Cross-validation of the RT-QuIC assay for the antemortem detection of chronic wasting disease in elk.

Chronic wasting disease is a progressively fatal, horizontally transmissible prion disease affecting several members of the cervid species. Conventional diagnosis relies on ELISA or IHC evaluation using tissues collected post-mortem; however, recent research has focused on newly developed amplification techniques using samples collected antemortem. The present study sought to cross-validate the real-time quaking-induced conversion assay (RT-QuIC) evaluation of rectal biopsies collected from an elk herd with endemic CWD, assessing both binary positive/negative test results as well as relative rates of amplification between laboratories. We found that results were correlative in both categories across all laboratories performing RT-QuIC, as well as to conventional IHC performed at a national reference laboratory. A significantly higher number of positive samples were identified using RT-QuIC, with results seemingly unhindered by low follicle counts. These findings support the continued development and implementation of amplification assays in the diagnosis of prion diseases of veterinary importance, targeting not just antemortem sampling strategies, but post-mortem testing approaches as well.

The anticancer drug imatinib induces cellular autophagy.

The tyrosine kinase inhibitor imatinib (Gleevec, Novartis Pharmaceuticals Corporation; Basel, Switzerland) is a powerful drug for treatment of chronic myelogenous leukemia (CML) and other malignancies. It selectively targets various tyrosine kinases, thereby leading to growth arrest of respective cancer cells. Given its wide application, it is of high importance to know all related underlying molecular mechanisms. We had previously found that imatinib increases the cellular clearance of intracellular protein aggregates by targeting the abl pathway and thereby upregulating lysosomal activity. Here, we describe that imatinib dose dependently activates the cellular autophagy machinery in mammalian cells, independently of tissue type, species origin or immortalization status of cells. Autophagy is an archetypical cellular degradation mechanism implicated in many physiological and pathophysiological conditions. Our data link for the first time the process of autophagy with the mode of action of imatinib. Induction of autophagy might represent an additional mechanism of imatinib to induce growth arrest, promote apoptosis in cancer cells and eventually even promote tumour regression.

Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein.

Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPcand the tertiary structure of PrPScare unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein. Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds. Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found. Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPcand of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of alpha-helices 1 and 3.

Travel-related vector-borne virus infections in Germany.

Laboratory diagnosis of imported, vector-borne virus diseases during a 22-month-period in Munich, Germany, is summarized. IN 13/317 Germans returning from the Mediterranean with suspected sandfly fever, acute sandfly fever, serotype Toscana, was confirmed serologically: 84.6% of the infections were acquired in Italy. Of 249 German tourists with febrile disease returning from the tropics, acute infection with dengue virus was diagnosed serologically in 26 (10.4%): most infections were acquired in Thailand (57.7%). In a seroepidemiological study of 670 German aid workers who had spent two years in the tropics, 49 (7.3%) were positive for antibodies to dengue, 9 (1.3%) to chikungunya, and 1 (0.1%) to Sindbis virus. Of 17 Middle Eastern patients with suspected viral haemorrhagic fever, genomic Crimean-Congo haemorrhagic fever virus RNA was amplified in 4 (23.5%) by semi-nested reverse transcriptase polymerase chain reaction, and confirmed by molecular characterization of nucleic acid. With the increase in travel to and from endemic areas, imported vector-borne virus infections are increasingly important in Germany.

Polymerase chain reaction for diagnosis and identification of distinct variants of Crimean-Congo hemorrhagic fever virus in the United Arab Emirates.

Viral hemorrhagic fever has re-emerged in the United Arab Emirates (UAE) since November 1993. Genomic RNA of Crimean-Congo hemorrhagic virus (C-CHFV) was detected by a newly developed, nested reverse transcriptase polymerase chain reaction (RT-PCR) in the sera of four (25.0%) of 16 suspected cases of viral hemorrhagic fever. The RT-PCR was based on oligonucleotide primers deducted from the small RNA segment encoding the nucleoprotein of the virus. By comparison with a nucleotide sequence of a C-CHFV isolate from a Chinese sheep, a divergence of 10.0-11.8% was detected in the C-CHFV variants causing the UAE outbreak. In the four positive sera, three phylogenetically distinct C-CHFV variants were amplified and confirmed by direct sequencing of the PCR fragments. These C-CHFV sequences were obtained directly from sera of infected humans without prior propagation in cell culture. The RT-PCR allows rapid detection of genomic C-CHFV RNA in clinical specimens and study of the molecular epidemiology of this infection.

