bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogenetic Analysis of
Salmonella
,
Shigella
, and
Escherichia coli
Strains on the Basis of the
gyrB
Gene Sequence
Authors: Fukushima Masao , Kakinuma Kenichi , Kawaguchi Ryuji
Published: October 2002

Phylogenetic analysis of about 200 strains of
Salmonella
,
Shigella
, and
Escherichia coli
was carried out using the nucleotide sequence of the gene for DNA gyrase B (
gyrB
), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of
Salmonella
,
Shigella
, and
Escherichia coli
in which
Salmonella
forms a cluster separate from but closely related to
Shigella
and
E. coli.
In comparison with 16S rRNA analysis, the
gyrB
sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the
gyrB
gene might be a useful tool for differentiating between closely related species of bacteria such as
Shigella
spp. and
E. coli
. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable
gyrB
sequence region can identify unknown bacteria to the species level. In summary, we have shown that
gyrB
sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.
Direct Detection of Enterovirus 71 (EV71) in Clinical Specimens from a Hand, Foot, and Mouth Disease Outbreak in Singapore by Reverse Transcription-PCR with Universal Enterovirus and EV71-Specific Primers
Authors: Singh Sunita , Chow Vincent T. K. , Phoon M. C. , Chan K. P. , Poh Chit Laa
Published: October 2002

A recent outbreak of hand, foot, and mouth disease in Singapore in 2000 affected several thousand children and resulted in four deaths. The aim of this study was to determine the applicability of reverse transcription-PCR (RT-PCR) with universal pan-enterovirus primers and enterovirus 71 (EV71) type-specific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. With the universal primers, EV71 RNA sequences were successfully detected by RT-PCR and direct sequencing in 71% of positive specimens. Three pairs of EV71 type-specific primers were evaluated for rapid detection of EV71 directly from clinical specimens and cell culture isolates. By using a seminested RT-PCR strategy, specific identification of EV71 sequences directly in clinical specimens was achieved, with a detection rate of 53%. In contrast, cell culture could isolate EV71 in only 20% of positive specimens. EV71 was detected directly from brain, heart, and lung specimens of two deceased siblings. Although more than one type of enterovirus was identified in clinical specimens from this outbreak, 90% of the enteroviruses were confirmed as EV71. The data demonstrate the clinical applicability of pan-enterovirus and seminested RT-PCR for the detection of EV71 RNA directly from clinical specimens in an outbreak situation.
1998-1999 Rotavirus Seasons in Juiz de Fora, Minas Gerais, Brazil: Detection of an Unusual G3P[4] Epidemic Strain
Authors: Rosa e Silva Maria Luzia , Pires de Carvalho Iná , Gouvea Vera
Published: October 2002

An epidemiologic survey on the rotavirus strains causing gastroenteritis in young children was conducted in Juiz de Fora, Minas Gerais, in Southern Brazil during two consecutive seasons. Rotavirus was detected in 94 of the 1,056 fecal specimens collected from January 1998 to December 1999. Among the 13 discernible long electrophoretic profiles found, one was highly prevalent (73.4%) and represented the rotavirus strain responsible for the May-August winter epidemic outbreak of 1998, as clearly shown in a three-dimensional graph. This epidemic strain, designated JF98, was characterized as subgroup II and genotype G3P[4] by the original reverse transcription-PCR typing assays. Besides the unusual combination of G and P types, this G3 strain lacked reactivity with anti-G3-specific monoclonal antibodies and presented an uncommon pattern upon digestion of its cDNA-copied VP7 gene with the
Bst
YI restriction enzyme. Strain JF98 affected primarily 6- to 24-month-old children and accounted for 85.5% of the severe rotavirus-associated dehydrating diarrhea cases that required hospitalization. As in our previous studies in neighboring Rio de Janeiro and São Paulo, a remarkably large proportion (44%) of mixed infections was detected, generating a complex set of circulating strains in the community, represented by the many distinct electropherotypes. Other common human types were detected as minor strains in single or in mixed infections, including the JF98 strain. Those were types G1, G4, G8, G9, P[8], and P[6], but not G2 or G5. One specimen contained a mixture of group A and C rotaviruses.
Differentiation of Foot-and-Mouth Disease Virus-Infected from Vaccinated Pigs by Enzyme-Linked Immunosorbent Assay Using Nonstructural Protein 3AB as the Antigen and Application to an Eradication Program
Authors: Chung Wen-Bin , Sorensen Karl Johan , Liao Pei-Chih , Yang Pin-Cheng , Jong Ming-Hwa
Published: October 2002

Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field. Positive reactions were found in sera from fattening pigs and sows 16 weeks and 3.5 years postoutbreak, respectively. There was, however, no positive reaction in sows with at least 10 vaccinations. Maternally derived antibodies to the 3AB antigen persisted in piglets up to 13 weeks of age. A high correlation coefficient (r= 0.93) was found between the test results from sow sera and those from their offspring. Therefore, piglet serum was a good substitute for sow serum to monitor the infection status of the dam. The application of this assay to serological surveillance in an FMD eradication program in Taiwan showed that the positive reactors steadily decreased over time in both finishers and sows, indicating that the pig population risk of infection by FMDV has decreased.
Role of Corpus Gastritis and
cagA
-Positive
Helicobacter pylori
Infection in Reflux Esophagitis
Authors: Queiroz Dulciene Maria Magalhães , Rocha Gifone Aguiar , Oliveira Celso Affonso de , Rocha Andreia Maria Camargos , Santos Adriana , Cabral Mônica Maria Demas Álvares , Nogueira Ana Margarida Miguel Ferreira
Published: October 2002

Considering that the role of
Helicobacter pylori
infection in gastroesophageal reflux and reflux esophagitis (GERD) is still controversial and that the role of virulence markers of the bacterium has not been evaluated in most studies of GERD, we investigated the association among
H. pylori
infection with
cagA
-positive and -negative strains, corpus gastritis, and GERD in a large group of patients by controlling for confounding factors. We studied prospectively 281 consecutive adult patients: 93 with GERD and 188 controls.
H. pylori
infection status was diagnosed by culture, by the preformed urease test, with a carbolfuchsin-stained smear, and by histology. The
cagA
status was determined by PCR of
H. pylori
isolates and gastric biopsy specimens.
H. pylori
infection was diagnosed in 191 (68.0%) of 281 patients. Among the 93 patients with GERD, 84 presented with mild or moderate esophagitis and 9 presented with severe esophagitis. In the multivariate analysis, the age of the patients and the degree of oxyntic gastritis were associated with GERD. Among the strains isolated from patients with GERD and from the control group, 24.4 and 66.9%, respectively, were positive for
cagA
(
P
< 0.001). Compared to infection with
cagA
-negative strains, infection with
cagA
-positive
H. pylori
strains was associated with a more intense gastritis in the corpus (
P
= 0.001).
cagA
status (odds ratio [OR] = 0.16, 95% confidence interval [CI] = 0.07 to 0.40), gastritis of the corpus (OR = 0.69, 95% CI = 0.48 to 0.99), and age (OR = 1.04, 95% CI = 1.01 to 1.07) were associated with GERD. In conclusion, the study provides evidence supporting the independent protective roles of
cagA
-positive
H. pylori
strains and the degree of corpus gastritis against GERD.
Molecular Epidemiology of Caliciviruses Causing Outbreaks and Sporadic Cases of Acute Gastroenteritis in Spain
Authors: Buesa J. , Collado B. , López-Andújar P. , Abu-Mallouh R. , Rodríguez Díaz J. , García Díaz A. , Prat J. , Guix S. , Llovet T. , Prats G. , Bosch A.
Published: October 2002

The molecular epidemiology of human caliciviruses (HuCVs) causing sporadic cases and outbreaks of acute gastroenteritis around eastern Spain (Catalonia and the Valencian Community) was studied by reverse transcription-PCR (RT-PCR) and by sequencing part of the RNA polymerase gene in open reading frame 1. HuCVs were detected in 44 of 310 stool specimens (14.19%) negative for other enteric pathogens obtained from children with acute gastroenteritis. Norwalk-like viruses (NLVs) were the most common cause of the gastroenteritis outbreaks investigated here. They were detected in 14 out of 25 (56%) outbreaks with an identified pathogen. Genotypes producing both sporadic cases and outbreaks were diverse, with a predominance of GGII strains related to genotypes Melksham and Lordsdale. Five strains clustered with a “new variant” designated GGIIb, which was detected circulating throughout quite a few European countries in the years 2000 and 2001. The emergence mechanism of these strains might be the occurrence of intertypic recombinations between different viruses. The nucleotide sequence of part of the capsid gene (ORF2) from three of these strains demonstrated their relationship with Mexico virus.
Detection of
Bacillus anthracis
DNA by LightCycler PCR
Authors: Bell Constance A. , Uhl James R. , Hadfield Ted L. , David John C. , Meyer Richard F. , Smith Thomas F. , Cockerill Franklin R.
Published: October 2002

Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of
Bacillus anthracis
, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of
B. anthracis
contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of
B. anthracis
by PCR. We developed a rapid-cycle real-time PCR detection assay for
B. anthracis
that utilizes the LightCycler instrument (LightCycler
Bacillus anthracis
kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (
pagA
) was detected in 29 of 29 virulent
B. anthracis
strains, and the gene encoding the capsular protein B (
capB
) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only
pagA
or
pagB
genes, could be detected and differentiated from virulent strains. The assays were specific for
B. anthracis
: the results were negative for 57 bacterial strains representing a broad range of organisms, including
Bacillus
species other than
anthracis
(
n
= 31) and other non-
Bacillus
species (
n
= 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per μl of sample. The LightCycler
Bacillus anthracis
assay appears to be a suitable method for rapid identification of cultured isolates of
B. anthracis
. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of
B. anthracis
directly from human specimens.
Qualitative Detection of Hepatitis C Virus RNA: Comparison of Analytical Sensitivity, Clinical Performance, and Workflow of the Cobas Amplicor HCV Test Version 2.0 and the HCV RNA Transcription-Mediated Amplification Qualitative Assay
Authors: Krajden Mel , Ziermann Rainer , Khan Asphani , Mak Annie , Leung Kimmy , Hendricks David , Comanor Lorraine
Published: October 2002

The qualitative Cobas Amplicor hepatitis C virus (HCV) version 2.0 assay (HCV PCR) and the Bayer Reference Testing Laboratory HCV RNA transcription-mediated amplification assay (HCV TMA) were compared for analytical sensitivity, clinical performance, and workflow. Limits of detection were determined by testing dilutions of the World Health Organization HCV standard in replicates of 15 at concentrations of from 1.0 to 70 IU/ml. The limit of detection of the HCV PCR assay was calculated to be 45 IU/ml on initial testing and 32 IU/ml after resolution of gray zone results. The calculated limit of detection for HCV TMA was 6 IU/ml. To compare clinical performance, 300 specimens, grouped as follows, were evaluated: 112 samples that were indeterminate in an anti-HCV enzyme immunoassay (EIA) and for which HCV RNA was not detected by HCV PCR; 79 samples that were EIA positive and for which HCV RNA was not detected by HCV PCR; and 105 samples that were both EIA and HCV PCR positive. For these groups, interassay concordance ranged from 96.2% to 100%. In addition, three HCV PCR gray zone specimens and one neonatal specimen were also evaluated. A 64-sample run (full run, 91 specimens) required 5 h for testing by HCV TMA, whereas almost 8 h were required to test a full run of 22 specimens by HCV PCR. HCV TMA demonstrated excellent concordance with HCV PCR when clinical samples were tested. However, HCV TMA was more sensitive than HCV PCR, required less time for test result completion, and had a greater throughput.
Multiplex PCR Assays for Simultaneous Detection of Six Major Serotypes and Two Virulence-Associated Phenotypes ofStreptococcus suisin Tonsillar Specimens from Pigs
Authors: Wisselink Henk J. , Joosten Jeroen J. , Smith Hilde E.
Published: October 2002

