Feline Coronaviruses: Pathogenesis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) belongs to the few animal virus diseases in which, in the course of a generally harmless persistent infection, a virus acquires a small number of mutations that fundamentally change its pathogenicity, invariably resulting in a fatal outcome. The causative agent of this deadly disease, feline infectious peritonitis virus (FIPV), arises from feline enteric coronavirus (FECV). The review summarizes our current knowledge of the genome and proteome of feline coronaviruses (FCoVs), focusing on the viral surface (spike) protein S and the five accessory proteins. We also review the current classification of FCoVs into distinct serotypes and biotypes, cellular receptors of FCoVs and their presumed role in viral virulence, and discuss other aspects of FIPV-induced pathogenesis. Our current knowledge of genetic differences between FECVs and FIPVs has been mainly based on comparative sequence analyses that revealed "discriminatory" mutations that are present in FIPVs but not in FECVs. Most of these mutations result in amino acid substitutions in the S protein and these may have a critical role in the switch from FECV to FIPV. In most cases, the precise roles of these mutations in the molecular pathogenesis of FIP have not been tested experimentally in the natural host, mainly due to the lack of suitable experimental tools including genetically engineered virus mutants. We discuss the recent progress in the development of FCoV reverse genetics systems suitable to generate recombinant field viruses containing appropriate mutations for in vivo studies.

Time management in radiation oncology: evaluation of time, attendance of medical staff, and resources during radiotherapy for prostate cancer: the DEGRO-QUIRO trial.

PURPOSE: In order to evaluate resource requirements, the German Society of Radiation Oncology (DEGRO) recorded the times needed for core procedures in the radio-oncological treatment of various cancer types within the scope of its QUIRO trial. The present study investigated the personnel and infrastructural resources required in radiotherapy of prostate cancer. METHODS: The investigation was carried out in the setting of definitive radiotherapy of prostate cancer patients between July and October 2008 at two radiotherapy centers, both with well-trained staff and modern technical facilities at their disposal. Personnel attendance times and room occupancy times required for core procedures (modules) were each measured prospectively by two independently trained observers using time measurements differentiated on the basis of professional group (physician, physicist, and technician), 3D conformal (3D-cRT), and intensity-modulated radiotherapy (IMRT). RESULTS: Total time requirements of 983 min for 3D-cRT and 1485 min for step-and-shoot IMRT were measured for the technician (in terms of professional group) in all modules recorded and over the entire course of radiotherapy for prostate cancer (72-76 Gy). Times needed for the medical specialist/physician were 255 min (3D-cRT) and 271 min (IMRT), times of the physicist were 181 min (3D-cRT) and 213 min (IMRT). The difference in time was significant, although variations in time spans occurred primarily as a result of various problems during patient treatment. CONCLUSION: This investigation has permitted, for the first time, a realistic estimation of average personnel and infrastructural requirements for core procedures in quality-assured definitive radiotherapy of prostate cancer. The increased time needed for IMRT applies to the step-and-shoot procedure with verification measurements for each irradiation planning.

[Biotechnological therapies for the treatment of back pain: alternatives to corticosteroids]. / Biotechnologische Konzepte zur Behandlung von Rückenschmerzen : Erste Erfahrungen mit Alternativen zu Glukokortikoiden.

In recent years, it is increasingly clear that back pain is not only caused by biomechanical problems. Currently, biologically-based local therapy concepts for the treatment of affected spinal regions as an alternative to the standard treatment with steroids are in development or in early stages of clinical application. The common features of these new therapies are to intervene in the regulation of homeostasis at various key points at the affected region and specifically to suppress or block catabolic influences as well as to provide with anti-inflammatory substances and growth factors. These include on one hand the genetically produced Biologicals such as TNF-α inhibitors and cytokine antagonists and on the other hand therapies with autologous blood preparations (Autologous Conditioned Serum [ACS], and Platelet Rich Plasma formulations [PRP]). This article presents the individual methods, gives an overview of developments and results of various studies and discusses current recommendations.

In vitro potency tests: challenges encountered during method development.

