Type-specific Assays Research Articles

Complete Genome Characterisation of a Novel 26th Bluetongue Virus Serotype from Kuwait

Bluetongue virus is the “type” species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing “bluetongue” (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen “VP7” showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein “VP2” identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other “eastern” or “western” BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection.

Seroepidemiology of herpes simplex virus type 1 and 2 in pregnant women in Switzerland: an obstetric clinic based study

Objective To assess the seroprevalence of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) IgG antibodies and the seroincidence of HSV-1 and HSV-2 infections in pregnant women attending the maternity clinic of the University Hospital Lausanne. Study design Blood samples from 1030 women were taken at the usual pregnancy visit in the first trimester to assess the prevalence rate of IgG antibodies against HSV-1 and HSV-2 using a type-specific assay. A second blood sample was taken 6–8 weeks postpartum from returning women who were seronegative for HSV-2 or HSV-1 to assess the incidence of seroconversion (primary infection). Results The seroprevalence rates were 79.4% (95% CI: 76.9–81.9) for HSV-1 and 21.2% (18.7–23.7) for HSV-2 in women 14–46 years old. Type-specific serostatus patterns were as follows: 17.3% HSV-1/-2: +/+, 62.1% HSV-1/-2: +/−, 3.9% HSV-1/-2: −/+, 16.7% HSV-1/-2: −/−. Two hundred and sixty five women (59 of the 212 seronegative for HSV-1 (27.8%) and 265 of the 812 seronegative for HSV-2 (32.6%)) returned to the outpatient clinic for the post-delivery check and a second blood sample was obtained. One HSV-1 seroconversion was detected (HSV-1 seroconversion rate 2.4%/100 patient × year (95% CI: 0.06–13.4)) in a patient who had symptoms compatible with primary genital herpes. No HSV-2 seroconversion was detected (HSV-2 seroconversion rate: 0/100 patient × year (97.5% one-sided CI: 0–2)). Conclusion Compared to a previous population-based study, our study results suggest a rise in the prevalence of HSV-2 among pregnant women in Switzerland. The low incidence of seroconversion detected during pregnancy is consistent with the very low reported incidence of neonatal herpes in Switzerland. Condensation This study in a public hospital in Western Switzerland suggests an increasing prevalence of HSV-2, but a low incidence of primary infections in women of childbearing age.

Efficacy of interferon alpha with ribavirin for treatment of chronic Hepatitis C

To investigate the prevalence and distribution of HCV genotypes and the clinical effect of interferon-alpha combined with ribavirin treatment in chronic hepatitis C patients in Kunming. 60 patients were divided into two groups based on drug therapies: PEG-interferon-a plus ribavirin treatment group for HCV 1b and interferon-a plus ribavirin treatment group for non-HCV-1b. Serum ALT levels and HCV RNA quantitations of the patients were detected during treatment and follow-up. The HCV genotypes of 60 patients were determined by type specific probe assay, and five different types were found. Their overall prevalence were 21.7% for type 1b, 5% for type 2a, 16.7% for type 3a, 48.3% for type 3b, and 8.3% for type 6a. Sustained viral response rates for PEG-interferon treatment group were 46.1%, for interferon treatment group were 74.4%. The abnormal rate of serum ALT after the treatment had no significant difference between HCV-1b and non-HCV-1b patients (P>0.05). All patients with early viral responses got sustained viral response. HCV-3b is the most dominant genotype in Kunming. The effect of PEG-interferon-a plus ribavirin treatment for genotype 1b is unsatisfactory. The early viral response is a good predictor for the responses to antiviral therapy in chronic hepatitis C patients.

