CP204L Is a Multifunctional Protein of African Swine Fever Virus That Interacts with the VPS39 Subunit of the Homotypic Fusion and Vacuole Protein Sorting Complex and Promotes Lysosome Clustering.

Virus replication depends on a complex interplay between viral and host proteins. In the case of African swine fever virus (ASFV), a large DNA virus, only a few virus-host protein-protein interactions have been identified to date. In this study, we demonstrate that the ASFV protein CP204L interacts with the cellular homotypic fusion and protein sorting (HOPS) protein VPS39, blocking its association with the lysosomal HOPS complex, which modulates endolysosomal trafficking and promotes lysosome clustering. Instead, CP204L and VPS39 are targeted to virus factories and localized at the periphery of the virus DNA replication sites. Furthermore, we show that loss of VPS39 reduces the levels of virus proteins synthesized in the early phase of infection and delays ASFV replication but does not completely inhibit it. Collectively, these results identify a novel virus-host protein interaction that modulates host membrane rearrangement during infection and provide evidence that CP204L is a multifunctional protein engaged in distinct steps of the ASFV life cycle. IMPORTANCE African swine fever virus (ASFV) was first identified over a hundred years ago. Since then, much effort has been made to understand the pathogenesis of ASFV. However, the specific roles of many individual ASFV proteins during the infection remain enigmatic. This study provides evidence that CP204L, one of the most abundant ASFV proteins, modulates endosomal trafficking during virus infection. Through protein-protein interaction, CP204L prevents the recruitment of VPS39 to the endosomal and lysosomal membranes, resulting in their accumulation. Consequently, CP204L and VPS39 become sequestered in the ASFV replication and assembly site, known as the virus factory. These results uncover a novel function of viral protein CP204L and extend our understanding of complex interaction between virus and host.

African swine fever whole-genome sequencing-Quantity wanted but quality needed.

The pandemic spread of African swine fever virus (ASFV) genotype II (GTII) has led to a global crisis. Since the circulating strains are almost identical, time and money have been mis-invested in whole-genome sequencing the last years. New methods, harmonised protocols for sample selection, sequencing, and bioinformatics are therefore urgently needed.

Identification of African swine fever virus-like elements in the soft tick genome provides insights into the virus' evolution.

BACKGROUND: African swine fever virus (ASFV) is a most devastating pathogen affecting swine. In 2007, ASFV was introduced into Eastern Europe where it continuously circulates and recently reached Western Europe and Asia, leading to a socio-economic crisis of global proportion. In Africa, where ASFV was first described in 1921, it is transmitted between warthogs and soft ticks of the genus Ornithodoros in a so-called sylvatic cycle. However, analyses into this virus' evolution are aggravated by the absence of any closely related viruses. Even ancient endogenous viral elements, viral sequences integrated into a host's genome many thousand years ago that have proven extremely valuable to analyse virus evolution, remain to be identified. Therefore, the evolution of ASFV, the only known DNA virus transmitted by arthropods, remains a mystery. RESULTS: For the identification of ASFV-like sequences, we sequenced DNA from different recent Ornithodoros tick species, e.g. O. moubata and O. porcinus, O. moubata tick cells and also 100-year-old O. moubata and O. porcinus ticks using high-throughput sequencing. We used BLAST analyses for the identification of ASFV-like sequences and further analysed the data through phylogenetic reconstruction and molecular clock analyses. In addition, we performed tick infection experiments as well as additional small RNA sequencing of O. moubata and O. porcinus soft ticks. CONCLUSION: Here, we show that soft ticks of the Ornithodoros moubata group, the natural arthropod vector of ASFV, harbour African swine fever virus-like integrated (ASFLI) elements corresponding to up to 10% (over 20 kb) of the ASFV genome. Through orthologous dating and molecular clock analyses, we provide data suggesting that integration could have occurred over 1.47 million years ago. Furthermore, we provide data showing ASFLI-element specific siRNA and piRNA in ticks and tick cells allowing for speculations on a possible role of ASFLI-elements in RNA interference-based protection against ASFV in ticks. We suggest that these elements, shaped through many years of co-evolution, could be part of an evolutionary virus-vector 'arms race', a finding that has not only high impact on our understanding of the co-evolution of viruses with their hosts but also provides a glimpse into the evolution of ASFV.

Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards.

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

Swift and Reliable "Easy Lab" Methods for the Sensitive Molecular Detection of African Swine Fever Virus.

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this "easy lab" workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the "easy lab" strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the "easy lab" concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the "easy lab" strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.

Re-emergence of porcine epidemic diarrhea virus in a piglet-producing farm in northwestern Germany in 2019.

