bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence of Extrahepatic Sites of Replication of the Hepatitis E Virus in a Swine Model
Authors: Williams T. P. E. , Kasorndorkbua C. , Halbur P. G. , Haqshenas G. , Guenette D. K. , Toth T. E. , Meng X. J. 
Published: July 2002

ABSTRACT
Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. Our recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication and pathogenesis in a swine model. In an early study, we experimentally infected specific-pathogen-free pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were inoculated intravenously with swine HEV, 19 pigs (group 2) were inoculated with the US-2 strain of human HEV, and 17 pigs (group 3) were used as uninoculated controls. The clinical and pathological findings have been previously reported. In this expanded study, we aim to identify the potential extrahepatic sites of HEV replication using the swine model. Two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days postinoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive-strand HEV RNA in each tissue collected during necropsy at different DPI. A negative-strand-specific RT-PCR was standardized and used to detect the replicative, negative strand of HEV RNA from tissues that tested positive for the positive-strand RNA. As expected, positive-strand HEV RNA was detected in almost every type of tissue at some time point during the viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine HEV- and human HEV-inoculated pigs. However, replicative, negative-strand HEV RNA was detected primarily in the small intestines, lymph nodes, colons, and livers. Our results indicate that HEV replicates in tissues other than the liver. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV.
Multicenter Proficiency Testing of Nucleic Acid Amplification Methods for the Detection of Enteroviruses
Authors: Van Vliet Karin E. , Muir Peter , Echevarria Jose M. , Klapper Paul E. , Cleator Graham M. , Van Loon Anton M. 
Published: July 2002

ABSTRACT
A multicenter study of molecular detection of enteroviruses was conducted using a proficiency panel. Of 70 data sets, 46 (66%) reported correct results for samples containing at least 1 50% infective dose per ml and for negative samples. Variation in performance between laboratories demonstrates the need for ongoing quality control.
Development and Evaluation of a Quantitative, Touch-Down, Real-Time PCR Assay for Diagnosing
Pneumocystis carinii
Pneumonia
Authors: Larsen Hans Henrik , Masur Henry , Kovacs Joseph A. , Gill Vee J. , Silcott Victoria A. , Kogulan Palaniandy , Maenza Janine , Smith Margo , Lucey Daniel R. , Fischer Steven H. 
Published: July 2002

ABSTRACT

A rapid (time to completion, <4 h, including DNA extraction) and quantitative touch-down (QTD) real-time diagnostic
Pneumocystis carinii
PCR assay with an associated internal control was developed, using fluorescence resonance energy transfer (FRET) probes for detection. The touch-down procedure significantly increased the sensitivity of the assay compared to a non-touch-down procedure. Tenfold serial dilutions of a cloned target were used as standards for quantification.
P. carinii
DNA has been detected in respiratory specimens from patients with
P. carinii
pneumonia (PCP) and from patients without clinical evidence of PCP. The latter probably represents colonization or subclinical infection. It is logical to hypothesize that quantification might prove helpful in distinguishing between infected and colonized patients: the latter group would have lower copy numbers than PCP patients. A blinded retrospective study of 98 respiratory samples (49 lower respiratory tract specimens and 49 oral washes), from 51 patients with 24 episodes of PCP and 34 episodes of other respiratory disease, was conducted. PCR-positive samples from colonized patients contained a lower concentration of
P. carinii
DNA than samples from PCP patients: lower respiratory tract samples from PCP and non-PCP patients contained a median of 938 (range, 2.4 to 1,040,000) and 2.6 (range, 0.3 to 248) (
P
< 0.0004) copies per tube, respectively. Oral washes from PCP and non-PCP patients contained a median of 49 (range, 2.1 to 2,595) and 6.5 (range, 2.2 to 10) (
P
< 0.03) copies per tube, respectively. These data suggest that this QTD PCR assay can be used to determine if
P. carinii
is present in respiratory samples and to distinguish between colonization and infection.
Detection of
seg
,
seh
, and
sei
genes in
Staphylococcus aureus
Isolates and Determination of the Enterotoxin Productivities of
S. aureus
Isolates Harboring
seg
,
seh
, or
sei
Genes
Authors: Omoe Katsuhiko , Ishikawa Machiko , Shimoda Yu , Hu Dong-Liang , Ueda Shigeko , Shinagawa Kunihiro 
Published: July 2002

