African swine fever (ASF) is a complex disease of swine, caused by a large DNA virus belonging to the family Asfarviridae. The disease shows variable clinical signs, with high case fatality rates, up to 100%, in the acute forms. ASF is currently present in Africa and Europe where it circulates in different scenarios causing a high socio-economic impact. In most affected regions, control has not been effective in part due to lack of a vaccine. The availability of an effective and safe ASFV vaccines would support and enforce control-eradication strategies. Therefore, work leading to the rational development of protective ASF vaccines is a high priority. Several factors have hindered vaccine development, including the complexity of the ASF virus particle and the large number of proteins encoded by its genome. Many of these virus proteins inhibit the host's immune system thus facilitating virus replication and persistence. We review previous work aimed at understanding ASFV-host interactions, including mechanisms of protective immunity, and approaches for vaccine development. These include live attenuated vaccines, and "subunit" vaccines, based on DNA, proteins, or virus vectors. In the shorter to medium term, live attenuated vaccines are the most promising and best positioned candidates. Gaps and future research directions are evaluated.

African swine fever (ASF) was introduced into the Eastern European Union in 2014 and led to considerable mortality among wild boar. In contrast, unexpected high antibody prevalence was reported in hunted wild boar in north-eastern Estonia. One of the causative virus strains was recently characterized. While it still showed rather high virulence in the majority of experimentally infected animals, one animal survived and recovered completely. Here, we report on the follow-up characterization of the isolate obtained from the survivor in the acute phase of infection. As a first step, three in vivo experiments were performed with different types of pigs: twelve minipigs (trial A), five domestic pigs (trial B), and five wild boar (trial C) were inoculated. 75% of the minipigs and all domestic pigs recovered after an acute course of disease. However, all wild boar succumbed to infection within 17 days. Representative samples were sequenced using NGS-technologies, and whole-genomes were compared to ASFV "Georgia 2007/1". The alignments indicated a deletion of 14560 base pairs at the 5' end, and genome reorganization by duplication. The characteristic deletion was confirmed in all trial samples and local field samples. In conclusion, an ASFV variant was found in Estonia that showed reduced virulence.

African swine fever (ASF) is among the most devastating viral diseases of pigs. In recent years, the disease has spread alarmingly. Despite intensive research activities, promising vaccine candidates are still lacking. For this reason, a study was undertaken to re-assess inactivated ASFV preparations with state-of-the-art adjuvants. Inactivated preparations of ASF virus (ASFV) "Armenia08" were adjuvanted with either Polygen™ or Emulsigen(®)-D, respectively, and used to immunize six weaner pigs two times with a three-week interval. Six weeks after the first immunization, animals were challenged with the homologues highly virulent ASFV. Although ASFV-specific antibodies were detectable in all but one vaccinated animal prior to challenge, no protective effect of immunization was observed. All animals developed acute-lethal ASF and had to be euthanized within eleven days post challenge. A slightly accelerated clinical course in vaccinees could even indicate an antibody dependent enhancement, which could also influence efficacy of other vaccine approaches.

The African swine fever virus (ASFV) j13L gene encodes a 177 amino acid protein (19.0 kDa) with a putative transmembrane domain between residues 32 and 52. There is a potential signal peptide cleavage site at residue 54 and several possible motifs for phosphorylation and myristylation. Rabbit antisera raised against a synthetic peptide from the C terminus of the j13L ORF identified proteins of 25-27 kDa in cells infected with a recombinant vaccinia virus expressing the j13L ORF, in ASFV-infected cells and in purified extracellular ASF virions. In ASFV-infected cells the j13L protein was expressed late during infection and exhibited size variation (25-27 kDa) between the different ASFV strains. Nucleotide sequence analysis of the gene in these strains showed that these size differences were due to variation in the number and sequence of tandemly repeated amino acid repeats. Although ASFV-infected animals made antibodies to the j13L protein, no protection was observed when pigs were vaccinated with a recombinant vaccinia virus expressing the j13L ORF.

