 62 Kuwaiti children with gastrointestinal symptoms found to be positive by microscopy were genotyped and subtyped with a small subunit rRNA-based PCR-restriction fragment length polymorphism analysis and a 60-kDa glycoprotein-based DNA sequencing tool. The median age of infected children was 4.5 years, and 77% of infections occurred during the cool season of November to April. Fifty-eight of the children (94%) had
Cryptosporidium parvum
, three (5%) had
Cryptosporidium hominis
, and one (1%) had both
C. parvum
and
C. hominis
. Altogether, 13 subtypes of
C. parvum
(belonging to four subtype allele families) and
C. hominis
(belonging to three subtype allele families) were observed, with 92% of specimens belonging to the common allele family IIa and the unusual allele family IId. Thus, the transmission of cryptosporidiosis in Kuwaiti children differed significantly from other tropical countries.
Matrix-Assisted Laser Desorption Ionization-Time of Flight (Mass Spectrometry) for Hepatitis C Virus Genotyping
Authors: Ilina Elena N. , Malakhova Maja V. , Generozov Edward V. , Nikolaev Eugene N. , Govorun Vadim M.
Published: June 2005

Determination of the hepatitis C virus (HCV) genotype has become accepted as the standard procedure in laboratory practice. Genotype assignment helps in disease prognosis and assists in establishing the appropriate duration of treatment. More than 10 types and 70 subtypes of HCV have been described. In Russia the most common subtypes are 1a, 1b, 2a, and 3a, and the types 4 and 5 are relatively rare. The “gold standard” for testing is gene sequencing. However, a variety of other assays had been developed to provide more rapid and cheaper forms of testing. The aim of this study was to determine the HCV genotype by minisequencing followed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Fragments of 5′ untranslated region of the HCV genome were amplified. Three oligonucleotide primers were designed to detect two sets of genotype-specific single nucleotide polymorphisms. The primer extension reaction was performed using modified thermostable DNA polymerase and in the presence of dideoxynucleosides. The molecular weights of the reaction products were analyzed with MALDI-TOF mass spectrometer. The HCV genotype was determined by registering the particles of the expected molecular weights. The method was used to genotype HCV from HCV-positive blood sera or plasma. The 1a, 1b, 2a, 3a, and 4 genotype HCVs were determined in the samples examined. The data were confirmed by direct sequencing. Thus, we propose a new accurate and efficient method for HCV genotyping based on minisequencing followed by mass spectrometry.
Analysis of
Bordetella pertussis
Populations in European Countries with Different Vaccination Policies
Authors: van Amersfoorth S. C. M. , Schouls L. M. , van der Heide H. G. J. , Advani A. , Hallander H. O. , Bondeson K. , von König C. H. W. , Riffelmann M. , Vahrenholz C. , Guiso N. , Caro V. , Njamkepo E. , He Q. , Mertsola J. , Mooi F. R.
Published: June 2005

Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of
Bordetella pertussis
and ultimately on the disease burden in Europe. In the present study, a total of 102
B. pertussis
isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (
ptxA
and
ptxC
), pertactin (
prn
), and tracheal colonization factor (
tcfA
); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the
ptxC
alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the
B. pertussis
population in Europe in the coming years.
Community-Wide Outbreaks of Clonally Related CTX-M-14 β-Lactamase-Producing
Escherichia coli
Strains in the Calgary Health Region
Authors: Pitout Johann D. D. , Gregson Daniel B. , Church Deirdre L. , Elsayed Sameer , Laupland Kevin B.
Published: June 2005