ABSTRACT

To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (
sea
to
sei
) in
Staphylococcus aureus
, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%)
S. aureus
isolates were found to be positive for one or more
se
genes. The
se
genotype was classified into 14 genotypes.
seg
and
sei
coexisted in the same
S. aureus
strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most
seh
-harboring
S. aureus
isolates were able to produce a significant amount of SEH. However, most of the
S. aureus
isolates harboring
seg
and about 60% of the isolates harboring
sei
did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in
S. aureus
strains harboring
seg
and
sei
genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.
Real-Time PCR for Detection and Quantitation of
Leishmania
in Mouse Tissues
Authors: Nicolas Luc , Prina Eric , Lang Thierry , Milon Geneviève 
Published: July 2002

ABSTRACT

Leishmania
spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the
Leishmania
burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative
Leishmania major
burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of
L. major
DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 10
4
parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of
L. donovani
and
L. amazonensis
, but it was 100-fold less sensitive for
L. mexicana
. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of
Leishmania
kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of
Leishmania
spp
.
Identification of the
Lymantria dispar
Nucleopolyhedrovirus Envelope Fusion Protein Provides Evidence for a Phylogenetic Division of the
Baculoviridae
Authors: Pearson Margot N. , Groten Christoph , Rohrmann George F. 
Published: July 2002

ABSTRACT

The complete genome sequences of a number of diverse members of the
Baculoviridae
including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of
Autographa californica
multinucleocapsid NPV (Ac
M
NPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses,
Lymantria dispar M
NPV (Ld
M
NPV), revealed a single open reading frame (
ld130
) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length
ld130
gene and of an
ld130
-enhanced green fluorescent protein gene (
egfp
) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with Ld
M
NPV or transfected with
ld130-egfp
. In addition, cells transfected with either
ld130
or
ld130-egfp
or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of
gp64
appears to be confined to a relatively closely related group of NPVs, homologs of
ld130
are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.
Evolution of Lamivudine Resistance in Human Immunodeficiency Virus Type 1-Infected Individuals: the Relative Roles of Drift and Selection
Authors: Frost Simon D. W. , Nijhuis Monique , Schuurman Rob , Boucher Charles A. B. , Brown Andrew J. Leigh 
Published: July 2002

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) rapidly develops resistance to lamivudine during monotherapy, typically resulting in the appearance at position 184 in reverse transcriptase (RT) of isoleucine instead of the wild-type methionine (M184I) early in therapy, which is later replaced by valine (M184V). M184V reduces viral susceptibility to drug in vitro by approximately 100-fold, but also results in a lower processivity of RT. We show that a drop in absolute viral fitness associated with the outgrowth of M184V results in a drop in viral load only in individuals with high CD4
+
counts, from whom we estimate the relative fitness of M184V in the presence of drug to be approximately 10% of that of the wild type prior to therapy. The timing of emergence of the M184V mutant varies widely between infected individuals. From analysis of the frequency of M184I and M184V mutants determined at multiple time points in seven individuals during lamivudine therapy, we estimated the fitness advantage of M184V over M184I during therapy to be approximately 23% on average. We have also estimated the average ratio of the frequencies of the two mutants prior to therapy to be 0.2:1, with a range from 0.12:1 to 0.33:1. We have found that the differences between individuals in the rate of evolution of lamivudine resistance arise due to genetic drift affecting the relative frequency of M184I and M184V prior to therapy. These results show that stochastic effects can be significant in HIV evolution, even when there is large fitness difference between mutant and wild-type variants.
Definition of Five New Simian Immunodeficiency Virus Cytotoxic T-Lymphocyte Epitopes and Their Restricting Major Histocompatibility Complex Class I Molecules: Evidence for an Influence on Disease Progression
Authors: Evans David T. , Jing Peicheng , Allen Todd M. , O'Connor David H. , Horton Helen , Venham John E. , Piekarczyk Marian , Dzuris John , Dykhuzen Marta , Mitchen Jacque , Rudersdorf Richard A. , Pauza C. David , Sette Alessandro , Bontrop Ronald E. , DeMars Robert , Watkins David I. 
Published: July 2002