Due to its impact on animal health and pig industry, African swine fever (ASF) is regarded as one of the most important viral diseases of pigs. Following the ongoing epidemic in the Transcaucasian countries and the Russian Federation, African swine fever virus was introduced into the Estonian wild boar population in 2014. Epidemiological investigations suggested two different introductions into the southern and the north-eastern part of Estonia. Interestingly, outbreak characteristics varied considerably between the affected regions. While high mortality and mainly virus-positive animals were observed in the southern region, mortality was low in the north-eastern area. In the latter, clinically healthy, antibody-positive animals were found in the hunting bag and detection of virus was rare. Two hypotheses could explain the different behaviour in the north-east: (i) the frequency of antibody detections combined with the low mortality is the tail of an older, so far undetected epidemic wave coming from the east, or (ii) the virus in this region is attenuated and leads to a less severe clinical outcome. To explore the possibility of virus attenuation, a re-isolated ASFV strain from the north-eastern Ida-Viru region was biologically characterized in European wild boar. Oronasal inoculation led to an acute and severe disease course in all animals with typical pathomorphological lesions. However, one animal recovered completely and was subsequently commingled with three sentinels of the same age class to assess disease transmission. By the end of the trial at 96 days post-initial inoculation, all animals were completely healthy and neither virus nor viral genomes were detected in the sentinels or the survivor. The survivor, however, showed high antibody levels. In conclusion, the ASFV strain from north-eastern Estonia was still highly virulent but nevertheless, one animal recovered completely. Under the experimental conditions, no transmission occurred from the survivor to susceptible sentinel pigs.

Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17 days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

BACKGROUND

In our recent studies, alternative splicing has been shown to have a major role in inflammation and autoimmune muscle diseases.

OBJECTIVE

To examine the novel hypothesis that the expression of an essential alternative splicing factor, alternative splicing factor 2 (ASF/SF2), is modulated in muscle inflammation.

METHODS

ASF/SF2 expression in muscle biopsy samples from eight patients with inflammatory myopathy and six non-myositic controls was determined by using western blot with anti-ASF/SF2 antibodies. To further elucidate the mechanism of reduced ASF/SF2 expression in inflamed muscle, differentiated C2C12 myotubes were stimulated with proinflammatory cytokine tumour necrosis factor alpha (TNFalpha), followed by western blot analysis of ASF/SF2 expression.

RESULTS

ASF/SF2 expression in the muscle biopsy samples from patients with inflammatory myopathy was found to be lower (mean of relative densitometric units 41.1 (2SD 20.7)) than that of the non-myositic controls (mean of relative densitometric units 76.7 (39.6); p<0.05). In addition to this, ASF/SF2 expression was seen to be significantly down regulated (sevenfold) in C2C12 myotubes compared with expression variations in the beta-actin control (0.62-fold; mean 1.22 (0.40); p<0.05).

CONCLUSIONS

Collectively, it is shown, for the first time, that alternative splicing factor ASF/SF2 is down regulated in autoimmune inflammatory myositis-potentially via a TNFalpha-mediated pathway. The development of (1) novel autoantigen isoform microarrays for disease diagnosis and prognosis; (2) novel autoantigen-tolerising treatments for autoimmune diseases; and (3) novel splicing-redirection treatments can be facilitated by the ongoing study of alternative splicing of autoantigen transcripts.

ASF/SF2 is a well-studied SR protein that plays important roles in premRNA splicing and other aspects of RNA metabolism. Genetic inactivation experiments have revealed the fundamental roles of ASF/SF2 and other SR proteins in cell viability and animal development. However, the nature of the events triggered by in vivo depletion of ASF/SF2 remained largely elusive. Recently, we have demonstrated a significant function of ASF/SF2 in the maintenance of genome stability by preventing the formation of R loops, which provided new insights into the essential roles of ASF/SF2 in cellular physiology.

Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-containing RNAs must be exported from the nucleus where they are normally retained, it must be ensured that the unspliced HIV-1 RNA is actively exported from the nucleus and protected from degradation by processes such as nonsense-mediated decay. Here we report the identification of a novel 178-nt-long exon located in the gag-pol gene of HIV-1 and its inclusion in at least two different mRNA species. Although efficiently spliced in vitro, this exon appears to be tightly repressed and infrequently used in vivo. The splicing is activated or repressed in vitro by the splicing factors ASF/SF2 and heterogeneous nuclear ribonucleoprotein A1, respectively, suggesting that splicing is controlled by these factors. Interestingly, mutations in the 5'-splice site resulted in a dramatic reduction in the steady-state level of HIV-1 RNA, and this effect was partially reversed by expression of U1 small nuclear RNA harboring the compensatory mutation. This implies that U1 small nuclear RNA binding to optimal but non-functional splice sites might have a role in protecting unspliced HIV-1 mRNA from degradation.

BACKGROUND

Among adrenergic receptor subtypes that regulate lipid mobilization, the alpha2-adrenergic receptor is involved in the inhibition of fatty acid mobilization from adipose tissue. A C-1291G polymorphism is located in the alpha2-adrenergic receptor gene (ADRA2A) but no association with body fat accumulation has been reported yet.

METHODS

Body mass index (BMI), fat mass (FAT), percentage body fat (%FAT), trunk-to-extremity skinfold ratio (TER), sum of eight skinfolds (SF8), and abdominal subcutaneous (ASF), visceral (AVF), and total (ATF) fat areas assessed by CT scan have been measured in adult sedentary white (n = 503) and black (n = 276) subjects participating in the HERITAGE Family Study. Association between the C-1291G polymorphism and each phenotype was tested separately in men and women of each race using ANCOVA with the effects of age as covariate in addition to the effects of BMI for TER and of FAT for AVF, ASF, and ATF.

RESULTS

The allele frequencies of the ADRA2A C-1291G polymorphism differed between races. No association was observed in white subjects, except for a moderate effect of the polymorphism accounting for less than 1% of the variance in AVF and ATF in women. In black subjects, however, the G-1291 allele was found to be associated with an increase of TER in men (3.8% of variance accounted for by the polymorphism), while in black women it was associated with a decrease in TER (2.9%) and in AVF (2.5%).

CONCLUSIONS

These results suggest a role for the ADRA2A gene in determining the propensity to store fat in the abdominal area, independently of total body fatness.

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Three laboratory colonies of the argasid tick Onithodoros moubata porcinus van der Merwe were started from collections made in 1983 at three different sites in Zimbabwe. All of the colonies contained ticks infected with African swine fever (ASF) virus that was readily transmitted by bite to domestic pigs. Although they were maintained on virus-free pig blood, ASF virus infections persisted in the colonies for at least 1 yr. Despite the fact that ASF virus passes transstadially, sexually, and transovarially in this tick species sometime during the following year, the virus disappeared from the colonies. Studies comparing fecundity in infected and uninfected lots of O. moubata porcinus showed that mortality rates were considerably higher among the infected ticks. A similar study with Ornithodoros erraticus Lucas, a tick that harbors and transmits ASF virus on the Iberian Peninsula, gave essentially the same results. This is probably a factor involved in the clearance of ASF virus from tick populations that are not subjected to reinfection. How this information may be applied in the eradication of African swine fever in Portugal and Spain is discussed.

