bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Detection of Enterohemorrhagic
Escherichia coli
by Real-Time PCR with Fluorescent Hybridization Probes
Authors: Bellin Tobias , Pulz Matthias , Matussek Andreas , Hempen Hans-Günther , Gunzer Florian
Published: July 2002

In this report, we present a PCR protocol for rapid identification of enterohemorrhagic
Escherichia coli
on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.
Identification of CTX-M-14 Extended-Spectrum β-Lactamase in Clinical Isolates of
Shigella sonnei
,
Escherichia coli
, and
Klebsiella pneumoniae
in Korea
Authors: Pai Hyunjoo , Choi Eun-Hwa , Lee Hoan-Jong , Hong Jung Yun , Jacoby George A.
Published: July 2002

CTX-M-14 β-lactamase was identified in a stool isolate of
Shigella sonnei
and in blood isolates of
Escherichia coli
(one isolate) and
Klebsiella pneumoniae
(two isolates) from different parts of Korea. The amino acid sequence differed by one amino acid from CTX-M-9 (Ala-231→ Val) and was identical to that of β-lactamases recently found in China and Japan.
Immunofluorescence Method for Detection of Ebola Virus Immunoglobulin G, Using HeLa Cells Which Express Recombinant Nucleoprotein
Authors: Saijo Masayuki , Niikura Masahiro , Morikawa Shigeru , Kurane Ichiro
Published: July 2002

A novel recombinant baculovirus which expresses Ebola virus (EBO) nucleoprotein (NP) under the control of the cytomegalovirus immediate-early promoter was constructed. HeLa cells abortively infected with the baculovirus expressed EBO NP, and this was used as an immunofluorescent (IF) antigen to detect EBO immunoglobulin G (IgG) antibody. This IF method has high efficacy in detecting EBO IgG antibody in clinical specimens, indicating its usefulness in the diagnosis of EBO infections and seroepidemiological studies.
Evaluation of United States-Licensed Human Immunodeficiency Virus Immunoassays for Detection of Group M Viral Variants
Authors: Koch Walter H. , Sullivan Patrick S. , Roberts Charles , Francis Kori , Downing Robert , Mastro Timothy D. , Nkengasong John , Hu Dale , Masciotra Silvina , Schable Charles , Lal Renu B.
Published: July 2002

Six Food and Drug Administration (FDA)-licensed human immunodeficiency virus type 1 (HIV-1) and HIV-1/2 immunoassays, including five enzyme immunoassays and one rapid test, were challenged with up to 250 serum samples collected from various global sites. The serum samples were from individuals known to be infected with variants of HIV-1 including group M subtypes A, B, B′, C, D, E, F, and G and group O. All immunoassays detected the vast majority of samples tested. Three samples produced low signal over cutoff values in one or more tests: a clade B sample, an untypeable sample with a low antibody titer, and a group O sample. It is concluded that HIV-1 immunoassays used in the United States are capable of detecting most HIV-1 group M variants.
Low Prevalence of Community-Acquired Methicillin-Resistant
Staphylococcus aureus
in Adults at a University Hospital in the Central United States
Authors: Suntharam Nila , Hacek Donna , Peterson Lance R.
Published: July 2002

Community-acquired MRSA (CA-MRSA) is potentially a new emerging pathogen with most strains susceptible to many antimicrobials except for β-lactam antibiotics. We retrospectively reviewed MRSA isolates during a 20-month study period (January 1998 through August 1999) and investigated those that were clindamycin susceptible. Patients were not considered to harbor CA-MRSA if they had been admitted to a hospital within the preceding 2 years or if their isolate had been obtained more than 72 h after admission. There were 2,817
S. aureus
isolates, with 1,071 (38%) being MRSA. Of these 1,071 isolates, 161 were clindamycin susceptible; these were recovered from 81 patients. Of these 81 patients, 20 appeared to have community-acquired strains, but only 2 could be confirmed as having CA-MRSA.
Detection of Amantadine-Resistant Influenza A Virus Strains in Nursing Homes by PCR-Restriction Fragment Length Polymorphism Analysis with Nasopharyngeal Swabs
Authors: Saito Reiko , Oshitani Hitoshi , Masuda Hiroki , Suzuki Hiroshi
Published: July 2002