ABSTRACTSimian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (borc). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypesbandcseparately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at theirMamu-DRBloci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.
Evasion of Host Defenses by Measles Virus: Wild-Type Measles Virus Infection Interferes with Induction of Alpha/Beta Interferon Production
Authors: Naniche Denise , Yeh Annie , Eto Danelle , Manchester Marianne , Friedman Robert M. , Oldstone Michael B. A. 
Published: July 2002

ABSTRACTMeasles is a highly contagious disease currently responsible for over one million childhood deaths, particularly in the developing world. Since alpha/beta interferons (IFNs) are pivotal players both in nonspecific antiviral immunity and in specific cellular responses, their induction or suppression by measles virus (MV) could influence the outcome of a viral infection. In this study we compare the IFN induction and sensitivity of laboratory-passaged attenuated MV strains Edmonston and Moraten with those of recent wild-type viruses isolated and passaged solely on human peripheral blood mononuclear cells (PBMC) or on the B958 marmoset B-cell line. We report that two PBMC-grown wild-type measles isolates and two B958-grown strains of MV induce 10- to 80-fold-lower production of IFN by phytohemagglutinin-stimulated peripheral blood lymphocytes (PBL) compared to Edmonston and Moraten strains of measles. Preinfection of PBL with these non-IFN-inducing MV isolates prevents Edmonston-induced but not double-stranded-RNA-induced IFN production. This suggests that the wild-type viruses can actively inhibit Edmonston-induced IFN synthesis and that this is not occurring by double-stranded RNA. Furthermore, the wild-type MV is more sensitive than Edmonston MV to the effect of IFN. MV is thus able to suppress the synthesis of the earliest mediator of antiviral immunity, IFN-α/β. This could have important implications in the virulence and spread of MV.
Characterization of the Coronavirus M Protein and Nucleocapsid Interaction in Infected Cells
Authors: Narayanan Krishna , Maeda Akihiko , Maeda Junko , Makino Shinji 
Published: July 2002

ABSTRACT
Coronavirus contains three envelope proteins, M, E and S, and a nucleocapsid, which consists of genomic RNA and N protein, within the viral envelope. We studied the macromolecular interactions involved in coronavirus assembly in cells infected with a murine coronavirus, mouse hepatitis virus (MHV). Coimmunoprecipitation analyses demonstrated an interaction between N protein and M protein in infected cells. Pulse-labeling experiments showed that newly synthesized, unglycosylated M protein interacted with N protein in a pre-Golgi compartment, which is part of the MHV budding site. Coimmunoprecipitation analyses further revealed that M protein interacted with only genomic-length MHV mRNA, mRNA 1, while N protein interacted with all MHV mRNAs. These data indicated that M protein interacted with the nucleocapsid, consisting of N protein and mRNA 1, in infected cells. The M protein-nucleocapsid interaction occurred in the absence of S and E proteins. Intracellular M protein-N protein interaction was maintained after removal of viral RNAs by RNase treatment. However, the M protein-N protein interaction did not occur in cells coexpressing M protein and N protein alone. These data indicated that while the M protein-N protein interaction, which is independent of viral RNA, occurred in the M protein-nucleocapsid complex, some MHV function(s) was necessary for the initiation of M protein-nucleocapsid interaction. The M protein-nucleocapsid interaction, which occurred near or at the MHV budding site, most probably represented the process of specific packaging of the MHV genome into MHV particles.
The 60-Residue C-Terminal Region of the Single-Stranded DNA Binding Protein of Herpes Simplex Virus Type 1 Is Required for Cooperative DNA Binding
Authors: Mapelli Marina , Mühleisen Martin , Persico Giorgia , van der Zandt Hans , Tucker Paul A. 
Published: July 2002