Enterobacteriaceae
producing extended-spectrum β-lactamases (ESBLs) typically cause nosocomial infections. Previous surveillance in the Calgary Health Region showed that
Escherichia coli
strains producing ESBLs were common among community patients. During the period (2000 to 2002): 23 of 157 (15%) of the strains were positive for
bla
CTX-M
genes from the CTX-M-I group (CTX-M-1-like) and 87 of 157 (55%) of the strains were positive for
bla
CTX-M
genes from the CTX-M-III group (CTX-M-14-like). The objective of this study was to investigate the molecular epidemiology of these strains. The β-lactamases were characterized, and the genetic relatedness of the isolates was analyzed by digesting genomic DNA with the restriction endonuclease XbaI and by performing pulse-field gel electrophoresis (PFGE). PFGE revealed two closely related restriction patterns (clusters CTXM14A and CTXM14AR) among 67 (77%) CTX-M-14 producers. These strains from CTXM14A had nearly identical susceptibility patterns and were isolated most often from urine samples obtained at community sites during 2000 and 2001. Strains from the CTX-M-1-like and CTX-M-negative groups were unrelated to clusters CTXM14, CTXM14AR, and CTXM14NR. We conclude that clonally related strains of
E. coli
producing CTX-M-14 β-lactamases were responsible for a predominantly community-wide outbreak. Further studies are warranted to investigate whether community-onset diseases caused by ESBL-producing
E. coli
are related to a point source or transmission within the community.
Genomic Analysis of Vaccine-Derived Poliovirus Strains in Stool Specimens by Combination of Full-Length PCR and Oligonucleotide Microarray Hybridization
Authors: Laassri Majid , Dragunsky Eugenia , Enterline Joan , Eremeeva Tatiana , Ivanova Olga , Lottenbach Kathleen , Belshe Robert , Chumakov Konstantin
Published: June 2005

Sabin strains of poliovirus used in the manufacture of oral poliovirus vaccine (OPV) are prone to genetic variations that occur during growth in cell cultures and the organisms of vaccine recipients. Such derivative viruses often have increased neurovirulence and transmissibility, and in some cases they can reestablish chains of transmission in human populations. Monitoring for vaccine-derived polioviruses is an important part of the worldwide campaign to eradicate poliomyelitis. Analysis of vaccine-derived polioviruses requires, as a first step, their isolation in cell cultures, which takes significant time and may yield viral stocks that are not fully representative of the strains present in the original sample. Here we demonstrate that full-length viral cDNA can be PCR amplified directly from stool samples and immediately subjected to genomic analysis by oligonucleotide microarray hybridization and nucleotide sequencing. Most fecal samples from healthy children who received OPV were found to contain variants of Sabin vaccine viruses. Sequence changes in the 5′ untranslated region were common, as were changes in the VP1-coding region, including changes in a major antigenic site. Analysis of stool samples taken from cases of acute flaccid paralysis revealed the presence of mixtures of recombinant polioviruses, in addition to the emergence of new sequence variants. Avoiding the need for cell culture isolation dramatically shortened the time needed for identification and analysis of vaccine-derived polioviruses and could be useful for preliminary screening of clinical samples. The amplified full-length viral cDNA can be archived and used to recover live virus for further virological studies.
Comparison of Immunohistochemistry and Virus Isolation for Diagnosis of West Nile Virus
Authors: Ellis Angela E. , Mead Daniel G. , Allison Andrew B. , Gibbs Samantha E. J. , Gottdenker Nicole L. , Stallknecht David E. , Howerth Elizabeth W.
Published: June 2005

Immunohistochemistry and virus isolation were performed on 1,057 birds. Immunohistochemistry, virus isolation, or both found 325 birds to be West Nile virus positive. Of these, 271 were positive by both methods. These results indicate that virus isolation and immunohistochemistry are approximately equal in their ability to detect West Nile virus.
Multicenter Evaluation of a
Candida albicans
Peptide Nucleic Acid Fluorescent In Situ Hybridization Probe for Characterization of Yeast Isolates from Blood Cultures
Authors: Wilson D. A. , Joyce M. J. , Hall L. S. , Reller L. B. , Roberts G. D. , Hall G. S. , Alexander B. D. , Procop G. W.
Published: June 2005

We evaluated aliquots from 244 clinical blood culture bottles that demonstrated yeasts on Gram stain using a
Candida albicans
peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) probe. The sensitivity, specificity, positive predictive value, and negative predictive value of the
C. albicans
PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively.
Clinical Relevance of the
vacA
,
iceA
,
cagA
, and
flaA
Genes of
Helicobacter pylori
Strains Isolated in Eastern Taiwan
Authors: Wu Chao-Chuan , Chou Pai-Yu , Hu Chi-Tan , Liu Zuo-Chai , Lin Chih-Yung , Tseng Yi-Hsiung , Lin Nien-Tsung
Published: June 2005

