bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transmission Studies of
Babesia microti
in
Ixodes ricinus
Ticks and Gerbils
Authors: Gray Jeremy , von Stedingk Lars Victor , Gürtelschmid Mikael , Granström Marta 
Published: July 2002

ABSTRACT

In order to investigate the possible role of
Ixodes ricinus
as a vector of zoonotic
Babesia microti
infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected
B. microti
in the blood and spleens of infected gerbils (
Meriones unguiculatus
) and also in laboratory-induced infections of
I. ricinus
ticks.
B. microti
DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that
I. ricinus
could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal
I. ricinus
as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a
B. microti
strain (GI) from a distant geographical region (United States) can infect and be transmitted by
I. ricinus
suggests that other European
B. microti
strains, in addition to the HK strain used here, are probably infective for
I. ricinus
, supporting the view that infection of humans with European
B. microti
may be a regular occurrence.
Usefulness of Multilocus Sequence Typing for Characterization of Clinical Isolates of
Candida albicans
Authors: Bougnoux M.-E. , Morand S. , d'Enfert C. 
Published: July 2002

ABSTRACT

Molecular characterization of
Candida albicans
isolates is essential for understanding the epidemiology of nosocomial infections caused by this yeast. Here, we investigated the potential value of multilocus sequence typing (MLST) for characterizing epidemiologically related or unrelated
C. albicans
strains of various clinical origins. Accordingly, we sequenced the internal regions (loci) of six selected housekeeping genes of 40
C. albicans
clinical isolates and 2 reference strains. In all, 68 polymorphic nucleotide sites were identified, of which 65 were found to be heterozygous in at least one isolate. Ten to 24 different genotypes were observed at the different loci, resulting, when combined, in 39 unique genotype combinations or diploid sequence types (DSTs). When MLST was applied to 26 epidemiologically unrelated isolates and the 2 reference strains, it allowed the identification of 27 independent DSTs, thus demonstrating a discriminatory power of 99.7. Using multidimensional scaling together with the minimum spanning tree method to analyze interstrain relationships, we identified six groups of genetically related isolates on the basis of bootstrap values of greater than 900. Application of MLST to 14 epidemiologically related isolates showed that those recovered from patients in the same hospital ward during the same 3 months had specific DSTs, although 73% of these isolates were genetically very close. This suggests that MLST can trace minute variations in the sequences of related isolates. Overall, MLST proved to be a highly discriminatory and stable method for unambiguous characterization of
C. albicans
.
Reovirus Nonstructural Protein μNS Binds to Core Particles but Does Not Inhibit Their Transcription and Capping Activities
Authors: Broering Teresa J. , McCutcheon Aimee M. , Centonze Victoria E. , Nibert Max L. 
Published: July 2002

ABSTRACT
Previous studies provided evidence that nonstructural protein μNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68:455–466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of μNS. Given the possible role of μNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant μNS can bind to cores in vitro. The μNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of μNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of μNS were bound. Thus, the μNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the μNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to μNS. The μNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5′ ends of the transcripts. In vitro competition assays showed that mixing μNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins μ1 and ς3. These findings are consistent with the presence of μNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, μNS may block or delay outer-capsid assembly and allow continued transcription by these particles.
Differential Pathogenicity of Two Feline Leukemia Virus Subgroup A Molecular Clones, pFRA and pF6A
Authors: Phipps Andrew J. , Chen Hang , Hayes Kathleen A. , Roy-Burman Pradip , Mathes Lawrence E. 
Published: July 2002

