bluuebunny/crossref_metadata_2025 huggingface dataset:
16S Ribosomal DNA Sequence Analysis Distinguishes Biotypes of
Streptococcus bovis
:
Streptococcus bovis
Biotype II/2 Is a Separate Genospecies and the Predominant Clinical Isolate in Adult Males
Authors: Clarridge Jill E. , Attorri Silvia M. , Zhang Qing , Bartell John
Published: July 2002

We characterized 22 human clinical strains of
Streptococcus bovis
by genotypic (16S rRNA gene sequence analysis [MicroSeq]; Applied Biosystems, Foster City, Calif.) and phenotypic (API 20 Strep and Rapid ID32 Strep systems (bioMerieux Vitek, Hazelton, Mo.) methods. The strains, isolated from blood, cerebrospinal fluid (CSF), and urine, formed two distinct 16S ribosomal DNA sequence clusters. Three strains which were associated with endocarditis urinary tract infection (UTI), and sepsis clustered with the
S. bovis
type strain ATCC 33317 (cluster 1); other closely related type strains were
S. equinus
and
S. infantarius
. Nineteen strains clustered at a distance of about 2.5% dissimilarity to the
S. bovis
type strain (cluster 2) and were associated with central nervous system (CNS) disease in addition to endocarditis, UTI, and sepsis. All strains were distinct from
S. gallolyticus
. Within cluster 2, a single strain grouped with ATCC strain 43143 (cluster 2a) and may be phenotypically distinct. All the other strains formed a second subgroup (cluster 2b) that was biochemically similar to
S. bovis
biotype II/2 (mannitol negative and beta galactosidase, alpha galactosidase, beta glucuronidase, and trehalose positive). The API 20 Strep system identified isolates of cluster 2b as
S. bovis
biotype II/2, those of cluster 1 as
S. bovis
biotype II/1, and that of cluster 2a as
S. bovis
biotype I. There was an excellent correlation of biotype and genotype:
S. bovis
biotype II/2 isolates form a separate genospecies distinct from the
S. bovis, S. gallolyticus
, and
S. infantarius
type strains and are the most common isolates in adult males.
Myxobolus
sp., Another Opportunistic Parasite in Immunosuppressed Patients?
Authors: Moncada Ligia I. , López Myriam C. , Murcia Martha I. , Nicholls Santiago , León Frecia , Guı́o Olga Lucia , Corredor Augusto
Published: July 2002

During a study of intestinal parasitic infections in human immunodeficiency virus-positive patients, a parasite belonging to the phylum
Myxozoa
, recently described from human samples, was identified in one sample. When this parasite was stained by the modified Ziehl-Neelsen staining method, the features of the spores were identified: they were pyriform in shape, had thick walls, and had one suture and two polar capsules, with each one having four or five coils. The suture and two polar capsules were observed with the chromotrope-modified stain. The number of stools passed was more than 30 per day, but oocysts of
Isospora belli
were also found. Upon reexamination of some formalin- or merthiolate-iodine-formaldehyde-preserved samples an identical parasite was found in another sample from a patient presenting with diarrhea.
Strongyloides stercoralis
larvae and eggs of
Hymenolepis nana
and
Ascaris lumbricoides
were also found in this sample. Given that both patients were also infected with other pathogens that cause diarrhea, the possible pathogenic role of this parasite could not be established. The probable route of infection also could not be established.
Simple and Efficient Method for Measuring Anti-Toxoplasma Immunoglobulin Antibodies in Human Sera Using Complement-Mediated Lysis of Transgenic Tachyzoites Expressing β-Galactosidase
Authors: Dando Caroline , Gabriel Katie E. , Remington Jack S. , Parmley Stephen F.
Published: July 2002

A simple and efficient method using transgenic
Toxoplasma gondii
tachyzoites expressing β-galactosidase was developed for detection of specific antibodies against the parasite in sera of patients. The titers obtained with the new test were similar to those obtained with the Sabin-Feldman dye test run in parallel. Although significant changes in endpoint titers were not observed when sera drawn sequentially at 2- to 3-week intervals were tested with both procedures, apparent differences in antibody affinity were observed with the new test which were not perceptible with the Sabin-Feldman dye test. Like the Sabin-Feldman dye test, the new test is based on complement lysis of tachyzoites, but it is much easier to perform and the reaction is read colorimetrically instead of visually.
Citrate Synthase Gene Sequence: a New Tool for Phylogenetic Analysis and Identification of
Ehrlichia
Authors: Inokuma Hisashi , Brouqui Philippe , Drancourt Michel , Raoult Didier
Published: July 2002