Annual consumption of amantadine increased abruptly after its approval for the treatment of influenza A virus infections in Japan in 1998, and the emergence of amantadine-resistant viruses is now a matter of concern. To detect resistant influenza A virus strains, we have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for nasopharyngeal swabs. Three different primer sets for nested PCR were designed to incorporate restriction sites into the amplicon to differentiate single-amino-acid substitutions at positions 27, 30, and 31 that confer resistance in the transmembrane domain of the M2 protein. Each PCR product was digested with respective endonucleases (
Bsp
LU11I for amino acid change at position 27,
Hha
I for position 30, and
Sca
I for position 31), and the polymorphisms were determined by electrophoresis. Thirty-four (24.1%) of 141 PCR-positive samples had resistance patterns in eight nursing homes in the 1998–1999 season. Thirty-one viruses (91.2%) showed a change at position 31 (serine to asparagine), three viruses (8.8%) showed a change at position 30 (alanine to threonine), and none showed a change at position 27. The incidence of resistant viruses did not show any significant difference between four facilities where amantadine was used mainly for influenza treatment and four other facilities where it was used only for Parkinson’s disease, values being 27.6 and 16.3%, respectively. We have confirmed that the PCR-RFLP method is useful for detecting amantadine-resistant strains directly from nasopharyngeal swabs and that resistant viruses were circulating in nursing homes where the drug was used not only for influenza virus but also for Parkinson’s disease.
Cryptococcosis in a Bottlenose Dolphin (
Tursiops truncatus
) Caused by
Cryptococcus neoformans
var.
gattii
Authors: Miller W. George , Padhye Arvind A. , van Bonn William , Jensen Eric , Brandt Mary E. , Ridgway Sam H.
Published: July 2002

We describe the first case of cryptococcosis caused by
Cryptococcus neoformans
var.
gattii
in a male Atlantic bottlenose dolphin (
Tursiops truncatus
). The dolphin showed clinical signs of tachypnea, transient dyspnea, and mild tachycardia and developed multiple hyperechoic nodules, parenchymal consolidation, and thickening of pleura. A diagnosis of bronchopneumonia with pleuritis was made. Itraconazole therapy was implemented for 120 days, and trough levels in serum were within or above the suggested therapeutic range. Titers of cryptococcal antigen in serum increased eightfold during therapy, and the case had a fatal outcome. Necropsy examination findings included enlarged pulmonary lymph nodes and extensive coalescing granulomatous lesions throughout both lungs. Histologic examination revealed numerous, spherical to ellipsoidal, mucicarmine-positive, 3- to 14-μm, encapsulated, budding cells consistent with
C. neoformans
. Culture of the lung tissue yielded colonies of
C. neoformans
. The isolate was urease positive and nitrate negative and exhibited phenoloxidase activity. It was positive on canavanine-glycine-bromothymol blue agar. When tested by the Iatron serodiagnostic reagent kit (Iatron Laboratories, Inc.), it was shown to belong to serotype B.
Characterization of Shiga Toxin-Producing
Escherichia coli
O26 Strains and Establishment of Selective Isolation Media for These Strains
Authors: Hiramatsu Reiji , Matsumoto Masakado , Miwa Yoshio , Suzuki Yasumoto , Saito Makoto , Miyazaki Yutaka
Published: July 2002

We characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing
Escherichia coli
(STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H−) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC ≥ 50 μg/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC ≤ 1.56 μg/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnose-nonfermented) colonies on both media; the vast majority of selected
E. coli
strains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.
Prevalence of Parvovirus B19 and Parvovirus V9 DNA and Antibodies in Paired Bone Marrow and Serum Samples from Healthy Individuals
Authors: Heegaard Erik D. , Petersen Bodil Laub , Heilmann Carsten J. , Hornsleth Allan
Published: July 2002