Animal source foods (ASF) have always been a constituent of human diets. Their pattern of use, however, changed in dramatic ways over the course of human evolution. Before 2 million years ago (mya), meat in particular was acquired opportunistically via hunting of small or young animals and scavenging of animals killed by other species. At some point after that time, humans began to hunt cooperatively, making possible the acquisition of meat from large game. The marked increase in human heights between 2.0 and 1.7 mya may be linked to more efficient means of acquiring meat, namely through hunting. The final pattern of meat (and other ASF) use before the modern era is associated with the shift from hunting and gathering beginning approximately 10,000 y ago. This fundamental dietary change resulted in a narrowing of diet, reduced consumption of meat and increased focus on domesticated grains. The study of archaeological human remains from around the world reveals that this period in human dietary history saw a decline in health, including increased evidence of morbidity (poorer dental health, increased occlusal abnormalities, increased iron deficiency anemia, increased infection and bone loss). Human populations living in developing and developed settings today rely on meats with lipid compositions that when eaten in excess promote cardiovascular disease. As humans become more sedentary and eat more high fat foods, we can expect to see increases in heart disease, osteoporosis and other diseases of "civilization."

The majority of mutations that cause isolated growth hormone deficiency type II are the result of aberrant splicing of transcripts encoding human growth hormone. Such mutations increase skipping of exon 3 and encode a 17.5-kDa protein that acts as a dominant negative to block secretion of full-length protein produced from unaffected alleles. Previously, we identified a splicing regulatory element in exon 3 (exonic splicing enhancer 2 (ESE2)), but we had not determined the molecular mechanism by which this element prevents exon skipping. Here, we show that two members of the serine/arginine-rich (SR) protein superfamily (ASF/SF2 and SC35) act antagonistically to regulate exon 3 splicing. ASF/SF2 activates exon 3 inclusion, but SC35, acting through a region just downstream of ESE2, can block such activation. These findings explain the disease-causing mechanism of a patient mutation in ESE2 that creates a functional SC35-binding site that then acts synergistically with the downstream SC35 site to produce pathological levels of exon 3 skipping. Although the precedent for SR proteins acting as repressors is established, this is the first example of a patient mutation that creates a site through which an SR protein represses splicing.

Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.

Embryonic stem cells (ESCs) possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells; however, whether ESCs can differentiate toward the odontogenic lineage is still unknown. In this study, we developed an efficient culture strategy to induce the differentiation of murine ESCs (mESCs) into dental epithelial cells. By culturing mESCs in ameloblasts serum-free conditioned medium (ASF-CM), we could induce their differentiation toward dental epithelial cell lineages; however, similar experiments with the tooth germ cell-conditioned medium (TGC-CM) did not yield effective results. After culturing the cells for 14days in the differentiation-inducing media, the expression of ameloblast-specific proteins such as cytokeratin (CK)14, ameloblastin (AMBN), and amelogenin (AMGN) was markedly higher in mESCs obtained with embryoid body (EB) formation than in mESCs obtained without EB formation. We observed that immunocompromised mice implanted with induced murine EBs (mEBs) showed tissue regenerative capacity and produced odontogenic epithelial-like structures, whereas those implanted with mSCE monolayer cells mainly formed connective tissues. Thus, for the first time, we report that ASF-CM provides a suitable microenvironment for inducing mESC differentiation along the odontogenic epithelial cell lineage. This result has important implications for tooth tissue engineering.

African swine fever (ASF) is an asymptomatic infection of warthogs and bushpigs, which has become an emergent disease of domestic pigs, characterized by hemorrhage, lymphopenia, and disseminated intravascular coagulation. It is caused by a large icosohedral double-stranded DNA virus, African swine fever virus (ASFV), with infection of macrophages well characterized in vitro and in vivo. This study shows that virulent isolates of ASFV also infect primary cultures of porcine aortic endothelial cells and bushpig endothelial cells (BPECs) in vitro. Kinetics of early and late gene expression, viral factory formation, replication, and secretion were similar in endothelial cells and macrophages. However, ASFV-infected endothelial cells died by apoptosis, detected morphologically by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and nuclear condensation and biochemically by poly(ADP-ribose) polymerase (PARP) cleavage at 4 h postinfection (hpi). Immediate-early proinflammatory responses were inhibited, characterized by a lack of E-selectin surface expression and interleukin 6 (IL-6) and IL-8 mRNA synthesis. Moreover, ASFV actively downregulated interferon-induced major histocompatibility complex class I surface expression, a strategy by which viruses evade the immune system. Significantly, Western blot analysis showed that the 65-kDa subunit of the transcription factor NF-kappaB, a central regulator of the early response to viral infection, decreased by 8 hpi and disappeared by 18 hpi. Both disappearance of NF-kappaB p65 and cleavage of PARP were reversed by the caspase inhibitor z-VAD-fmk. Interestingly, surface expression and mRNA transcription of tissue factor, an important initiator of the coagulation cascade, increased 4 h after ASFV infection. These data suggest a central role for vascular endothelial cells in the hemorrhagic pathogenesis of the disease. Since BPECs infected with ASFV also undergo apoptosis, resistance of the natural host must involve complex pathological factors other than viral tropism.