The genotypes of
Helicobacter pylori flaA
,
cagA
,
vacA
, and
iceA
were determined for DNA isolated from patients with chronic gastritis or peptic ulcer in eastern Taiwan. The
vacA
gene encoding the s1a subtype was found to predominate in peptic ulcer patients, and the
iceA1
genotype was associated with chronic gastritis.
cagA
and
flaA
genes were not found to be associated with these types of disease.
Surveillance ofStaphylococcus aureusin Veterinary Teaching Hospitals
Authors: Middleton John R. , Fales William H. , Luby Christopher D. , Oaks J. Lindsay , Sanchez Susan , Kinyon Joann M. , Wu Ching Ching , Maddox Carol W. , Welsh Ronald D. , Hartmann Faye
Published: June 2005

Staphylococcus aureusisolates (n= 70) from 65 patients (36 canine, 18 equine, 7 bovine, 2 avian, and 2 feline) at seven veterinary teaching hospitals in the United States were studied. The majority of patients (83%) with anS. aureusinfection were canine and equine, but this may have reflected a sample bias based on clinic case loads and diagnostic lab submissions at the participating institutions. Fourteen percent of patients with anS. aureusinfection were infected with a methicillin-resistantS. aureus(MRSA) isolate. Six of seven institutions had at least one MRSA infection during the study. Pulsed-field gel electrophoresis on 63 of the 70 isolates yielded 58 unique strains ofS. aureus. None of the strain types of the MRSA isolates matched each other or the type of any otherS. aureusisolate. The proportions of patients infected with an MRSA isolate were not significantly different between institutions or animal species (P≥ 0.222). Methicillin-resistantS. aureusisolates in this study seemed to be community acquired rather than hospital acquired.
Evaluation of a New Molecular System for Simultaneous Identification of Four
Enterococcus
Species and Their Glycopeptide Resistance Genotypes
Authors: Eigner U. , Fahr A. , Weizenegger M. , Witte W.
Published: June 2005

We compared the performances of a new DNA-based strip assay and the VITEK 2 system for identification of 105 enterococcal strains and differentiation of their
van
gene-associated resistance levels. Both methods provided excellent results. The molecular assay showed advantages in time to result for identification of
van
-associated genes.
Dissemination of Multisusceptible Methicillin-Resistant
Staphylococcus aureus
in Singapore
Authors: Hsu Li-Yang , Koh Tse-Hsien , Singh Kamaljit , Kang Mei-Ling , Kurup Asok , Tan Ban-Hock
Published: June 2005

Analysis of hospital-acquired methicillin-resistant
Staphylococcus aureus
strains isolated from a tertiary public hospital in Singapore revealed that multisusceptible strains had gradually started to replace the endemic multiresistant strain (ST239-MRSA-III) since 2002. Molecular typing showed that this was a predominantly clonal outbreak of a UK-EMRSA-15 strain (ST22-MRSA-IV).
Distribution of
Burkholderia cepacia
Complex Species among Isolates Recovered from Persons with or without Cystic Fibrosis
Authors: Reik Rebecca , Spilker Theodore , LiPuma John J.
Published: June 2005

We analyzed
Burkholderia cepacia
complex isolates recovered from 1,218 cystic fibrosis (CF) patients and 90 patients without CF. Although all
B. cepacia
complex species were found, some were rarely identified. The distribution of species differed between the CF and non-CF populations and appears to be changing over time among CF patients.
Molecular Detection of a New
Anaplasma
Species Closely Related to
Anaplasma phagocytophilum
in Canine Blood from South Africa
Authors: Inokuma Hisashi , Oyamada Maremichi , Kelly Patrick J. , Jacobson Linda A. , Fournier Pierre-Edouard , Itamoto Kazuhito , Okuda Masaru , Brouqui Philippe
Published: June 2005

Canine DNA samples from South Africa were found to contain 16S rRNA gene nucleotide and citrate synthase gene nucleotide and deduced amino acid sequences that were most similar to
Anaplasma phagocytophilum
: 98%, 66%, and 69% similarity, respectively. This suggests that a new
Anaplasma
species closely related to
A. phagocytophilum
occurs in Africa.
Four Laboratory-Associated Cases of Infection with
Escherichia coli
O157:H7
Authors: Spina Nancy , Zansky Shelley , Dumas Nellie , Kondracki Stan
Published: June 2005