Visual Detection of High-Risk Human Papillomavirus Genotypes 16, 18, 45, 52, and 58 by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye

A simple, rapid, sensitive, qualitative, colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB) was established to detect high-risk human papillomavirus (HPV) genotypes 16, 18, 45, 52, and 58. All initial validation studies with the control DNA proved to be type specific. The colorimetric type-specific LAMP assay could achieve a sensitivity of 10 to 100 copies at 63°C for 65 min, comparable to that of real-time PCR. In order to evaluate the reliability of HPV type-specific LAMP, the assay was further evaluated with HPV DNAs from a panel of 294 clinical specimens whose HPV status was previously determined with a novel one-step typing method with multiplex PCR. The tested panel comprised 108 HPV DNA-negative samples and 186 HPV-DNA-positive samples of 14 genotypes. The results showed that the sensitivity of HPV type-specific LAMP for HPV types 16, 18, 45, 52, and 58 was 100%, 100%, 100%, 100%, and 100%, respectively, and the specificity was 100%, 98.5%, 100%, 98.8%, and 99.2%, respectively, compared with a novel one-step typing method with multiplex PCR. No cross-reactivity with other HPV genotypes was observed. In conclusion, this qualitative and colorimetric LAMP assay has potential usefulness for the rapid screening of HPV genotype 16, 18, 45, 52, and 58 infections, especially in resource-limited hospitals or rural clinics of provincial and municipal regions in China.

MassARRAY Spectrometry Is More Sensitive than PreTect HPV-Proofer and Consensus PCR for Type-Specific Detection of High-Risk Oncogenic Human Papillomavirus Genotypes in Cervical Cancer

Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance.

Characterization of herpes simplex virus clinical isolate Y3369 as a glycoprotein G variant and its bearing on virus typing.

BackgroundHerpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Several tests are available for typing HSV, including a monoclonal antibody specific for glycoprotein G and several PCR assays.FindingsA clinical isolate was identified as herpes simplex virus, but tested negative for both HSV-1 and HSV-2 antigens using type-specific monoclonal antibody assays. The isolate was determined to be HSV-1 by PCR analysis. A mutation which likely caused the monoclonal antibody non-reactivity was found in glycoprotein G. Phylogenetic analysis revealed two groups of HSV, one with the mutation and one without. Three population studies examining mutations in HSV-1 glycoprotein G were analyzed by chi-squared test. To this point, the epitope which the monoclonal antibody recognizes was only found in HSV-1 isolates from human European populations (p < 0.0001).ConclusionsThese findings suggest that the PCR-based methods for HSV typing may be more useful than the standard monoclonal antibody test in areas of the world where the variant in glycoprotein G is more prevalent.

Detection of seasonal H3N2 influenza A virus by type-specific TaqMan minor groove binder probe assay

Despite the emergence of the pandemic H1N1 influenza A virus in 2009, seasonal H3N2 viruses continue to co-circulate in the population and may even predominate in the coming influenza season. We describe a specific minor groove binder TaqMan assay for H3N2 viruses with a detection limit of 16.5 standard DNA copies.

Genital Herpes / Genitalni herpes

Abstract Genital herpes is a chronic, nearly always active herpes simplex virus (HSV) infection of sacral ganglia, that may appear bilaterally and in more ganglia than previously thought. It represents one of the most prevalent sexually transmitted infections, and the most frequent cause of genital ulcer disease in the general populations of developed countries. It is caused by HSV type-2 (HSV-2) in 60-80% of cases, with HSV-1 infections causing the remainder. Genital herpes caused by HSV-1 is on the rise. Since genital HSV-1 infections have higher risk for transmission from mother to infant during delivery than HSV-2, they account for 30% of all cases of neonatal herpes. Serological studies have found prevalence of HSV-2 in the general population of developed contries to be up to 25%. Thirty years ago, herpes was defined as “Today’s Scarlet Letter”in the absence of reliable serological tests and highly effective medications, for diagnosis and treatment of genital herpes. In 2000, apart from virus isolation in cell culture (70% sensitivity), that has long been regarded as the diagnostic gold standard, type specific serological tests and higly effective antiviral agents have evolved. However, the following questions were raised: should serological testing be routinely recommended in asymptomatic patients; can antiviral therapy reduce asymptomatic shedding of the virus; can antiviral therapy reduce sexual transmission of infection; can antiviral therapy reduce acquisitation of viral copathogens, such as human immunodeficiency virus (HIV)? Now, ten years later, we know the answers. Type specific HSV DNA detection by real-time PCR assays (100% sensitivity) are tests of choice for every person with recurrent genital ulcers lasting more than 4 days, and must be available in those laboratories currently performing a significant number of PCR tests for different purposes. Type specific IgG serology assays are indicated in all asymptomatic persons who are at increased risk for HSV infection. In sexually active patients experiencing ≥ 6 recurrences per year, daily supressive dose of acyclovir, valacyclovir or famciclovir should be discontinued after a maximum of a year of continuous antiviral therapy in order to reassess recurrence frequency. If necessary, the therapy should be restarted after at least two recurrences. With such expansive diagnostic possibilities and more aggressive therapeutic approaches, we can define genital herpes not as a “Scarlet Letter”, but as a “widespread untoward consequence of human sexuality”.