Multiplex PCR assays for the detection and identification of variousStreptococcus suisstrains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on theS. suiscapsular polysaccharide (cps) genes specific for serotypes 1 (and 14), 7, and 9, were amplified in multiplex PCR I. Two other targets, based on the serotype 2- (and 1/2-) specificcpsgene and theepfgene, encoding the EF proteins of virulent serotype 2 and highly virulent serotype 1 strains, were amplified in multiplex PCR II. To identify false-negative results, firefly luciferase (luc) DNA and primers based on thelucgene were included in the assay. The multiplex PCR assays were evaluated with tonsillar specimens from pigs infected withS. suisstrains. The results obtained with the PCR assays were compared with the results obtained with a bacteriological examination. Most (94%) of the results obtained with multiplex PCR assays were confirmed by the bacteriological examination. The PCR method seems to be more sensitive compared to the bacteriological method, since the remaining 6% of the samples were positive by PCR and negative by bacteriological examination. These results indicate that the PCR method is highly specific for the detection ofS. suisstrains most frequently involved in clinical disease in infected pig herds. The serotypes found by PCR in tonsillar specimens from diseased pigs were compared with the serotypes of the strains isolated from the affected tissues of the same pigs. The results showed that there is significant association between carriership and clinical illness forS. suisserotype 9 and EF-positive serotype 2 strains and not for serotype 7 and EF-negative serotype 2 (or 1/2) strains.
Monitoring of Viral Load by Quantitative Plasma PCR during Active Cytomegalovirus Infection of Individual Liver Transplant Patients
Authors: Piiparinen Heli , Höckerstedt Krister , Lappalainen Maija , Suni Jukka , Lautenschlager Irmeli
Published: October 2002

A quantitative PCR test, the Cobas Amplicor CMV Monitor, was used for the monitoring of viral load in the peripheral blood of 27 individual liver transplant patients and correlated with cytomegalovirus (CMV) pp65 antigenemia. Altogether, 243 specimens were analyzed. During the first 3 months, 20 patients showed PCR positivity which correlated with pp65 antigenemia. Of those, 13 patients developed symptomatic CMV infection 27 to 52 days after transplantation, with a significantly higher peak viral load in PCR and in pp65 assay compared with the seven asymptomatic infections (median 10,200 versus 2,240 copies/ml,
P
< 0.05, and median 100 versus 30 pp65-positive cells/50,000 leukocytes,
P
< 0.01). Five were primary infections of D+/R− cases (donor CMV seropositive and recipient seronegative) and demonstrated, except in one case, a high peak viral load (>10,000 copies/ml; range, 10,200 to 21,600 copies, and ≥50 positive cells, range, 50 to 800 cells). The peak viral loads of the six D+/R+ patients with symptomatic infection varied widely (range, 2,290 to 126,000 copies and 50 to 300 positive cells). Two D−/R+ patients developed symptomatic infection with a lower viral load (range, 1,120 to 6,510 copies and 25 to 100 positive cells). All symptomatic infections were successfully treated with ganciclovir. The asymptomatic infections all in D+/R+ patients with low copy numbers (<5,500 copies) were monitored until CMV disappeared. One of the seven PCR-negative patients had one sample with low antigenemia, but the subsequent specimens were all negative. The time-related correlation of the two methods was also good. In summary, quantitative PCR could equally well be used as the CMV pp65 assay for the monitoring of viral load in individual transplant patients.
Genotyping of
Francisella tularensis
Strains by Pulsed-Field Gel Electrophoresis, Amplified Fragment Length Polymorphism Fingerprinting, and 16S rRNA Gene Sequencing
Authors: García Del Blanco N. , Dobson M. E. , Vela A. I. , De La Puente V. A. , Gutiérrez C. B. , Hadfield T. L. , Kuhnert P. , Frey J. , Domínguez L. , Rodríguez Ferri E. F.
Published: October 2002

We evaluated three molecular methods for identification of
Francisella
strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54
Francisella tularensis
subsp.
holarctica
, 5
F. tularensis
subsp.
tularensis
, 2
F. tularensis
subsp.
novicida
, and 1
F. philomiragia
strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes
Xho
I and
Bam
HI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of
F. tularensis
subsp.
holarctica.
The AFLP analysis technique produced some degree of discrimination among
F. tularensis
subsp.
holarctica
strains (one primary cluster with three major subclusters and minor variations within subclusters) when
Eco
RI-C and
Mse
I-A,
Eco
RI-T and
Mse
I-T,
Eco
RI-A and
Mse
I-C, and
Eco
RI-0 and
Mse
I-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of
F. tularensis
subsp.
tularensis
,
F. tularensis
subsp.
novicida
, and
F. philomiragia
were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all
F. tularensis
subsp.
holarctica
isolates compared in this study. These results suggest that although limited genetic heterogeneity among
F. tularensis
subsp.
holarctica
isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of
F. tularensis
and suitable techniques for the differentiation of individual strains.
Isolation from a Healthy Carrier and Characterization of a
Neisseria meningitidis
Strain That Is Deficient in γ-Glutamyl Aminopeptidase Activity
Authors: Takahashi Hideyuki , Tanaka Hiroshi , Inouye Hiroo , Kuroki Toshiro , Watanabe Yuko , Yamai Shiro , Watanabe Haruo
Published: October 2002