Parvovirus B19 (hereafter referred to as B19) exhibits a marked tropism to human bone marrow (BM), and infection may lead to erythema infectiosum, arthropathy, hydrops fetalis, and various hematologic disorders. Recently, a distinct parvovirus isolate termed V9 with an unknown clinical spectrum was discovered. In contrast to the many studies of B19 serology and viremia, valid information on the frequency of B19 or V9 DNA in the BM of healthy individuals is limited. To develop a reference value, paired BM and serum samples from healthy subjects were tested for the presence of B19 and V9 DNA and specific antibodies. Immunoglobulin M (IgM) was not found in any of the serum samples. The prevalence of IgG showed a gradual and steady increase from 37% in children aged 1 to 5 years to 87% in people aged >50 years. When 190 well-characterized subjects were examined, B19 DNA was detected in the BM of 4 individuals (2.1%; 95% confidence interval, 0.58 to 5.3%) while none of the paired serum samples showed evidence of circulating viral DNA. V9 DNA was not found in any of the BM or serum samples. The finding of B19 DNA probably indicated a primary infection in one 7-year-old individual and reinfection or reactivation of persistent infection in the remaining three persons, aged 47 to 58 years. Serving as a benchmark for future studies, these findings are useful when interpreting epidemiologic data, performing BM transplantation, or considering clinical implications of parvovirus infection.
Biofilm Production by Isolates of
Candida
Species Recovered from Nonneutropenic Patients: Comparison of Bloodstream Isolates with Isolates from Other Sources
Authors: Shin Jong Hee , Kee Seung Jung , Shin Myung Geun , Kim Soo Hyun , Shin Dong Hyeon , Lee Sang Ku , Suh Soon Pal , Ryang Dong Wook
Published: July 2002

Biofilm production has been implicated as a potential virulence factor of some
Candida
species responsible for catheter-related fungemia in patients receiving parenteral nutrition. We therefore compared clinical bloodstream isolates representing seven different
Candida
species to each other and to those from other anatomical sites for the capacity to form biofilms in glucose-containing medium. Potential associations between the capacity to form biofilms and the clinical characteristics of fungemia were also analyzed. Isolates included the following from nonneutropenic patients: 101 bloodstream isolates (35
C. parapsilosis
, 30
C. albicans
, 18
C. tropicalis
, 8
C. glabrata
, and 10 other
Candida
species isolates) and 259 clinical isolates from other body sites (116
C. albicans
, 53
C. glabrata
, 43
C. tropicalis
, 17
C. parapsilosis
, and 30 other
Candida
species isolates). Organisms were grown in Sabouraud dextrose broth (SDB) containing a final concentration of 8% glucose to induce biofilm formation, as published previously. Biofilm production was determined by both visual and spectrophotometric methods. In this medium, biofilm production by
C. albicans
isolates was significantly less frequent (8%) than that by non-
C. albicans Candida
species (61%;
P
< 0.0001). The overall proportion of non-
C. albicans Candida
species isolates from the blood that produced biofilms was significantly higher than that of non-
C. albicans Candida
isolates obtained from other sites (79% versus 52%;
P
= 0.0001). Bloodstream isolates of
C. parapsilosis
alone were significantly more likely to be biofilm positive than were
C. parapsilosis
isolates from other sites (86% versus 47%;
P
= 0.0032). Non-
C. albicans Candida
species, including
C. parapsilosis
, were more likely to be biofilm positive if isolates were derived from patients whose candidemia was central venous catheter (CVC) related (95%;
P
< 0.0001) and was associated with the use of total parenteral nutrition (TPN) (94%;
P
< 0.005). These data suggest that the capacity of
Candida
species isolates to produce biofilms in vitro in glucose-containing SDB may be a reflection of the pathogenic potential of these isolates to cause CVC-related fungemia in patients receiving TPN.
Detection of a Human Rotavirus with G12 and P[9] Specificity in Thailand
Authors: Pongsuwanna Y. , Guntapong R. , Chiwakul M. , Tacharoenmuang R. , Onvimala N. , Wakuda M. , Kobayashi N. , Taniguchi K.
Published: July 2002

G12 rotavirus has not been detected anywhere in the world since the first detection of a human strain, L26 (G12, P1B[4]), in the Philippines in 1990. In this study, we isolated a human rotavirus (strain T152) with a VP7 of G12 specificity from the stool of an 11-month-old diarrheic patient in Thailand. The strain T152 exhibited a long RNA pattern and subgroup I specificity. In the comparison of the nucleotide and amino acid sequences of the VP7 gene of strain T152 with those of rotaviruses with different G type specificities, strain T152 showed the highest identity, 90.9 and 93.9%, respectively, to G12 prototype strain L26. In contrast, the VP4 gene of strain T152 showed the highest identity with P[9] specificity of human strains K8 and AU-1 and feline strains Cat2 and FRV-1, with homologies of 89.3 to 90.6% at the nucleotide level and 93.9 to 95.6% at the amino acid level. Thus, strain T152 was found to be a natural reassortant strain with G12 and P[9] specificities.
Comparison of Eleven Commercial Tests for
Chlamydia pneumoniae
-Specific Immunoglobulin G in Asymptomatic Healthy Individuals
Authors: Hermann Corinna , Graf Kathrin , Groh Annemarie , Straube Eberhard , Hartung Thomas
Published: July 2002

