bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology
Authors: Esteves F. , Gaspar J. , De Sousa B. , Antunes F. , Mansinho K. , Matos O. 
Published: March 2011

ABSTRACT

Pneumocystis jirovecii
pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct
P. jirovecii
loci encoding dihydrofolate reductase (
DHFR
), mitochondrial large-subunit rRNA (
mtLSU rRNA
), and superoxide dismutase (
SOD
) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (
DHFR312
,
mt85
,
SOD215
, and
SOD110
), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied:
mt85C-SOD110T
,
SOD110T-SOD215C
, and
SOD110C-SOD215T
. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of
DHFR312C
,
mt85C
,
SOD110T
, and
SOD215C
were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with
DHFR312T
,
mt85T
,
SOD110C
, and
SOD215T
. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping
P. jirovecii
SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP.
Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB
Authors: Woo Patrick C. Y. , Teng Jade L. L. , Yeung Juilian M. Y. , Tse Herman , Lau Susanna K. P. , Yuen Kwok-Yung 
Published: March 2011

ABSTRACT

Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (
http://147.8.74.24/16SpathDB
) based on the 16S rRNA gene sequences of all medically important bacteria listed in the
Manual of Clinical Microbiology
and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (
Gordonibacter pamelaeae
) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the
Manual of Clinical Microbiology
. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories.
Assembly of Alphavirus Replication Complexes from RNA and Protein Components in a Novel
trans
-Replication System in Mammalian Cells
Authors: Spuul Pirjo , Balistreri Giuseppe , Hellström Kirsi , Golubtsov Andrey V. , Jokitalo Eija , Ahola Tero 
Published: March 2011

ABSTRACT

For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in
trans
, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the
trans
-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the
trans
-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.
Subclinical Brain Injury Caused by H5N1 Influenza Virus Infection
Authors: Shinya Kyoko , Makino Akiko , Hatta Masato , Watanabe Shinji , Kim Jin Hyun , Hatta Yasuko , Gao Peng , Ozawa Makoto , Le Quynh Mai , Kawaoka Yoshihiro 
Published: March 2011

ABSTRACT
Although H5N1 influenza A viruses can cause systemic infection, their neurotropism and long-term effects on the central nervous system (CNS) are not fully understood. We assessed H5N1viral invasion of the CNS and its long-term effects in a ferret model. An H5N1 virus caused nonsuppurative encephalitis, which lasted for 3 months without neurologic signs. Further, another H5N1 virus caused nonsuppurative vasculitis with brain hemorrhage. Three-dimensional analysis of viral distribution in the brain identified the olfactory system as a major route for brain invasion. The efficient growth of virus in the upper respiratory tract may thus facilitate viral brain invasion.
Investigation of Koi Herpesvirus Latency in Koi
Authors: Eide Kathleen E. , Miller-Morgan Tim , Heidel Jerry R. , Kent Michael L. , Bildfell Rob J. , LaPatra Scott , Watson Gregory , Jin Ling 
Published: March 2011

ABSTRACT

Koi herpesvirus (KHV) has recently been classified as a member of the family of
Alloherpesviridae
within the order of
Herpesvirales
. One of the unique features of
Herpesviridae
is latent infection following a primary infection. However, KHV latency has not been recognized. To determine if latency occurs in clinically normal fish from facilities with a history of KHV infection or exposure, the presence of the KHV genome was investigated in healthy koi by PCR and Southern blotting. KHV DNA, but not infectious virus or mRNAs from lytic infection, was detected in white blood cells from investigated koi. Virus shedding was examined via tissue culture and reverse transcription-PCR (RT-PCR) testing of gill mucus and feces from six koi every other day for 1 month. No infectious virus or KHV DNA was detected in fecal secretion or gill swabs, suggesting that neither acute nor persistent infection was present. To determine if KHV latent infections can be reactivated, six koi were subjected to a temperature stress regime. KHV DNA and infectious virus were detected in both gill and fecal swabs by day 8 following temperature stress. KHV DNA was also detectable in brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas in euthanized koi 1 month post-temperature stress. Our study suggests that KHV may become latent in leukocytes and other tissues, that it can be reactivated from latency by temperature stress, and that it may be more widespread in the koi population than previously suspected.
Structure and Functional Relevance of a Transcription-Regulating Sequence Involved in Coronavirus Discontinuous RNA Synthesis
Authors: Dufour David , Mateos-Gomez Pedro A. , Enjuanes Luis , Gallego José , Sola Isabel 
Published: March 2011