γ-Glutamyl aminopeptidase (GGT) activity is used as a specific marker for the identification of
Neisseria meningitidis
. We isolated from a healthy carrier and characterized an
N. meningitidis
isolate which lacked the activity due to the insertional mutation of the
ggt
gene, suggesting that naturally occurring
N. meningitidis
isolates do not always possess GGT activity.
Real-Time PCR Detection of
Salmonella
in Suspect Foods from a Gastroenteritis Outbreak in Kerr County, Texas
Authors: Daum Luke T. , Barnes William J. , McAvin James C. , Neidert Margaret S. , Cooper Lynn A. , Huff William B. , Gaul Linda , Riggins W. S. , Morris Sandra , Salmen Ann , Lohman Kenton L.
Published: October 2002

In June 2001, an outbreak of acute gastroenteritis among 109 attendees of a church picnic in Kerr County, Texas, was reported. A 5′-nuclease PCR assay was used to screen for
Salmonella
in nine food items from the buffet line. Barbeque chicken B tested positive for
Salmonella
, and no amplification was detected in the remaining food items. These PCR findings were consistent with culture results and were confirmed by direct nucleotide sequencing.
Salmonella enterica
serotype Panama was cultured from both food and patient stool samples.
First Reported Isolation of
Cryptococcus neoformans
var.
gattii
from a Patient in Singapore
Authors: Taylor M. B. , Chadwick D. , Barkham T.
Published: October 2002

Cryptococcal meningitis has long been known to afflict immunocompetent patients in Singapore. We report the first identification of an isolate of
Cryptococcus neoformans
var.
gattii
from a Singaporean resident; this variety can cause invasive disease in the immunocompetent. Meningitis in a traveler returning from this area may be cryptococcal.
Modeling Viral Genome Fitness Evolution Associated with Serial Bottleneck Events: Evidence of Stationary States of Fitness
Authors: Lázaro Ester , Escarmís Cristina , Domingo Esteban , Manrubia Susanna C.
Published: October 2002

Evolution of fitness values upon replication of viral populations is strongly influenced by the size of the virus population that participates in the infections. While large population passages often result in fitness gains, repeated plaque-to-plaque transfers result in average fitness losses. Here we develop a numerical model that describes fitness evolution of viral clones subjected to serial bottleneck events. The model predicts a biphasic evolution of fitness values in that a period of exponential decrease is followed by a stationary state in which fitness values display large fluctuations around an average constant value. This biphasic evolution is in agreement with experimental results of serial plaque-to-plaque transfers carried out with foot-and-mouth disease virus (FMDV) in cell culture. The existence of a stationary phase of fitness values has been further documented by serial plaque-to-plaque transfers of FMDV clones that had reached very low relative fitness values. The statistical properties of the stationary state depend on several parameters of the model, such as the probability of advantageous versus deleterious mutations, initial fitness, and the number of replication rounds. In particular, the size of the bottleneck is critical for determining the trend of fitness evolution.
Direct and Quantitative Single-Cell Analysis of Human Immunodeficiency Virus Type 1 Reactivation from Latency
Authors: Kutsch Olaf , Benveniste Etty N. , Shaw George M. , Levy David N.
Published: October 2002

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.
Inhibition of Nuclear Import and Alteration of Nuclear Pore Complex Composition by Rhinovirus
Authors: Gustin Kurt E. , Sarnow Peter
Published: October 2002

Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.
Identification and Characterization of the Pseudorabies Virus Tegument Proteins UL46 and UL47: Role for UL47 in Virion Morphogenesis in the Cytoplasm
Authors: Kopp Martina , Klupp Barbara G. , Granzow Harald , Fuchs Walter , Mettenleiter Thomas C.
Published: October 2002