The seroprevalence of anti-
Chlamydia pneumoniae
-specific immunoglobulin G (IgG) antibodies is high in the adult population. Experience is required to perform a microimmunofluorescence test (MIF), the current “gold standard” for serological diagnosis, and the assay still lacks standardization. Partially automated enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs), which are more standardized and for which the reading of results is less subjective, have been developed. The different commercially available serological tests differ in their sensitivities and specificities, depending primarily on the antigen used. Therefore, we evaluated 11 different tests (10 were species specific, 1 was genus specific) for IgG antibodies using serum samples of 80 apparently healthy volunteers. The interpretation of the results was based on the results of the gold standard, MIF: a sample was judged positive if it was positive by at least three of the four different MIFs. Based on this internal standard, we found that 71% of the samples were positive, while 8% were false positive by some tests. The correlations between the results of the different MIFs ranged from 83 to 99%, and the correlations between the results of the MIFs and the different ELISAs and EIAs ranged from 78 to 98%. Comparison of the IgG titers measured by MIF showed good agreement (
r
= 0.76 to 0.91). This analysis revealed that some ELISAs and EIAs fail to detect low IgG titers. The specificities of the species-specific tests varied from 95 to 100%, and the sensitivities varied from 58 to 100%. These results indicate that serological assays for the detection of anti-
C. pneumoniae
-specific IgG vary greatly in their sensitivities and specificities. MIF must still be considered the best method for the detection of IgG in apparently healthy subjects, but the sensitivities and specificities of new ELISAs approximate those of MIFs.
Candida glabrataOropharyngeal Candidiasis in Patients Receiving Radiation Treatment for Head and Neck Cancer
Authors: Redding Spencer W. , Kirkpatrick William R. , Coco Brent J. , Sadkowski Lee , Fothergill Annette W. , Rinaldi Michael G. , Eng Tony Y. , Patterson Thomas F.
Published: July 2002

Candida glabratacolonization is common in patients receiving radiation treatment for head and neck cancer, but to our knowledge has never been described as the infecting organism with oropharyngeal candidiasis (OPC). This study presents the first three patients described withC.glabrataOPC in this patient population. Patient 1 developedC.glabrataOPC and required fluconazole, 800 mg/day, for clinical resolution. Antifungal susceptibility testing revealed a MIC of fluconazole of >64 μg/ml. Elapsed time from initial culturing to treatment decision was 7 days. Patients 2 and 3 developedC.glabrataOPC. They were patients in a study evaluating OPC infections, and cultures were taken immediately. CHROMagarCandidaplates with 0, 8, and 16 μg of fluconazole/ml were employed for these cultures. Lavender colonies, consistent withC.glabrata, grew on the 0- and 8-μg plates but not on the 16-μg plate from patient 2 and grew on all three plates from patient 3. Based on these data, a fluconazole dose of 200 mg/day was chosen for patient 2 and a dose of 400 mg/day was chosen for patient 3, with clinical resolution in both. Elapsed time from initial culturing to treatment decision was 2 days.C.glabratadoes cause OPC in head and neck radiation treatment patients, and the use of fluconazole-impregnated chromogenic agar may significantly reduce treatment decision time compared to that with conventional culturing and antifungal susceptibility testing.
Characterization of Unusual Bacteria Isolated from Respiratory Secretions of Cystic Fibrosis Patients and Description of
Inquilinus limosus
gen. nov., sp. nov
Authors: Coenye Tom , Goris Johan , Spilker Theodore , Vandamme Peter , LiPuma John J.
Published: July 2002

Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include
Acinetobacter
sp.,
Bordetella hinzii
,
Burkholderia fungorum
,
Comamonas testosteroni
,
Chryseobacterium
sp.,
Herbaspirillum
sp.,
Moraxella osloensis
,
Pandoraea
genomospecies 4,
Ralstonia gilardii
,
Ralstonia mannitolilytica
,
Rhizobium radiobacter
, and
Xanthomonas
sp. In addition, one isolate most likely represents a novel
Ralstonia
species, whereas nine isolates belong to novel taxa within the α-
Proteobacteria
. Eight of these latter isolates are classified into the novel genus
Inquilinus
gen. nov. as
Inquilinus limosus
gen. nov., sp. nov., or as
Inquilinus
sp. The remaining 17 isolates are characterized as members of the family
Enterobacteriaceae
. The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.
Fatal Case of Pneumonia Caused by a Nonhemolytic Strain of
Streptococcus pyogenes
Authors: Taylor Mark B. , Barkham Timothy
Published: July 2002