Vaccines play a key role in the control of viral diseases both in humans and in animals. In order to ensure the quality and consistency of vaccines they are extensively tested, including potency control of individual batches. In the case of vaccines against rabies the most widely used test for batch potency control is the National Institutes of Health (NIH) test. The NIH test is performed in mice leading to the consumption of thousands of animals every year. Protection against rabies after vaccination is associated with neutralizing antibodies directed against the viral glycoprotein (G). Therefore the amount of G-protein in vaccine preparations is an important parameter with regard to potency. Additionally the structural integrity of virus particles in vaccine preparations may be crucial for their immunogenicity. The objective of our work is the development of in vitro methods to determine the potency of vaccines against rabies. The result of this ongoing project shall be an assay panel including measurement of the antigenic content as well as parameters of antigen quality in a vaccine preparation allowing a precise prediction of the potency of rabies vaccines without using animal experiments.

Time management in radiation oncology: development and evaluation of a modular system based on the example of rectal cancer treatment. The DEGRO-QUIRO trial.

PURPOSE: The goal was to develop and evaluate a modular system for measurement of the work times required by the various professional groups involved in radiation oncology before, during, and after serial radiation treatment (long-term irradiation with 25-28 fractions of 1.8 Gy) based on the example of rectal cancer treatment. MATERIALS AND METHODS: A panel of experts divided the work associated with providing radiation oncology treatment into modules (from the preparation of radiotherapy, RT planning and administration to the final examination and follow-up). The time required for completion of each module was measured by independent observers at four centers (Rostock, Bamberg, Düsseldorf, and Offenbach, Germany). RESULTS: A total of 1,769 data sets were collected from 63 patients with 10-489 data sets per module. Some modules (informed consent procedure, routine treatments, CT planning) exhibited little deviation between centers, whereas others (especially medical and physical irradiation planning) exhibited a wide range of variation (e.g., 1 h 49 min to 6 h 56 min for physical irradiation planning). The mean work time per patient was 12 h 11 min for technicians, 2 h 59 min for physicists, and 7 h 6 min for physicians. CONCLUSION: The modular system of time measurement proved to be reliable and produced comparable data at the different centers. Therefore, the German Society of Radiation Oncology (DEGRO) decided that it can be extended to other types of cancer (head and neck, prostate, and breast cancer) with appropriate modifications.

Factors influencing the antibody response to vaccination against rabies.

Preventive vaccination against rabies virus is a highly effective method for preventing rabies in humans and animals. For travel purposes, vaccination of domestic carnivores is obligatory. In addition, some countries require testing for neutralizing antibodies against rabies. The minimal threshold level accepted by WHO/OIE is 0.5 IU/ml. Despite proper vaccination some animals do not reach the threshold. The objective of this study was to identify specific risk factors in dogs and cats for post-vaccination rabies antibody titres below 0.5 IU/ml by FAVN test. Rabies vaccination protocols and recommendations were reviewed with regard to travel regulations. Comprehensive data was collected on animals tested for rabies antibodies via a questionnaire sent to veterinarians who submitted sera for rabies titration. The questionnaire included data on species, age, sex, breed, vaccine used, date of last vaccination and blood sampling, vaccination history and further medical treatments at time of vaccination. Data on around 1,200 animals was analysed. Most animals older than one year had already received more than one rabies vaccination. The influence of breed and sex on antibody titre seems to be insignificant. Young dogs have a high risk of results below 0.5 IU/ml after their first vaccination. This risk can be minimised by the application of a second vaccination and blood sampling according to the manufacturer's recommendations. An important factor for the test outcome might be the virus strain used in the vaccine.

Phenotypic variability in Meesmann's dystrophy: clinical review of the literature and presentation of a family genetically identical to the original family.

PURPOSE: To describe the phenotypic variability in Meesmann's microcystic dystrophy of the corneal epithelium based on a review of the literature and the presentation of a Danish family. METHODS: We carried out a clinical examination of the family and genetic sequencing of DNA. RESULTS: Subjective symptoms often include blurred vision and ocular irritation. Typical cases may be entirely free of complaints. Intermittent pain episodes, such as occur in recurrent erosion syndrome, are not the rule. Genetic sequencing indicated a familial relationship with the originally described Meesmann family. Clinical variability was similar. Approximately 85% of cases showed microcysts in the entire epithelium. The remaining 15% demonstrated variants with microcysts in the upper or lower part of the cornea, or in the central or peripheral cornea, as well as subepithelial opacities. CONCLUSIONS: Meesmann's dystrophy occurs worldwide. The largest family described is the original German one, now supplemented with a Danish branch. Despite the presence of an identical genetic defect, the clinical phenotype varies. This suggests that non-KRT12-related mechanisms are responsible for the variation.

