The C-half of cisplatin resistance-associated overexpressed protein (CROP), an SR-related protein, comprises domains rich in arginine and glutamate residues (RE domain), and is rich in arginine and serine residues (RS domain). We analyzed the role of the individual domains of CROP in cellular localization, subnuclear localization, and protein-protein interaction. CROP fused with green fluorescent protein, GFP-CROP, localized exclusively to the nucleus and showed a speckled intranuclear distribution. The yeast two-hybrid system revealed that CROP interacted with SF2/ASF, an SR protein involved in RNA splicing, as well as CROP itself. The RE and RS domains were necessary for both the intranuclear speckled distribution and the protein-protein interaction. CROP was phosphorylated by mSRPK1, mSRPK2, and Clk1 in vitro, and when cells were treated with cisplatin the subnuclear distribution of GFP-CROP was changed. These results suggest that cisplatin affects RNA splicing by changing the subnuclear distribution of SR proteins including CROP.

OBJECTIVE

To investigate whether the C-60G polymorphism and other markers in the hormone-sensitive lipase (LIPE) gene are associated with baseline body composition and free-fatty acid (FFA) concentrations measured at rest and during low-intensity exercise in white and black subjects participating in the HERITAGE Family Study.

METHODS

Adult sedentary white (245 men and 258 women) and black (91 men and 185 women) subjects.

METHODS

body mass index (BMI); fat mass (FAT); percentage body fat (%FAT); fat-free mass (FATFR); sum of eight skinfolds (SF8); subcutaneous (ASF), visceral (AVF) and total (ATF) abdominal fat areas assessed by CT scan; plasma FFA concentrations measured at rest (FFAR), at a power output of 50 W (FFA50) and at a relative power output of 60% of VO(2max) (FFA60%); and fasting insulin (INS).

METHODS

Association between the C-60G polymorphism of the LIPE gene and each phenotype was tested separately in men and women using ANCOVA with the effects of age and race as covariates and with further adjustment for FAT for ASF, AVF, ATF, FFAR, FFA50 and FFA60%. Secondly, owing to significant gene-by-race interaction, associations were investigated separately in each of the two race groups. Linkage was tested with the C-60G polymorphism, a dinucleotide repeat polymorphism in the intron 7 of the LIPE gene and two microsatellites markers (D19S178 and D19S903) flanking the LIPE gene.

RESULTS

There were no race differences in the allele frequencies of the C-60G polymorphism of the LIPE gene. No association or gene-by-race interaction was observed in men. However, in women, strong gene-by-race interactions were observed for BMI (P=0.0005), FAT (P=0.0007), %FAT (P=0.0003), SF8 (P=0.0001), ASF (P=0.03) and ATF (P=0.01). When the analysis was performed separately in each race, white women carriers of the -60G allele exhibited lower %FAT (P=0.005) and SF8 (P=0.01) than non-carriers, while in black women, the -60G allele was associated with higher BMI (P=0.004), FAT (P=0.009), %FAT (P=0.01) and SF8 (P=0.0009). These associations were no longer significant after adjusting for INS. Evidence of linkage was observed in whites with ATF, FFAR, FFA50 and FFA60%.

CONCLUSIONS

These results suggest that the C-60G polymorphism in the LIPE gene plays a role in determining body composition and that its effect is sex-, race- and insulin-dependent.

To investigate the relationship between cellular microelasticity and the structural features of cytoskeletons (CSKs), a microindentation test for apical cell membranes and observation of the spatio-distribution of actin CSKs of fibroblasts were performed by fluorescence and atomic force microscopy (FM/AFM). The indentation depths of apical cell membranes were measured from AFM force-indentation (f-i) curves under equal final loads and mapped two-dimensionally to show the relative distribution of local microelasticity on cell membranes. Intracellular spatial distribution of actin CSKs was visualized fluorescently by high Z-resolution cross-sectional observation of a cell on which indentation mapping analysis had been performed in advance. Structural features of stress fibers (SFs) were observed as three typical patterns of dense SF, sparse SF and sparser SF cell groups, which were quantitated using the degree of orientation in apical SFs (ASFs) that had been defined using two-dimensional Fourier analysis. In indentation depth maps, the upper nuclear region was markedly softer than the pseudopodium region. The mean indentation depth of the upper nuclear region decreased with increased SF density in whole cells and the degree of orientation of ASF, although the pseudopodium region did not exhibit such a trend. The apical membrane of adhered cells was found to tend to stiffen with the increase in both density and degree of orientation of SFs.