The sequence of the citrate synthase gene (
gltA
) of 13 ehrlichial species (
Ehrlichia chaffeensis
,
Ehrlichia canis
,
Ehrlichia muris
, an
Ehrlichia
species recently detected from
Ixodes ovatus
,
Cowdria ruminantium
,
Ehrlichia phagocytophila
,
Ehrlichia equi
, the human granulocytic ehrlichiosis [HGE] agent,
Anaplasma marginale
,
Anaplasma centrale
,
Ehrlichia sennetsu
,
Ehrlichia risticii
, and
Neorickettsia helminthoeca
) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial
gltA
genes are 1,197 bp (
E. sennetsu
and
E. risticii
) to 1,254 bp (
A. marginale
and
A. centrale
) long, and GC contents of the gene vary from 30.5% (
Ehrlichia
sp. detected from
I. ovatus
) to 51.0% (
A. centrale
). The percent identities of the
gltA
nucleotide sequences among ehrlichial species were 49.7% (
E. risticii
versus
A. centrale
) to 99.8% (HGE agent versus
E. equi
). The percent identities of deduced amino acid sequences were 44.4% (
E. sennetsu
versus
E. muris
) to 99.5% (HGE agent versus
E. equi
), whereas the homology range of 16S rRNA genes was 83.5% (
E. risticii
versus the
Ehrlichia
sp. detected from
I. ovatus
) to 99.9% (HGE agent,
E. equi
, and
E. phagocytophila
). The architecture of the phylogenetic trees constructed by
gltA
nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial
gltA
sequences, two sets of primers were designed to amplify tick-borne
Ehrlichia
and
Neorickettsia
genogroup
Ehrlichia
(
N. helminthoeca
,
E. sennetsu
, and
E. risticii
), respectively. Tick-borne
Ehrlichia
species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of
Acs
I and
Xho
I with the exception of
E. muris
and the very closely related ehrlichia derived from
I. ovatus
for which sequence analysis of the PCR product is needed. Similarly,
Neorickettsia
genogroup
Ehrlichia
species were specifically identified by RFLP patterns of
Rca
I digestion. If confirmed this technique will be useful in rapidly identifying
Ehrlichia
spp.
Diversity of
Babesia
Infecting European Sheep Ticks (
Ixodes ricinus
)
Authors: Duh Darja , Petrovec Miroslav , Avsic-Zupanc Tatjana
Published: July 2002

Questing
Ixodes ricinus
(Acari: Ixodidae) adult and nymphal ticks collected in various parts of Slovenia were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-ribosomal DNA [rDNA]). Thirteen of 135 ticks were found to contain babesial DNA. Sequence determination and analysis of amplified portions of nss-rDNA revealed their identity with
Babesia microti
and a high degree of homology with
Babesia odocoilei
and
Babesia divergens
. The results of this study represent the first genetic evidence of
B. microti
and
B. divergens
-like parasites in
I. ricinus
ticks in Europe.
Comparison of Six PCR Methods Using Peripheral Blood for Detection of Canine Visceral Leishmaniasis
Authors: Lachaud Laurence , Marchergui-Hammami Sarah , Chabbert Elisabeth , Dereure Jacques , Dedet Jean Pierre , Bastien Patrick
Published: July 2002

The objectives of this study were to compare the sensitivities and reliabilities of different PCR methods for the diagnosis and epidemiological study of canine visceral leishmaniasis (CVL) using dog blood. We chose to work with peripheral blood, as this type of sampling is noninvasive, straightforward, and easy to repeat. Six PCR methods were compared: three primer pairs target genomic DNA, and the other three target kinetoplast (mitochondrial) DNA. Sensitivity, specificity, reproducibility, and ease of interpretation without hybridization were evaluated for each method. The assessment was first performed using artificial samples. All methods could detect less than one parasite per reaction tube. However, the sensitivities varied among the different methods by a factor of 500 on purified cultivated parasites and by a factor of 10,000 on seeded dog blood samples (i.e., from 10 to 10−3parasite per ml of blood for the latter). Only four methods were found sufficiently reliable for the diagnosis of CVL. They were tested on 37 dogs living in an area of endemicity and grouped according to clinical status and specific serology. Only the two methods targeting kinetoplast DNA (K13A-K13B and RV1-RV2) could detect the parasite in 100% of symptomatic infected dogs. Similarly, all seropositive dogs were found PCR positive by these methods versus 62% by the genomic-DNA-based methods. Finally, these kinetoplast-based methods proved clearly superior to the others in the detection ofLeishmaniain asymptomatic dogs. Our data allow the discussion of the advantages and drawbacks of highly sensitive versus moderately sensitive PCR methods in diagnosis and prevalence studies of CVL.
Discordance between Genotypic and Phenotypic Drug Resistance Profiles in Human Immunodeficiency Virus Type 1 Strains Isolated from Peripheral Blood Mononuclear Cells
Authors: Sarmati Loredana , Nicastri Emanuele , Parisi Saverio G. , d'Ettorre Gabriella , Mancino Giorgio , Narciso Pasquale , Vullo Vincenzo , Andreoni Massimo
Published: July 2002

The aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern.
Pseudoepidemic Due to a Unique Strain of
Mycobacterium szulgai
: Genotypic, Phenotypic, and Epidemiological Analysis
Authors: Zhang Qing , Kennon Randa , Koza Maureen A. , Hulten Kristina , Clarridge Jill E.
Published: July 2002

In mid-1999, we noted multiple isolations at the Veterans Affairs Medical Center (VAMC) Houston Tex. of an unusual nonpigmented
Mycobacterium
species. Since, on the basis of 16S rRNA gene sequence analysis, the strains were identical to the
Mycobacterium szulgai
type strain and since
M. szulgai
has been reported only rarely as a commensal or environmental isolate, we were concerned about laboratory contamination, nosocomial spread, or even the possibility that this could be a novel organism associated with disease. Our investigation found that from 1999 to 2000, 37 strains of
M. szulgai
were isolated from patients at the VAMC (the base rate for the previous 10 years had been <1 isolation per year). We compared the phenotypic properties and genetic relatedness of these 37 strains (31 of which were nonpigmented) as well as eight stock strains and the
M. szulgai
type strain. All strains were similar in cellular fatty acid patterns, growth rates, and biochemical characteristics. However, we found three genogroups by gene sequence analysis. Genogroup I comprised the
M. szulgai
type strain, all the tested nonpigmented strains (27 of the 31 strains were tested), two pigmented strains isolated in 1999 and 2000, and five pigmented stock strains. Genogroup II comprised five pigmented strains: three were isolated from 1999 to 2000 and two were stock strains. The single strain (isolated in 1996) in genogroup III was pigmented and was the only strain associated with disease. Whereas the randomly amplified polymorphic DNA (RAPD) patterns of all nonpigmented strains were identical, indicating that they came from a common source (the pseudoepidemic strain), the RAPD patterns of the other strains were varied. In our investigation for a possible source, we found that there were no common reagents, specimen-processing or patient locations, or procedures linking the 31 pseudoepidemic strains. However, a nonpigmented
M. szulgai
strain with a gene sequence and RAPD pattern identical to those of the pseudoepidemic strain was recovered from a water storage tank serving the hospital. We concluded that the strains most likely originated from hospital water, which transiently inoculated our patients. Although no disease was associated with this cluster of isolates, the event was costly because identification was problematic and we could not easily discount the isolations, since most of the patients were immunocompromised and were candidates for opportunistic infection.
Population Structure and Genetic Diversity of
Actinobacillus actinomycetemcomitans
Strains Isolated from Localized Juvenile Periodontitis Patients
Authors: Kaplan Jeffrey B. , Schreiner Helen C. , Furgang David , Fine Daniel H.
Published: July 2002

The phylogeny of 20
Actinobacillus actinomycetemcomitans
strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (
lkt
), cytolethal distending toxin (
cdt
), major fimbrial subunit (
flp-1
), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that
A. actinomycetemcomitans
strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and
lkt
,
cdt
,
flp-1
, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of
A. actinomycetemcomitans
carry pathogenic potential.
New Yeast Species,
Malassezia dermatis
, Isolated from Patients with Atopic Dermatitis
Authors: Sugita Takashi , Takashima Masako , Shinoda Takako , Suto Hajime , Unno Tetsushi , Tsuboi Ryoji , Ogawa Hideoki , Nishikawa Akemi
Published: July 2002

Malassezia
species are considered to be one of the exacerbating factors in atopic dermatitis (AD). During examination of the cutaneous colonization of
Malassezia
species in AD patients, we found a new species on the surface of the patients' skin. Analysis of ribosomal DNA sequences suggested that the isolates belonged to the genus
Malassezia
. They did not grow in Sabouraud dextrose agar but utilized specific concentrations of Tween 20, 40, 60, and 80 as a lipid source. Thus, we concluded that our isolates were new members of the genus
Malassezia
and propose the name
Malassezia dermatis
sp. nov. for these isolates.
Equine Abortion Associated with the
Borrelia parkeri
-
B. turicatae
Tick-Borne Relapsing Fever Spirochete Group
Authors: Walker R. L. , Read D. H. , Hayes D. C. , Nordhausen R. W.
Published: July 2002

Direct amplification and sequencing of the 16S rRNA gene and a variable region of the flagellin gene from fetal liver-associated spirochetes belonging to the
Borrelia parkeri
-
B. turicatae
tick-borne relapsing fever spirochete group with a late-term abortion in a mare are described.
Rapid PCR-Based Identification of Methicillin-Resistant
Staphylococcus aureus
from Screening Swabs
Authors: Jonas D. , Speck M. , Daschner F. D. , Grundmann H.
Published: July 2002