[Tick-borne encephalitis (TBE) with special emphasis on infection in horses]. / Die tick-borne Enzephalitis (TBE) unter besonderer Berücksichtigung der Infektion beim Pferd.

The tick-borne encephalitis (TBE), also known as early summer meningo-encephalitis, is a geographically limited virus infection transmitted mainly by ticks. The importance of TBE is largely underestimated. The causative agent TBE-Virus (TBEV) is grouped into the genus Flavivirus of the virus family Flaviviridae. Clinical disease including fatal outcomes has been described for men and dogs. With regard to horses only a limited number of case reports is available. In a study performed at the Institute of Virology, Justus-Liebig-University Giessen serum samples from the German endemic region of Marburg-Biedenkopf were tested for antibodies against TBEV. From 240 sera tested 7 (2.9%) were regarded as positive in a serum neutralization test (SNT). In an ELISA, performed in parallel to confirm the SNT results, 5 out of 7 positive sera from the SNT were also positive. The remaining two samples with low SNT-titres and all sera from horses negative in the SNT were also negative in the ELISA. This article is focussed on TBE of horses. In this context different aspects of TBE are included such as properties of the causative agent, interactions between causative agent, host animals and environment, spread of TBEV, pathogenesis, clinical symptoms, diagnosis and control.

Dissection of a viral autoprotease elucidates a function of a cellular chaperone in proteolysis.

Replication of positive-strand RNA viruses involves translation of polyproteins which are proteolytically processed into functional peptides. These maturation steps often involve virus-encoded autoproteases specialized in generating their own N or C termini. Nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus represents such an enzyme. Bovine viral diarrhea virus NS2 creates in cis its own C terminus and thereby releases an essential viral replication factor. As a unique feature, this enzyme requires for proteolytic activity stoichiometric amounts of a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) or its fragment Jiv90. To obtain insight into the structural organization of the NS2 autoprotease, the basis for its restriction to cis cleavage, as well as its activation by Jiv, we dissected NS2 into functional domains. Interestingly, an N-terminal NS2 fragment covering the active center of the protease, cleaved in trans an artificial substrate composed of a C-terminal NS2 fragment and two downstream amino acids. In the authentic NS2, the 4 C-terminal amino acids interfered with binding and cleavage of substrates offered in trans. These findings strongly suggest an intramolecular product inhibition for the NS2 autoprotease. Remarkably, the chaperone fragment Jiv90 independently interacted with protease and substrate domain and turned out to be essential for the formation of a protease/substrate complex that is required for cleavage. Thus, the function of the cell-derived protease cofactor Jiv in proteolysis is regulation of protease/substrate interaction, which ultimately results in positioning of active site and substrate peptide into a cleavage-competent conformation.

Persistence of bovine viral diarrhea virus is determined by a cellular cofactor of a viral autoprotease.

Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.

[Noroviruses and sapoviruses in man and farm animals]. / Noroviren und Sapoviren beim Menschen und bei Nutztieren.

Noro- and Sapoviruses belong to the virus family Caliciviridae and are important causative agents of acute epidemic gastroenteritis in man. In many cases transmission of Noro- and Sapoviruses occurs via contaminated food or water and the respective diseases are therefore designated as food borne. Recently, the presence of Noro- and Sapoviruses in farm animals has attracted increasing attention. Clinical symptoms were observed after experimental infection of cattle and swine with members of the two virus groups. Thus far it is not known, whether virus transmission from animals to man does occur.

Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus.

Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.

Bovine viral diarrhea virus with deletions in the 5'-nontranslated region: reduction of replication in calves and induction of protective immunity.

