• Blood-borne viruses
• Diagnosis
• High-resolution melting
• Multiplex polymerase chain reaction

• KIPyV
• PCR
• Viral respiratory diseases
• WUPyV

• Child
• Humans
• Nasopharynx/virology
• Polyomavirus/genetics
• Polyomavirus/isolation & purification
• Real-Time Polymerase Chain Reaction/methods
• Reference Standards
• Reproducibility of Results
• Respiratory Tract Infections/virology
• Suction

In this report, a DNA hybridization based chemiluminescent detection method has been proposed for reliable detection of multiple pathogens. The use of surface modified magnetic nanoparticles can help to integrate this system into an automated platform for high throughput applications.

• Co-infection
• Genotyping techniques
• Hepatitis viruses
• Quantitative real-time polymerase chain reaction
• Serotyping
• Viral genome

• DNA, Viral/blood
• DNA, Viral/genetics
• Hepatitis Viruses/classification
• Hepatitis Viruses/genetics
• Hepatitis Viruses/immunology
• Hepatitis, Viral, Human/blood
• Hepatitis, Viral, Human/diagnosis
• Hepatitis, Viral, Human/genetics
• Hepatitis, Viral, Human/immunology
• Humans
• Multiplex Polymerase Chain Reaction
• Predictive Value of Tests
• Reproducibility of Results
• Serogroup
• Serologic Tests/methods
• Serotyping

A novel class of probes for fluorescence detection was developed and combined to microgel particles for a high sensitive fluorescence detection of nucleic acids.

• Cervical cancer
• Genotyping
• Multiplex PCR
• Screening test

• Adolescent
• Adult
• Aged
• DNA Primers/genetics
• DNA, Viral/genetics
• Female
• Genotype
• Human Papillomavirus DNA Tests/standards
• Humans
• Middle Aged
• Multiplex Polymerase Chain Reaction/standards
• Papillomaviridae/genetics
• Papillomavirus Infections/diagnosis
• Papillomavirus Infections/virology
• Real-Time Polymerase Chain Reaction/standards
• Sensitivity and Specificity
• Young Adult

• Co-infection
• HCV
• HIV-1
• Multiplex RT-PCR

AIM: To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment.

METHODS: We developed a stable S3-green fluorescence protein (GFP) cell line that replicated the GFP-tagged HCV sub-genomic RNA derived from a highly efficient JFH1 virus. S3-GFP replicon cell line was injected subcutaneously into γ-irradiated SCID mice. We showed that the S3-GFP replicon cell line formed human HCC xenografts in SCID mice. Cells were isolated from subcutaneous tumors and then serially passaged multiple times in SCID mice by culturing in growth medium supplemented with G-418. The mouse-adapted S3-GFP replicon cells were implanted subcutaneously and also into the liver of SCID mice via intrasplenic infusion to study the replication of HCV in the HCC xenografts. The tumor model was validated for antiviral testing after intraperitoneal injection of interferon-α (IFN-α).

RESULTS: A highly tumorigenic S3-GFP replicon cell line was developed that formed subcutaneous tumors within 2 wk and diffuse liver metastasis within 4 wk in SCID mice. Replication of HCV in the subcutaneous and liver tumors was confirmed by cell colony assay, detection of the viral RNA by ribonuclease protection assay and real-time quantitative reverse transcription polymerase chain reaction. High-level replication of HCV sub-genomic RNA in the tumor could be visualized by GFP expression using fluorescence microscopy. IFN-α cleared HCV RNA replication in the subcutaneous tumors within 2 wk and 4 wk in the liver tumor model.

CONCLUSION: A non-infectious mouse model allows us to study replication of HCV in subcutaneous and metastatic liver tumors. Clearance of HCV by IFN-α supports use of this model to test other anti-HCV drugs.

• Hepatitis C virus
• Hepatocellular carcinoma
• Tumor xenograft
• SCID mouse
• Interferon-α
• Antiviral agent
• Virus replication

• Cell Line
• Cytoplasm/genetics
• Cytoplasm/metabolism
• Cytoplasm/virology
• Down-Regulation
• Gene Expression
• Hepacivirus/enzymology
• Hepacivirus/immunology
• Hepacivirus/physiology
• Hepatitis C/genetics
• Hepatitis C/metabolism
• Hepatitis C/virology
• Humans
• Immunoglobulin G/genetics
• Immunoglobulin G/metabolism
• Immunoglobulin G/pharmacology
• Viral Nonstructural Proteins/immunology
• Virus Replication

• CA129776 NCI NIH HHS
• CA127481 NCI NIH HHS
• R21DK070551 NIDDK NIH HHS
• R56AI64617 NIAID NIH HHS
• R44AI082778 NIAID NIH HHS

The sustained virological response to interferon-alpha (IFN-α) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-α responsiveness between HCV 1 and other genotypes have not been elucidated.

Virus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-α effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-α against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-α induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones.

The cellular Jak-Stat pathway is critical for a successful IFN-α antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.

• Adult
• Benzothiazoles
• Blood/virology
• Desiccation
• Diamines
• HIV Infections/diagnosis
• HIV Infections/virology
• HIV-1/genetics
• HIV-1/isolation & purification
• Hepacivirus/genetics
• Hepacivirus/isolation & purification
• Hepatitis C/diagnosis
• Hepatitis C/virology
• Humans
• Organic Chemicals
• Quinolines
• Reverse Transcriptase Polymerase Chain Reaction/methods
• Sensitivity and Specificity
• Specimen Handling/methods
• Staining and Labeling/methods
• Time Factors
• Transition Temperature
• Young Adult

The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.

Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences.

The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods.

Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.

• Antiretroviral Therapy, Highly Active
• HIV Infections/drug therapy
• HIV Infections/virology
• HIV-1/isolation & purification
• HIV-1/physiology
• Humans
• Reverse Transcriptase Polymerase Chain Reaction/methods
• Sensitivity and Specificity
• Viral Load/methods

• R01 AI058736 NIAID NIH HHS
• 1219A153217-01 PHS HHS
• R01A1058736-01A1 PHS HHS