The changing epidemiology pattern and frequency distribution of hepatitis C virus in Pakistan

Information regarding the changing pattern in hepatitis C virus (HCV) genotypes/subtypes and resulting disease outcome is not well known. The specific objective of this study was to find out the frequency distribution of HCV genotypes and changing pattern of various HCV genotypes overtime in well-characterized Pakistani HCV isolates. The genotype distribution of HCV from all the four provinces of Pakistan was tracked for a period of 10 years (2000–2009) on total 20,552 consecutive anti-HCV and HCV RNA positive patients sample using type-specific genotyping assay. Of these, 16,891 (82.2%) samples were successfully genotyped. Of these 11,189 (54.4%) were males and 9363 (45.55%) were females. Of the successfully genotyped samples, 12,537 (74.2%) were with 3a, 1834 (10.9%) with 3b, 50 (0.24%) with 3c, 678 (3.3%) with 1a, 170 (0.83%) with 1b, 49 (0.24%) with 1c, 431 (2.1%) with 2a, 48 (0.23%) with 2b, 3 (0.01%) with 2c, 13 (0.06%) with 5a, 12 (0.06%) with 6a, 101 (0.49%) with 4, and 965 (4.7%) were with mixed-genotype infection. A changing pattern of HCV genotypes prevalence was observed in Pakistan overtime, with an increase in the relative proportion of genotype 3a and mixed genotypes and a decrease of genotypes 3b, 2b, 4, 5a and 2a. This changed HCV genotype pattern might have direct impact on HCV disease outcome and new therapeutic strategies may be needed.

Evaluation of Multiplex Type-Specific Real-Time PCR Assays Using the LightCycler and Joint Biological Agent Identification and Diagnostic System Platforms for Detection and Quantitation of Adult Human Respiratory Adenoviruses

Every year, thousands of basic military trainees in each service of the U.S. Armed Forces experience acute respiratory disease. The majority of this disease burden results from infection with human adenoviruses. We designed single- and multiplex assays that detect and discriminate adenovirus types B3, E4, B7, B11, B14, and B21. A total of 116 oropharyngeal swab specimens obtained from patients at the Naval Health Research Center were used to validate the new assays. Type-specific singleplex assays were designed and used independently to successfully identify 94 representative patient specimens. The lower limits of detection for our singleplex real-time PCR assays were calculated to be 50, 500, 500, 50, 50, and 50 genomic copies per reaction for human adenovirus type B3 (HAdV-B3), HAdV-E4, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21, respectively. These were then multiplexed to increase efficiency and tested against singleplex assays using titrated controls. The HAdV-B3/B11 and HAdV-E4/B7 multiplex assays were as sensitive and specific as they were individually. The HAdV-B14/B21 multiplex assay was not as efficient at detecting HAdV-B14 as the singleplex assay. Interestingly, a statistically significant difference was found between the viral loads of HAdV-B14 and those of HAdV-B3, -E4, -B7, and -B21 (P < 0.001). The assays did not cross-react with other adenoviruses, influenza virus, respiratory syncytial virus, or respiratory disease-causing bacteria. These assays have the potential to be useful as clinical diagnostic tools for the detection of HAdV infection in adult populations.

