Porcine low-density lipoprotein receptor plays an important role in classical swine fever virus infection.

Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus.

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

Infections with highly pathogenic avian influenza A virus (HPAIV) H5N8 in harbor seals at the German North Sea coast, 2021.

In brain tissue of three harbor seals of the German North Sea coast, high virus loads of highly pathogenic avian influenza virus (HPAIV) H5N8 were detected. Identification of different virus variants indicates high exposure to HPAIV circulating in wild birds, but there is no evidence for H5 specific antibodies in healthy seals. Replication of avian viruses in seals may allow HPAIV to acquire mutations needed to adapt to mammalian hosts as shown by PB2 627K variants detected in these cases.

In the Search of Marine Pestiviruses: First Case of Phocoena Pestivirus in a Belt Sea Harbour Porpoise.

Pestiviruses are widespread pathogens causing severe acute and chronic diseases among terrestrial mammals. Recently, Phocoena pestivirus (PhoPeV) was described in harbour porpoises (Phocoena phocoena) of the North Sea, expanding the host range to marine mammals. While the role of the virus is unknown, intrauterine infections with the most closely related pestiviruses- Bungowannah pestivirus (BuPV) and Linda virus (LindaV)-can cause increased rates of abortions and deaths in young piglets. Such diseases could severely impact already vulnerable harbour porpoise populations. Here, we investigated the presence of PhoPeV in 77 harbour porpoises, 277 harbour seals (Phoca vitulina), grey seals (Halichoerus grypus) and ringed seals (Pusa hispida) collected in the Baltic Sea region between 2002 and 2019. The full genome sequence of a pestivirus was obtained from a juvenile female porpoise collected along the coast of Zealand in Denmark in 2011. The comparative Bayesian phylogenetic analyses revealed a close relationship between the new PhoPeV sequence and previously published North Sea sequences with a recent divergence from genotype 1 sequences between 2005 and 2009. Our findings provide further insight into the circulation of PhoPeV and expand the distribution from the North Sea to the Baltic Sea region with possible implications for the vulnerable Belt Sea and endangered Baltic Proper harbour porpoise populations.

Atypical porcine pestivirus-A widespread virus in the Swedish wild boar population.

The recently identified causative agent of congenital tremor in domestic piglets, atypical porcine pestivirus (APPV), was detected in serum from Swedish wild boar. A previous study from Sweden described APPV in domestic piglets suffering from congenital tremor, but the APPV situation in the wild boar population was unknown. In this study, 595 serum samples from wild boar originating from 13 counties in the south and central parts of Sweden, collected between 2000 and 2018, were analysed for the presence of the APPV-genome and for antibodies against the APPV-glycoprotein Erns . The results revealed that APPV is highly abundant in the Swedish wild boar population; 12% (73/595) were APPV-genome positive in serum and 72% (433/595) of the tested wild boars displayed APPV-specific antibodies. The present study also shows that APPV has been present in the Swedish wild boar population since at least the year 2000. The viral sequences obtained from the wild boars were highly similar to those obtained from Swedish domestic pigs positive for APPV and suffering from congenital tremor, suggesting a viral exchange between wild boars and domestic pigs. The high proportion of viraemic and seropositive wild boar is indicative of wild boar being an important reservoir for APPV.

Different impact of bovine complement regulatory protein 46 (CD46bov) as a cellular receptor for members of the species Pestivirus H and Pestivirus G.