A PCR identification of methicillin-resistant
Staphylococcus aureus
(MRSA), obviating the need for subculture on agar media, was investigated. The combination of MRSA detection by
mecA femB
PCR with prior enrichment in selective broth was tested for 439 swabs. PCR identified 36 MRSA-positive samples, in concordance with conventional methods.
Genotypic Characteristics of Two Serotypes ofBartonella henselae
Authors: La Scola Bernard , Liang Zhongxing , Zeaiter Zaher , Houpikian Pierre , Grimont Patrick A. D. , Raoult Didier
Published: July 2002

The study of 16S rRNA gene sequences of all isolates ofBartonella henselaeobtained in our laboratory and others from human patients or cats has revealed two genotypes according to the sequence of the 16S rRNA gene. Two isolates of these genotypes have previously been related to two different serotypes, and lack of cross-protection of the two serotypes has been demonstrated in cats. We investigated the grouping of eight strains ofB. henselaeon the basis of 16S ribosomal DNA, 35-kDa protein, Pap 31 protein, and internal transcribed spacer (ITS) gene sequencing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles; and monoclonal antibody reactivity studies. Houston-1, 90-615, and SA2 strains showed the same patterns in SDS-PAGE, but they differed from the patterns ofB. henselaeisolates URBHLLY8, URBHLIE9, Cat6, Fizz, and CAL-1. Nine monoclonal antibodies derived from BALB/c mice immunized withB. henselaeHouston-1 strain reacted only with strains Houston-1, 90-615, and SA2, and not with any otherBartonellastrains. The two serogroups corresponded with two genotypes based on differences in the sequences of the genes encoding 16S rRNA, 35-kDa protein, and Pap 31 protein. Sequences of ITS genes were highly divergent among strains, as each had a unique sequence and the subdivision was not supported by DNA-DNA relatedness study. Study of 22 additional strains ofB. henselaeisolated from French bacteremic cats demonstrated that they all belong to one or the other of the proposed serotype or genotype.
Antibiotic Resistance, Virulence Gene, and Molecular Profiles of Shiga Toxin-Producing
Escherichia coli
Isolates from Diverse Sources in Calcutta, India
Authors: Khan Asis , Das S. C. , Ramamurthy T. , Sikdar A. , Khanam J. , Yamasaki S. , Takeda Y. , Nair G. Balakrish
Published: July 2002

Antibiotic resistance, virulence gene, and molecular profiles of Shiga toxin-producing
Escherichia coli
(STEC) non-O157 strains isolated from human stool samples, cow stool samples, and beef samples over a period of 2 years in Calcutta, India, were determined. Resistance to one or more antibiotics was observed in 49.2% of the STEC strains, with some of the strains exhibiting multidrug resistance. The dominant combinations of virulence genes present in the strains studied were
stx
1
and
stx
2
(44.5% of strains) and
stx
1
,
stx
2
, and
hlyA
(enterohemorrhagic
E. coli
hemolysin gene) (19% of strains). Only 6.4% of the STEC strains harbored
eae
. The diversity of STEC strains from various sources was assessed by random amplification of polymorphic DNA (RAPD). STEC strains that gave identical or nearly similar DNA fingerprints in RAPD-PCR and had similar virulence genotypes were further characterized by pulsed-field gel electrophoresis (PFGE). Identical RAPD and PFGE profiles were observed in four sets of strains, with each set comprising two strains. There was no match in the RAPD and PFGE profiles between strains of STEC isolated from cows and those isolated from humans. It appears that the clones present in bovine sources are not transmitted to humans in the Calcutta setting although these strains showed evolutionary relatedness. Maybe for this reason, STEC has still not become a major problem in India.
Human Immunodeficiency Virus Type 1 Induces Apoptosis in CD4+but Not in CD8+T Cells in Ex Vivo-Infected Human Lymphoid Tissue
Authors: Grivel Jean-Charles , Malkevitch Nina , Margolis Leonid
Published: July 2002

Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4+T cells along with death and/or dysfunction of CD8+T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4+and CD8+T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4+T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8+T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4+T cells, neither viral isolate induced apoptosis in CD8+T cells. Moreover, in both infected and control tissues we found similar numbers of CD8+T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3–anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4+T cells, the death of CD8+T cells apparently requires additional factors.
Immunogenicity of Mutations Induced by Nucleoside Reverse Transcriptase Inhibitors for Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Cells
Authors: Samri Assia , Haas Gaby , Duntze Joerg , Bouley Jean-Marc , Calvez Vincent , Katlama Christine , Autran Brigitte
Published: July 2002