Novel and Expanded Roles for MAPK Signaling inArabidopsisStomatal Cell Fate Revealed by Cell Type–Specific Manipulations

Mitogen-activated protein kinase (MAPK) signaling networks regulate numerous eukaryotic biological processes. In Arabidopsis thaliana, signaling networks that contain MAPK kinases MKK4/5 and MAPKs MPK3/6 function in abiotic and biotic stress responses and regulate embryonic and stomatal development. However, how single MAPK modules direct specific output signals without cross-activating additional downstream processes is largely unknown. Studying relationships between MAPK components and downstream signaling outcomes is difficult because broad experimental manipulation of these networks is often lethal or associated with multiple phenotypes. Stomatal development in Arabidopsis follows a series of discrete, stereotyped divisions and cell state transitions. By expressing a panel of constitutively active MAPK kinase (MAPKK) variants in discrete stomatal lineage cell types, we identified a new inhibitory function of MKK4 and MKK5 in meristemoid self-renewal divisions. Furthermore, we established roles for MKK7 and MKK9 as both negative and (unexpectedly) positive regulators during the major stages of stomatal development. This has expanded the number of known MAPKKs that regulate stomatal development and allowed us to build plausible and testable subnetworks of signals. This in vivo cell type-specific assay can be adapted to study other protein families and thus may reveal insights into other complex signal transduction pathways in plants.

The Use of Human Papillomavirus Seroepidemiology to Inform Vaccine Policy

In this issue, Dunne et al. (1) report on the seroprevalence of four types of human papillomavirus (HPV) in a study of 499 men from Tucson, Arizona and Tampa, Florida. They observed increasing seropositivity with age for HPV6/11, the major causes of genital warts and respiratory papillomatosis, and for HPV16 and HPV 18, the major causes of anogenital cancers and some oropharyngeal cancers. Men who smoked were more likely to be seropositive to at least one of the types, as were those who had a current sexual partner in the past 3 months. Interestingly, in contrast to findings from studies in women, the association of seropositivity with lifetime sexual partners was weak. Overall, seroprevalence to HPV 16 was 12.1%, to HPV 6/11 combined was 9.7%, and to HPV 18 was 5.4%; only 1% of men were positive on all three type-specific assays. HPV6, 11, 16, and 18 are the four types prevented by the currently licensed HPV prophylactic vaccine (Merck’s Gardasil). The results suggest that benefit from quadrivalent vaccination would decrease with age due to increasing previous exposure resulting in immunity to re-infection. Viewed uncritically, the data also would suggest that considerable benefit in men might remain well into adulthood because seroprevalence did not increase substantially until the 35+ year old age group. Any tool to guide the planning of vaccine programs is welcome, and this study represented a fruitful collaboration of excellent epidemiology and serology. As the authors state, vaccine recommendations in men are both topical and important. Policies must weigh the high cost of male vaccination programs that would cost billions of dollars against possible benefits that include prevention of genital warts and a fraction of anogenital and oral cancers in men and their sexual partners. Adding male vaccination to female vaccination programs would also help prevent, to a still unknown amount, the great burden of cervical cancer/precancer in women. It is important for readers to put the paper by Dunne et al. in context, with realization of the complexity of HPV serology compared with other viral infections in which viremia leads reliably to antibody titers that predict subsequent immunity. The proper and exact use of cross-sectional HPV serosurveys in formulation of vaccination policy is made difficult by 1) diverse assays that are not yet fully developed or comparable and 2) our incomplete understanding of what different serology assays are measuring with regard to HPV exposure and immunity, especially in men.