The genus Pestivirus within the family Flaviviridae comprises highly relevant animal pathogens such as bovine viral diarrhoea virus 1 and 2 (BVDV-1 and -2) classified into the two species Pestivirus A and Pestivirus B, respectively. First described in 2004, HoBi-like pestiviruses (HoBiPeV) represent emerging bovine pathogens that belong to a separate species (Pestivirus H), but share many similarities with BVDV-1 and -2. Additionally, two giraffe pestivirus (GPeV) strains both originating from Kenya represent another distinct species (Pestivirus G), whose members replicate very efficiently in bovine cells. In this study, we investigated the role of bovine complement regulatory protein 46 (CD46bov), the receptor of BVDV-1 and -2, in the entry of HoBiPeV and GPeV. For this purpose, bovine CD46-knockout and CD46-rescue cell lines were generated by CRISPR/Cas9 technology and subsequent trans-complementation, respectively. Our results provide strong evidence that the impact of CD46bov differs between viruses belonging to Pestivirus H and viruses representing Pestivirus G: CD46bov revealed to be a major cellular entry factor for HoBiPeV strain HaVi-20. In contrast, GPeV strain PG-2 presented as largely independent of CD46bov, suggesting a different entry mechanism involving other molecular determinants which remain to be identified. In addition, we demonstrated that, similar to BVDV-1 and -2, virus isolates of both Pestivirus H and Pestivirus G are able to adapt to cell culture conditions by using heparan sulfate to enter the host cell. In conclusion, our findings show that different bovine pestiviruses use diverse mechanisms of host cell entry.

Pathogen Exposure in White Whales (Delphinapterus leucas) in Svalbard, Norway.

The Svalbard white whale (Delphinapterus leucas) population is one of the smallest in the world, making it particularly vulnerable to challenges such as climate change and pathogens. In this study, serum samples from live captured (2001−2016) white whales from this region were investigated for influenza A virus (IAV) antibodies (Abs) (n = 27) and RNA (n = 25); morbillivirus (MV) Abs (n = 3) and RNA (n = 25); Brucella spp. Abs; and Toxoplasma gondii Abs (n = 27). IAV Abs were found in a single adult male that was captured in Van Mijenfjorden in 2001, although no IAV RNA was detected. Brucella spp. Abs were found in 59% of the sample group (16/27). All MV and T. gondii results were negative. The results show that Svalbard white whales have been exposed to IAV and Brucella spp., although evidence of disease is lacking. However, dramatic changes in climate and marine ecosystems are taking place in the Arctic, so surveillance of health parameters, including pathogens, is critical for tracking changes in the status of this vulnerable population.

Comparative Analysis of Tunisian Sheep-like Virus, Bungowannah Virus and Border Disease Virus Infection in the Porcine Host.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Proposed Update to the Taxonomy of Pestiviruses: Eight Additional Species within the Genus Pestivirus, Family Flaviviridae.

Pestiviruses are plus-stranded RNA viruses belonging to the family Flaviviridae. They comprise several important pathogens like classical swine fever virus and bovine viral diarrhea virus that induce economically important animal diseases. In 2017, the last update of pestivirus taxonomy resulted in demarcation of 11 species designated Pestivirus A through Pestivirus K. Since then, multiple new pestiviruses have been reported including pathogens associated with disease in pigs or small ruminants. In addition, pestivirus sequences have been found during metagenomics analysis of different non-ungulate hosts (bats, rodents, whale, and pangolin), but the consequences of this pestivirus diversity for animal health still need to be established. To provide a systematic classification of the newly discovered viruses, we analyzed the genetic relationship based on complete coding sequences (cds) and deduced polyprotein sequences and calculated pairwise distances that allow species demarcation. In addition, phylogenetic analysis was performed based on a highly conserved region within the non-structural protein NS5B. Taking into account the genetic relationships observed together with available information about antigenic properties, host origin, and characteristics of disease, we propose to expand the number of pestivirus species to 19 by adding eight additional species designated Pestivirus L through Pestivirus S.

Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus.

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus.

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as a cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture-adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig-expressing cells and areas of nonexpressing cells, and one single cell line revealed not to express any CD46pig The CD46pig-deficient porcine lymphoma cell line, known to facilitate CSFV replication, was the only porcine cell line nonpermissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig-independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized.IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and is highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in the absence of CD46pig Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

Classical swine fever virus: the past, present and future.

Classical swine fever (CSF) is among the most relevant viral epizootic diseases of swine. Due to its severe economic impact, CSF is notifiable to the world organisation for animal health. Strict control policies, including systematic stamping out of infected herds with and without vaccination, have permitted regional virus eradication. Nevertheless, CSF virus (CSFV) persists in certain areas of the world and has re-emerged regularly. This review summarizes the basic established knowledge in the field and provides a comprehensive and updated overview of the recent advances in fundamental CSFV research, diagnostics and vaccine development. It covers the latest discoveries on the genetic diversity of pestiviruses, with implications for taxonomy, the progress in understanding disease pathogenesis, immunity against acute and persistent infections, and the recent findings in virus-host interactions and virulence determinants. We also review the progress and pitfalls in the improvement of diagnostic tools and the challenges in the development of modern and efficacious marker vaccines compatible with serological tests for disease surveillance. Finally, we highlight the gaps that require research efforts in the future.

Genetically distinct pestiviruses pave the way to improved classical swine fever marker vaccine candidates based on the chimeric pestivirus concept.

Classical swine fever (CSF) is one of the most important viral diseases of pigs. In many countries, the use of vaccines is restricted due to limitations of subunit vaccines with regard to efficacy and onset of protection as well as failure of live vaccines to differentiate infected from vaccinated animals (DIVA principle). Chimeric pestiviruses based on CSF virus (CSFV) and the related bovine viral diarrhea virus (BVDV) have been licensed as live marker vaccines in Europe and Asia, but cross-reactive antibodies can cause problems in DIVA application due to close antigenic relationship. To develop marker vaccine candidates with improved DIVA properties, three chimeric viruses were generated by replacing Erns of CSFV Alfort-Tübingen with homologue proteins of only distantly related pestiviruses. The chimeric viruses "Ra", "Pro", and "RaPro" contained Erns sequences of Norway rat and Pronghorn pestiviruses or a combination of both, respectively. In porcine cells, the "Pro" chimera replicated to high titers, while replication of the "Ra" chimera was limited. The "RaPro" chimera showed an intermediate phenotype. All vaccine candidates were attenuated in a vaccination/ challenge trial in pigs, but to different extents. Inoculation induced moderate to high levels of neutralizing antibodies that protected against infection with a genetically heterologous, highly virulent CSFV. Importantly, serum samples of vaccinated animals did not show any cross-reactivity in a CSFV Erns antibody ELISA. In conclusion, the Erns antigen from distantly related pestiviruses can provide a robust serological negative marker for a new generation of improved CSFV marker vaccines based on the chimeric pestivirus concept.

Characterization of the Humoral Immune Response Induced after Infection with Atypical Porcine Pestivirus (APPV).

Atypical porcine pestivirus (APPV) is a widely distributed pathogen causing congenital tremor (CT) in piglets. So far, no data are available regarding the humoral immune response against APPV. In this study, piglets and their sows from an affected herd were tested longitudinally for viral genome and antibodies. APPV genome was detected in the majority of the piglets (14/15) from CT affected litters. Transient infection of gilts was observed. Kinetics of Erns- and E2-specific antibodies and their neutralizing capacity were determined by recently (Erns) and newly (E2) developed antibody ELISAs and virus neutralization assays. Putative maternally derived antibodies (MDA) were detected in most piglets, but displayed only low to moderate neutralizing capacity (ND50 ≤ 112). Horizontal APPV transmission occurred when uninfected and infected piglets were mingled on the flat deck. Horizontally infected piglets were clinically inapparent and showed only transient viremia with subsequently consistently high E2 antibody levels. For piglets from CT affected litters, significantly lower neutralizing antibody titers were observed. Results indicate that E2 represents the main target of neutralizing antibodies. Characterization of the humoral immune response against APPV will help to provide valuable serological diagnosis, to understand the epidemiology of this novel pathogen, and to implement tailored prevention strategies.

Genetic variability of porcine pegivirus in pigs from Europe and China and insights into tissue tropism.

Pegiviruses belong to the family Flaviviridae and have been found in humans and other mammalian species. To date eleven different pegivirus species (Pegivirus A-K) have been described. However, little is known about the tissue tropism and replication of pegiviruses. In 2016, a so far unknown porcine pegivirus (PPgV, Pegivirus K) was described and persistent infection in the host, similar to human pegivirus, was reported. In this study, qRT-PCR, phylogenetic analyses and fluorescence in situ hybridization (FISH) were implemented to detect and quantify PPgV genome content in serum samples from domestic pigs from Europe and Asia, in tissue and peripheral blood mononuclear cell (PBMC) samples and wild boar serum samples from Germany. PPgV was detectable in 2.7% of investigated domestic pigs from Europe and China (viral genome load 2.4 × 102 to 2.0 × 106 PPgV copies/ml), while all wild boar samples were tested negative. Phylogenetic analyses revealed pairwise nucleotide identities >90% among PPgVs. Finally, PPgV was detected in liver, thymus and PBMCs by qRT-PCR and FISH, suggesting liver- and lymphotropism. Taken together, this study provides first insights into the tissue tropism of PPgV and shows its distribution and genetic variability in Europe and China.