We report a case of pneumonia with a fatal outcome caused by a nonhemolytic strain of
Streptococcus pyogenes
. This strain was isolated in pure growth from blood cultures and was fully identified biochemically. Such strains will be difficult to recognize and isolate from sites with heavy growth of normal flora. This phenomenon has been reported sporadically before, and it is unknown how common such strains may be in pharyngeal samples.
The Spread of Herpes Simplex Virus Type 1 from Trigeminal Neurons to the Murine Cornea: an Immunoelectron Microscopy Study
Authors: Ohara Peter T. , Chin Marian S. , LaVail Jennifer H.
Published: July 2002

An animal model has been developed to clarify the mechanism for spread of herpes simplex virus (HSV) from neuron to epithelial cells in herpetic epithelial keratitis. HSV was introduced into the murine trigeminal ganglion via stereotaxic guided injection. After 2 to 5 days, the animals were euthanized. Ganglia and corneas were prepared for light and electron microscopic immunocytochemistry with antisera to HSV. At 2 days, labeled axons were identified in the stromal layer. At 3 days, we could detect immunoreactive profiles of trigeminal ganglion cell axons that contained many vesicular structures. By 3 and 4 days, the infection had spread to all layers of epithelium, and the center of a region of infected epithelium appeared thinned. At 5 day, fewer basal cells appeared infected, although infection persisted in superficial cells where it had expanded laterally. Mature HSV was found in the extracellular space surrounding wing and squamous cells. Viral antigen was expressed in small pits along the apical surfaces of wing and squamous cells but not at the basal surface of these cells or on basal cells. This polarized expression of viral antigen resulted in the spread of HSV to superficial cells and limited lateral spread to neighboring basal cells. The pathogenesis of HSV infection in these mice may serve as a model of the human recurrent epithelial disease in the progression of focal sites of infection and transfer from basal to superficial cells.
Functional Analysis of the Genomic and Antigenomic Promoters of Human Respiratory Syncytial Virus
Authors: Fearns Rachel , Collins Peter L. , Peeples Mark E.
Published: July 2002

The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon system. The 3′ ends of the genome and antigenome, which, respectively, contain the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) regions, should each contain a promoter for RNA replication. The 3′ genome end also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3′-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when juxtaposed with a transcription gene-start (GS) signal, this sequence was also able to direct transcription. It was also shown that the region of Le immediately preceding the GS signal of the first gene could be deleted with either no effect or with a slight decrease in transcription initiation. Thus, the TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3′ end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription. Increasing the length of the introduced TrC sequence incrementally to 147 nt resulted in a fourfold increase in replication and a nearly complete inhibition of transcription. These two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but rather are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enhancing
cis
-acting element. In contrast, the inhibitory effect on transcription was due solely to the altered spacing between the 3′ end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor the inhibition of transcription was due to increased base-pairing potential between the 3′ and 5′ ends. The relative strengths of the Le and TrC promoters for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity between the RSV antigenomic and genomic promoters, but provided a further distinction between promoter requirements for transcription and replication.
Gene Gun-Mediated DNA Immunization Primes Development of Mucosal Immunity against Bovine Herpesvirus 1 in Cattle
Authors: Loehr B. I. , Willson P. , Babiuk L. A. , van Drunen Littel-van den Hurk S.
Published: July 2002