ABSTRACTTransmissible gastroenteritis coronavirus (TGEV) genomic RNA transcription generates 5′- and 3′-coterminal subgenomic mRNAs. This process involves a discontinuous step during the synthesis of minus-sense RNA that is modulated by transcription-regulating sequences located at the 3′ end of the leader (TRS-L) and also preceding each viral gene (TRS-Bs). TRSs include a highly conserved core sequence (CS) (5′-CUAAAC-3′) and variable flanking sequences. It has been previously proposed that TRS-Bs act as attenuation or stop signals during the synthesis of minus-sense RNAs. The nascent minus-stranded RNA would then be transferred by a template switch process to the TRS-L, which acts as the acceptor RNA. To study whether the TRS-L is structured and to determine whether this structure has a functional impact on genomic and subgenomic viral RNA synthesis, we have used a combination of nuclear magnetic resonance (NMR) spectroscopy and UV thermal denaturation approaches together with site-directed mutagenesis andin vivotranscriptional analyses. The results indicated that a 36-nucleotide oligomer encompassing the wild-type TRS-L forms a structured hairpin closed by an apical AACUAAA heptaloop. This loop contains most of the CS and is isolated from a nearby internal loop by a short Watson-Crick base-paired stem. TRS-L mutations altering the structure and the stability of the TRS-L hairpin affected replication and transcription, indicating the requirement of a functional RNA hairpin structure in these processes.
Identification of a Human SCARB2 Region That Is Important for Enterovirus 71 Binding and Infection
Authors: Yamayoshi Seiya , Koike Satoshi 
Published: March 2011

ABSTRACT
We previously identified human scavenger receptor class B, member 2 (SCARB2), as a cellular receptor for enterovirus 71 (EV71). Expression of human SCARB2 (hSCARB2) permitted mouse L929 cells to efficiently bind to virions and to produce both viral proteins and progeny viruses upon EV71 infection. Mouse Scarb2 (mScarb2) exhibited 85.8% amino acid identity and 99.9% similarity to hSCARB2. The expression of mScarb2 in L929 cells conferred partial susceptibility. Very few virions bound to mScarb2-expressing cells. The viral titer in L929 cells expressing mScarb2 was approximately 40- to 100-fold lower than that in L929 cells expressing hSCARB2. Using hSCARB2-mScarb2 chimeric mutants, we attempted to map the region that was important for efficient EV71 infection. L929 cells expressing chimeras that carried amino acids 142 to 204 from the human sequence were susceptible to EV71, while chimeras that carried the mouse sequence in this region were not. Moreover, this region was also critical for binding to virions. The determination of this region in hSCARB2 that is important for EV71 binding and infection greatly contributes to the understanding of virus-receptor interactions. Further studies will clarify the early steps of EV71 infection.
A Respiratory Syncytial Virus Replicon That Is Noncytotoxic and Capable of Long-Term Foreign Gene Expression
Authors: Malykhina Olga , Yednak Mark A. , Collins Peter L. , Olivo Paul D. , Peeples Mark E. 
Published: March 2011