An investigation of four cases of infection with
Escherichia coli
O157:H7 among laboratorians from different clinical laboratories revealed that the DNA fingerprint pattern of each case isolate was indistinguishable from that of an isolate handled in the laboratory prior to illness. These data suggest that the infections were laboratory acquired, and they demonstrate the importance of laboratorians strictly adhering to biosafety practices recommended for the handling of infectious materials.
Molecular Epidemiology Investigation of Outbreaks of Fowl Cholera in Geographically Related Poultry Flocks
Authors: Kardos G. , Kiss I.
Published: June 2005

The present work describes two cases of consecutive fowl cholera outbreak series occurring in goose and turkey flocks. Most strains isolated from epidemiologically related outbreaks showed genetic relatedness, as revealed by enterobacterial repetitive intergenic consensus-PCR and pulsed-field gel electrophoresis, suggesting that the consecutive outbreaks were due to recurrences rather than reinfections.
Prevalence ofHelicobacter pullorumamong Patients with Gastrointestinal Disease and Clinically Healthy Persons
Authors: Ceelen Liesbeth , Decostere Annemie , Verschraegen Gerda , Ducatelle Richard , Haesebrouck Freddy
Published: June 2005

Feces from 531 patients with gastroenteritis and from 100 clinically healthy individuals were tested forHelicobacter pullorumby use of PCR. Samples positive by PCR were qualified for isolation.H. pullorumDNA was demonstrated to be present in feces from 4.3% of patients with gastrointestinal disease but also in feces from 4.0% of clinically healthy persons. One strain was isolated from one patient with gastrointestinal disease.
Species-Level Molecular Identification of Invasive “
Streptococcus milleri
” Group Clinical Isolates by Nucleic Acid Sequencing in a Centralized Regional Microbiology Laboratory
Authors: Daley Peter , Church Deirdre L. , Gregson Daniel B. , Elsayed Sameer
Published: June 2005

Organisms belonging to the “
Streptococcus milleri
” group are important invasive human pathogens. A detailed understanding of their pathogenesis in human infection has only recently been facilitated by the use of molecular methods to study this group of organisms.
Multicenter Evaluation of a Semiautomated, Standardized Assay for Detection of Hepatitis B Virus DNA in Blood Donations
Authors: Romanò Luisa , Velati Claudio , Baruffi Lorella , Fomiatti Laura , Colucci Giuseppe , Zanetti Alessandro R. ,   the Italian Group for the Study of Transfusion Transmissible Diseases
Published: June 2005

We evaluated the COBAS Ampliscreen hepatitis B virus (HBV) test using standards, seroconversion panels, consecutive donations, and samples from patients with abnormal alanine aminotransferase and chronic hepatitis C. Specificity was 100% and sensitivity was 20 IU/ml. In seroconversion panels, HBV DNA was detected up to 4 to 18 days before HBsAg, suggesting that this assay is useful in shortening the infectious window phase.
First Identification of
Clostridium celerecrescens
in Liquid Drained from an Abscess
Authors: Glazunova Olga O. , Raoult Didier , Roux Véronique
Published: June 2005

We present the first report on the characterization of
Clostridium celerecrescens
isolated from liquid drained via abscess puncture from a 45-year-old man. The organism was identified by sequence comparison of 16S rRNA genes.
Human Immunodeficiency Virus Type 1 Vpr Impairs Dendritic Cell Maturation and T-Cell Activation: Implications for Viral Immune Escape
Authors: Majumder Biswanath , Janket Michelle L. , Schafer Elizabeth A. , Schaubert Keri , Huang Xiao-Li , Kan-Mitchell June , Rinaldo Charles R. , Ayyavoo Velpandi
Published: June 2005

Antigen presentation and T-cell activation are dynamic processes involving signaling molecules present in both APCs and T cells. Effective APC function and T-cell activation can be compromised by viral immune evasion strategies, including those of human immunodeficiency virus type 1 (HIV-1). In this study, we determined the effects of HIV-1 Vpr on one of the initial target of the virus, dendritic cells (DC), by investigating DC maturation, cytokine profiling, and CD8-specific T-cell stimulation function followed by a second signal. Vpr impaired the expression of CD80, CD83, and CD86 at the transcriptional level without altering normal cellular transcription. Cytokine profiling indicated that the presence of Vpr inhibited production of interleukin 12 (IL-12) and upregulated IL-10, whereas IL-6 and IL-1β were unaltered. Furthermore, DC infected with HIV-1vpr+significantly reduced the activation of antigen-specific memory and recall cytotoxic-T-lymphocyte responses. Taken together, these results indicate that HIV-1 Vpr may in part be responsible for HIV-1 immune evasion by inhibiting the maturation of costimulatory molecules and cytokines essential for immune activation.
Regulation of the Translation Initiation Factor eIF4F by Multiple Mechanisms in Human Cytomegalovirus-Infected Cells
Authors: Walsh Derek , Perez Cesar , Notary Joanna , Mohr Ian
Published: June 2005

