Screening for Viruses in Indigenous Greek Black Pigs.

The successful advancement of xenotransplantation has led to the development of highly sensitive detection systems for the screening of potentially zoonotic viruses in donor pigs and preventing their transmission to the recipient. To validate these methods, genetically modified pigs generated for xenotransplantation, numerous minipigs and other pig breeds have been tested, thereby increasing our knowledge concerning the pig virome and the distribution of pig viruses. Of particular importance are the porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV) and the hepatitis E virus genotype 3 (HEV3). PCMV/PRV has been shown to reduce the survival time of pig transplants in non-human primates and was also transmitted in the first pig heart transplantation to a human patient. The main aim of this study was to determine the sensitivities of our methods to detect PCMV/PRV, HEV3, porcine lymphotropic herpesvirus-1 (PLHV-1), PLHV-2, PLHV-3, porcine circovirus 2 (PCV2), PCV3, PCV4 and porcine parvovirus 1 (PPV1) and to apply the methods to screen indigenous Greek black pigs. The high number of viruses found in these animals allowed for the evaluation of numerous detection methods. Since porcine endogenous retroviruses (PERVs) type A and B are integrated in the genome of all pigs, but PERV-C is not, the animals were screened for PERV-C and PERV-A/C. Our detection methods were sensitive and detected PCMV/PRV, PLHV-1, PLHV-1, PLHV-3, PVC3 and PERV-C in most animals. PPV1, HEV3, PCV4 and PERV-A/C were not detected. These data are of great interest since the animals are healthy and resistant to diseases.

Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL + -Staphylococcus aureus infections.

Staphylococcus aureus strains that produce the toxin Panton-Valentine leukocidin (PVL-SA) frequently cause recurrent skin and soft tissue infections. PVL binds to and kills human neutrophils, resulting in the formation of neutrophil extracellular traps (NETs), but the pathomechanism has not been extensively studied. Furthermore, it is unclear why some individuals colonized with PVL-SA experience recurring infections whereas others are asymptomatic. We thus aimed to (1) investigate how PVL exerts its pathogenicity on neutrophils and (2) identify factors that could help to explain the predisposition of patients with recurring infections. We provide genetic and pharmacological evidence that PVL-induced NET formation is independent of NADPH oxidase and reactive oxygen species production. Moreover, through NET proteome analysis we identified that the protein content of PVL-induced NETs is different from NETs induced by mitogen or the microbial toxin nigericin. The abundance of the proteins cathelicidin (CAMP), elastase (NE), and proteinase 3 (PRTN3) was lower on PVL-induced NETs, and as such they were unable to kill S. aureus. Furthermore, we found that neutrophils from affected patients express higher levels of CD45, one of the PVL receptors, and are more susceptible to be killed at a low PVL concentration than control neutrophils. Neutrophils from patients that experience recurring PVL-positive infections may thus be more sensitive to PVL-induced NET formation, which might impair their ability to combat the infection.

Comparison of methods for the detection of porcine cytomegalovirus/roseolovirus in relation to biosafety monitoring of xenotransplantation products.

BACKGROUND: The porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV), is widely distributed in pig populations. It has been shown that PCMV/PRV was transmitted by pig xenotransplants to non-human primates, and significantly reduced the survival time of the recipient. PCMV/PRV was also transmitted during the first transplantation of a pig heart into a human patient. PCMV/PRV establishes a lifelong persistent infection (latency) in the host, is difficult to detect in this stage, and consequential poses a threat to future clinical xenotransplantations. Therefore, sensitive and specific methods and goal-oriented strategies how, when, and where to test should be used for screening donor pigs. METHODS: In this study we compared experimentally the PCMV/PRV detection methods including PCR-based (real-time PCR, nested PCR) and immunological methods (Western blot assay, ELISA) recently published by Halecker et al. (Sci. Rep. 2022;12(1):21545) and Fischer et al. (Xenotransplantation 2023:e12803). We also compared the antigens used for antibody detection (a recombinant protein and synthetic peptides corresponding to a conserved region of the glycoprotein B, gB). RESULTS: The published methods can be used for screening donor pigs, with the results being similar. The antigens used for the detection of PCMV/PRV-specific antibodies are almost identical and give comparable results. Overall, the optimal diagnostic tests, the samples used for testing and the time of sampling play a crucial role in preventing the transmission of PCMV/PRV during xenotransplantation. CONCLUSION: Sensitive methods are available to screen donor pigs for PCMV/PRV, but a rational application of a combination of PCR-based and immunological methods as well as rational detection strategies are important for the detection of the virus during latency.

Evidence for Microchimerism in Baboon Recipients of Pig Hearts.

Xenotransplantation, like allotransplantation, is usually associated with microchimerism, i.e., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans, as well as in most preclinical trials in nonhuman primates (for review, see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. This makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate the appropriate virological methods to be used for the identification of microchimerism, first by screening for porcine cellular genes, and then how to detect infection of the host. Using porcine short interspersed nuclear sequences (SINEs), which have hundreds of thousands of copies in the pig genome, significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Lastly, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated and infection excluded in all animals. These methods can be used in future clinical trials.