Hepatitis C and arboviral antibodies in the island populations of Mauritius and Rodrigues.

A serological survey for antibodies to hepatitis C virus (HCV), dengue viruses (DEN), West Nile virus (WN), and sindbis virus (SIN) was carried out in sera of selected groups of the population of the Islands of Mauritius (n = 449) and Rodrigues (n = 115), Indian Ocean. 8.3% of 564 sera were positive for anti-HCV. In Mauritius, 2.1% of sera of healthy individuals were found with anti-HCV. The highest prevalence was found in sexually transmitted disease (STD) patients and prison inmates with 46.2% and 43.8%, respectively. None of the sera from blood donors sampled from Rodrigues Island had anti-HCV. Antibodies to arboviruses were detected in sera of individuals from both islands. Anti-DEN IgG was detected in 3.8% of sera from Mauritius and 0.9% from Rodrigues. Anti-WN IgG was detected in 2.2% of sera from Mauritius and 0.9% from Rodrigues. All sera from Rodrigues were without anti-SIN IgG, 1.1% of those from Mauritius were positive. This suggests that arboviruses occur on these islands.

Aseptic meningitis caused by sandfly fever virus, serotype Toscana.

Sandfly fever virus, serotype Toscana (TOS), is endemic in some Mediterranean countries and causes sandfly fever (pappataci fever). In some patients, TOS may cause meningitis and meningoencephalitis. We report on two German adults returning from Italy with TOS-related meningitis, complicated in one case by abducens nerve palsy. TOS infection should be considered as a cause of acute central nervous system disorders in patients returning from areas of endemicity.

Serosurvey and laboratory diagnosis of imported sandfly fever virus, serotype Toscana, infection in Germany.

Of eight acute infections in German tourists caused by sandfly fever virus, serotype Toscana (TOS), and diagnosed clinically and serologically, seven were acquired during visits to Tuscany, Italy, and one to Coimbra, Portugal. An indirect immunofluorescence assay (IFA) using infected cells, and a newly developed enzyme-immunoassay (EIA) using crude virus antigen prepared from infected Vero-E6 cells was used to detect anti-TOS IgM and IgG. In a seroepidemiological survey of 859 health care workers and medical students, anti-TOS IgG was detected in 1.0% by IFA, and in 0.7% by EIA. In 2034 German patients hospitalized for various diseases, 1.6% were positive for anti-TOS IgG by IFA, and 0.8% by EIA. Anti-TOS IgG was detected in 43 samples of commercial immunoglobulins at titres of 10-1000 by EIA. Although the seroprevalence of antibodies to TOS is low in Germany, TOS infection should be considered in patients returning from endemic areas who complain of fever, and headaches, and have symptoms of meningitis.

Nested RT-PCR for detection of sandfly fever virus, serotype Toscana, in clinical specimens, with confirmation by nucleotide sequence analysis.

A single tube, reverse transcriptase/polymerase chain reaction (RT-PCR) was developed and evaluated for detecting a 400-bp product of the small RNA of sandfly fever virus, serotype Toscana (TOS). For more sensitive detection of genomic TOS RNA, a nested PCR amplifying a 243-bp cDNA within the RT-PCR product was established. Nucleotide sequence analysis of first- and second-round PCR products using the dideoxy cycle sequencing technique confirmed a previously published sequence of the TOS reference strain (ISS. Phl.3). By nested PCR, genomic TOS RNA was amplified from two consecutive sera taken 3 and 7 weeks after the onset of illness in one patient, and from CSF of a second patient obtained at the onset of meningitis. Authenticity of amplified PCR products was confirmed by nucleotide sequence analysis, revealing a sequence identical to the TOS reference strain. RT-PCR and nested PCR are useful for laboratory diagnosis and studies of the molecular epidemiology of TOS infection.

Imported dengue virus infections in German tourists.