Association Between The ACTN3 (R577X) Genotype And Oxidative Capacity Of Fast Muscle Fibers In Humans

About 18% of Western European populations have a complete deficiency of the alpha-actinin-3 protein due to homozygosity for a stop codon mutation (577X) in the ACTN3 gene. The alpha-actinin-3 protein has an important function in the Z-line of the sarcomere where it binds the thin actin filaments. The 577XX null genotype seems to be beneficial for sustained muscle contractions, indicated by increased X-allele carriers in elite endurance athletes, while RR genotype carriers outperform XX carriers for high velocity contractions. Recently MacArthur et al. provided a mechanistic explanation for these findings with detailed studies of an Actn3-/- mouse model. Mice lacking alpha-actinin-3 showed reduced fast fiber diameter, increased activity of multiple enzymes in the aerobic metabolic pathway, altered contractile properties, and enhanced recovery from fatigue. PURPOSE: To replicate findings in Actn3-/- mice fast muscle -showing a shift towards those characteristic of slow fibers- in human muscle. The purpose of this study was to compare the fiber-specific oxidative capacity of fast and slow muscle fibers in humans with a different ACTN3 R577X genotype. METHODS: 90 healthy young men (18-29 yr) were genotyped for ACTN3 R577X. Twenty-two XX and twenty-two RR subjects underwent a muscle biopsy of the right vastus lateralis muscle at rest. The oxidative capacity marker succinate dehydrogenase (SDH) was measured in a fiber type specific assay using immunohistochemistry. RESULTS: We found no significant differences in SDH staining in fast fibers comparing XX and RR carriers: XX carriers show only 0.5% increased staining for IIa fibers and 6% for IIx fibers (P > 0.37). Furthermore, non-significant group differences (P = 0.27) were found for SDH staining in the slow type I fibers. A significantly higher SDH activity was found in slow fibers compared to fast muscle fibers (P < 0.01), with equal differences for both genotype groups (type I fibers 44% higher than type II fibers in XX vs. 43% higher in RR carriers). CONCLUSIONS: At present, we are unable to confirm a shift in oxidative capacity of fast muscle fibers as reported in an Actn3-/- knockout mouse in our human samples. The presence of different physiological mechanisms and morphological characteristics of muscle fibers in rodents compared to humans might explain these findings.

Herpes Simplex

Herpes Simplex

Two real-time RT-PCR assays of classical swine fever virus, developed for the genetic differentiation of naturally infected from vaccinated wild boars

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a disease notifiable to the Office International des Epizooties (OIE). A live marker vaccine would be the ultimate choice for controlling CSF, which enables serological and genetic differentiation of vaccine from wild type CSFV. Recently, a marker vaccine CP7_E2alf has been reported [Koenig, P., Lange, E., Reimann, I., Beer, M., 2007. CP7_E2alf: a safe and efficient marker vaccine strain for oral immunisation of wild boar against classical swine fever virus (CSFV). Vaccine 25, 3391–3399]. A vaccine-specific TaqMan real-time RT-PCR assay was developed and evaluated, and a second, wild type-specific assay was modified from an established one in such a way that both can be performed in two wells side-by-side in a microplate in a single run. The detection limit is 50 viral RNA copies per reaction for the vaccine-specific assay, and 20 copies per reaction for the wild type assay. The two assays have been shown to be highly specific and reproducible, with potential application for genetic differentiation of wild type CSFV from the marker vaccine CP7_E2alf in wild boar vaccination programs.

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producingClostridium botulinum,Clostridium baratiiandClostridium butyricum

To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism. Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin (bont) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A, bont/B, bont/E, bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg-1000 fg of total DNA in the PCR tube (25-250 genome equivalents) which correspond to 10(3) to 10(4) cells ml(-1). After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak. These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism. Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.