As a viral opportunistic pathogen associated with serious disease among the immunocompromised and congenital defects in newborns, human cytomegalovirus (HCMV) must engage the translational machinery within its host cell to synthesize the viral proteins required for its productive growth. However, unlike many viruses, HCMV does not suppress the translation of host polypeptides. Here, we examine how HCMV regulates the cellular cap recognition complex eIF4F, a critical component of the cellular translation initiation apparatus that recruits the 40S ribosome to the 5′ end of the mRNA. This study establishes that the cap binding protein eIF4E, together with the translational repressor 4E-BP1, are both phosphorylated early in the productive viral growth cycle and that the activity of the cellular eIF4E kinase, mnk, is critical for efficient viral replication. Furthermore, HCMV replication also induces an increase in the overall abundance of eIF4F components and promotes assembly of eIF4F complexes. Notably, increasing the abundance of select eIF4F core components and associated factors alters the ratio of active eIF4F complexes in relation to the 4E-BP1 translational repressor, illustrating a new strategy through which members of the herpesvirus family enhance eIF4F activity during their replicative cycle.
Role of the Hypervariable Hinge Region of Phosphoprotein P of Vesicular Stomatitis Virus in Viral RNA Synthesis and Assembly of Infectious Virus Particles
Authors: Das Subash C. , Pattnaik Asit K.
Published: June 2005

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using a series of deletion mutants spanning the hinge region of the protein, we observed that amino acid residues 201 through 220 are required for the activity of P protein in both replication and transcription. Neither insertion nor deletion had any effect on the interaction of P protein with N or L proteins. Infectious VSVs with a deletion in the hinge region possessed retarded growth characteristics and exhibited small-plaque morphology. Interestingly, VSV containing one P protein deletion mutant (PΔ7, with amino acids 141 through 200 deleted), which possessed significant levels of replication and transcription activity, could be amplified only by passage in cells expressing the wild-type P protein. We conclude that the hypervariable hinge region of the P protein plays an important role in viral RNA synthesis. Furthermore, our results provide a previously unidentified function for the P protein: it plays a critical role in the assembly of infectious VSV.
Longitudinal Assessment of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Gamma Interferon Responses during the First Year of Life in HIV-1-Infected Infants
Authors: Lohman Barbara L. , Slyker Jennifer A. , Richardson Barbra A. , Farquhar Carey , Mabuka Jenniffer M. , Crudder Christopher , Dong Tao , Obimbo Elizabeth , Mbori-Ngacha Dorothy , Overbaugh Julie , Rowland-Jones Sarah , John-Stewart Grace
Published: June 2005

Human immunodeficiency virus type 1 (HIV-1) infection results in different patterns of viral replication in pediatric compared to adult populations. The role of early HIV-1-specific responses in viral control has not been well defined, because most studies of HIV-1-infected infants have been retrospective or cross-sectional. We evaluated the association between HIV-1-specific gamma interferon (IFN-γ) release from the cells of infants of 1 to 3 months of age and peak viral loads and mortality in the first year of life among 61 Kenyan HIV-1-infected infants. At 1 month, responses were detected in 7/12 (58%) and 6/21 (29%) of infants infected in utero and peripartum, respectively (P= 0.09), and in ∼50% of infants thereafter. Peaks of HIV-specific spot-forming units (SFU) increased significantly with age in all infants, from 251/106peripheral blood mononuclear cells (PBMC) at 1 month of age to 501/106PBMC at 12 months of age (P= 0.03), although when limited to infants who survived to 1 year, the increase in peak HIV-specific SFU was no longer significant (P= 0.18). Over the first year of life, infants with IFN-γ responses at 1 month had peak plasma viral loads, rates of decline of viral load, and mortality risk similar to those of infants who lacked responses at 1 month. The strength and breadth of IFN-γ responses at 1 month were not significantly associated with viral containment or mortality. These results suggest that, in contrast to HIV-1-infected adults, in whom strong cytotoxic T lymphocyte responses in primary infection are associated with reductions in viremia, HIV-1-infected neonates generate HIV-1-specific CD8+-T-cell responses early in life that are not clearly associated with improved clinical outcomes.
Promoters, Transcripts, and Regulatory Proteins of Mungbean Yellow Mosaic Geminivirus
Authors: Shivaprasad P. V. , Akbergenov Rashid , Trinks Daniela , Rajeswaran R. , Veluthambi K. , Hohn Thomas , Pooggin Mikhail M.
Published: June 2005

Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of
Mungbean yellow mosaic virus-Vigna
(MYMV), a bipartite geminivirus in the genus
Begomovirus
. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.
Isolation of a Bovine Plasma Fibronectin-Containing Complex Which Inhibits the Expression of Bovine Leukemia Virus p24
Authors: van den Heuvel Marianne J. , Jefferson Barbara J. , Jacobs Robert M.
Published: June 2005

Bovine leukemia virus (BLV) is a deltaretrovirus that infects cattle worldwide. In agriculturally intensive regions, approximately 30% of dairy cows are BLV infected. Like the human T-cell leukemia virus (HTLV), there is a lengthy period of viral quiescence after initial infection with BLV. Unlike HTLV, BLV resides predominantly in B cells. Lymphoma is observed in less than 10% of BLV-infected adult cattle. Although viremia is undetectable in vivo, BLV-infected peripheral blood mononuclear cells readily become productive when cultured in vitro. Productivity is markedly diminished when cultures are supplemented with bovine plasma. This inhibitory activity of bovine plasma has been attributed to the “plasma blocking factor” (PBF). Here, we describe the purification of a PBF whose activity was resistant to heating to 65°C for 10 min and was attributable to a fibronectin-containing complex of approximately 320 kDa under nonreducing conditions. By use of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry), a protein with a size of 220 kDa and a pI of 5.4 was identified as a member of the fibronectin group of molecules. Both the purified protein and the commercially available bovine fibronectin inhibited BLV production in naturally infected peripheral blood mononuclear cells, although the fibronectin was less biologically active.
Development of a Novel System To Study Hepatitis Delta Virus Genome Replication
Authors: Chang Jinhong , Gudima Severin O. , Tarn Chi , Nie Xingcao , Taylor John M.
Published: June 2005

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (δAg). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of δAg species that are nonfunctional or even inhibitory. A replication system was devised where all δAg was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small δAg, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express δAg. Thus, replication of this RNA was under control of the TET-inducible δAg. In the absence of TET, there was sufficient δAg to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both δAg and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G
1
/G
0
cells relative to both S and G
2
/M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.
Differential Sensitivity of Murine Leukemia Virus to APOBEC3-Mediated Inhibition Is Governed by Virion Exclusion
Authors: Doehle Brian P. , Schäfer Alexandra , Wiegand Heather L. , Bogerd Hal P. , Cullen Bryan R.
Published: June 2005

While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deficient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members.
An Interplay between Hypervariable Region 1 of the Hepatitis C Virus E2 Glycoprotein, the Scavenger Receptor BI, and High-Density Lipoprotein Promotes both Enhancement of Infection and Protection against Neutralizing Antibodies
Authors: Bartosch Birke , Verney Géraldine , Dreux Marlène , Donot Peggy , Morice Yoann , Penin François , Pawlotsky Jean-Michel , Lavillette Dimitri , Cosset Francois-Loïc
Published: June 2005

Hepatitis C virus (HCV) circulates in the bloodstream in different forms, including complexes with immunoglobulins and/or lipoproteins. To address the significance of such associations, we produced or treated HCV pseudoparticles (HCVpp), a valid model of HCV cell entry and its inhibition, with naïve or patient-derived sera. We demonstrate that infection of hepatocarcinoma cells by HCVpp is increased more than 10-fold by human serum factors, of which high-density lipoprotein (HDL) is a major component. Infection enhancement requires scavenger receptor BI, a molecule known to mediate HDL uptake into cells as well as HCVpp entry, and involves conserved amino acid positions in hypervariable region 1 (HVR1) of the E2 glycoprotein. Additionally, we show that the interaction with human serum or HDL, but not with low-density lipoprotein, leads to the protection of HCVpp from neutralizing antibodies, including monoclonal antibodies and antibodies present in patient sera. Finally, the deletion or mutation of HVR1 in HCVpp abolishes infection enhancement and leads to increased sensitivity to neutralizing antibodies/sera compared to that of parental HCVpp. Altogether, these results assign to HVR1 new roles which are complementary in helping HCV to survive within its host. Besides immune escape by mutation, HRV1 can mediate the enhancement of cell entry and the protection of virions from neutralizing antibodies. By preserving a balance between these functions, HVR1 may be essential for the viral persistence of HCV.
Avian Sarcoma and Leukosis Virus Cytopathic Effect in the Absence of TVB Death Domain Signaling
Authors: Klucking Sara , Collins Asha S. , Young John A. T.
Published: June 2005