Bovine viral diarrhea virus (BVDV) with deletions in the 5'-nontranslated region (5'-NTR) were tested for their suitability as live BVD vaccines. Firstly, the genetic stability of the mutants was established by culturing over 15 passages in bovine cells. Secondly, two deletion mutants and the parent strain CP7-5A were characterised with respect to in vivo replication competence, attenuation and induction of protective immunity against BVDV. Naïve calves (n = 5 per group) were inoculated with mutants d2-31 and d5-57 or CP7-5A and 5 weeks later, a challenge with the BVDV type 1 strain New York was performed. The mutants were found to be genetically and phenotypically stable. Moreover, the results indicate that the mutants were attenuated with regard to effects including pyrexia and drop in leucocyte counts. Infection with the mutants induced moderate to high titers of BVDV neutralizing antibodies and completely prevented viremia after challenge infection with a heterologous BVDV strain. Taken together, the 5'-NTR deletion mutants combine a good safety profile with good efficacy and are therefore well suited as candidate live vaccines.

Genetic heterogeneity of bovine viral diarrhoea viruses from Spain.

Forty-four bovine viral diarrhoea virus (BVDV) isolates collected from the north of Spain between 1989 and 2000 were characterised at the molecular level. For 24 of these isolates the entire N(pro) gene sequence was determined. Phylogenetic analysis revealed that 21 isolates belong to subgroup BVDV-1b, while two BVDV-1c isolates and one BVDV-1h isolate were found. Furthermore, 20 additional virus isolates were analysed by differential reverse transcription-polymerase chain reaction (RT-PCR) and classified as BVDV-1. The results from our study demonstrate that BVDV-1b is the most prevalent subgroup present in bovine dairy herds of Spain, while there is no evidence for the presence of BVDV-2.

Cell-derived sequences in the N-terminal region of the polyprotein of a cytopathogenic pestivirus.

Efficient proteolytic release of nonstructural protein 3 (NS3) from the viral polyprotein is considered to be crucial for the cytopathogenicity of pestiviruses. Here we describe a novel cytopathogenic (cp) bovine viral diarrhea virus strain (BVDV CP8) with a complex insertion composed of viral and cell-derived sequences, including two fragments of the cellular J-domain protein Jiv (J-domain protein interacting with viral protein) located in the N-terminal region of the polyprotein. BVDV CP8 expresses a Jiv fusion protein of 513 amino acids in addition to a complete set of viral proteins. This protein has the capacity to induce NS2-3 cleavage in trans. Accordingly, CP8 is a representative of a novel type of cp pestivirus with a cp-specific mutation located outside of the NS2-3 gene.

Cytopathogenicity of pestiviruses: cleavage of bovine viral diarrhea virus NS2-3 has to occur at a defined position to allow viral replication.

Despite of highly divergent genome organizations, the N terminus of nonstructural protein 3 (NS3) is highly conserved between cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains. Generation of NS3, often by NS2-3 cleavage, is a marker of cp BVDV. The significance of the cleavage site within NS2-3 for viral replication was addressed by the use of BVDV replicons. Our results demonstrate that elongation as well as truncation of NS3 strongly interfere with viral RNA replication. This finding strongly suggests that the observed conservation of the N terminus of NS3 between cp BVDV is caused by functional selection and not by the presence of a hotspot of recombination.

Characterization of sealpox virus, a separate member of the parapoxviruses.

A disease outbreak characterized by lesions of the skin and mucosa of the oral cavity was recognized in harbor seals ( Phoca vitulina) from the German North Sea. Using electron microscopy typical parapoxvirus particles were observed. The presence of parapoxvirus was confirmed by PCR and nucleotide sequencing of part of the putative major envelope protein coding gene. Comparative sequence analysis revealed that the virus from seal is significantly different from the established parapoxvirus species Orf virus, Bovine papular stomatitis virus, Pseudocowpox virus, and Parapoxvirus of red deer in New Zealand. The results of our analysis provide evidence for inclusion of the seal parapoxvirus as member of a separate species within the genus Parapoxvirus.

[Recall dermatitis caused by re-exposure to docetaxel following irradiation of the brain. Case report and review of the literature]. / Recall-Dermatitis durch Docetaxel-Reexposition nach Gehirnbestrahlung. Fallbeobachtung und Literaturübersicht.