Model cell systems representing expression regulation and signal transduction of thymic stromal lymphopoietin (TSLP) in the development of asthmatic symptoms

Thymic stromal lymphopoietin (TSLP) is a novel interleukin-7-like cytokine, which triggers dendritic cell-mediated inflammatory responses ultimately executed by Th2 helper cells. TSLP is a central player in the development of allergic, especially asthmatic symptoms, and has been suggested to represent a missing link in the information flow from antigen-exposed epithelial cells via dendritic cells (DCs) to T helper cells. We have challenged the lung epithelial cell line A549 with various allergens such as extracts from dust mites, pollen and fungi, and could show by real time PCR for the first time an allergen-dependent specific transcriptional upregulation of TSLP. To approach the as yet poorly defined TSLP-induced signal transduction in dendritic cells, we established a cellular model system on the basis of the murine pro-B cell line Ba/F3. The heterodimeric human TSLP receptor (hTSLPR) consisting of the novel TSLP receptor chain and the IL-7 receptor alpha chain was functionally reconstituted in factor-dependent Ba/F3 cells. We could demonstrate by phosphotyrosine protein analysis and by STAT type-specific reporter gene assays, that the ligand-stimulated TSLPR triggers activation of STAT3 and STAT5 as well as of STAT1. Employment of specific inhibitors proved the functional involvement of Janus kinases (JAKs) in TSLP-dependent, receptor-mediated cellular responses. These achievements provide tools for research towards a better molecular understandimg of atopic asthma and for the future development of new therapeutics interfering with TSLP function.

Mise au point de nouvelles techniques de diagnostic pour les maladies à orbivirus chez les animaux domestiques et sauvages

The recent emergence and spread of bluetongue virus (BTV) across the whole of Europe suggests that other orbiviruses could also emerge to threaten livestock species and wildlife popula­tions in Europe and other parts of the world. The genus Orbivirus is the largest within the family Reoviridae, containing 22 virus species, as well as 14 unclassified orbiviruses. The orbiviruses are transmitted primarily by arthropod vectors (e.g. Culicoides, mosquitoes or ticks) and several are associated with severe and economically important diseases of livestock, including BTV in cattle and sheep, African horse sickness virus (AHSV), equine encephalosis virus (EEV) and Peruvian horse sickness virus (PHSV) in equids, as well as epizootic haemorrhagic disease virus (EHDV) in wild ungulates or cattle. Recent incursions of BTV in Europe, Southeastern USA, Australia and Asia, EHDV in North Africa, the Middle East and the Mediterranean region, AHSV in sub-Saharan Africa, and EEV in Israel and Gambia, indicate a need for the development of faster, more sensitive and more reliable diagnostic assays. These are required to detect and identify rapidly the viruses and virus types involved, monitor their incidence and movement, and identify infected animals. The Orbivirus genome is composed of 10 linear segments of double-stranded ribonucleic acid (dsRNA), each segment coding for at least one viral protein. The outer capsid proteins VP2 and VP5 are situated on or near the surface of the virus particle and are more variable than components of the virus core, or the non-structural proteins. VP2 (encoded by Seg- 2) is the outermost of the BTV capsid proteins and represents the primary target antigen for neutralising antibodies, and hence Seg-2 is a target for the development of type-specific nucleic-acid-based diagnostic assays. In contrast, the genome segments coding for protein components of the virus core and/or the non-structural proteins can be used as targets for development of serogroup (virus-species) specific, reverse transcription - poly­merase chain reaction (RT-PCR) based diagnostic assays. Virus species-specific and type-specific conventional (gel based) RT-PCR diagnostic assays, for the detection, identification and typing of some of these viruses (BTV, EHDV and AHSV), have been developed using the sequence data for segments 7 and 2, respectively. Initial evaluation studies indicate that these assays are reliable, specific, do not cross-react with related orbiviruses (group/species specific) or with related types (type specific). Although they are labour intensive, the results obtained can be confirmed by sequence analyses of the resulting complemen­tary deoxyribonucleic acid complementary (c) DNA amplicons, and phylogenetic comparisons to determine the strain of virus involved. However, conventional RT-PCR assays are prone to cross-contamination, potentially leading to false positive results. The authors also describe group-specific real-time RT-PCR assays that use a ‘closed-tube’ format, which are therefore less suscep­tible to cross-contamination. These assays target the conserved genome segment 9, or genome segment 1, which can be used to detect all of the serotypes, as well as geographic variants (different topotypes) of BTV, EHDV, AHSV, EEV and PHSV. Type-specific real-time RT-PCR assays that target the most variable genome segment 2 can be used to differentiate 25 serotypes of BTV or the seven serotypes of EHDV. These diagnostic assays were found to be very sensitive, reproducible and suitable for rapid screening of field samples. Results will be presented from studies to optimise these RT-PCR assays.