The impact of drug resistance mutations induced by nucleoside reverse transcriptase (RT) inhibitors (NRTI) on cytotoxic T-lymphocyte (CTL) recognition of human immunodeficiency virus type 1 strain LAI (HIV-1LAI) RT was addressed in 35 treated or untreated patients. Two HIV-1LAIRT regions encompassing mutation M41L, L74V, M184V, and T215Y/F were recognized in 75 and 83% mutated and in 33 and 42% unmutated samples, respectively. A total of 41 new CTL epitopes overlapping these mutations were predicted. Mutations enhanced HLA-binding scores of 17 epitopes, decreased scores of 5, and had no effect in 19. Four predicted epitopes containing mutations 41, 74, and 184 were tested and recognized by CD8 cells from mutated or unmutated samples, with frequencies up to 270 gamma interferon spot-forming cells per 106peripheral blood mononuclear cells. Therefore, RT mutations induced by NRTI can increase the immunogenicity of RT for CTL and might allow a better immune control of resistant viruses in vivo, suggesting that specific immune therapy might help prevent these mutations.
Efficacy of Postexposure Prophylaxis after Intravaginal Exposure of Pig-Tailed Macaques to a Human-Derived Retrovirus (Human Immunodeficiency Virus Type 2)
Authors: Otten Ron A. , Smith Dawn K. , Adams Debra R. , Pullium Jennifer K. , Jackson Eddie , Kim Caryn N. , Jaffe Harold , Janssen Robert , Butera Sal , Folks Thomas M.
Published: July 2002

Postexposure prophylaxis (PEP) after intravaginal exposure to human immunodeficiency virus (HIV) was investigated using the HIV type 2 (HIV-2)/pig-tailed macaque transmission model. PEP for 28 days with the reverse transcriptase inhibitor (
R
)-9-(2-phosphonylmethoxypropyl)adenine (PMPA; tenofovir) was initiated 12 to 72 h following HIV-2 exposure. Systemic infection was not evident in the 12- and 36-h groups, as defined by plasma viremia, cell-associated provirus, antibody responses, and lymph node virus. Breakthrough infection in the 72-h group was detected at week 16 post-virus exposure. These results demonstrate for the first time using a vaginal transmission model that early intervention after high-risk sexual exposures may prevent infection.
Evolution of Bovine Respiratory Syncytial Virus
Authors: Valarcher Jean-François , Schelcher François , Bourhy Hervé
Published: July 2002

Until now, the analysis of the genetic diversity of bovine respiratory syncytial virus (BRSV) has been based on small numbers of field isolates. In this report, we determined the nucleotide and deduced amino acid sequences of regions of the nucleoprotein (N protein), fusion protein (F protein), and glycoprotein (G protein) of 54 European and North American isolates and compared them with the sequences of 33 isolates of BRSV obtained from the databases, together with those of 2 human respiratory syncytial viruses and 1 ovine respiratory syncytial virus. A clustering of BRSV sequences according to geographical origin was observed. We also set out to show that a continuous evolution of the sequences of the N, G, and F proteins of BRSV has been occurring in isolates since 1967 in countries where vaccination was widely used. The exertion of a strong positive selective pressure on the mucin-like region of the G protein and on particular sites of the N and F proteins is also demonstrated. Furthermore, mutations which are located in the conserved central hydrophobic part of the ectodomain of the G protein and which result in the loss of four Cys residues and in the suppression of two disulfide bridges and an α helix critical to the three-dimensional structure of the G protein have been detected in some recent French BRSV isolates. This conserved central region, which is immunodominant in BRSV G protein, thus has been modified in recent isolates. This work demonstrates that the evolution of BRSV should be taken into account in the rational development of future vaccines.
The EICP22 Protein of Equine Herpesvirus 1 Physically Interacts with the Immediate-Early Protein and with Itself To Form Dimers and Higher-Order Complexes
Authors: Derbigny Wilbert A. , Kim Seong K. , Caughman Gretchen B. , O'Callaghan Dennis J.
Published: July 2002

The EICP22 protein (EICP22P) of
Equine herpesvirus 1
(EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione
S
-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.
The Ends on Herpesvirus DNA Replicative Concatemers Contain
pac2 cis
Cleavage/Packaging Elements and Their Formation Is Controlled by Terminal
cis
Sequences
Authors: McVoy Michael A. , Nixon Daniel E. , Hur Jay K. , Adler Stuart P.
Published: July 2002

Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that
cis
elements control the direction of packaging. Two highly conserved
cis
elements named
pac1
and
pac2
lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that
pac2
elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of
pac2
ends on concatemers depended on terminal
cis
sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of
pac2
ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which
pac2
elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.
Measles Virus Spread between Neurons Requires Cell Contact but Not CD46 Expression, Syncytium Formation, or Extracellular Virus Production
Authors: Lawrence Diane M. P. , Patterson Catherine E. , Gales Tracy L. , D'Orazio Joseph L. , Vaughn Melinda M. , Rall Glenn F.
Published: July 2002