Proteins encoded by the UL46 and UL47 genes of herpes simplex virus type 1 (HSV-1) constitute major components of the viral tegument. However, their functions have so far not been elucidated in detail. By use of monospecific antisera directed against bacterially expressed glutathione-
S
-transferase fusion proteins, the homologous UL46 and UL47 proteins of the alphaherpesvirus pseudorabies virus (PrV) were identified in virus-infected cells and in virions. The PrV UL46 gene product of 693 amino acids (aa) exhibits an apparent molecular mass of 95 kDa, whereas the UL47 product of 750 aa was identified as a 97-kDa protein. Both are present in purified virions, correlating with their role as tegument proteins. Immunofluorescence analysis by confocal laser scan microscopy showed that late in infection the UL46 product is detectable in the cytoplasm, whereas the UL47 product was observed to be diffuse in the cytoplasm and speckled in the nucleus. Virus mutants lacking either the UL46 or the UL47 gene or both were isolated on noncomplementing cells, demonstrating that these genes either singly or in combination are not required for productive viral replication. However, plaque sizes were decreased. Interestingly, in one-step growth analysis, UL47 deletion mutants exhibited an approximately 10-fold decrease in final titers, whereas the UL46 deletion mutant was not affected. This finding correlated with ultrastructural observations which showed unimpaired virion morphogenesis in the absence of the UL46 protein, whereas in the absence of the UL47 protein intracytoplasmic aggregates of partially tegumented capsids were observed. In summary, we identified the PrV UL46 and UL47 proteins and show that the UL47 protein plays an important role in virion assembly in the cytoplasm.
Hantaan Virus Infection Causes an Acute Neurological Disease That Is Fatal in Adult Laboratory Mice
Authors: Wichmann Dominic , Gröne Hermann-Josef , Frese Michael , Pavlovic Jovan , Anheier Bärbel , Haller Otto , Klenk Hans-Dieter , Feldmann Heinz
Published: October 2002

Hantaan virus, the etiological agent of Korean hemorrhagic fever, is transmitted to humans from persistently infected mice (
Apodemus agrarius
), which serve as the primary reservoir. Here we demonstrate that several strains of adult
Mus musculus domesticus
(C57BL/6, BALB/c, AKR/J, and SJL/J) were susceptible to Hantaan virus infection when infected intraperitoneally. First clinical signs were loss of weight, ruffled fur, and reduced activity, which were followed by neurological symptoms, such as paralyses and convulsions. Within 2 days of disease onset, the animals died of acute encephalitis. PCR analysis indicated a systemic infection with viral RNA present in all major organs. Immunohistochemical and in situ hybridization analyses of postmortem material detected viral antigen and RNA in the central nervous system (predominantly brain), liver, and spleen. In the central nervous system, viral antigen and RNA colocalized with perivascular infiltrations, the predominant pathological finding. To investigate the involvement of the interferon system in Hantaan virus pathogenesis, we infected alpha/beta interferon receptor knockout mice. These animals were more susceptible to Hantaan virus infection, indicating an important role of interferon-induced antiviral defense mechanisms in Hantaan virus pathogenesis. The present model may help to overcome shortcomings in the development of therapeutic and prophylactic measurements against hantavirus infections.
Analysis of the Complete Genome Sequence of the Hz-1 Virus Suggests that It Is Related to Members of the
Baculoviridae
Authors: Cheng Chia-Hsiung , Liu Su-Mei , Chow Teh-Yuan , Hsiao Yu-Yun , Wang Dan-Ping , Huang Jiann-Jang , Chen Hong-Hwa
Published: October 2002

We report the complete sequence of a large rod-shaped DNA virus, called the Hz-1 virus. This virus persistently infects the
Heliothis zea
cell lines. The Hz-1 virus has a double-stranded circular DNA genome of 228,089 bp encoding 154 open reading frames (ORFs) and also expresses a persistence-associated transcript 1, PAT1. The G+C content of the Hz-1 virus genome is 41.8%, with a gene density of one gene per 1.47 kb. Sequence analysis revealed that a 9.6-kb region at 43.6 to 47.8 map units harbors five cellular genes encoding proteins with homology to dUTP pyrophosphatase, matrix metalloproteinase, deoxynucleoside kinase, glycine hydroxymethyltransferase, and ribonucleotide reductase large subunit. Other cellular homologs were also detected dispersed in the viral genome. Several baculovirus homologs were detected in the Hz-1 virus genome. These include PxOrf-70, PxOrf-29, AcOrf-81, AcOrf-96, AcOrf-22, VLF-1, RNA polymerase LEF-8 (orf50), and two structural proteins, p74 and p91. The Hz-1 virus p74 homolog shows high structural conservation with a double transmembrane domain at its C terminus. Phylogenetic analysis of the p74 revealed that the Hz-1 virus is evolutionarily distant from the baculoviruses. Another distinctive feature of the Hz-1 virus genome is a gene that is involved in insect development. However, the remainder of the ORFs (81%) encoded proteins that bear no homology to any known proteins. In conclusion, the sequence differences between the Hz-1 virus and the baculoviruses outnumber the similarities and suggest that the Hz-1 virus may form a new family of viruses distantly related to the
Baculoviridae
.
The Antiviral Response to Gamma Interferon
Authors: Costa-Pereira Ana P. , Williams Timothy M. , Strobl Birgit , Watling Diane , Briscoe James , Kerr Ian M.
Published: October 2002