The cytopathic effect (CPE) seen with some subgroups of avian sarcoma and leukosis virus (ASLV) is associated with viral Env activation of the death-promoting activity of TVB (a tumor necrosis factor receptor-related receptor that is most closely related to mammalian TNF-related apoptosis-inducing ligand [TRAIL] receptors) and with viral superinfection leading to unintegrated viral DNA (UVD) accumulation, which is presumed to activate a cellular DNA damage response. In this study, we employed cells that express signaling-deficient ASLV receptors to demonstrate that an ASLV CPE can be uncoupled from the death-promoting functions of the TVB receptor. However, these cell-killing events were associated with much higher levels of viral superinfection and DNA accumulation than those seen when the virus used signaling-competent TVB receptors. These findings suggest that a putative cellular DNA damage response that is activated by UVD accumulation might act in concert with the death-signaling pathways activated by Env-TVB interactions to trigger cell death. Such a model is consistent with the well-established synergy that exists between TRAIL-signaling pathways and DNA damage responses which is currently being exploited in cancer therapy regimens.
Innate STAT1-Dependent Genomic Response of Neurons to the Antiviral Cytokine Alpha Interferon
Authors: Wang Jianping , Campbell Iain L.
Published: June 2005

Alpha/beta interferons (IFNs-α/β) are cytokines that play an essential role in the host defense against viral infection. Our previous studies have shown that the key IFN signaling molecule STAT1 is highly elevated and activated in central nervous system neurons during viral infection and in transgenic mice with astrocyte production of IFN-α (glial fibrillary acidic protein [GFAP]-IFN-α), suggesting that neurons are a very responsive target cell population for IFNs. To elucidate the genomic response of neurons to IFN-α, we undertook studies both in vitro and in vivo. Gene chip analysis was applied to RNA from IFN-α-treated or untreated primary cortical neuronal cultures derived from embryonic day 15 fetal wild-type or STAT1 knockout (KO) mice. The expression of 51 known and 5 unknown genes was increased significantly by more than twofold after exposure of wild-type but not STAT1 KO neurons to IFN-α. Some more highly expressed genes included IFN-induced 15-kDa protein, ubiquitin-specific protease 18, glucocorticoid attenuated response genes, IFN-induced GTPases, and the chemokine CXCL10. For several of these genes, the gene chip findings were confirmed by RNase protection assays. In addition, examination of the expression of some of these selected genes revealed that they were increased in neurons in the brain of either GFAP-IFN-α mice or mice infected with lymphocytic choriomeningitis virus. In conclusion, our study revealed a robust STAT1-dependent genomic response of neurons to IFN-α, highlighting an innate potential of these cells to defend against viral infection in the brain.
Herpes Simplex Virus ICP27 Activation of Stress Kinases JNK and p38
Authors: Hargett Danna , McLean Tim , Bachenheimer Steven L.
Published: June 2005

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutanttsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutantvi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, thed100 (0−/4−) andd103 (4−/22−/47−) mutants activated p38 and JNK, while thed106 (4−/22−/27−/47−) andd107 (4−/27−) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.
Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors
Authors: Hanawa Hideki , Persons Derek A. , Nienhuis Arthur W.
Published: June 2005

Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3′ long terminal repeat (LTR) is copied over into the 5′ LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.
A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta
Authors: Vega-Arreguín Julio C. , Timchenko Tatiana , Gronenborn Bruno , Ramírez Bertha Cecilia
Published: June 2005

Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus
Faba bean necrotic yellows virus
(FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in
Nicotiana benthamiana
leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected
Vicia faba
plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.