In patients with subacute sclerosing panencephalitis (SSPE), which is associated with persistent measles virus (MV) infection in the brain, little infectious virus can be recovered despite the presence of viral RNA and protein. Based on studies of brain tissue from SSPE patients and our work with MV-infected NSE-CD46
+
mice, which express the measles receptor CD46 on neurons, several lines of evidence suggest that the mechanism of viral spread in the central nervous system differs from that in nonneuronal cells. To examine this alternate mechanism of viral spread, as well as the basis for the loss of normal transmission mechanisms, infection and spread of MV Edmonston was evaluated in primary CD46
+
neurons from transgenic mice and differentiated human NT2 neurons. As expected, unlike that between fibroblasts, viral spread between neurons occurred in the absence of syncytium formation and with minimal extracellular virus. Electron microscopy analysis showed that viral budding did not occur from the neuronal surface, although nucleocapsids were present in the cytoplasm and aligned at the cell membrane. We observed many examples of nucleocapsids present in the neuronal processes and aligned at presynaptic neuronal membranes. Cocultures of CD46
+
and CD46
−
neurons showed that cell contact but not CD46 expression is required for MV spread between neurons. Collectively, these results suggest that the neuronal environment prevents the normal mechanisms of MV spread between neurons at the level of viral assembly but allows an alternate, CD46-independent mechanism of viral transmission, possibly through the synapse.
Interaction of the Cauliflower Mosaic Virus Coat Protein with the Pregenomic RNA Leader
Authors: Guerra-Peraza Orlene , de Tapia Marc , Hohn Thomas , Hemmings-Mieszczak Maja
Published: July 2002

Using the yeast three-hybrid system, the interaction of theCauliflower mosaic virus(CaMV) pregenomic 35S RNA (pgRNA) leader with the viral coat protein, its precursor, and a series of derivatives was studied. The purine-rich domain in the center of the pgRNA leader was found to specifically interact with the coat protein. The zinc finger motif of the coat protein and the preceding basic domain were essential for this interaction. Removal of the N-terminal portion of the basic domain led to loss of specificity but did not affect the strength of the interaction. Mutations of the zinc finger motif abolished not only the interaction with the RNA but also viral infectivity. In the presence of the very acidic C-terminal domain, which is part of the preprotein but is not present in the mature CP, the interaction with the RNA was undetectable.
Genetic Evidence for an Interaction between Human Immunodeficiency Virus Type 1 Matrix and α-Helix 2 of the gp41 Cytoplasmic Tail
Authors: Murakami Tsutomu , Freed Eric O.
Published: July 2002

The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (α-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of α-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction.
Interaction between the Open Reading Frame III Product and the Coat Protein Is Required for Transmission of Cauliflower Mosaic Virus by Aphids
Authors: Leh Véronique , Jacquot Emmanuel , Geldreich Angèle , Haas Muriel , Blanc Stéphane , Keller Mario , Yot Pierre
Published: July 2002

Transmission of cauliflower mosaic virus (CaMV) by aphids requires two viral nonstructural proteins, the open reading frame (ORF) II and ORF III products (P2 and P3). An interaction between a C-terminal domain of P2 and an N-terminal domain of P3 is essential for transmission. Purified particles of CaMV are efficiently transmitted only if aphids, previously fed a P2-containing solution, are allowed to acquire a preincubated mixture of P3 and virions in a second feed, thus suggesting a direct interaction between P3 and coat protein. Herein we demonstrate that P3 directly interacts with purified viral particles and unassembled coat protein without the need for any other factor and that P3 mediates the association of P2 with purified virus particles. The interaction domain of P3 is located in its C-terminal half, downstream of the P3-P2 interaction domain but overlapping a region which binds nucleic acids. Mutagenesis of P3 which interferes with the interaction between P3 and virions is correlated with the loss of transmission by aphids. Taken together, our results demonstrate that P3 plays a crucial role in the formation of the CaMV transmissible complex by serving as a bridge between P2 and virus particles.
Alteration of Zinc-Binding Residues of Simian Immunodeficiency Virus p8
NC
Results in Subtle Differences in Gag Processing and Virion Maturation Associated with Degradative Loss of Mutant NC
Authors: Yovandich Jason L. , Chertova Elena N. , Kane Brad P. , Gagliardi Tracy D. , Bess Julian W. , Sowder Raymond C. , Henderson Louis E. , Gorelick Robert J.
Published: July 2002

