bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Chimeric Antibodies as Positive Controls in an Enzyme-Linked Immunosorbent Assay for Diagnosis of Scrub Typhus (Infection by
Orientia tsutsugamushi
)
Authors: Jones Martina L. , Barnard Ross T. 
Published: August 2007

ABSTRACT

The use of human sera collected from individuals of known infected and noninfected status is necessary for the validation of diagnostic assays and for the determination of cutoff values. However, the routine inclusion of pooled human sera from infected individuals for use as positive controls in commercial assay kits has many disadvantages. Sufficient quantities of sera can be difficult to obtain, and there are ethical and safety issues to be considered. Additionally, each batch of control material requires standardization, as each will differ in antibody titer. We have genetically engineered chimeric immunoglobulin G (IgG), IgM, and IgA antibodies consisting of mouse-derived variable regions and human constant regions derived from peripheral blood lymphocytes. The chimeric nature of these antibodies allows the desired antigen specificity created through mouse immunization and hybridoma technology while retaining a human constant region required for recognition by the enzyme-conjugated antihuman signal antibody. We have investigated the potential use of chimeric IgG with specificity for the major surface antigen of
Orientia tsutsugamushi
as an alternative positive control for inclusion in a commercial enzyme-linked immunosorbent assay kit for the diagnosis of rickettsia scrub typhus (caused by infection with
O. tsutsugamushi
). Chimeric IgG was expressed in stably transfected CHO cells, allowing production of unlimited quantities. The purified protein was found to have a much greater specificity for the scrub typhus antigen than the serum-derived controls. The methods described could be applied to other assay kits for the detection of antibodies against infectious agents.
Salmonella enterica
Serovar Typhi Ty21a Expressing Human Papillomavirus Type 16 L1 as a Potential Live Vaccine against Cervical Cancer and Typhoid Fever
Authors: Fraillery Dominique , Baud David , Pang Susana Yuk-Ying , Schiller John , Bobst Martine , Zosso Nathalie , Ponci Françoise , Nardelli-Haefliger Denise 
Published: August 2007

ABSTRACT

Human papillomavirus (HPV) vaccines based on L1 virus-like particles (VLPs) can prevent HPV-induced genital neoplasias, the precursors of cervical cancer. However, most cervical cancers occur in developing countries, where the implementation of expensive vaccines requiring multiple injections will be difficult. A live
Salmonella
-based vaccine could be a lower-cost alternative. We previously demonstrated that high HPV type 16 (HPV16)-neutralizing titers are induced after a single oral immunization of mice with attenuated
Salmonella enterica
serovar Typhimurium strains expressing a codon-optimized version of HPV16 L1 (L1S). To allow the testing of this type of vaccine in women, we constructed a new L1-expressing plasmid, kanL1S, and tested kanL1S recombinants of three
Salmonella enterica
serovar Typhi vaccine strains shown to be safe in humans, i.e., Ty21a, the actual licensed typhoid vaccine, and two highly immunogenic typhoid vaccine candidates, Ty800 and CVD908-
htrA
. In an intranasal mouse model of
Salmonella
serovar Typhi infection, Ty21a kanL1S was unique in inducing HPV16-neutralizing antibodies in serum and genital secretions, while anti-
Salmonella
responses were similar to those against the parental Ty21a vaccine. Electron microscopy examination of Ty21a kanL1S lysates showed that L1 assembled in capsomers and capsomer aggregates but not well-ordered VLPs. Comparison to the neutralizing antibody response induced by purified HPV16 L1 VLP immunizations in mice suggests that Ty21a kanL1S may be an effective prophylactic HPV vaccine. Ty21a has been widely used against typhoid fever in humans with a remarkable safety record. These finds encourage clinical testing of Ty21a kanL1S as a combined typhoid fever/cervical cancer vaccine with the potential for worldwide application.
Differential B-Cell Responses Are Induced byMycobacterium tuberculosisPE Antigens Rv1169c, Rv0978c, and Rv1818c
Authors: Narayana Yeddula , Joshi Beenu , Katoch V. M. , Mishra Kanhu Charan , Balaji Kithiganahalli N. 
Published: August 2007