Dengue viruses (DEN) are increasingly becoming endemic and epidemic in tropical and subtropical countries. In this study, 17 serologically confirmed clinical cases of DEN infection diagnosed in German tourists over the period from May 1993 until May 1994 are presented. Thirteen out of 17 (76.5%) infections occurred in southeast Asia (Thailand 58.9%), and 4/17 (23.5%) in Central and South America. All sera screened by means of the indirect immunofluorescence assay (IIFA) were positive for anti-DEN IgM. Acute infection was confirmed by demonstrating anti-DEN IgM using a "mu-capture assay". Sixteen out of 17 (94.1%) of patients presenting with fever upon admission were positive for anti-DEN IgG, with titres of > or = 128 in the IIFA. This report indicates the rising significance of DEN as a travel-associated infection in German tourists.

Immunoblot detection of antibodies to Toscana virus.

Sera from patients with sandfly fever caused by Toscana virus (TOSV) infection were tested by immunoblot for specific antibody response to TOSV derived from infected Vero-E6 cells. The 28 kDa TOSV nucleoprotein (N) was identified as the major immunodominant protein recognized by immunoblot. In sera of patients with acute TOSV infection, specific antibodies of the IgM, IgA, and IgG class were detected. Using sandfly fever virus, serotypes Sicilian (SFSV) and Naples (SFNV), as antigens for immunoblot, TOSV antibody-positive sera cross-reacted with the corresponding N proteins. These sera reacted for IgM and IgG by SFSV immunoblot, and for IgM by SFNV immunoblot. The diagnosis of sandfly fever may be confirmed by TOSV immunoblot.

Shortest known prion protein allele in highly BSE-susceptible lemurs.

We describe the shortest prion protein allele known to date. Surprisingly, it is found as a polymorphism exactly in a species (prosimian lemurs) which seems highly susceptible to oral infection with BSE-derived prions. The truncation of the prion protein we found raises several questions. First, is the truncated octarepeat structure we describe, consisting of two octarepeats, still functional in copper binding? A second question is whether this truncation is related to the remarkable oral infectibility of lemurs with BSE-derived prions. And finally, one could argue that this genotype alone might favour development of a prion disease, even in the absence of exogenous infection.

A recombinant Toscana virus nucleoprotein in a diagnostic immunoblot test system.

Sandfly fever, a vector-borne disease endemic in the Mediterranean region, is caused by Toscana virus (TOS). The disease is increasingly important as a travel-related infection. Serological diagnosis is currently dependent on viral antigens derived from TOS-infected cell cultures. In this study, we report the cloning and expression of the TOS nucleoprotein (N) in Escherichia coli and evaluation of the recombinant (r) TOS N protein as an antigen for immunoblot assays. The TOS N gene was amplified by reverse-transcriptase polymerase chain reaction and cloned into the bacterial expression vector pTrcHis-A. Sera with known TOS antibody status were used to evaluate the immunoblot assay. The expressed rTOS N protein was purified and used as antigen for immunoblots. By recombinant immunoblot, the TOS antibody status (IgM and/or IgG) of the test panel was correctly identified. No cross-reactivity was detected. The rTOS N protein is useful as an antigen for immunoblot assays, and will enable more laboratories to perform TOS antibody diagnosis.

Comparison of seven commercial tests for the detection of parvovirus B19-specific IgM.

Several enzyme immunoassays (EIA) for the detection of parvovirus B19 IgM (anti-B19 IgM) are now commercially available. In this study, seven commercial EIAs (Biotrin, DAKO, Viramed, Viratech, R-Biopharm, Mast) were compared with an in-house EIA (MvP-EIA) using native viral B19 particles and the reference IgM radioimmunoassay (MACRIA). A total of 88 sera were tested. Results agreed in 39/88 (44.3%) sera, whereas 47/88 (53.4%) were discrepant and 2/88 (2.3%) gave an equivocal result. Assay sensitivity ranged from 70.3 to 100% and specificity, from 75.9 to 100%. The best results were obtained with two EIAs (Biotrin, DAKO) using baculovirus-expressed B19 proteins as antigen. This study has shown that baculovirus-expressed B19 antibody tests are suitable tools for detecting anti-B19 IgM.

Intracellular re-routing of prion protein prevents propagation of PrP(Sc) and delays onset of prion disease.

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.