ABSTRACT

Respiratory syncytial virus (RSV) infection of most cultured cell lines causes cell-cell fusion and death. Cell fusion is caused by the fusion (F) glycoprotein and is clearly cytopathic, but other aspects of RSV infection may also contribute to cytopathology. To investigate this possibility, we generated an RSV replicon that lacks all three of its glycoprotein genes and so cannot cause cell-cell fusion or virus spread. This replicon includes a green fluorescent protein gene and an antibiotic resistance gene to enable detection and selection of replicon-containing cells. Adaptive mutations in the RSV replicon were not required for replicon maintenance. Cells containing the replicon could be cloned and passaged many times in the absence of antibiotic selection, with 99% or more of the cells retaining the replicon after each cell division. Transient expression of the F and G (attachment) glycoproteins supported the production of virions that could transfer the replicon into most cell lines tested. Since the RSV replicon is not toxic to these cultured cells and does not affect their rate of cell division, none of the 8 internal viral proteins, the viral RNA transcripts, or the host response to these molecules or their activities is cytopathic. However, the level of replicon genome and gene expression is controlled in some manner well below that of complete virus and, as such, might avoid cytotoxicity. RSV replicons could be useful for cytoplasmic gene expression
in vitro
and
in vivo
and for screening for compounds active against the viral polymerase.
Electroporation Enhances Immunogenicity of a DNA Vaccine Expressing Woodchuck Hepatitis Virus Surface Antigen in Woodchucks
Authors: Liu Katherine H. , Ascenzi Mary A. , Bellezza Christine A. , Bezuidenhout Abraham J. , Cote Paul J. , Gonzalez-Aseguinolaza Gloria , Hannaman Drew , Luxembourg Alain , Evans Claire F. , Tennant Bud C. , Menne Stephan 
Published: March 2011

ABSTRACTThe development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection.
A Novel Respiratory Model of Infection with Monkeypox Virus in Cynomolgus Macaques
Authors: Goff Arthur J. , Chapman Jennifer , Foster Chad , Wlazlowski Carly , Shamblin Joshua , Lin Kenny , Kreiselmeier Norman , Mucker Eric , Paragas Jason , Lawler James , Hensley Lisa 
Published: March 2011

ABSTRACT
Variola, the causative agent of smallpox, and the related monkeypox virus are both select agents that, if purposefully released, would cause public panic and social disruption. For this reason research continues in the areas of animal model and therapeutic development. Orthopoxviruses show a widely varying degree of host specificity, making development of accurate animal models difficult. In this paper, we demonstrate a novel respiratory infection technique that resulted in “classic” orthopox disease in nonhuman primates and takes the field of research one step closer to a better animal model.
Development of a Glycoprotein D-Expressing Dominant-Negative and Replication-Defective Herpes Simplex Virus 2 (HSV-2) Recombinant Viral Vaccine against HSV-2 Infection in Mice
Authors: Akhrameyeva Natalie V. , Zhang Pengwei , Sugiyama Nao , Behar Samuel M. , Yao Feng 
Published: March 2011

ABSTRACTUsing the T-REx (Invitrogen, California) gene switch technology and a dominant-negative mutant polypeptide of herpes simplex virus 1 (HSV-1)-origin binding protein UL9, we previously constructed a glycoprotein D-expressing replication-defective and dominant-negative HSV-1 recombinant viral vaccine, CJ9-gD, for protection against HSV infection and disease. It was demonstrated that CJ9-gD is avirulent following intracerebral inoculation in mice, cannot establish detectable latent infection following different routes of infection, and offers highly effective protective immunity against primary HSV-1 and HSV-2 infection and disease in mouse and guinea pig models of HSV infections. Given these favorable safety and immunological profiles of CJ9-gD, aiming to maximize levels of HSV-2 glycoprotein D (gD2) expression, we have constructed an ICP0 null mutant-based dominant-negative and replication-defective HSV-2 recombinant, CJ2-gD2, that contains 2 copies of the gD2 gene driven by the tetracycline operator (tetO)-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2 expresses gD2 as efficiently as wild-type HSV-2 infection and can lead to a 150-fold reduction in wild-type HSV-2 viral replication in cells coinfected with CJ2-gD2 and wild-type HSV-2 at the same multiplicity of infection. CJ2-gD2 is avirulent following intracerebral injection and cannot establish a detectable latent infection following subcutaneous (s.c.) immunization. CJ2-gD2 is a more effective vaccine than HSV-1 CJ9-gD and a non-gD2-expressing dominant-negative and replication-defective HSV-2 recombinant in protection against wild-type HSV-2 genital disease. Using recall response, we showed that immunization with CJ2-gD2 elicited strong HSV-2-specific memory CD4+and CD8+T-cell responses. Collectively, given the demonstrated preclinical immunogenicity and its unique safety profiles, CJ2-gD2 represents a new class of HSV-2 replication-defective recombinant viral vaccines in protection against HSV-2 genital infection and disease.
The ZIIR Element of the Epstein-Barr Virus BZLF1 Promoter Plays a Central Role in Establishment and Maintenance of Viral Latency
Authors: Yu Xianming , McCarthy Patrick J. , Lim Hui-Jun , Iempridee Tawin , Kraus Richard J. , Gorlen Daniel A. , Mertz Janet E. 
Published: March 2011