ABSTRACTThe multigene PE and PPE family represents about 10% of the genome ofMycobacterium tuberculosis. Here, we report that three members of the PE family, namely, Rv1169c, Rv0978c, and Rv1818c, elicit a strong, but differential, B-cell humoral response among different clinical categories of tuberculosis patients. The study population (n= 211) was comprised of different clinical groups of both adult and child patients: group 1 (n= 94) patients with pulmonary infection, group 2 (n= 30) patients with relapsed infection, group 3 (n= 31) patients with extrapulmonary infections, and clinically healthy donors (n= 56). Among the PE proteins studied, group 1 adult patient sera reacted to Rv1818c and Rv0978c, while Rv1169c elicited immunoreactivity in group 3 children. However, all three PE antigens studied as well as the 19-kDa antigen did not demonstrate humoral reactivity with sera from group 2 patients with relapsed infection. The current study shows that while responsiveness to all three PE antigens is a good marker forM. tuberculosisinfection, a strong response to Rv0978c or to Rv1818c by group 1 adult patients with pulmonary infection or largely restricted reactivity to Rv1169c antigen in child patients with extrapulmonary infections offers the possibility of differential utility in the serodiagnosis of tuberculosis.
Comparative Analyses of Phenotypic and Genotypic Methods for Detection of Enterotoxigenic
Escherichia coli
Toxins and Colonization Factors
Authors: Sjöling Å. , Wiklund G. , Savarino S. J. , Cohen D. I. , Svennerholm A.-M. 
Published: August 2007

ABSTRACT

Enterotoxigenic
Escherichia coli
(ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers. However, this pathogen has often not been reported in surveys of diarrheal pathogens, due to lack of simple standardized methods to detect ETEC in many laboratories. ETEC expresses one or both of two different enterotoxin subtypes: heat-stable toxins, a heat-labile toxin (LT), and more than 22 different colonization factors (CFs) that mediate adherence to the intestinal cell wall. Here we compare established phenotypic and genotypic detection methods and newly developed PCR detection methods with respect to sensitivity, specificity, positive predictive value, and ease of performance. The methods include GM1-enzyme-linked immunosorbent assay and dot blot techniques using specific monoclonal antibodies (MAbs) for phenotypic detection of the toxins and CFs, respectively, as well as different PCR and DNA/DNA hybridization techniques, including new PCR assays, for genotypic identification of the toxin and CF genes, respectively. We found very good general agreement in results derived from genotypic and phenotypic methods. In a few strains, LT and CFs were identified genetically but not phenotypically. Based on our analyses, we recommend initial screening for ETEC in clinical samples by multiplex toxin gene PCR. Toxin-positive strains may then be analyzed by dot blot tests for detection of the CFs expressed on the bacterial surface and by PCR for determination of additional CFs for which MAbs are currently lacking as well as for strains that harbor silent CF genes.
Persistently Infected Calves as Reservoirs for Acquisition and Transovarial Transmission of
Babesia bovis
by
Rhipicephalus
(
Boophilus
)
microplus
Authors: Howell Jeanne M. , Ueti Massaro W. , Palmer Guy H. , Scoles Glen A. , Knowles Donald P. 
Published: August 2007