Vaccination by a mucosal route is an excellent approach to the control of mucosally acquired infections. Several reports on rodents suggest that DNA vaccines can be used to achieve mucosal immunity when applied to mucosal tissues. However, with the exception of one study with pigs and another with horses, there is no information on mucosal DNA immunization of the natural host. In this study, the potential of inducing mucosal immunity in cattle by immunization with a DNA vaccine was demonstrated. Cattle were immunized with a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein B, which was delivered with a gene gun either intradermally or intravulvomucosally. Intravulvomucosal DNA immunization induced strong cellular immune responses and primed humoral immune responses. This was evident after BHV-1 challenge when high levels of both immunoglobulin G (IgG) and IgA were detected. Intradermal delivery resulted in lower levels of immunity than mucosal immunization. To determine whether the differences between the immune responses induced by intravulvomucosal and intradermal immunizations might be due to the efficacy of antigen presentation, the distributions of antigen and Langerhans cells in the skin and mucosa were compared. After intravulvomucosal delivery, antigen was expressed early and throughout the mucosa, but after intradermal administration, antigen expression occurred later and superficially in the skin. Furthermore, Langerhans cells were widely distributed in the mucosal epithelium but found primarily in the basal layers of the epidermis of the skin. Collectively, these observations may account for the stronger immune response induced by mucosal administration.
Sequences of
Citrus Tristeza Virus
Separated in Time and Space Are Essentially Identical
Authors: Albiach-Martí María R. , Mawassi Munir , Gowda Siddarame , Satyanarayana Tatineni , Hilf Mark E. , Shanker Savita , Almira Ernesto C. , Vives María C. , López Carmelo , Guerri Jose , Flores Ricardo , Moreno Pedro , Garnsey Steve M. , Dawson William O.
Published: July 2002

The first
Citrus tristeza virus
(CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.
The Karyophilic Properties of Human Immunodeficiency Virus Type 1 Integrase Are Not Required for Nuclear Import of Proviral DNA
Authors: Petit Caroline , Schwartz Olivier , Mammano Fabrizio
Published: July 2002

Integrase (IN) is a key component of the preintegration nucleoprotein complex (PIC), which transports the retroviral genome from the cytoplasm to the nucleus of newly infected cells. Retroviral IN proteins have intrinsic karyophilic properties, which for human immunodeficiency virus type 1 (HIV-1) are currently attributed to regions that display sequence homology to previously characterized nuclear localization signals. We asked here whether the karyophilic properties of HIV-1 IN are involved in the nuclear import of PIC. We mutated three conserved basic regions in the C-terminal domain of IN and analyzed the effects of mutations on subcellular localization of the protein, viral particle composition, IN dimerization within virions, and infectivity. Alteration of two sequences caused the loss of nuclear accumulation of IN and drastically reduced the capacity of the protein to multimerize. Mutation of the most C-terminal sequence had no effect on the subcellular localization and dimerization of IN. Nevertheless, conservation of all three sequences was required for viral infectivity. Despite the perturbation of IN subcellular localization, all mutant viruses displayed normal reverse transcription and nuclear transport of PICs in newly infected cells. The replicative defect was instead at the level of integration, for which all mutants were markedly affected in vivo. Besides reinforcing the association between dimerization of IN and nuclear accumulation of the enzyme, our data demonstrate that subcellular localization of IN alone cannot predict the fate of the PICs.
Effect of Coexpression of Interleukin-2 by Recombinant Respiratory Syncytial Virus on Virus Replication, Immunogenicity, and Production of Other Cytokines
Authors: Bukreyev Alexander , Whitehead Stephen S. , Prussin Calman , Murphy Brian R. , Collins Peter L.
Published: July 2002

We constructed rRSV/mIL-2, a recombinant respiratory syncytial virus (rRSV) containing the coding sequence of murine interleukin-2 (mIL-2) in a transcription cassette inserted into the G-F intergenic region. The recovered virus (rRSV/mIL-2) expressed high levels (up to 2.8 μg/ml) of mIL-2 in cell culture. Replication of rRSV/mIL-2 in vitro was reduced up to 13.6-fold from that of wild-type (wt) rRSV, an effect that was due to the presence of the foreign insert but was not specific to mIL-2. Replication of the rRSV/mIL-2 virus in the upper and lower respiratory tracts of BALB/c mice was reduced up to 6.3-fold, an effect that was specific to mIL-2. The antibody response, including the levels of RSV-specific serum immunoglobulin G1 (IgG1), IgG2a, IgA, and total IgG, and the level of protective efficacy against wt RSV challenge were not significantly different from those of wt rRSV. Analysis of total pulmonary cytokine mRNA isolated 1 and 4 days following infection with rRSV/mIL-2 revealed elevated levels of mRNA for IL-2, gamma interferon (IFN-γ), IL-4, IL-5, IL-6, IL-10, IL-13, and IL-12 p40 compared to those for wt rRSV. Flow cytometry of total pulmonary mononuclear cells isolated 10 days following infection with rRSV/mIL-2 revealed increased levels of CD4+T lymphocytes expressing either IFN-γ or IL-4 compared to those of wt rRSV. These elevations in cytokine mRNA or cytokine-expressing CD4+cells relative to those of wt rRSV-primed animals were not observed following challenge with wt RSV on day 28. Thus, the expression of mIL-2 by rRSV was associated with a modest attenuation of virus growth in vivo, induction of serum antibodies at levels comparable to that of wt rRSV, and transient increases in both the Th1 and Th2 CD4+lymphocytes and cytokine mRNAs compared to those of wt rRSV.
Overexpression of Essential Splicing Factor ASF/SF2 Blocks the Temporal Shift in Adenovirus Pre-mRNA Splicing and Reduces Virus Progeny Formation
Authors: Molin Magnus , Akusjärvi Göran
Published: July 2002