ABSTRACT
F6A, a molecular clone of subgroup A feline leukemia virus (FeLV) is considered to be highly infectious but weakly pathogenic. In recent studies with a closely related subgroup A molecular clone, FRA, we demonstrated high pathogenicity and a strong propensity to undergo recombination with endogenous FeLV (enFeLV), leading to a high frequency of transition from subgroup A to A/B. The present study was undertaken to identify mechanisms of FeLV pathogenesis that might become evident by comparing the two closely related molecular clones. F6A was shown to have an infectivity similar to that of FRA when delivered as a provirus. Virus load and antibody responses were also similar, although F6A-infected cats consistently carried higher virus loads than FRA-infected cats. However, F6A-infected cats were slower to undergo de novo recombination with enFeLV and showed slower progression to disease than FRA-infected cats. Tumors collected from nine pF6A- or pFRA-inoculated cats expressed lymphocyte markers for T cells (seven tumors) and B cells (one tumor), and non-T/B cells (one tumor). One cat with an A-to-A/C conversion developed erythrocyte hypoplasia. Genomic mapping of recombinants from pF6A- and pFRA-inoculated cats revealed similar crossover sites, suggesting that the genomic makeup of the recombinants did not contribute to increased progression to neoplastic disease. From these studies, the mechanism most likely to account for the pathologic differences between F6A and FRA is the lower propensity for F6A to undergo de novo recombination with enFeLV in vivo. A lower recombination rate is predicted to slow the transition from subgroup A to A/B and slow the progression to disease.
Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo
Authors: Zheng Zhi-Ming , Quintero Jesse , Reid Eric S. , Gocke Christian , Baker Carl C. 
Published: July 2002

ABSTRACT
Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.
A Carboxy-Terminally Truncated Form of the Human Immunodeficiency Virus Type 1 Vpr Protein Induces Apoptosis via G
1
Cell Cycle Arrest
Authors: Nishizawa Masako , Kamata Masakazu , Katsumata Ryoichi , Aida Yoko 
Published: July 2002

ABSTRACT

Viral protein R (Vpr) of human immunodeficiency virus type 1 inhibits cell proliferation by arresting the cell cycle at the G
2
phase and inducing to apoptosis after G
2
arrest. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation via a novel pathway that is distinct from G
2
arrest. However, the mechanism of this effect of C81 is unknown. We demonstrate here that C81 can induce apoptosis via G
1
arrest of the cell cycle. Immunostaining for various markers of stages of the cell cycle and flow cytometry analysis of DNA content showed that most HeLa cells that had been transiently transfected with a C81 expression vector were arrested at the G
1
phase and not at the G
2
or S phase of the cell cycle. Staining for annexin V, which binds phosphatidylserine on the plasma membrane, as an early indicator of apoptosis and measurement of the activity of caspase-3, a signaling molecule in apoptotic pathways, indicated that C81 is a strong inducer of apoptosis. Expression of C81 induced the condensation, fragmentation, and clumping of chromatin that are typical of apoptosis. Furthermore, the kinetics of the C81-induced G
1
arrest were closely correlated with changes in the number of annexin V-positive cells and the activity of caspase-3. Replacement of Ile or Leu residues by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of C81 revealed that Ile60, Leu67, and Ile74 play important roles both in the C81-induced G
1
arrest and in apoptosis. Thus, it appears that C81 induces apoptosis through pathways that are identical to those utilized for G
1
arrest of the cell cycle. It has been reported that Ile60, Leu67, and Ile74 also play an important role in the C81-induced suppression of growth. These results suggest that the suppression of growth induced by C81 result in apoptosis that is independent of G
2
arrest of the cell cycle.
Deletion Mutagenesis Downstream of the 5′ Long Terminal Repeat of Human Immunodeficiency Virus Type 1 Is Compensated for by Point Mutations in both the U5 Region and
gag
Gene
Authors: Liang Chen , Rong Liwei , Russell Rodney S. , Wainberg Mark A. 
Published: July 2002

ABSTRACT

We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5′ long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA
3
Lys
primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the
gag
gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels.
Trafficking of Varicella-Zoster Virus Glycoprotein gI: T
338
-Dependent Retention in the
trans
-Golgi Network, Secretion, and Mannose 6-Phosphate-Inhibitable Uptake of the Ectodomain
Authors: Wang Zuo-Hong , Gershon Michael D. , Lungu Octavian , Zhu Zhenglun , Gershon Anne A. 
Published: July 2002