BACKGROUND: Together with radiation therapy the taxanes Paclitaxel and Docetaxel are more and more integrated into multimodal therapy regimens concerning breast- and lung cancer as well as squamous cell carcinoma of the head and neck. Especially in palliative situations we have to be aware of increasing side effects caused by interaction of the different treatment components. Therefore we report on a severe recall dermatitis that occurred in two breast-cancer patients following irradiation of the brain and reexposition to Docetaxel. PATIENTS AND METHOD: From January until March 1999 two female patients suffering from metastatic breast cancer and newly diagnosed cerebral metastases respectively carcinomatous meningitis underwent irradiation of the whole brain (2 Gy 5 days/week up to a reference dose of 50 Gy) in our department. Both patients had several courses of Docetaxel (Taxotere) 30 mg/m2 BSA weekly respectively 100 mg/m2 BSA/month since October and November 1998. After completion of radiotherapy chemotherapy with Docetaxel was continued. RESULTS: Both patients tolerated Docetaxel well before and during radiotherapy. However, after having finished irradiation of the brain and receiving Docetaxel again a severe erythema of the irradiated skin and large areas of moist epitheliolysis with crust occurred (CTC grade IV). CONCLUSION: The dermatitis related to irradiation and reexposition to Docetaxel observed in our two cases is interpreted as a recall reaction. The basic initiating pathologic mechanism has not been solved completely. Further investigation is needed to find out how the taxanes can be used in combination radiochemotherapy regimens without causing severe toxicity to the irradiated skin or mucosa.

Efficacy and safety of contact transscleral diode laser cyclophotocoagulation for advanced glaucoma.

BACKGROUND: This prospective study was conducted to evaluate the efficacy and safety of transscleral diode laser cyclophotocoagulation in refractory, advanced glaucoma. PATIENTS AND METHODS: One hundred eyes of 100 patients with advanced glaucoma refractory to medical treatment were consecutively treated by transscleral diode laser cyclophotocoagulation. Success was defined as a final intraocular pressure between 5 and 21 mm Hg in eyes with a visual acuity of more than hard movements, relief of pain in eyes with a visual acuity of hand movements or less including blind eyes, and reduction of carbonic anhydrase inhibitor use in all eyes. RESULTS: Ninety-three patients were followed up for 1 year after initial treatment. The overall success rate was 74.2%. Of 60 eyes with a visual acuity of more than hand movements, intraocular pressure between 5 and 21 mm Hg was achieved in 41 (68.3%) eyes. Relief of pain was achieved in 28 (84.8%) of 33 eyes. Reduction of systemic carbonic anhydrase inhibitor use was highly significant (P < 0.0001). Within 1 year, 173 laser procedures (mean, 1.9 per patient) were performed. The probability of success increased significantly (P = 0.004) with the age of the patients, from 55% for patients younger than the age of 50 years to 83% for patients older than the age of 50 years. Previous ocular surgery decreased the success probability from 95% to 68% (P = 0.02). A high success rate was achieved in inflammatory glaucoma (75%), primary open-angle glaucoma (89.5%), and neovascular glaucoma (86.7%). The results were relatively poor in traumatic glaucoma (57.1%), aphakic glaucoma (57.1%), and congenital or juvenile glaucoma (62.5%). No significant relationship between loss of visual acuity and failure of treatment (P = 0.3) could be detected. No phthisis bulbi or persistent hypotonia developed. CONCLUSIONS: Transscleral diode laser cyclophotocoagulation is an effective and safe method for the treatment of advanced, refractory glaucoma. However, repeated treatments are often necessary. Success of treatment depends on the age of patients, previous surgery, and the type of glaucoma.

A cellular J-domain protein modulates polyprotein processing and cytopathogenicity of a pestivirus.

Pestiviruses are positive-strand RNA viruses closely related to human hepatitis C virus. Gene expression of these viruses occurs via translation of a polyprotein, which is further processed by cellular and viral proteases. Here we report the formation of a stable complex between an as-yet-undescribed cellular J-domain protein, a member of the DnaJ-chaperone family, and pestiviral nonstructural protein NS2. Accordingly, we termed the cellular protein Jiv, for J-domain protein interacting with viral protein. Jiv has the potential to induce in trans one specific processing step in the viral polyprotein, namely, cleavage of NS2-3. Efficient generation of its cleavage product NS3 has previously been shown to be obligatory for the cytopathogenicity of the pestiviruses. Regulated expression of Jiv in cells infected with noncytopathogenic bovine viral diarrhea virus disclosed a direct correlation between the intracellular level of Jiv, the extent of NS2-3 cleavage, and pestiviral cytopathogenicity.