BACKGROUND: Porcine epidemic diarrhea (PED) is a viral enteric disease of pigs. It affects all age classes of animals but lethality is mainly seen in suckling piglets. After its first appearance in England in 1971, Porcine epidemic diarrhea virus (PEDV) has spread worldwide. While sporadic outbreaks prevailed in Europe, the disease had high impact in Asia. Following particularly severe outbreaks in 2011, high impact cases were also reported in the United States and neighboring countries in 2013. Subsequently, outbreaks were also reported in several European countries including Germany. These outbreaks were less severe. This case report describes a recent case of PED re-emergence in Germany and the sequence analyses of the causative PEDV. CASE PRESENTATION: In spring 2019 5 years after re-introduction of PED into Central Europe, a piglet-producer in northwestern Germany experienced an outbreak that affected sows, their suckling piglets, and weaners. After initial confirmation of PEDV by real-time RT-PCR, fecal material and small intestine samples from affected pigs were subjected to metagenomic analyses employing next-generation sequencing. Phylogenetic analyses showed high identities among the PEDV sequences obtained from samples of different animals and a close relation to recent strains from Hungary and France. Compared to the PEDV strains analyzed in 2014, genetic drift could be confirmed. Changes were mainly observed in the spike protein encoding S gene segment. In addition, metagenomic analyses showed multiple Picobirnavirus reads in all investigated samples. CONCLUSION: This case report shows that PEDV is still circulating in Europe. The causative strains are moderately virulent and are still closely related to the so-called INDEL strains reported previously in Europe, including Germany. However, a genetic drift has taken place that can be seen in a novel cluster comprising strains from Germany, Hungary and France in 2019. Relevance and impact of the detected Picobirna sequences need further investigations.

Comparative Analysis of Whole-Genome Sequence of African Swine Fever Virus Belgium 2018/1.

We analyzed the whole-genome sequence of African swine fever virus Belgium 2018/1. The strain fits into the European genotype II (>99.98% identity). The high-coverage sequence revealed 15 differences compared with an improved African swine fever virus Georgia 2007/1 sequence. However, in the absence of genetic markers, no spatial or temporal correlations could be defined.

Detection of African swine fever virus in the tissues of asymptomatic pigs in smallholder farming systems along the Kenya-Uganda border: implications for transmission in endemic areas and ASF surveillance in East Africa.

The persistence of African swine fever virus (ASFV) in endemic areas, with small-scale but regular outbreaks in domestic pigs, is not well understood. ASFV has not been detected using conventional diagnosis in these pigs or adjacent populations of resistant African wild pigs, that could act as potential carriers during the outbreaks. However, such data are crucial for the design of evidence-based control strategies. We conducted cross-sectional (1107 pigs) and longitudinal (100 pigs) monitoring of ASFV prevalence in local pigs in Kenya and Uganda. The horizontal survey revealed no evidence of ASFV in the serum or blood using either conventional or real-time PCR. One pig consistently tested positive using ELISA, but negative using PCR assays on blood. Interestingly, the isotype of the antibodies from this animal were strongly IgA biased relative to control domestic pigs and warthogs, suggesting a role for mucosal immunity. The tissues from this pig were positive by PCR following post-mortem. Internal organ tissues of 44 healthy pigs (28 sentinel pigs and 16 pigs from slaughter slabs) were tested with four different PCR assays; 15.9 % were positive for ASFV suggesting that healthy pigs carrying ASFV exist in the swine population in the study area. P72 and p54 genotyping of ASFV revealed very limited diversity: all were classified in genotype IX at both loci, as were virtually all viruses causing recent ASF outbreaks in the region. Our study suggests that carrier pigs may play a role in ASF disease outbreaks, although the triggers for outbreaks remain unclear and require further investigation. This study significantly increases scientific knowledge of the epidemiology of ASF in the field in Africa, which will contribute to the design of effective surveillance and control strategies.

African and classical swine fever: similarities, differences and epidemiological consequences.

For the global pig industry, classical (CSF) and African swine fever (ASF) outbreaks are a constantly feared threat. Except for Sardinia, ASF was eradicated in Europe in the late 1990s, which led to a research focus on CSF because this disease continued to be present. However, ASF remerged in eastern Europe in 2007 and the interest in the disease, its control and epidemiology increased tremendously. The similar names and the same susceptible species suggest a similarity of the two viral diseases, a related biological behaviour and, correspondingly, similar epidemiological features. However, there are several essential differences between both diseases, which need to be considered for the design of control or preventive measures. In the present review, we aimed to collate differences and similarities of the two diseases that impact epidemiology and thus the necessary control actions. Our objective was to discuss critically, if and to which extent the current knowledge can be transferred from one disease to the other and where new findings should lead to a critical review of measures relating to the prevention, control and surveillance of ASF and CSF. Another intention was to identify research gaps, which need to be closed to increase the chances of a successful eradication of ASF and therefore for a decrease of the economic threat for pig holdings and the international trade.

A decade of research into classical swine fever marker vaccine CP7_E2alf (Suvaxyn® CSF Marker): a review of vaccine properties.

Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. However, until recently, the available live vaccines did not allow a serological marker concept that is essentially important to circumvent long-term trade restrictions. In 2014, a new live attenuated marker vaccine, Suvaxyn® CSF Marker (Zoetis), was licensed by the European Medicines Agency. This vaccine is based on pestivirus chimera "CP7_E2alf" that carries the main immunogen of CSF virus "Alfort/187", glycoprotein E2, in a bovine viral diarrhea virus type 1 backbone ("CP7"). This review summarizes the available data on design, safety, efficacy, marker diagnostics, and its possible integration into control strategies.

Quasispecies composition and diversity do not reveal any predictors for chronic classical swine fever virus infection.

Classical swine fever (CSF) can run acute, chronic, and prenatal courses in both domestic pigs and wild boar. Although chronic infections are rare events, their epidemiological impact is very high due to the long-term shedding of virus. So far, little is known about the factors that influence disease course and outcome from either the host or virus's perspective. To elucidate the viral determinants, we analyzed the role of the viral populations for the development of chronic CSF virus (CSFV) infections. Three different animal trials that had led to both chronic and acute infections were chosen for a detailed analysis by deep sequencing. The three inocula represented sub-genogroups 2.1 and 2.3, and two viruses were wild-type CSFV, one derived from an infectious cDNA clone. These viruses and samples derived from acutely and chronically infected animals were subjected to next-generation sequencing. Subsequently, the derived full-length genomes were compared at both the consensus and the quasispecies level. At consensus level, no differences were observed between the parental viruses and the viruses obtained from chronically infected animals. Despite a considerable level of variability at the quasispecies level, no indications were found for any predictive pattern with regard to the chronicity of the CSFV infections. While there might be no direct marker for chronicity, moderate virulence of some CSFV strains in itself seems to be a crucial prerequisite for the establishment of long-term infections which does not need further genetic adaption. Thus, general host and virus factors need further investigation.

Evolution and molecular epidemiology of classical swine fever virus during a multi-annual outbreak amongst European wild boar.

Classical swine fever is a viral disease of pigs that carries tremendous socio-economic impact. In outbreak situations, genetic typing is carried out for the purpose of molecular epidemiology in both domestic pigs and wild boar. These analyses are usually based on harmonized partial sequences. However, for high-resolution analyses towards the understanding of genetic variability and virus evolution, full-genome sequences are more appropriate. In this study, a unique set of representative virus strains was investigated that was collected during an outbreak in French free-ranging wild boar in the Vosges-du-Nord mountains between 2003 and 2007. Comparative sequence and evolutionary analyses of the nearly full-length sequences showed only slow evolution of classical swine fever virus strains over the years and no impact of vaccination on mutation rates. However, substitution rates varied amongst protein genes; furthermore, a spatial and temporal pattern could be observed whereby two separate clusters were formed that coincided with physical barriers.

African swine fever virus transmission cycles in Central Europe: Evaluation of wild boar-soft tick contacts through detection of antibodies against Ornithodoros erraticus saliva antigen.

BACKGROUND: African swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format. RESULTS: Out of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely. CONCLUSION: In conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.

Comparison of porcine epidemic diarrhea viruses from Germany and the United States, 2014.

Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States.

Porcine complement regulatory protein CD46 and heparan sulfates are the major factors for classical swine fever virus attachment in vitro.

Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.

Classical swine fever virus marker vaccine strain CP7_E2alf: genetic stability in vitro and in vivo.

Recently, CP7_E2alf (SuvaxynCSF Marker), a live marker vaccine against classical swine fever virus, was licensed through the European Medicines Agency. For application of such a genetically engineered virus under field conditions, knowledge about its genetic stability is essential. Here, we report on stability studies that were conducted to assess and compare the mutation rate of CP7_E2alf in vitro and in vivo. Sequence analyses upon passaging confirmed the high stability of CP7_E2alf, and no recombination events were observed in the experimental setup. The data obtained in this study confirm the genetic stability of CP7_E2alf as an important safety component.

Course and transmission characteristics of oral low-dose infection of domestic pigs and European wild boar with a Caucasian African swine fever virus isolate.

In 2007, African swine fever virus (ASFV) was introduced into the Transcaucasian countries and Russia. Since then, it has spread alarmingly and reached the European Union. ASFV strains are highly virulent and lead to almost 100% mortality under experimental conditions. However, the possibility of dose-dependent disease courses has been discussed. For this reason, a study was undertaken to assess the risk of chronic disease and the establishment of carriers upon low-dose oronasal infection of domestic pigs and European wild boar. It was demonstrated that very low doses of ASFV are sufficient to infect especially weak or runted animals by the oronasal route. Some of these animals did not show clinical signs indicative of ASF, and they developed almost no fever. However, no changes were observed in individual animal regarding the onset, course and outcome of infection as assessed by diagnostic tests. After amplification of ASFV by these animals, pen- and stablemates became infected and developed acute lethal disease with similar characteristics in all animals. Thus, we found no indication of prolonged or chronic individual courses upon low-dose infection in either species. The scattered onset of clinical signs and pathogen detection within and among groups confirms moderate contagiosity that is strongly linked with blood contact. In conclusion, the prolonged course at the "herd level" together with the exceptionally low dose that proved to be sufficient to infect a runted wild boar could be important for disease dynamics in wild-boar populations and in backyard settings.

Emergence of porcine epidemic diarrhea virus in southern Germany.

BACKGROUND: Over the last years, porcine epidemic diarrhea virus (PEDV) has caused devastating enteric diseases in the US and several countries in Asia, while outbreaks in Europe have only been reported sporadically since the 1980s. At present, only insufficient information is available on currently circulating PEDV strains in Europe and their impact on the European swine industry. In this case report, we present epidemic outbreaks of porcine epidemic diarrhea in three farms in South-Western Germany. CASE PRESENTATION: Epidemic outbreaks of diarrhea affecting pigs of all age groups were reported in three farms, one fattening farm and two piglet producing farms, in South-Western Germany between May and November 2014. In the fattening farm yellowish, watery diarrhea without evidence of mucus or blood was associated with a massive reduction of feed consumption. Severity of clinical signs and mortality in young suckling pigs varied significantly between the two affected sow farms. While mortality in suckling piglets reached almost 70 % in one sow herd, no increase in suckling piglet mortality was observed in the second sow farm. In all three cases, PEDV was confirmed in feces and small intestines by RT-qPCR. Phylogenetic analyses based on full-length PEDV genomes revealed high identity among strains from all three herds. Moreover, the German strains showed very high nucleotide identity (99.4 %) with a variant of PEDV (OH851) that was isolated in the United States in January 2014. This strain with insertions and deletions in the S-gene (so called INDEL strains) was reported to show lower virulence. Slightly lower identities were found with other strains from the US and Asia. CONCLUSION: Phylogenetic information on the distribution of PEDV strains in Europe is severely lacking. In this case report we demonstrate that acute outbreaks of PEDV occurred in southern Germany in 2014. Current strains were clearly different from isolates found in the 1980s and were closely related to a PEDV variant found in the US in 2014. Moreover, the present case report indicates that variant strains of PEDV, containing insertions and deletions in the S gene, which were reported to be of lower virulence, might be able to cause high mortality in suckling piglets.

The core protein of classical Swine Fever virus is dispensable for virus propagation in vitro.

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447(Δc)), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447(Δc). Upon infection of the natural host, Vp447(Δc) was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.

Comparative analyses of host responses upon infection with moderately virulent classical swine fever virus in domestic pigs and wild boar.

BACKGROUND: Classical swine fever (CSF) is one of the most important viral diseases of pigs. Clinical signs may vary from almost inapparent infection to a hemorrhagic fever like illness. Among the host factors leading to different disease courses are age, breed, and immune status. The aim of this study was to compare host responses of different pig breeds upon infection with a recent moderately virulent CSF virus (CSFV) strain, and to assess their impact on the clinical outcome and the efficiency of immune responses. To this means, two domestic pig types (German Landrace and hybrids), were compared to European wild boar. Along with clinical and pathological assessments and routine virological and serological methods, kinetics of immune-cellular parameters were evaluated. FINDINGS: All animals were susceptible to infection and despite clinical differences, virus could be detected in all infected animals to similar amounts. All but one animal developed an acute disease course, two landrace animals recovered after a transient infection. One wild boar got chronically infected. Changes in the percentages of lymphocyte subsets in peripheral blood did not show a clear correlation with the clinical outcome. High and early titers of neutralizing antibodies were especially detected in wild boar and German Landrace pigs. CONCLUSIONS: While differences among breeds did not have the expected impact on course and outcome of CSFV infection, preload with facultative pathogens and even small differences in age seemed to be more relevant. Future studies will target the characterization of responses observed during different disease courses including cytokine reactions and further analyses of lymphocyte subsets.