ABSTRACT

The
trans
-Golgi network (TGN) is putatively the site where varicella-zoster virus is enveloped. gE is targeted to the TGN by selective retrieval from the plasmalemma in response to signaling sequences in its endodomain. gI lacks these sequences but forms a complex with gE. We now find that gI is targeted to the TGN and plasma membrane when expressed in Cos-7 cells; nevertheless, surface labeling revealed that gI is not retrieved from the plasma membrane. TGN targeting of gI depended on the T
338
of its endodomain and was lost when T
338
was deleted or mutated to A, S, or D. The endodomain of gI was sufficient, if it contained T
338
, to target a fusion protein containing the ectodomain of the human interleukin-2 receptor to the TGN. A truncated protein consisting only of the gI ectodomain was secreted and taken up by nontransfected cells. This uptake of the secreted gI ectodomain was blocked by mannose 6-phosphate. Following cotransfection, both gI and gE were retrieved to the TGN from the plasma membrane in 26.7% of cells, neither gI nor gE was internalized in 18.3%, and gE was retrieved to the TGN while gI remained at the plasma membrane in 55%. We suggest that the T
338
of its endodomain is necessary to retain gI in the TGN; moreover, because gI and gE interact, the signaling sequences of each glycoprotein reinforce one another in ensuring that both glycoproteins are concentrated in the TGN yet remain on the cell surface.
Human Parainfluenza Virus Type 3 (PIV3) Expressing the Hemagglutinin Protein of Measles Virus Provides a Potential Method for Immunization against Measles Virus and PIV3 in Early Infancy
Authors: Durbin Anna P. , Skiadopoulos Mario H. , McAuliffe Josephine M. , Riggs Jeffrey M. , Surman Sonja R. , Collins Peter L. , Murphy Brian R. 
Published: July 2002

ABSTRACT

Recombinant human parainfluenza virus type 3 (PIV3) was used as a vector to express the major protective antigen of measles virus, the hemagglutinin (HA) glycoprotein, in order to create a bivalent PIV3-measles virus that can be administered intranasally. The measles virus HA open reading frame (ORF) was inserted as an additional transcriptional unit into the N-P, P-M, or HA-neuraminidase (HN)-L gene junction of wild-type PIV3 or into the N-P or P-M gene junction of an attenuated derivative of PIV3, termed r
cp
45L. The recombinant PIV3 (rPIV3) viruses bearing the HA inserts replicated more slowly in vitro than their parental viruses but reached comparable peak titers of ≥10
7.5
50% tissue culture infective doses per ml. Each of the wild-type or cold-passaged 45L (
cp
45L) PIV3(HA) chimeric viruses replicated 5- to 10-fold less well than its respective parent virus in the upper respiratory tract of hamsters. Thus, insertion of the ∼2-kb ORF itself conferred attenuation, and this attenuation was additive to that conferred by the
cp
45L mutations. The attenuated
cp
45L PIV3(HA) recombinants induced a high level of resistance to replication of PIV3 challenge virus in hamsters and induced very high levels of measles virus neutralizing antibodies (>1:8,000) that are well in excess of those known to be protective in humans. rPIV3s expressing the HA gene in the N-P or P-M junction induced about 400-fold more measles virus-neutralizing antibody than did the rPIV3 with the HA gene in the HN-L junction, indicating that the N-P or P-M junction appears to be the preferred insertion site. Previous studies indicated that the PIV3
cp
45 virus, a more attenuated version of r
cp
45L, replicates efficiently in the respiratory tract of monkeys and is immunogenic and protective even when administered in the presence of very high titers of passively transferred PIV3 antibodies (A. P. Durbin, C. J. Cho, W. R. Elkins, L. S. Wyatt, B. Moss, and B. R. Murphy, J. Infect. Dis. 179:1345–1351, 1999). This suggests that this intranasally administered PIV3(HA) chimeric virus can be used to immunize infants with maternally acquired measles virus antibodies in whom the current parenterally administered live measles virus vaccine is ineffective.
Role of Human Cytomegalovirus Immediate-Early Proteins in Cell Growth Control
Authors: Castillo Jonathan P. , Yurochko Andrew D. , Kowalik Timothy F. 
Published: July 2002

