Viviparity is made possible by the placenta, a structure acquired relatively recently in the evolutionary history of eutherian mammals. Compared to oviparity, it increases the survival rate of the fetus, owing to the eutherian placenta. Questions such as “How was the placenta acquired?” and “Why is there diversity in placental morphology among mammalian species?” remain largely unsolved. Our present understanding of the molecules regulating placental development remains unclear, owing in no small part to the persistent obscurity surrounding the molecular mechanisms underlying placental acquisition. Many genes that characterize eutherian placental morphology likely evolved to be expressed at the fetal-maternal interface concurrently with those during embryogenesis. Therefore, identifying these genes, how they were acquired, and how they came to be expressed specifically at the fetal-maternal interface will shed light on some crucial molecular mechanisms underlying placental evolution. Exhaustive studies support the hypothesis that endogenous retroviruses (ERVs) could be evolutional driving forces for trophoblast cell fusion and placental structure in mammalian placentas including those of the bovine species. This review focuses on bovine ERVs (BERVs) and their expression and function in the placenta.

Microchimerism is the presence of cells in an individual that have originated from another individual. The most common form of microchimerism is fetomaternal microchimerism, i.e., cells from a fetus pass through the placenta and establish cell lineages within the mother. Microchimerism was also described after transplantation of human organs in human recipients. Consequently, microchimerism may also be expected in xenotransplantation using pig cells or organs. Indeed, microchimerism was described in patients after xenotransplantations as well as in non-human primates after transplantation of pig organs. Here for the first time a comprehensive review of microchimerism in xenotransplantation is given. Since pig cells contain porcine endogenous retroviruses (PERVs) in their genome, detection of proviral DNA in the transplant recipients may be misinterpreted as infection of the recipient with PERV. To prevent this, methods discriminating between infection and microchimerism are described. This knowledge will be important for the interpretation of screening results in forthcoming human xenotransplantations.

Xenotransplantation is, like allotransplantation, usually associated with microchimerism, e.g., 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 non-human 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. That makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate which virological methods should be used to identify microchimerism, first of all screening for porcine cellular genes. Using porcine short interspersed nuclear sequences (SINES), which have hundred 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. Finally, 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 in all animals. These methods can be used in future clinical trials.

The epitranscriptomic modification m6A is a prevalent RNA modification that plays a crucial role in the regulation of various aspects of RNA metabolism. It has been found to be involved in a wide range of physiological processes and disease states. Of particular interest is the role of m6A machinery and modifications in viral infections, serving as an evolutionary marker for distinguishing between self and non-self entities. In this review article, we present a comprehensive overview of the epitranscriptomic modification m6A and its implications in the interplay between viruses and their host, focusing on immune responses and viral replication. We outline future research directions that highlight the role of m6A in viral nucleic acid recognition, initiation of antiviral immune responses, and modulation of antiviral signaling pathways. Additionally, we discuss the potential of m6A as a prognostic biomarker and a target for therapeutic interventions in viral infections.

Auckland Island pigs represent an inbred population of feral pigs isolated on the subantarctic island for over 100 years. The animals have been maintained under pathogen-free conditions in New Zealand, they are well characterized virologically and have been used as donor sources in first clinical trials of porcine neonatal islet cell transplantation for the treatment of human diabetes patients. The animals do not carry any of the xenotransplantation-relevant viruses and in the first clinical trials no porcine viruses including porcine endogenous retroviruses (PERVs) have been transmitted to the human recipients. PERVs pose a special risk in xenotransplantation since they are part of the pig genome. When the copy number of PERVs in these animals was analyzed using droplet digital PCR and primers binding to a conserved region of the polymerase gene (PERVpol), a copy number typical for Western pigs was found, confirming previous phylogenetic analyses of microsatellites and mitochondrial analyses showing a closer relationship to European pigs than to Chinese pigs. When kidney cells from very young piglets were analyzed, only around 20 PERVpol copies were detected. Using these cells as donors in somatic cell nuclear transfer (SCNT), animals were born showing a PERVpol copy numbers between 35 and 56. This data indicates that Auckland Is-land pigs have a similar copy number in comparison with other Western pig breeds and that the copy number is higher in adult animals compared with cells from young piglets. Most im-portantly, PERV-C free animals were selected and the absence of additional eight porcine viruses was demonstrated.