Expression of cytoplasmic mRNA from most adenovirus transcription units is subjected to a temporal regulation at the level of alternative pre-mRNA splicing. The general tendency is that splice site selection changes from proximal to distal late after infection. Interestingly, ASF/SF2, which is a prototypical member of the SR family of splicing factors, has the opposite effect on splice site selection, inducing an increase in proximal splice site usage. We have previously shown that SR proteins late during an adenovirus infection become partially inactivated as splicing regulatory proteins. A prediction from these results is that overexpression of an SR protein, such as ASF/SF2, during virus growth will interfere with virus replication by disturbing the balance of functional and nonfunctional ASF/SF2 in the infected cell. To test this hypothesis, we reconstructed a recombinant adenovirus expressing ASF/SF2 under the transcriptional control of a regulated promoter. The results show that, as predicted, induction of ASF/SF2 during lytic virus growth prevents the early to late shift in mRNA expression from both early (E1A and E1B) and late (L1) transcription units. Furthermore, ASF/SF2 overexpression blocks viral DNA replication and reduces selectively cytoplasmic accumulation of major late mRNA, resulting in a lower virus yield. Collectively, our results provide additional support for the hypothesis that viral control of SR protein function is important for the proper expression of viral proteins during lytic virus growth.
Uneven Distribution of Hepatitis C Virus Quasispecies in Tissues from Subjects with End-Stage Liver Disease: Confounding Effect of Viral Adsorption and Mounting Evidence for the Presence of Low-Level Extrahepatic Replication
Authors: Laskus Tomasz , Radkowski Marek , Wang Lian-Fu , Nowicki Marek , Rakela Jorge
Published: July 2002

We have found differences among the populations of hepatitis C virus sequences in serum, peripheral blood mononuclear cells (PBMCs), and various tissues in patients with chronic hepatitis C. These results are compatible with the existence of independent viral compartments in the infected host. Our results also suggest that PBMCs, and probably various tissues, can selectively adsorb viral subpopulations differing in the E2 region.
Intrinsic Susceptibility of Rhesus Macaque Peripheral CD4+T Cells to Simian Immunodeficiency Virus In Vitro Is Predictive of In Vivo Viral Replication
Authors: Goldstein Simoy , Brown Charles R. , Dehghani Houman , Lifson Jeffrey D. , Hirsch Vanessa M.
Published: July 2002

Previous studies with simian immunodeficiency virus (SIV) infection of rhesus macaques suggested that the intrinsic susceptibility of peripheral blood mononuclear cells (PBMC) to infection with SIV in vitro was predictive of relative viremia after SIV challenge. The present study was conducted to evaluate this parameter in a well-characterized cohort of six rhesus macaques selected for marked differences in susceptibility to SIV infection in vitro. Rank order relative susceptibility of PBMC to SIVsmE543-3-infection in vitro was maintained over a 1-year period of evaluation. Differential susceptibility of different donors was maintained in CD8+T-cell-depleted PBMC, macrophages, and CD4+T-cell lines derived by transformation of PBMC with herpesvirus saimiri, suggesting that this phenomenon is an intrinsic property of CD4+target cells. Following intravenous infection of these macaques with SIVsmE543-3, we observed a wide range in plasma viremia which followed the same rank order as the relative susceptibility established by in vitro studies. A significant correlation was observed between plasma viremia at 2 and 8 weeks postinoculation and in vitro susceptibility (P< 0.05). The observation that the two most susceptible macaques were seropositive for simian T-lymphotropic virus type 1 may suggests a role for this viral infection in enhancing susceptibility to SIV infection in vitro and in vivo. In summary, intrinsic susceptibility of CD4+target cells appears to be an important factor influencing early virus replication patterns in vivo that should be considered in the design and interpretation of vaccine studies using the SIV/macaque model.
Gene Transfer Using Recombinant Rabbit Hemorrhagic Disease Virus Capsids with Genetically Modified DNA Encapsidation Capacity by Addition of Packaging Sequences from the L1 or L2 Protein of Human Papillomavirus Type 16
Authors: El Mehdaoui Slimane , Touzé Antoine , Laurent Sylvie , Sizaret Pierre-Yves , Rasschaert Denis , Coursaget Pierre
Published: July 2002