Invader human papillomavirus (HPV) type 16 and 18 assays as adjuncts to HPV screening of cervical papanicolaou smears with atypical squamous cells of undetermined significance

High-risk (HR) human papillomavirus (HPV) testing is standard practice for triaging women who have Papanicolaou (Pap) smears with atypical squamous cells of undetermined significance (ASC-US), however, only 5% to 17% of these women have underlying cervical intraepithelial neoplasia 2 (CIN-2)/CIN-3. Recent reports have demonstrated that the presence of either HPV type 16 (HPV-16) or HPV-18 confers an elevated risk for CIN-2/CIN-3. The current study was designed to determine the prevalence of HPV-16 and HPV-18 in ASC-US Pap smears and to determine whether further typing would enhance the risk stratification of patients for CIN-2/CIN-3. One hundred seventy-eight Pap smears with ASC-US were screened retrospectively for HR HPV by using the proprietary Invader screening assay followed by typing for HPV-16 and HPV-18 by using Invader type-specific probes on 100 of the samples. Clinical follow-up results were correlated with HPV types. Fifty-one percent of the ASC-US samples were positive for HR HPV, the majority of which (70%) harbored non-HPV-16/HPV-18 HR HPV types; 27% were associated with HPV-16, whereas only 3% contained HPV-18. The screening assay indicated that 46% of women who had Pap smears with ASC-US were in need of further HPV-16/HPV-18 typing. Testing for HPV-16 stratified women with ASC-US into 3 groups: 1) 14% of women were positive for HPV-16 and had a high risk (54%) of CIN-2/CIN-3 on follow-up biopsy, 2) 35% of women were positive for non-HPV-16 HPV types and had an intermediate risk (9%), and 3) 51% of women were negative for HPV and had a negligible risk for CIN-2/CIN-3. The combined application of a proprietary screening assay and a type-specific HPV-16 assay demonstrated global potential for the development of tailored management protocols for women who have Pap smears with ASC-US.

Expression Pattern and Subcellular Localization of Human Papillomavirus Minor Capsid Protein L2

The expression pattern of human papillomavirus (HPV) capsid antigen L2 is poorly described, and the significance of its localization with both promyelocytic leukemia protein (PML) and Daxx in a subnuclear domain, nuclear domain 10 (ND-10), when ectopically expressed in tissue culture cells is controversial. To address whether ND-10 localization of L2 occurs in natural cervical lesions, we used a HPV16 and HPV18 L2-specific monoclonal antibody (RG-1), in addition to rabbit antiserum to HPV6 L2, to localize L2. Immunohistochemical staining with RG-1 produced diffuse staining in the nuclei of some cells located within the superficial epithelial layers in eight of nine cases of HPV16/18(+) cervical intraepithelial neoplasia grade 1 (CIN1); however, no staining was observed in HPV16/18(+) high-grade CIN (0 of 8 cases), normal cervical epithelium (0 of 20 cases), cervical squamous cell carcinoma (0 of 102 cases), adenocarcinoma (0 of 51 cases), or adenosquamous carcinoma (0 of 6 cases). HPV16/18(+) cervical lesions that express L2 exhibit higher HPV16/18 genome copies per cell compared with those that do not positively stain with RG-1 (P = 0.04). RG-1 staining of HeLa cells transfected with L2 expression constructs was frequently concentrated in the ND-10, particularly in cells expressing high levels of L2, and co-localized with the cellular markers of ND-10, PML, and Daxx. In contrast, L2 was primarily diffuse within the nucleus and distinct from ND-10 as defined by PML immunofluorescent staining in CIN lesions, condylomata, and HPV16-transduced organotypic cultures.