ABSTRACT

Babesia bovis
is a deadly disease of cattle resulting in severe economic losses in the vast regions of the world where it is endemic. If reintroduced into the United States, babesiosis would cause significant mortality in the naïve cattle population. In order to address the risk to U.S. cattle, it is essential to quantify the transovarial transmission efficiency in adult female
Boophilus microplus
ticks following acquisition feeding on persistently infected cattle. This study tested the hypothesis that infection rates are the same for larval progeny derived from females fed to repletion during persistent or acute infection. Increasing parasite levels during acute infection correlated with an increasing number of females harboring kinetes detectable in hemolymph (
r
= 0.9). The percent infected larvae ranged from 0 to 20% when derived from females fed to repletion on persistently infected calves and from 4 to 6% when derived from females fed to repletion during acute parasitemia. There was no significant difference in infection rates of larval progeny, implying that the risk associated with the introduction of either persistently infected or acutely infected cattle is equal. Parasite levels ranged from 2.4 × 10
2
to 1.9 × 10
5
in 3-day-fed larvae derived from females fed to repletion on persistently infected cattle. One group of larvae failed to transmit the parasite, suggesting that a threshold level of parasites must be obtained by larval progeny via transovarial transmission in order for larvae to deliver sufficient parasites to infect a naïve host.
Identification of Less-Common
Streptococcus pneumoniae
Serotypes by a Multiplex PCR-Based Reverse Line Blot Hybridization Assay
Authors: Zhou Fei , Kong Fanrong , Tong Zhongsheng , Gilbert Gwendolyn L. 
Published: August 2007

ABSTRACT
We developed a multiplex PCR-based reverse line blot (mPCR/RLB) assay to identify 50 uncommon pneumococcal serotypes. In combination with a previously described mPCR/RLB assay (3), all 90 pneumococcal serotypes can be identified individually (32 serotypes) or, because of predictable cross-reactions, to within small groups of two to five related serotypes (58 serotypes), which can be distinguished using serotype-specific antisera.
Longitudinal Molecular Epidemiological Analysis of Feline Calicivirus Infection in an Animal Shelter: a Model for Investigating Calicivirus Transmission within High-Density, High-Turnover Populations
Authors: Coyne Karen P. , Edwards David , Radford Alan D. , Cripps Peter , Jones David , Wood James L. N. , Gaskell Rosalind M. , Dawson Susan 
Published: August 2007

ABSTRACT
The control of outbreaks of calicivirus infection in high-density, high-throughput populations is a challenge to both human and veterinary medicine. In such populations, the prevalence of infection is, in part, dependent on the levels of biosecurity and how this affects virus transmission. Here we show how longitudinal analysis of feline calicivirus (FCV) infection in an animal rescue shelter can be used as a model to examine the dynamics of calicivirus transmission and evolution in such environments. FCV was isolated from 33 of 116 cats sampled over a 15-month period (overall prevalence, 28%). Sequence analysis of the immunodominant variable regions of the viral capsid gene identified 16 strains circulating in the shelter, with no single strain appearing to predominate. The majority of these strains were introduced into the shelter from the community and did not appear to be transmitted within the population. However, for three of these strains, putative transmission events within the shelter were identified. The rates of evolution within hypervariable regions of the FCV capsid gene in individual cats ranged from 0.05 to 1.4% per week, with the highest rates generally being found in animals that either acquired the virus while in the shelter or were undergoing acute infection. These data suggest that despite the high prevalence and presence of multiple strains of FCV within the shelter, the spread of such pathogens may be restricted by various control measures, including good hygiene and biosecurity.
Chronological Study of Antibiotic Resistances and Their Relevant Genes in Korean Avian Pathogenic
Escherichia coli
Isolates
Authors: Kim Tae-Eun , Jeong Yong-Wun , Cho Sun-Hee , Kim Sun-Joong , Kwon Hyuk-Joon 
Published: August 2007