In all retroviruses analyzed to date (except for the spumaretroviruses), the Zn
2+
-coordinating residues of nucleocapsid (NC) perform or assist in crucial reactions necessary to complete the retrovirus life cycle. Six replication-defective mutations have been engineered in the two NC Zn
2+
fingers (ZFs) of simian immunodeficiency virus [SIV(Mne)] that change or delete specific Zn
2+
-interacting Cys residues and were studied by using electron microscopy, reversed-phase high-performance liquid chromatography, immunoblotting, and RNA quantification. We focused on phenotypes of produced particles, specifically morphology, Gag polyprotein processing, and genomic RNA packaging. Phenotypes were similar among viruses containing a point or deletion mutation involving the same ZF. Mutations in the proximal ZF (ZF1) resulted in near-normal Gag processing and full-length genomic RNA incorporation and were most similar to wild-type (WT) virions with electron-dense, conical cores. Mutation of the distal ZF, as well as point mutations in both ZFs, resulted in more unprocessed Gag proteins than a deletion or point mutation in ZF1, with an approximate 30% reduction in levels of full-length genomic RNA in virions. These mutant virions contained condensed cores; however, the cores typically appeared less electron dense and more rod shaped than WT virions. Surprisingly, deletion of both ZFs, including the basic linker region between the ZFs, resulted in the most efficient Gag processing. However, genomic RNA packaging was ∼10% of WT levels, and those particles produced were highly abnormal with respect to size and core morphology. Surprisingly, all NC mutations analyzed demonstrated a significant loss of processed NC in virus particles, suggesting that Zn
2+
-coordinated NC is protected from excessive proteolytic cleavage. Together, these results indicate that Zn
2+
coordination is important for correct Gag precursor processing and NC protein stability. Additionally, SIV particle morphology appears to be the result of proper and complete Gag processing and relies less on full-length genomic RNA incorporation, as dictated by the Zn
2+
coordination in the ZFs of the NC protein.
The Ability of Integrin αvβ3To Function as a Receptor for Foot-and-Mouth Disease Virus Is Not Dependent on the Presence of Complete Subunit Cytoplasmic Domains
Authors: Neff Sherry , Baxt Barry
Published: July 2002

The integrin αvβ3has been shown to function as one of the integrin receptors on cultured cells for foot-and-mouth disease virus (FMDV), and high-efficiency utilization of the bovine homolog of this integrin is dependent on the cysteine-rich repeat region of the bovine β3subunit. In this study we have examined the role of the cytoplasmic domains of the αvand β3subunits in FMDV infection. We have found that truncations or extensions of these domains of either subunit, including deletions removing almost all of the cytoplasmic domains, had little or no effect on the ability of the integrin to function as a receptor for FMDV. The lysosomotropic agent monensin inhibited viral replication in cells transfected with either intact or cytoplasmic domain-truncated αvβ3. In addition, viral replication in transfected cells was inhibited by an αvβ3function-blocking antibody but not by function-blocking antibodies to three other RGD-directed integrins, suggesting that these integrins are not involved in the infectious process. These results indicate that alterations to the cytoplasmic domains of either subunit, which lead to the inability of the integrin receptor to function normally, do not abolish the ability of the integrin to bind and internalize this viral ligand.
Residue 627 of PB2 Is a Determinant of Cold Sensitivity in RNA Replication of Avian Influenza Viruses
Authors: Massin Pascale , van der Werf Sylvie , Naffakh Nadia
Published: July 2002

Human influenza A viruses replicate in the upper respiratory tract at a temperature of about 33°C, whereas avian viruses replicate in the intestinal tract at a temperature close to 41°C. In the present study, we analyzed the influence of low temperature (33°C) on RNA replication of avian and human viruses in cultured cells. The kinetics of replication of the NP segment were similar at 33 and 37°C for the human A/Puerto-Rico/8/34 and A/Sydney/5/97 viruses, whereas replication was delayed at 33°C compared to 37°C for the avian A/FPV/Rostock/34 and A/Mallard/NY/6750/78 viruses. Making use of a genetic system for the in vivo reconstitution of functional ribonucleoproteins, we observed that the polymerase complexes derived from avian viruses but not human viruses exhibited cold sensitivity in mammalian cells, which was determined mostly by residue 627 of PB2. Our results suggest that a reduced ability of the polymerase complex of avian viruses to ensure replication of the viral genome at 33°C could contribute to their inability to grow efficiently in humans.
Genetic Diversity in RNA Virus Quasispecies Is Controlled by Host-Virus Interactions
Authors: Schneider William L. , Roossinck Marilyn J.
Published: July 2002