A role for alpha/beta interferon (IFN-α/β) in the IFN-γ antiviral response has long been suggested. Accordingly, possible roles for autocrine or double-stranded-RNA (dsRNA)-induced IFN-α/β in the IFN-γ response were investigated. Use was made of wild-type and a variety of mutant human fibrosarcoma cell lines, including mutant U5A cells, which lack a functional IFN-α/β receptor and hence an IFN-α/β response. IFN-γ did not induce detectable levels of IFN-α/β in any of the cell lines, nor was the IFN-γ response per se dependent on autocrine IFN-α/β. On the other hand, a number of responses to dsRNA [poly(I) · poly(C)] and encephalomyocarditis virus were greatly enhanced by IFN-γ pretreatment (priming) of wild-type cells or of mutant cells lacking an IFN-α/β response; these include the primary induction of dsRNA-inducible mRNAs, including IFN-β mRNA, and, to a lesser extent, the dsRNA-mediated activation of the p38 mitogen-activated protein (MAP) kinase(s). IFN-γ priming of mRNA induction by dsRNA is dependent on JAK1 and shows biphasic kinetics, with an initial rapid (<30-min) response being followed by a more substantial effect on overnight incubation. The IFN-γ-primed dsRNA responses appear to be subject to modulation through the p38, phosphatidylinositol 3-kinase, and ERK1/ERK2 MAP kinase pathways. It can be concluded that despite efficient priming of IFN-β production, the IFN-α/β pathways play no significant role in the primary IFN-γ antiviral response in these cell-virus systems. The observed IFN-γ priming of dsRNA responses, on the other hand, will likely play a significant role in combating virus infection in vivo.
C-Terminal gp40 Peptide Analogs Inhibit Feline Immunodeficiency Virus: Cell Fusion and Virus Spread
Authors: Medinas R. J. , Lambert D. M. , Tompkins W. A.
Published: October 2002

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1), gp160, is synthesized as a protein precursor that when proteolytically cleaved yields two subunits, gp120 and gp41. gp120 is the surface glycoprotein on HIV-1 responsible for binding to CD4, and gp41 is the transmembrane glycoprotein involved in the membrane fusion process. gp41 is divided into the N-terminal fusion peptide, the heptad repeat 1 (HR1) and HR2 regions, and the C-terminal transmembrane region, which are collectively responsible for virus fusion and entry into the cell. Synthetic peptides derived from the HR2 and HR1 regions of HIV-1LAIhave been shown to prevent virus-cell fusion and infection in vitro. In phase II clinical trials in HIV patients, data revealed that T20 has antiviral efficacy and is well tolerated. Similar results were obtained in vitro with HIV-2 and simian immunodeficiency virus, supporting the conservation of the gp41 ectodomain among lentiviruses. Feline immunodeficiency virus (FIV) infection in the cat has been used as a model to develop potential antivirals for HIV. To determine if synthetic gp40 analogs capable of inhibiting FIV infection could be identified, 15 overlapping 35-amino-acid peptides derived from the C-terminal HR2 domain of FIV gp40 were synthesized. These peptides were tested for efficacy against FIV in a syncytium-forming assay with FIV-infected CrFK cells and HeLa cells expressing the FIV receptor CXCR4. Several peptides exhibited activity at the nanogram level. Antiviral activity was confirmed by suppression of reverse transcriptase in a FIV feline CD4+-T-cell (FCD4-E) acute-infection assay. These data demonstrate that synthetic peptides derived from the HR2 domain of the FIV gp41 protein are effective inhibitors of FIV infection.
Intravirion Processing of the Human Immunodeficiency Virus Type 1 Vif Protein by the Viral Protease May Be Correlated with Vif Function
Authors: Khan Mohammad A. , Akari Hirofumi , Kao Sandra , Aberham Claudia , Davis David , Buckler-White Alicia , Strebel Klaus
Published: October 2002