ABSTRACT

Antibiograms and relevant genotypes of Korean avian pathogenic
Escherichia coli
(APEC) isolates (
n
= 101) recovered between 1985 and 2005 were assessed via disc diffusion test, PCR, restriction enzyme analysis, and sequencing. These isolates were highly resistant to tetracycline (84.2%), streptomycin (84.2%), enrofloxacin (71.3%), and ampicillin (67.3%), and most of the tetracycline, streptomycin, enrofloxacin, and ampicillin resistances were associated with
tetA
and/or
tetB
,
aadA
and/or
strA-strB
, mutations in
gyrA
and/or
parC
, and
TEM
, respectively. Class 1 integrons were detected in 40 isolates (39.6%), and a variety of gene cassettes conferring streptomycin (
aadA
), gentamicin (
aadB
), and trimethoprim (
dfr
) resistances were identified:
aadA1a
(27.5%),
dfrV
-
orfD
(2.5%),
aadB
-
aadA1a
(2.5%),
dfrI
-
aadA1a
(47.5%),
dfrXVII
-
aadA5
(12.5%), and
dfrXII
-
orfF
-
aadA2
(7.5%). In addition, several types of common promoters (P
ant
) of the gene cassettes (hybrid P1, weak P1, or weak P1 plus P2) and single-nucleotide polymorphisms in
aadA1a
were identified. The results of a chronological analysis demonstrated significant and continuous increases in the frequencies of resistances to several antibiotics (tetracycline, streptomycin, enrofloxacin, ampicillin, and trimethoprim-sulfamethoxazole) and of the relevant resistance genes (
tetA
,
strA-strB
, and
TEM
), mutations in
gyrA
and
parC
, and multidrug-resistant APEC strains during the period 2000 to 2005.
Antisense Transcription in the Human Cytomegalovirus Transcriptome
Authors: Zhang Guojuan , Raghavan Bindu , Kotur Mark , Cheatham Jacquelyn , Sedmak Daniel , Cook Charles , Waldman James , Trgovcich Joanne 
Published: August 2007

ABSTRACT
Human cytomegalovirus (HCMV) infections are prevalent in human populations and can cause serious diseases, especially in those with compromised or immature immune systems. The HCMV genome of 230 kb is among the largest of the herpesvirus genomes. Although the entire sequence of the laboratory-adapted AD169 strain of HCMV has been available for 18 years, the precise number of viral genes is still in question. We undertook an analysis of the HCMV transcriptome as an approach to enumerate and analyze the gene products of HCMV. Transcripts of HCMV-infected fibroblasts were isolated at different times after infection and used to generate cDNA libraries representing different temporal classes of viral genes. cDNA clones harboring viral sequences were selected and subjected to sequence analysis. Of the 604 clones analyzed, 45% were derived from genomic regions predicted to be noncoding. Additionally, at least 55% of the cDNA clones in this study were completely or partially antisense to known or predicted HCMV genes. The remarkable accumulation of antisense transcripts during infection suggests that currently available genomic maps based on open-reading-frame and other in silico analyses may drastically underestimate the true complexity of viral gene products. These findings also raise the possibility that aspects of both the HCMV life cycle and genome organization are influenced by antisense transcription. Correspondingly, virus-derived noncoding and antisense transcripts may shed light on HCMV pathogenesis and may represent a new class of targets for antiviral therapies.
Fowlpox Virus Encodes a Bcl-2 Homologue That Protects Cells from Apoptotic Death through Interaction with the Proapoptotic Protein Bak
Authors: Banadyga Logan , Gerig Jenna , Stewart Tara , Barry Michele 
Published: August 2007

ABSTRACT

Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the
Avipoxvirus
genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.
Pathogenic Potential of Borna Disease Virus Lacking the Immunodominant CD8 T-Cell Epitope
Authors: Richter Kirsten , Baur Karen , Ackermann Andreas , Schneider Urs , Hausmann Jürgen , Staeheli Peter 
Published: August 2007

ABSTRACTBorna disease virus (BDV) is a highly neurotropic, noncytolytic virus. Experimentally infected B10.BR mice remain healthy unless specific antiviral T cells that infiltrate the infected brain are triggered by immunization. In contrast, infected MRL mice spontaneously mount an antiviral T-cell response that can result in meningoencephalitis and neurological disease. The antiviral T cells may, alternatively, eliminate the virus without inducing disease if they are present in sufficient numbers before the virus replicates to high titers. Since the immune response of H-2kmice is directed mainly against the epitope TELEISSI located in the viral nucleoprotein N, we generated BDV mutants that feature TQLEISSI in place of TELEISSI. We show that adoptive transfer of BDV N-specific CD8 T cells induced neurological disease in B10.BR mice persistently infected with wild-type BDV but not with the mutant virus expressing TQLEISSI. Surprisingly, the mutant virus replicated less well in adult MRL wild-type mice than in mutant mice lacking mature CD8 T cells. Furthermore, when MRL mice were infected with the TQLEISSI-expressing BDV mutant as newborns, neurological disease was observed, although at a lower rate and with slower kinetics than in mice infected with wild-type virus. These results confirm that TELEISSI is the major CD8 T-cell epitope in H-2kmice and suggest that unidentified minor epitopes are present in the BDV proteome which are recognized rather efficiently by antiviral T cells if the dominant epitope is absent.
Cytomegalovirus UL131-128 Products Promote gB Conformational Transition and gB-gH Interaction during Entry into Endothelial Cells
Authors: Patrone Marco , Secchi Massimiliano , Bonaparte Eleonora , Milanesi Gabriele , Gallina Andrea 
Published: August 2007