ABSTRACTICP8 is the major single-stranded DNA (ssDNA) binding protein of the herpes simplex virus type 1 and is required for the onset and maintenance of viral genomic replication. To identify regions responsible for the cooperative binding to ssDNA, several mutants of ICP8 have been characterized. Total reflection X-ray fluorescence experiments on the constructs confirmed the presence of one zinc atom per molecule. Comparative analysis of the mutants by electrophoretic mobility shift assays was done with oligonucleotides for which the number of bases is approximately that occluded by one protein molecule. The analysis indicated that neither removal of the 60-amino-acid C-terminal region nor Cys254Ser and Cys455Ser mutations qualitatively affect the intrinsic DNA binding ability of ICP8. The C-terminal deletion mutants, however, exhibit a total loss of cooperativity on longer ssDNA stretches. This behavior is only slightly modulated by the two-cysteine substitution. Circular dichroism experiments suggest a role for this C-terminal tail in protein stabilization as well as in intermolecular interactions. The results show that the cooperative nature of the ssDNA binding of ICP8 is localized in the 60-residue C-terminal region. Since the anchoring of a C- or N-terminal arm of one protein onto the adjacent one on the DNA strand has been reported for other ssDNA binding proteins, this appears to be the general structural mechanism responsible for the cooperative ssDNA binding by this class of protein.
Bovine Parainfluenza Virus Type 3 (BPIV3) Fusion and Hemagglutinin-Neuraminidase Glycoproteins Make an Important Contribution to the Restricted Replication of BPIV3 in Primates
Authors: Schmidt Alexander C. , McAuliffe Josephine M. , Huang Anne , Surman Sonja R. , Bailly Jane E. , Elkins William R. , Collins Peter L. , Murphy Brian R. , Skiadopoulos Mario H. 
Published: July 2002

ABSTRACT

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F
H
HN
H
) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F
H
HN
H
replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F
H
HN
H
conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F
H
HN
H
as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.
The Nuclear Export Signal within the E4orf6 Protein of Adenovirus Type 5 Supports Virus Replication and Cytoplasmic Accumulation of Viral mRNA
Authors: Weigel Silke , Dobbelstein Matthias 
Published: July 2002

ABSTRACT
During the late phase of adenovirus infection, viral mRNA is efficiently transported from the nucleus to the cytoplasm while most cellular mRNA species are retained in the nucleus. Two viral proteins, E1B-55 kDa and E4orf6, are both necessary for these effects. The E4orf6 protein of adenovirus type 5 binds and relocalizes E1B-55 kDa, and the complex of the two proteins was previously shown to shuttle continuously between the nucleus and cytoplasm. Nucleocytoplasmic transport of the complex is achieved by a nuclear export signal (NES) within E4orf6. Mutation of this signal sequence severely reduces the ability of the E1B-55 kDa–E4orf6 complex to leave the nucleus. Here, we examined the role of functional domains within E4orf6 during virus infection. E4orf6 or mutants derived from it were transiently expressed, followed by infection with recombinant adenovirus lacking the E4 region and determination of virus yield. An arginine-rich putative alpha helix near the carboxy terminus of E4orf6 contributes to E1B-55 kDa binding and relocalization as well as to the synthesis of viral DNA, mRNA, and proteins. Further mutational analysis revealed that mutation of the NES within E4orf6 considerably reduces its ability to support virus production. The same effect was observed when nuclear export was blocked with a competitor. Further, a functional NES within E4orf6 contributed to the efficiency of late virus protein synthesis and viral DNA replication, as well as total and cytoplasmic accumulation of viral late mRNA. Our data support the view that NES-mediated nucleocytoplasmic shuttling strongly enhances most, if not all, intracellular activities of E4orf6 during the late phase of adenovirus infection.