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.
Rotavirus Infection Induces Cytoskeleton Disorganization in Human Intestinal Epithelial Cells: Implication of an Increase in Intracellular Calcium Concentration
Authors: Brunet Jean-Philippe , Jourdan Nathalie , Cotte-Laffitte Jacqueline , Linxe Catherine , Géniteau-Legendre Monique , Servin Alain , Quéro Anne-Marie
Published: July 2002

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca
2+
-dependent microtubule disassembly and Ca
2+
-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.
The S Segment of Rift Valley Fever Phlebovirus (
Bunyaviridae
) Carries Determinants for Attenuation and Virulence in Mice
Authors: Vialat P. , Billecocq A. , Kohl A. , Bouloy M.
Published: July 2002

Unlike all the other Rift Valley fever virus strains (
Bunyaviridae
,
Phlebovirus
) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the NSs nonstructural protein in the nuclei of infected cells (R. Muller, J. F. Saluzzo, N. Lopez, T. Drier, M. Turell, J. Smith, and M. Bouloy, Am. J. Trop. Med. Hyg. 53:405–411, 1995). This defect is correlated with a large in-frame deletion in the NSs coding region of the S segment of the tripartite genome. Here, we show that the truncated NSs protein of clone 13 is expressed and remains in the cytoplasm, where it is degraded rapidly by the proteasome. Through the analysis of reassortants between clone 13 and a virulent strain, we localized the marker(s) of attenuation in the S segment of this attenuated virus. This result raises questions regarding the role of NSs in pathogenesis and highlights, for the first time in the
Bunyaviridae
family, a major role of the S segment in virulence and attenuation, possibly associated with a defect in the nonstructural protein.
Identification and Analysis of the K5 Gene of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Haque Muzammel , Chen Jiguo , Ueda Keiji , Mori Yasuko , Nakano Kazusi , Hirata Yuko , Kanamori Shiro , Uchiyama Yasuo , Inagi Reiko , Okuno Toshiomi , Yamanishi Koichi
Published: July 2002

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes ∼80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-
O
-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5′ and 3′ ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.
Functional Reconstitution of Thymopoiesis after Human Immunodeficiency Virus Infection
Authors: Kitchen Scott G. , Killian Scott , Giorgi Janis V. , Zack Jerome A.
Published: July 2002

We have utilized combination antiretroviral therapy following human immunodeficiency virus type 1-induced human CD4+thymocyte depletion in the SCID-hu mouse to examine the immune competence of reconstituting thymocytes which appear following administration of combination therapy. These cells express a normal distribution of T-cell receptor variable gene families and are responsive to costimulatory signals. These results suggest that normal thymic function may be restored following antiretroviral treatment.
A Group B Coxsackievirus/Poliovirus 5′ Nontranslated Region Chimera Can Act as an Attenuated Vaccine Strain in Mice
Authors: Chapman Nora M. , Ragland Anna , Leser J. Smith , Höfling Katja , Willian Sandra , Semler Bert L. , Tracy Steven
Published: July 2002

The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5′ NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors.
Apoptosis in Coxsackievirus B3-Caused Diseases: Interaction between the Capsid Protein VP2 and the Proapoptotic Protein Siva
Authors: Henke Andreas , Launhardt Heike , Klement Katrin , Stelzner Axel , Zell Roland , Munder Thomas
Published: July 2002

Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. Apoptotic events are present in CVB3-induced disease, but it is unclear how CVB3 is involved in apoptosis and which viral proteins may induce the apoptotic pathway. In this report we demonstrate that the human and murine proapoptotic protein Siva specifically interact with the CVB3 capsid protein VP2. Furthermore, the transcription of Siva is strongly induced in tissue of CVB3-infected mice and is present in the same area which is positively stained for apoptosis, CD27, and CD70. It has been proposed that Siva is involved in the CD27/CD70-transduced apoptosis. Therefore, we suggest a molecular mechanism through which apoptotic events contributes to CVB3-caused pathogenesis.