Auckland Island pigs represent an inbred population of feral pigs isolated on the subantarctic island for over 100 years. The animals have been maintained under pathogen-free conditions in New Zealand, they are well characterized virologically and have been used as donor sources in first clinical trials of porcine neonatal islet cell transplantation for the treatment of human dia-betes patients. The animals do not carry any of the xenotransplantation-relevant viruses and in the first clinical trials no porcine viruses including porcine endogenous retroviruses (PERVs) have been transmitted to the human recipients. PERVs pose a special risk in xenotransplantation since they are part of the pig genome. When the copy number of PERVs in these animals was analyzed using droplet digital PCR, a copy number typical for Western pigs was found, confirming previous phylogenetic analyses of microsatellites and mitochondrial analyses showing a closer relationship to European pigs than to Chinese pigs. When kidney cells from very young piglets were analyzed, only around 20 PERVpol copies were detected. Using these cells as nuclear donors in a somatic cell nuclear transfer (SCNT), animals were born showing PERVpol copy numbers between 35 and 56. This data indicates that Auckland Island pigs have a lower copy number in comparison with other Western pig breeds and that the copy number is higher in adult animals compared with cells from young piglets. Most importantly, PERV-C free animals were selected and the absence of additional eight porcine viruses was demonstrated.

The high mutation rate of human immunodeficiency virus type 1 (HIV-1) plays a major role in treatment resistance from the development of vaccines to long-lasting drugs. In addressing the crux of the issue, various attempts to estimate the mutation rate of HIV-1 resulted in a large range of 10-5 - 10-3 errors/bp/cycle due to the use of different types of investigation methods. In this review, we discuss the different assay methods, their findings on the mutation rates of HIV-1 and how the location of these mutations can be further analyzed for their potential allosteric effects to reveal potentially new inhibitors with different pharmacodynamics that can be used to circumvent fast occurring HIV drug resistance. Given that HIV is one of the fastest mutating viruses, it is a good model for comprehensive study of its mutations that can give rise to much horizontal understanding towards overall viral drug resistance as well as emerging viral diseases.

Increasing evidence points to the role of endogenous retroviruses (ERVs) in driving cancer cell proliferation. The purpose of this study was to explore the possibility of repurposing antiretroviral agents to inhibit ERVs as a new approach in cancer treatment. We found that an integrase strand-transfer inhibitor, dolutegravir (DTG), effectively inhibited the proliferation of multiple cancer cell lines and its antiproliferative potency was positively correlated with the expression levels of the human endogenous retrovirus type K (HERV-K). DTG inhibited the expression of HERV-K in multiple human cancer cell lines and the mouse mammary tumor virus (MMTV) in the murine 4T1 mammary cancer cell line. We chose the fast-growing BT-20 cell line as a model to study the in vitro antiproliferative mechanisms of DTG. BT-20 cells overexpressing both HERV-K env and pol genes became more resistant to DTG than cells transduced with vector alone. Knockdown of HERV-K also increased DTG resistance of BT-20 cells. The antiproliferative effect of DTG correlated with enhanced expression of E-cadherin and reduction in cell motility and invasiveness. Surprisingly, DTG stimulated expression of the env gene of MMTV in vivo and promoted metastasis of 4T1 tumor cells to the lungs. Taken together, our data support a role of ERVs in tumor development and encourage further search for antiretroviral agents to treat malignancies in which endogenous retroviruses are active.

The successful advancement of xenotransplantation has led to the development of highly sensitive detection systems for the screening for potentially zoonotic viruses in donor pigs and preventing their transmission to the recipient. To validate these methods, genetically modified pigs generated for xenotransplantation, numerous minipig 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. Here, we determined 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) efficiently and used the methods to screen indigenous Greek black pigs. Since porcine endogenous retroviruses (PERVs) of 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 very resistant to diseases.

The increasing frequency of male cancer coupled with the reduction in male fertility seen worldwide motivated us to seek a potential evolutionary link between these two phenomena, concerning the reproductive transcriptional modules observed in cancer and the expression of cancer-testis-antigens (CTA). The phylostratigraphy analysis of the human genome allowed us to link the early evolutionary origin of cancer by reproductive life cycles of the unicellulars and early multicellulars, potentially driving soma-germ transition, female meiosis and parthenogenesis of polyploid giant cancer cells (PGCCs), with the expansion of the CTA multi-families, very late during evolution. CTA adaptation was aided by retrovirus domestication in the unstable genomes of mammals, for protecting male fertility in stress conditions, particularly that of humans, as compensation for the energy consumption by a large complex brain which also exploited retrotransposition. We found that the early and late evolutionary branches of human cancer are united by the immunity-proto-placental network, which evolved in the Cambrian and shares stress regulators with the finely-tuned sex determination system. We further propose that social stress and endocrine disruption caused by environmental pollution with organic materials, which alter sex determination in male foetuses and further spermatogenesis in adults, bias the development of PGCC-parthenogenetic cancer by default.