Multiple logistic regression was used on serological data collected in the context of the Sardinian African swine fever (ASF) eradication program from pig farms in the province of Nuoro, Sardinia. The monthly percentage of ASFV-positive herds decreased significantly from October 1994 through March 1996 (P < 0.001). The farm-level risk of seropositivity to African swine fever virus (ASFV) was higher in free-range farms than in partial-confinement farms (odds ratios (OR) varied between 4.9 in October 1994, and 5.7 in March 1996, P < 0.001). The risk of infection for total-confinement farms was one-fifth of the risk for partial-confinement farms in October 1994 (OR = 0.2, P < 0.001), whereas in March 1996, the estimated OR was 0.57 and not significant (upper confidence limit = 1.1). The maintenance of ASFV in Sardinia was primarily associated with free-range pig farms. The natural logarithm of the number of pigs tested per visit in a farm was positively associated with the risk of herd seropositivity (OR = 2.6, P < 0.001).

The role of the ancestral sylvatic cycle of the African swine fever virus (ASFV) is not well understood in the endemic areas of eastern Africa. We therefore analysed the ASF infection status on samples collected from 51 free-ranging warthogs (Phacocherus africanus) and 1576 Ornithodorus porcinus ticks from 26 independent warthog burrows at a single ranch in Kenya. Abattoir samples from 83 domestic pigs without clinical symptoms, originating from specific locations with no recent reported ASF outbreaks were included in this study. All samples were derived from areas of central Kenya, where ASF outbreaks have been reported in the past. Infection with ASFV was confirmed in 22 % of O. porcinus pools, 3.22 % of adult warthog serum samples and 49 % of domestic pig serum samples by using p72-based PCR. All of the warthog sera were positive for anti-ASFV antibodies, investigated by using ELISA, but none of the domestic pig sera were positive. Twenty O. porcinus-, 12 domestic pig- and three warthog-derived viruses were genotyped at four polymorphic loci. The ASFV isolates from ticks and domestic pigs clustered within p72 genotype X. By contrast, ASF viruses genotyped directly from warthog sera, at same locality as the tick isolates, were within p72 genotype IX and genetically similar to viruses causing recent ASF outbreaks in Kenya and Uganda. This represents the first report of the co-existence of different ASFV genotypes in warthog burrow-associated ticks and adult wild warthogs. The data from this and earlier studies suggest transfer of viruses of at least two different p72 genotypes, from wild to domestic pigs in East Africa.

African swine fever (ASF) entered Georgia in 2007 and the EU in 2014. In the EU, the virus primarily spread in wild boar (Sus scrofa) in the period from 2014-2018. However, from the summer 2018, numerous domestic pig farms in Romania were affected by ASF. In contrast to the existing knowledge on ASF transmission routes, the understanding of risk factors and the importance of different transmission routes is still limited. In the period from May to September 2019, 655 Romanian pig farms were included in a matched case-control study investigating possible risk factors for ASF incursion in commercial and backyard pig farms. The results showed that close proximity to outbreaks in domestic farms was a risk factor in commercial as well as backyard farms. Furthermore, in backyard farms, herd size, wild boar abundance around the farm, number of domestic outbreaks within 2 km around farms, short distance to wild boar cases and visits of professionals working on farms were statistically significant risk factors. Additionally, growing crops around the farm, which could potentially attract wild boar, and feeding forage from ASF affected areas to the pigs were risk factors for ASF incursion in backyard farms.