Equine Parvovirus-Hepatitis Frequently Detectable in Commercial Equine Serum Pools.

An equine parvovirus-hepatitis (EqPV-H) has been recently identified in association with equine serum hepatitis, also known as Theiler's disease. This disease was first described by Arnold Theiler in 1918 and is often observed after applications with blood products in equines. So far, the virus has only been described in the USA and China. In this study, we evaluated the presence of EqPV-H in several commercial serum samples to assess the potential risk of virus transmission by equine serum-based products for medical and research applications. In 11 out of 18 commercial serum samples, EqPV-H DNA was detectable with a viral load up to 105 copies/mL. The same serum batches as well as three additional samples were also positive for antibodies against the EqPV-H VP1 protein. The countries of origin with detectable viral genomes included the USA, Canada, New Zealand, Italy, and Germany, suggesting a worldwide distribution of EqPV-H. Phylogenetic analysis of the EqPV-H NS1 sequence in commercial serum samples revealed high similarities in viral sequences from different geographical areas. As horse sera are commonly used for the production of anti-sera, which are included in human and veterinary medical products, these results implicate the requirement for diagnostic tests to prevent EqPV-H transmission.

Reemergence of Classical Swine Fever, Japan, 2018.

In September 2018, classical swine fever reemerged in Japan after 26 years, affecting domestic pigs and wild boars. The causative virus belongs to the 2.1 subgenotype, which caused repeated outbreaks in eastern and Southeast Asia. Intensive surveillance of swine and vaccination of wild boars will help control and eradicate this disease in Japan.

[The newly discovered "atypical porcine pestivirus" (APPV): An old player in the "shaking piglets" disease complex?] / Das neu entdeckte "atypische porzine Pestivirus" (APPV): ein alter Akteur im "Zitterferkelkrankheit"-Komplex?

Congenital tremor (CT) or "shaking piglet" syndrome of newborn piglets is a well-known disease caused by different factors and resulting in different pathological alterations. In addition to non-infectious causes (like intoxication and genetic alterations), viral infections of the sow during gestation are of utmost importance. It has long time been known that classical swine fever virus, a virus belonging to the genus Pestivirus within the family Flaviviridae, induces CT. Very recently, a novel porcine pestivirus was discovered, which is also capable to induce the disease and was designated as "atypical porcine pestivirus" (APPV). APPV infection is apparently highly prevalent in pig populations worldwide. This article reviews the different forms of CT and summarizes recent studies of the newly discovered virus.

Comparison of Different In Situ Hybridization Techniques for the Detection of Various RNA and DNA Viruses.

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time.

Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges.

Classical swine fever (CSF) represents a major health and trade problem for the pig industry. In endemic countries or those with a wild boar reservoir, CSF remains a priority for Veterinary Services. Surveillance as well as stamping out and/or vaccination are the principle tools of prevention and control, depending on the context. In the past decades, marker vaccines and accompanying diagnostic tests allowing the discrimination of infected from vaccinated animals have been developed. In the European Union, an E2 subunit and a chimeric live vaccine have been licensed and are available for the use in future disease outbreak scenarios. The implementation of commonly accepted and globally harmonized concepts could pave the way to replace the ethically questionable stamping out policy by a vaccination-to-live strategy and thereby avoid culling of a large number of healthy animals and save food resources. Although a number of vaccines and diagnostic tests are available worldwide, technological advancement in both domains is desirable. This work provides a summary of an analysis undertaken by the DISCONTOOLS group of experts on CSF. Details of the analysis can be downloaded from the web site at http://www.discontools.eu/.