ABSTRACT
Herpesviruses use gB and gH-gL glycoproteins to execute fusion. Other virus-specific glycoproteins are required for receptor binding and fusion activation. The human cytomegalovirus (HCMV) UL131-128 proteins are essential for the infection of leukocytes, endothelial cells (ECs), and many epithelial cell lines. Here we show that UL131-128 play a role in a chain of events involving gB and gH during HCMV entry into ECs. An HCMV strain bearing the wild-type (wt) UL131-128 locus exhibited a gB transition from a protease-resistant to protease-sensitive form, a conformational change that was suppressed by a thiourea inhibitor of fusion (WY1768); in contrast, gH was susceptible to proteolysis throughout entry. Moreover, gB and gH transiently interacted, and a lipid mixing assay showed that the wt strain had carried out fusion by 60 min postinfection. However, these events were greatly altered when UL131-128-defective strains were used for infection or when there was an excess of soluble pUL128 during wt infection: the gB conformational change became WY1768 resistant, the gB-gH complex was no longer observed, and fusion was prevented. Both gB and gH in this case showed late protease resistance, related to their endocytic uptake. Our data point to the involvement of UL131-128 proteins in driving gB through a WY1768-sensitive fold transition, thus promoting a short-lived gB-gH complex and fusion; they also suggest that HCMV fuses with the EC plasma membrane and that endocytosis ensues only when the virus cannot trigger UL131-128-dependent steps.
Human Transcriptome Subtraction by Using Short Sequence Tags To Search for Tumor Viruses in Conjunctival Carcinoma
Authors: Feng Huichen , Taylor Jennifer L. , Benos Panayiotis V. , Newton Robert , Waddell Keith , Lucas Sebastien B. , Chang Yuan , Moore Patrick S. 
Published: August 2007

ABSTRACT
Digital transcript subtraction (DTS) was developed to subtract in silico known human sequences from expression library data sets, leaving candidate nonhuman sequences for further analysis. This approach requires precise discrimination between human and nonhuman cDNA sequences. Database comparisons show high likelihood that small viral sequences can be successfully distinguished from human sequences. DTS analysis of 9,026 20-bp tags from an expression library of BCBL-1 cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) resolved all but three candidate sequences. Two of these sequences belonged to KSHV transcripts, and the third belonged to an unannotated human expression sequence tag. Overall, 0.24% of transcripts from this cell line were of viral origin. DTS analysis of 241,122 expression tags from three squamous cell conjunctival carcinomas revealed that only 21 sequences did not align with sequences from human databases. All 21 candidates amplify human transcripts and have secondary evidence for being of human origin. This analysis shows that it is unlikely that distinguishable viral transcripts are present in conjunctival carcinomas at 20 transcripts per million or higher, which is the equivalent of approximately 4 transcripts per cell. DTS is a simple screening method to discover novel viral nucleic acids. It provides, for the first time, quantitative evidence against some classes of viral etiology when no viral transcripts are found, thereby reducing the uncertainty involved in new pathogen discovery.
Rescue of Influenza C Virus from Recombinant DNA
Authors: Crescenzo-Chaigne Bernadette , van der Werf Sylvie 
Published: August 2007

ABSTRACTThe rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3′ and 5′ noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 103and 104PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.