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Available data from vaccination/challenge experiments in pigs indicate that ASF protective immunity may be haemadsorption inhibition (HAI) serotype-specific. Recently, we have shown that two ASFV proteins, CD2v (EP402R) and C-type lectin (EP153R), are necessary and sufficient for mediating HAI serological specificity (Malogolovkin et al., 2015).. Here, using ASFV inter-serotypic chimeric viruses and vaccination/challenge experiments in pigs, we demonstrate that serotype-specific CD2v and/or C-type lectin proteins are important for protection against homologous ASFV infection. Thus, these viral proteins represent significant protective antigens for ASFV that should be targeted in future vaccine design and development. Additionally, these data support the concept of HAI serotype-specific protective immunity.

Maximising the ability of piglets to survive exposure to pathogens is essential to reduce early piglet mortality, an important factor in efficient commercial pig production. Mortality rates can be influenced by many factors, including early colonization by microbial commensals. Here we describe the development of an intestinal microbiota, the Bristol microbiota, for use in gnotobiotic pigs and its influence on synthesis of systemic immunoglobulins. Such a microbiota will be of value in studies of the consequences of early microbial colonization on development of the intestinal immune system and subsequent susceptibility to disease. Gnotobiotic pig studies lack a well-established intestinal microbiota. The use of the Altered Schaedler Flora (ASF), a murine intestinal microbiota, to colonize the intestines of Caesarean-derived, gnotobiotic pigs prior to gut closure, resulted in unreliable colonization with most (but not all) strains of the ASF. Subsequently, a novel, simpler porcine microbiota was developed. The novel microbiota reliably colonized the length of the intestinal tract when administered to gnotobiotic piglets. No health problems were observed, and the novel microbiota induced a systemic increase in serum immunoglobulins, in particular IgA and IgM. The Bristol microbiota will be of value for highly controlled, reproducible experiments of the consequences of early microbial colonization on susceptibility to disease in neonatal piglets, and as a biomedical model for the impact of microbial colonization on development of the intestinal mucosa and immune system in neonates.

BACKGROUND

Tissue factor (TF) is the primary initiator of blood coagulation. In response to tumor necrosis factor (TNF)-alpha human umbilical vein endothelial cells (HUVECs) express 2 TF isoforms: a soluble alternatively spliced isoform (asHTF) and membrane-bound "full length" (fl)TF. How the differential TF isoform expression is regulated is still unknown. This study compared the impact of PI3K/Akt pathway inhibition on alternative splicing of TF in HUVECs, to the influence of transcriptional regulation by inhibiting nuclear factor kappa B (NFkappaB).

RESULTS

The mRNA expression of TF isoforms was assessed by real-time PCR, the thrombogenic activity was measured by a chromogenic TF activity assay and the phosphorylation state of serine/arginine-rich (SR) proteins was analyzed by western blotting. Transfection of HUVECs was done 72 h before the inhibition experiments were performed. PI3K/Akt pathway inhibition reduced the mRNA expression of asHTF but not flTF. Inhibition of NFkappaB reduced the expression of both isoforms. Moreover, the PI3K/Akt pathway inhibition, but not that of NFkappaB, modified the phosphorylation of the SR proteins SRp75, SRp55 and SF2/ASF. Additionally, overexpression of SF2/ASF and SRp75 influenced the differential TF-isoform expression in HUVECs.

CONCLUSIONS

The PI3K/Akt pathway modulates alternative splicing of TF in HUVECs, distinct from transcriptional regulation.