ABSTRACT

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in several disorders, including an association between HCMV reactivation and the overproliferation of arterial smooth muscle cells observed in restenosis. Although HCMV can mediate a growth-arrest phenotype in infected cells, the virus can also promote an environment conducive to proliferation. Here, we present evidence that the HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, may be responsible for inducing this proliferative environment by altering cell cycle control. We find that expression of either of these IE proteins can alter the cell cycle distribution of randomly cycling cells towards S and G
2
/M phases. Additionally, we find that expression of IE2-86, but not IE1-72, induces quiescent cells into S phase and delays cell cycle exit. In the absence of p53, IE1-72 expression can induce S phase and delay cell cycle exit. We also demonstrate that p53 protein levels increase in fibroblasts following the expression of IE1-72. The observed accumulation of p53 protein in IE1-72-expressing cells may account for the inability of IE1-72 to induce S phase and delay cell cycle exit. Our data suggest that expression of HCMV IE1-72 and IE2-86 is sufficient to alter the cell cycle to generate an environment conducive to proliferation.
Membrane Targeting Properties of a Herpesvirus Tegument Protein-Retrovirus Gag Chimera
Authors: Bowzard J. Bradford , Visalli Robert J. , Wilson Carol B. , Loomis Joshua S. , Callahan Eric M. , Courtney Richard J. , Wills John W. 
Published: July 2002

ABSTRACT

The retroviral Gag protein is capable of directing the production and release of virus-like particles in the absence of all other viral components. Budding normally occurs after Gag is transported to the plasma membrane by its membrane-targeting and -binding (M) domain. In the Rous sarcoma virus (RSV) Gag protein, the M domain is contained within the first 86 amino acids. When M is deleted, membrane association and budding fail to occur. Budding is restored when M is replaced with foreign membrane-binding sequences, such as that of the Src oncoprotein. Moreover, the RSV M domain is capable of targeting heterologous proteins to the plasma membrane. Although the solution structure of the RSV M domain has been determined, the mechanism by which M specifically targets Gag to the plasma membrane rather than to one or more of the large number of internal membrane surfaces (e.g., the Golgi apparatus, endoplasmic reticulum, and nuclear, mitochondrial, or lysosomal membranes) is unknown. To further investigate the requirements for targeting proteins to discrete cellular locations, we have replaced the M domain of RSV with the product of the unique long region 11 (U
L
11) gene of herpes simplex virus type 1. This 96-amino-acid myristylated protein is thought to be involved in virion transport and envelopment at internal membrane sites. When the first 100 amino acids of RSV Gag (including the M domain) were replaced by the entire UL11 sequence, the chimeric protein localized at and budded into the Golgi apparatus rather than being targeted to the plasma membrane. Myristate was found to be required for this specific targeting, as were the first 49 amino acids of UL11, which contain an acidic cluster motif. In addition to shedding new light on UL11, these experiments demonstrate that RSV Gag can be directed to internal cellular membranes and suggest that regions outside of the M domain do not contain a dominant plasma membrane-targeting motif.
Effects of Genomic Length on Translocation of Hepatitis B Virus Polymerase-Linked Oligomer
Authors: Ho Tsung-Chuan , Jeng King-Song , Hu Cheng-Po , Chang Chungming 
Published: July 2002

ABSTRACT
Accurate translocation of the polymerase-linked oligomer to the acceptor site (DR1*) in reverse transcription is crucial for maintaining the correct size of the hepatitis B virus (HBV) genome. Various sizes of foreign sequences were inserted at different sites of the HBV genome, and their effects on accurate translocation of polymerase-linked oligomer to DR1* were tested. Three types of replicate DNA products were observed in these insertion mutants: RC (relaxed circle) and type I and type II DL (duplex linear) DNA. Our results indicated that the minus strand of RC and type I DL form was elongated from DR1*, while the minus strand of the type II DL form was elongated from multiple internal acceptor sites (IAS), such as IAS2. These IASs were also found to be used by wild-type HBV but with a very low frequency. Mutation of IAS2 by base substitution abrogated polymerase-linked oligomer transferring to IAS2, demonstrating that base pairing also plays an important role in the function of IAS2 as a polymerase-linked oligomer acceptor site. Data obtained from our insertion mutants also demonstrate that the distance between the polymerase-linked oligomer priming site and the acceptor is important. The polymerase-linked oligomer prefers to translocate to an acceptor, DR1* or IAS2, which are ca. 3.2 kb apart. However, it will translocate to both DR1* and IAS2 if they are not located 3.2 kb apart. These results suggest that the polymerase-linked oligomer may be able to scan bidirectionally for appropriate acceptor sites at a distance of 3.2 kb. A model is proposed to discuss the possible mechanism of polymerase-linked oligomer translocation.