Many RNA viruses have genetically diverse populations known as quasispecies. Important biological characteristics may be related to the levels of diversity in the quasispecies (quasispecies cloud size), including adaptability and host range. Previous work using
Tobacco mosaic virus
and
Cucumber mosaic virus
indicated that evolutionarily related viruses have very different levels of diversity in a common host. The quasispecies cloud size for these viruses remained constant throughout serial passages. Inoculation of these viruses on a number of hosts demonstrated that quasispecies cloud size is not constant for these viruses but appears to be dependent on the host. The quasispecies cloud size remained constant as long as the viruses were maintained on a given host. Shifting the virus between hosts resulted in a change in cloud size to levels associated with the new host. Quasispecies cloud size for these viruses is related to host-virus interactions, and understanding these interactions may facilitate the prediction and prevention of emerging viral diseases.
Evolutionary Relationships and Systematics of the Alphaviruses
Authors: Powers Ann M. , Brault Aaron C. , Shirako Yukio , Strauss Ellen G. , Kang WenLi , Strauss James H. , Weaver Scott C.
Published: July 2002

Partial E1 envelope glycoprotein gene sequences and complete structural polyprotein sequences were used to compare divergence and construct phylogenetic trees for the genus
Alphavirus
. Tree topologies indicated that the mosquito-borne alphaviruses could have arisen in either the Old or the New World, with at least two transoceanic introductions to account for their current distribution. The time frame for alphavirus diversification could not be estimated because maximum-likelihood analyses indicated that the nucleotide substitution rate varies considerably across sites within the genome. While most trees showed evolutionary relationships consistent with current antigenic complexes and species, several changes to the current classification are proposed. The recently identified fish alphaviruses salmon pancreas disease virus and sleeping disease virus appear to be variants or subtypes of a new alphavirus species. Southern elephant seal virus is also a new alphavirus distantly related to all of the others analyzed. Tonate virus and Venezuelan equine encephalitis virus strain 78V3531 also appear to be distinct alphavirus species based on genetic, antigenic, and ecological criteria. Trocara virus, isolated from mosquitoes in Brazil and Peru, also represents a new species and probably a new alphavirus complex.
Interaction of Zyxin, a Focal Adhesion Protein, with the E6 Protein from Human Papillomavirus Type 6 Results in Its Nuclear Translocation
Authors: Degenhardt Yan Yan , Silverstein Saul
Published: July 2002

Zyxin, a focal adhesion molecule, interacts specifically with the E6 protein from human papillomavirus (HPV) type 6 in a yeast two-hybrid screen of a cDNA library prepared from human keratinocytes. Zyxin does not interact significantly with E6 proteins from HPV types 11, 16, or 18. The interaction was confirmed by in vitro and in vivo analyses and it requires the LIM domains (Lin-11, Isl-1, and Mec-3 [G. Freyd, S. K. Kim, and H. R. Horvitz, Nature 344:876–879, 1990]) found at the carboxyl terminus of zyxin. Cotransfection of E6 from HPV (
6
E6) and zyxin results in the accumulation of zyxin in the nucleus where it can function as a transcriptional activator.
6
E6 can also mobilize endogenous zyxin to the nucleus.
Molecular Evolution of Puumala Hantavirus
Authors: Sironen Tarja , Vaheri Antti , Plyusnin Alexander
Published: July 2002

Puumala virus (PUUV) is a negative-stranded RNA virus in the genus
Hantavirus
, family
Bunyaviridae.
In this study, detailed phylogenetic analysis was performed on 42 complete S segment sequences of PUUV originated from several European countries, Russia, and Japan, the largest set available thus far for hantaviruses. The results show that PUUV sequences form seven distinct and well-supported genetic lineages; within these lineages, geographical clustering of genetic variants is observed. The overall phylogeny of PUUV is star-like, suggesting an early split of genetic lineages. The individual PUUV lineages appear to be independent, with the only exception to this being the Finnish and the Russian lineages that are closely connected to each other. Two strains of PUUV-like virus from Japan form the most ancestral lineage diverging from PUUV. Recombination points within the S segment were searched for and evidence for intralineage recombination events was seen in the Finnish, Russian, Danish, and Belgian lineages of PUUV. Molecular clock analysis showed that PUUV is a stable virus, evolving slowly at a rate of 0.7 × 10
−7
to 2.2 × 10
−6
nt substitutions per site per year.
Nuclear Covalently Closed Circular Viral Genomic DNA in the Liver of Hepatocyte Nuclear Factor 1α-Null Hepatitis B Virus Transgenic Mice
Authors: Raney Anneke K. , Eggers Carrie M. , Kline Eric F. , Guidotti Luca G. , Pontoglio Marco , Yaniv Moshe , McLachlan Alan
Published: July 2002

The role of hepatocyte nuclear factor 1α (HNF1α) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1α-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1α transcription factor. However, intracellular viral replication intermediates were increased two- to fourfold in mice lacking functional HNF1α protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1α-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1α-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.