The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral Pr and was sensitive to Pr inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala
150
. Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function, suggesting that intravirion processing of Vif is important for the production of infectious viruses.
Blockade of Human Immunodeficiency Virus Type 1 Expression by Caveolin-1
Authors: Llano Manuel , Kelly Tara , Vanegas Maria , Peretz Mary , Peterson Timothy E. , Simari Robert D. , Poeschla Eric M.
Published: October 2002

Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.
Truncation of Herpes Simplex Virus Type 2 Glycoprotein B Increases Its Cell Surface Expression and Activity in Cell-Cell Fusion, but These Properties Are Unrelated
Authors: Fan Zhenghong , Grantham Michael L. , Smith M. Shane , Anderson Eric S. , Cardelli James A. , Muggeridge Martin I.
Published: October 2002

Formation of small polykaryons by cell-cell fusion is characteristic of herpes simplex virus (HSV) lesions, but the great majority of viruses isolated from such lesions produce only limited cell fusion in tissue culture. Because of this, HSV laboratory strains that produce extensive cell fusion (syncytium formation) in culture are regarded as variants or mutants. Furthermore, the rarity of clinical isolates able to produce syncytia in culture suggests that extensive cell fusion is deleterious in vivo. Mutations that confer a syncytial phenotype can then be regarded as bypassing a mechanism that normally limits cell fusion. Determination of how these mutations, some of which are in the cytoplasmic tail of glycoprotein B (gB), lead to syncytium formation will likely reveal how fusion is controlled. Here we show the following. (i) Truncation of the cytoplasmic tail of HSV type 2 gB (gB-2) by a minimum of 25 residues or a maximum of 49 residues produces a syncytial phenotype. (ii) Truncation by 20 to 49 residues increases cell fusion when gB-2 is coexpressed with only gD-2, gH-2, and gL-2. (iii) Truncation by 25 or more residues removes a potential endocytosis motif and increases gB-2 cell surface expression. (iv) Mutation of this motif increases gB-2 cell surface expression but does not increase fusogenic activity, whereas mutation of another potential endocytosis motif does not increase surface expression but does increase fusogenic activity. Therefore, syncytial mutations in the cytoplasmic tail of gB-2 do not act by increasing cell surface levels of the protein.
Genetic Requirements for Homologous Recombination in
Autographa californica
Nucleopolyhedrovirus
Authors: Crouch Erin A. , Passarelli A. Lorena
Published: October 2002

It is known that baculovirus infection promotes high-frequency recombination between its genomes and plasmid DNA during the construction of recombinant viruses for foreign gene expression. However, little is known about the viral genes necessary to promote homologous recombination (HR). We developed an assay to identify viral genes that are necessary to stimulate HR. In this assay, we used two plasmids containing extensive sequence homology that yielded a visible and quantifiable phenotype if HR occurred. The plasmids contained the green fluorescent protein gene (
gfp
) that was mutated at either the N or the C terminus and a viral origin of DNA replication. When the plasmids containing these mutant
gfp
genes were transfected into insect cells alone or together, few green fluorescent protein (GFP)-positive cells were observed, confirming that the host cell machinery alone was not able to promote high levels of HR. However, if viral DNA or viral genes involved in DNA replication were cotransfected into cells along with the mutant
gfp
-containing plasmids, a dramatic increase in GFP-positive cells was observed. The viral genes
ie-1
,
ie-2
,
lef-7
, and
p35
were found to be important for efficient HR in the presence of all other DNA replication genes. However,
ie-1
and
ie-2
were sufficient to promote HR in the absence of other viral genes. Recombination substrates lacking a viral origin of replication had similar genetic requirements for recombination but were less dependent on
ie-1
. Interestingly, even though HR was stimulated by the presence of a viral origin of DNA replication, virally stimulated HR could proceed in the presence of the DNA synthesis inhibitor aphidicolin.
Overexpression of Promyelocytic Leukemia Protein Precludes the Dispersal of ND10 Structures and Has No Effect on Accumulation of Infectious Herpes Simplex Virus 1 or Its Proteins
Authors: Lopez Pascal , Jacob Robert J. , Roizman Bernard
Published: October 2002

A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing theMr69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of α, β, or γ groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the α0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication.