T-cell responses in domestic pigs and wild boar upon infection with the moderately virulent African swine fever virus strain 'Estonia2014'.

Infection with African swine fever virus (ASFV) causes a highly lethal haemorrhagic disease in domestic and Eurasian wild pigs. Thus, it is a major threat to pig populations worldwide and a cause of substantial economic losses. Recently, less virulent ASFV strains emerged naturally, which showed higher experimental virulence in wild boar than in domestic pigs. The reason for this difference in disease progression and outcome is unclear but likely involves different immunological responses. Unfortunately, besides the importance of CD8α+ lymphocytes, little is known about the immune responses against ASFV in suids. Against this background, we used a multicolour flow cytometry platform to investigate the T-cell responses in wild boar and domestic pigs after infection with the moderately virulent ASFV strain 'Estonia2014' in two independent trials. CD4- /CD8α+ and CD4+ /CD8α+ αß T-cell frequencies increased in both subspecies in various tissues, but CD8α+ γδ T cells differentiated and responded in wild boar only. Proliferation in CD8α+ T cells was found 10 days post infectionem only. Frequencies of T-bet+ T cells increased in wild boar but not in domestic pigs. Of note, we found a considerable loss of perforin expression in cytotoxic T cells, 5 and 7 dpi. Both subspecies established a regulatory T-cell response 10 dpi. In domestic pigs, we show increasing levels of ICOS+ and CD8α+ invariant Natural Killer T cells. These disparities in T-cell responses might explain some of the differences in disease progression in wild boar and domestic pigs and should pave the way for future studies.

Stability of African Swine Fever Virus in Carcasses of Domestic Pigs and Wild Boar Experimentally Infected with the ASFV "Estonia 2014" Isolate.

Europe is currently experiencing a long-lasting African swine fever (ASF) epidemic, both in domestic pigs and wild boar. There is great concern that carcasses of infected wild boar may act as long-term virus reservoirs in the environment. We evaluated the tenacity of ASF virus (ASFV) in tissues and body fluids from experimentally infected domestic pigs and wild boar, which were stored on different matrices and at different temperatures. Samples were analysed at regular intervals for viral genome and infectious virus. ASFV was most stable in spleen or muscles stored at -20 °C and in blood stored at 4 °C. In bones stored at -20 °C, infectious virus was detected for up to three months, and at 4 °C for up to one month, while at room temperature (RT), no infectious virus could be recovered after one week. Skin stored at -20 °C, 4 °C and RT remained infectious for up to three, six and three months, respectively. In urine and faeces, no infectious virus was recovered after one week, irrespective of the matrix. In conclusion, tissues and organs from decomposing carcasses that persist in the environment for a long time can be a source of infection for several months, especially at low temperatures.

Impaired T-cell responses in domestic pigs and wild boar upon infection with a highly virulent African swine fever virus strain.

Since African swine fever (ASF) first appeared in the Caucasus region in 2007, it has spread rapidly and is now present in numerous European and Asian countries. In Europe, mainly wild boar populations are affected and pose a risk for domestic pigs. In Asia, domestic pigs are almost exclusively affected. An effective and safe vaccine is not available, and correlates of protection are far from being understood. Therefore, research on immune responses, immune dysfunction and pathogenesis is mandatory. It is acknowledged that T cells play a pivotal role. Thus, we investigated T-cell responses of domestic pigs and wild boar upon infection with the highly virulent ASF virus (ASFV) strain 'Armenia08'. For this purpose, we used a flow cytometry-based multicolour analysis to identify T-cell subtypes (cytotoxic T cells, T-helper cells, γδ T cells) and their functional impairment in ASFV-infected pigs. Domestic pigs showed lymphopaenia, and neither in the blood nor in the lymphoid organs was a proliferation of CD8+ effector cells observed. Furthermore, a T-bet-dependent activation of the remaining CD8 T cells did not occur. In contrast, a T-cell response could be observed in wild boar at 5 days post-inoculation in the blood and in tendency also in some organs. However, this cytotoxic response was not beneficial as all wild boars showed a severe acute lethal disease and a higher proportion died spontaneously or was euthanized at the humane endpoint.

Experimental H1N1pdm09 infection in pigs mimics human seasonal influenza infections.

Pigs are anatomically, genetically and physiologically comparable to humans and represent a natural host for influenza A virus (IAV) infections. Thus, pigs may represent a relevant biomedical model for human IAV infections. We set out to investigate the systemic as well as the local immune response in pigs upon two subsequent intranasal infections with IAV H1N1pdm09. We detected decreasing numbers of peripheral blood lymphocytes after the first infection. The simultaneous increase in the frequencies of proliferating cells correlated with an increase in infiltrating leukocytes in the lung. Enhanced perforin expression in αß and γδ T cells in the respiratory tract indicated a cytotoxic T cell response restricted to the route of virus entry such as the nose, the lung and the bronchoalveolar lavage. Simultaneously, increasing frequencies of CD8αα expressing αß T cells were observed rapidly after the first infection, which may have inhibited uncontrolled inflammation in the respiratory tract. Taking together, the results of this study demonstrate that experimental IAV infection in pigs mimics major characteristics of human seasonal IAV infections.

Porcine Invariant Natural Killer T Cells: Functional Profiling and Dynamics in Steady State and Viral Infections.

Pigs are important livestock and comprehensive understanding of their immune responses in infections is critical to improve vaccines and therapies. Moreover, similarities between human and swine physiology suggest that pigs are a superior animal model for immunological studies. However, paucity of experimental tools for a systematic analysis of the immune responses in pigs represent a major disadvantage. To evaluate the pig as a biomedical model and additionally expand the knowledge of rare immune cell populations in swine, we established a multicolor flow cytometry analysis platform of surface marker expression and cellular responses for porcine invariant Natural Killer T cells (iNKT). In humans, iNKT cells are among the first line defenders in various tissues, respond to CD1d-restricted antigens and become rapidly activated. Naïve porcine iNKT cells were CD3+/CD4-/CD8+ or CD3+/CD4-/CD8- and displayed an effector- or memory-like phenotype (CD25+/ICOS+/CD5hi/CD45RA-/CCR7 ± /CD27+). Based on their expression of the transcription factors T bet and the iNKT cell-specific promyelocytic leukemia zinc finger protein (PLZF), porcine iNKT cells were differentiated into functional subsets. Analogous to human iNKT cells, in vitro stimulation of porcine leukocytes with the CD1d ligand α-galactosylceramide resulted in rapid iNKT cell proliferation, evidenced by an increase in frequency and Ki-67 expression. Moreover, this approach revealed CD25, CD5, ICOS, and the major histocompatibility complex class II (MHC II) as activation markers on porcine iNKT cells. Activated iNKT cells also expressed interferon-γ, upregulated perforin expression, and displayed degranulation. In steady state, iNKT cell frequency was highest in newborn piglets and decreased with age. Upon infection with two viruses of high relevance to swine and humans, iNKT cells expanded. Animals infected with African swine fever virus displayed an increase of iNKT cell frequency in peripheral blood, regional lymph nodes, and lungs. During Influenza A virus infection, iNKT cell percentage increased in blood, lung lymph nodes, and broncho-alveolar lavage. Our in-depth characterization of porcine iNKT cells contributes to a better understanding of porcine immune responses, thereby facilitating the design of innovative interventions against infectious diseases. Moreover, we provide new evidence that endorses the suitability of the pig as a biomedical model for iNKT cell research.

Pathogenesis of African swine fever in domestic pigs and European wild boar - Lessons learned from recent animal trials.

Over the last decade, African swine fever (ASF) has changed from an exotic disease of Sub-Saharan Africa to a considerable and serious threat to pig industry in Central Europe and Asia. With the introduction of genotype II strains into the European Union in 2014, the disease has apparently found a fertile breeding ground in the abundant wild boar population. Upon infection with highly virulent ASF virus (ASFV), a haemorrhagic fever like illness with high lethality is seen in naïve domestic pigs and wild boar. Despite intensive research, virulence factors, host-virus interactions and pathogenesis are still far from being understood, and neither vaccines nor treatment exist. However, to better understand the disease, and to work towards a safe and efficacious vaccine, this information is needed. The presented review targets the knowledge gained over the last five years with regard to ASF pathogenesis in the broader sense but with a focus on the pandemic genotype II strains. In this way, it is designed as an update and supplement to existing review articles on the same topic.