First virological and pathological study of Göttingen Minipigs with Dippity Pig Syndrome (DPS).

Dippity Pig Syndrome (DPS) is a well-known but rare complex of clinical signs affecting minipigs, which has not been thoroughly investigated yet. Clinically affected animals show acute appearance of red, exudating lesions across the spine. The lesions are painful, evidenced by arching of the back (dipping), and the onset of clinical signs is generally sudden. In order to understand the pathogenesis, histological and virological investigations were performed in affected and unaffected Göttingen Minipigs (GöMPs). The following DNA viruses were screened for using PCR-based methods: Porcine cytomegalovirus (PCMV), which is a porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, PLHV-3), porcine circoviruses (PCV1, PCV2, PCV3, PCV4), porcine parvovirus 1 (PPV1), and Torque Teno sus viruses (TTSuV1, TTSuV2). Screening was also performed for integrated porcine endogenous retroviruses (PERV-A, PERV-B, PERV-C) and recombinant PERV-A/C and their expression as well as for the RNA viruses hepatitis E virus (HEV) and SARS-CoV-2. Eight clinically affected and one unaffected GöMPs were analyzed. Additional unaffected minipigs had been analyzed in the past. The analyzed GöMPs contained PERV-A and PERV-B integrated in the genome, which are present in all pigs and PERV-C, which is present in most, but not all pigs. In one affected GöMPs recombinant PERV-A/C was detected in blood. In this animal a very high expression of PERV mRNA was observed. PCMV/PRV was found in three affected animals, PCV1 was found in three animals with DPS and in the unaffected minipig, and PCV3 was detected in two animals with DPS and in the unaffected minipig. Most importantly, in one animal only PLHV-3 was detected. It was found in the affected and unaffected skin, and in other organs. Unfortunately, PLHV-3 could not be studied in all other affected minipigs. None of the other viruses were detected and using electron microscopy, no virus particles were found in the affected skin. No porcine virus RNA with exception of PERV and astrovirus RNA were detected in the affected skin by next generation sequencing. This data identified some virus infections in GöMPs with DPS and assign a special role to PLHV-3. Since PCMV/PRV, PCV1, PCV3 and PLHV-3 were also found in unaffected animals, a multifactorial cause of DPS is suggested. However, elimination of the viruses from GöMPs may prevent DPS.

In vitro and in vivo characterization of the interaction, proinflammatory, immunomodulatory and antigenic properties of capsular polysaccharide from Streptococcus pneumoniae serotype 1.

Pneumococcal capsular polysaccharide (PCP) is the major virulence determinant of Streptococcus pneumoniae (pneumococcus). Strains devoid of the capsule are avirulent or highly attenuated. PCP is present in soluble form and on pneumococci in infected individuals. The present study was undertaken to study the interaction of PCP from serotype 1 (PCP1) with immune cells, and its proinflammatory, immunomodulatory and antigenic properties. Binding of PCP1 to the surface of immune cells led to proinflammatory cytokine production which was not cell line or cytokine restricted. HEK293T transfectants expressing TLR1 and TLR2 produced IL-8 upon stimulation with PCP1, untransfected cells did not do so. PCP1 failed to induce TNF-α production from RAW264.7 cells when pre-incubated with a TLR2 blocking antibody. The surface binding of PCP1 was abrogated in the presence of TLR2 blocking antibody. PCP1 failed to bind TLR2 deficient RAW264.7 cells and induce TNF-α production. Unlike PCP1, alkali-treated PCP1 failed to stimulate RAW264.7 cells to produce TNF-α indicating the importance of alkali-sensitive moieties like O-acetyl groups. Alkali-treated PCP1 elicited lower anti-PCP1 antibody response. Mice experiments suggested that alkali-sensitive groups are significant target of protective antibodies in PCP1 immunized mice. Our findings demonstrate that PCP1 is an important modulator of immune response against pneumococci.

A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence.

Streptococcus pneumoniae (pneumococcus) is a major bacterial pathogen that causes pneumonia and septicemia in humans. Pneumococci are cleared from the host primarily by antibody dependent opsonophagocytosis by phagocytes like neutrophils. Neutrophils release neutrophil extracellular traps (NETs) on contacting pneumococci. NETs immobilize pneumococci and restrict its dissemination in the host. One of the strategies utilized by pneumococci to evade the host immune response involves use of DNase(s) to degrade NETs. We screened the secretome of autolysin deficient S. pneumoniae to identify novel DNase(s). Zymogram analysis revealed 3 bands indicative of DNase activity. Mass spectrometric analysis led to the identification of TatD as a potential extracellular DNase. Recombinant TatD showed nucleotide sequence-independent endodeoxyribonuclease activity. TatD was associated with extracellular vesicles. Pneumococcal secretome degraded NETs from human neutrophils. Extracellular vesicle fraction from tatD deficient strain showed little NET degrading activity. Recombinant TatD efficiently degraded NETs. tatD deficient pneumococci showed lower bacterial load in lungs, blood and spleen in a murine sepsis model compared to wildtype strain, and showed less severe lung pathology and compromised virulence. This study provides insights into the role of a novel extracellular DNase in evasion of the innate immune system.