ABSTRACT

The Epstein-Barr virus (EBV)
BZLF1
gene encodes the immediate-early (IE) protein Zta, which plays a central role in regulating the switch between viral latency and lytic replication. A silencing element, ZIIR, is located between the ZID and ZII positive regulatory elements in the
BZLF1
promoter Zp. We report here the phenotypes of variants of EBV strain B95.8 containing base substitution mutations in this ZIIR element. HEK293 cells infected with ZIIR mutant (ZIIRmt) virus produced at least 20-fold more viral IE Zta and Rta and early (E) EAD protein than did cells infected with the parental wild-type (WT) virus, leading to viral DNA replication and production of infectious virus. However, ZIIR mutant virus was 1/10 as efficient as WT virus in establishing proliferating B-cell clones following infection of human primary blood B cells. The ZIIRmt-infected lymphoblastoid cell lines (LCLs) that did grow out exhibited a phenotype similar to the one observed in 293 cells, including marked overproduction of IE and E gene products relative to WT-infected LCLs and lytic replication of the viral genome. Incubation of the ZIIRmt-infected LCLs with the chemical inducer 12-
O
-tetradecanoyl-phorbol-13-acetate (TPA) led to much greater activation of Zp than did the same treatment of WT- or ZVmt-infected LCLs. Furthermore, a protein kinase C (PKC) inhibitor, bis-indolylmaleimide, eliminated this activation by TPA. Thus, we conclude that ZIIR is a potent silencing element of Zp; it plays a key role in establishment and maintenance of EBV latency by inhibiting activation of Zp through the PKC signal transduction pathway.
Sclerosing polidocanol injections or arthroscopic shaving to treat patellar tendinopathy/jumper's knee? A randomised controlled study
Authors: Willberg Lotta , Sunding Kerstin , Forssblad Magnus , Fahlström Martin , Alfredson Håkan 
Published: March 2011

BackgroundProximal patellar tendinopathy/jumper's knee (PT/JK) is well known to be difficult to treat. Recent studies using an ultrasound and colour Doppler-based treatment approach on the dorsal side of the tendon, sclerosing polidocanol injections and ultrasound-guided arthroscopic shaving, have shown promising clinical results.ObjectivesTo compare the clinical effects after treatment with sclerosing polidocanol injections and arthroscopic shaving.Material and methods52 patellar tendons (43 men and two women) with ultrasound and colour Doppler-verified diagnosis of PT/JK were randomly assigned to treatment with ultrasound and colour Doppler-guided sclerosing polidocanol injections (group A) or ultrasound and colour Doppler-guided arthroscopic shaving (group B). All patients were involved in patellar tendon loading sports or recreational activities, and had had a long duration of pain symptoms from the proximal patellar tendon. Pain during patellar tendon loading activity, and at rest, before and after treatment (visual analogue scale; VAS), and patient satisfaction with the result of the treatment, was registered.ResultsAfter treatment, the patients treated with arthroscopic shaving had a significantly lower VAS score at rest and during activity, and were significantly more satisfied compared with the patients in the sclerosing injection group.ConclusionsBoth treatment with ultrasound and colour Doppler-guided sclerosing polidocanol injections and arthroscopic shaving showed good clinical results, but patients treated with arthroscopic shaving had less pain and were more satisfied with the treatment result. Because surgical treatment is a one-stage treatment return to sports was faster in this group.