The adenovirus E4orf4 protein regulates the switch from early to late gene expression during the adenoviral replication cycle. Here we report that overexpression of adenovirus E4orf4 induces human papillomavirus type 16 (HPV-16) late gene expression from subgenomic expression plasmids. E4orf4 specifically overcomes the negative effects of two splicing silencers at the two late HPV-16 splice sites SD3632 and SA5639. This results in the production of HPV-16 spliced L1 mRNAs. We show that the interaction of E4orf4 with protein phosphatase 2A (PP2A) is necessary for induction of HPV-16 late gene expression. Also an E4orf4 mutant that fails to bind the cellular splicing factor ASF/SF2 fails to induce L1 mRNA production. Collectively, these results suggest that dephosphorylation of SR proteins by E4orf4 activates HPV-16 late gene expression. Indeed, a mutant ASF/SF2 protein in which the RS-domain had been deleted could itself induce HPV-16 late gene expression, whereas wild type ASF/SF2 could not.

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.

BACKGROUND

The vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes.

RESULTS

We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO) statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events.

CONCLUSIONS

Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatibility gene HLA-DRB1 and interleukin (IL)19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF(2) and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH), Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and mass spectrometry (MS) followed by peptide sequencing. Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in MG-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches.

A protein fraction has been isolated from normal human urine which upon chronic administration produced hypertension in rats. The hypertension is associated with retention of sodium and increased circulating aldosterone. The protein fraction has been purified to homogeneity, and its molecular weight has been determined to be 26,134 daltons by equilibrium ultracentrifugation. The compound has been identified to be clearly different from ACTH, angiotensin II, and beta-lipotropin. It stimulated aldosterone production from rat glomerulosa cells in vitro in a dose-dependent fashion from 10(-9) to 10(-4)M with a maximum stimulation at 10(-7) where a fourfold increase was obtained during 2 hours of incubation. Removal of some carbohydrate moieties by insoluble neuraminidase caused a twofold increase in aldosterone production in vitro. The protein fraction has been named "aldosterone-stimulating factor" or "ASF." Further studies are in progress to define its physiological role.

Asialofetuin (ASF) coupled to Sepharose has been used to isolate a Mr 30,000 protein from Triton X-100 extracts of the baby hamster kidney cell line BHK21 C13. Binding to ASF-Sepharose was specific for terminal beta-galactosyl residues. The lectin requires detergent for optimal solubilization and binding is independent of Ca2+ or reducing reagents. The lectin was labelled in a lactoperoxidase-catalysed iodination of intact BHK21 C13 cells, suggesting that it is associated with the cell surface. Antibodies to the lectin identify in Western blotting cross-reactive components in established cell lines of kidney (MDCK, NRK) and non-kidney (L, CHO, 3T3) origin. In young adult hamsters, the lectin is expressed in colon and duodenum and in lesser amounts in ileum, stomach, lung, liver and testes but is absent in kidney. The lectin is expressed in late embryonic and newborn hamster kidney but expression declines during 14 days after birth. Immunofluorescent staining of cryostat sections of newborn hamster kidney and intestine show that the lectin is expressed at apical epithelial surfaces. The presence of the lectin at the luminal surface of kidney tubules suggests a tubular origin for the BHK21 C13 cell line. Possible functions of the Mr 30,000 lectin in kidney development are discussed.

Prohibitin (PHB) is a ubiquitous protein with a number of different molecular functions. PHB is involved in tumorigenesis by exerting either a permissive or blocking action on tumor growth, depending on the cell context. In the present study, we investigated the effects of the histone deacetylase inhibitors (HDACis), trichostatin A (TSA) and sodium butyrate (NaB), on PHB expression in the thyroid tumor cell lines, TPC-1 and FRO. Both TSA and NaB increased PHB mRNA levels. Transfection experiments showed that the overexpression of HDAC1 or 2, but not 3, inhibited PHB promoter activity. The effects of TSA and NaB on the two major PHB mRNA splicing isoforms, were also investigated. Both TSA and NaB decreased the mRNA levels of the shorter isoform, but increased those of the longer isoform. Only the latter isoform contains a 3'UTR, which has been reported to exert a growth suppressive action. Thus, our data demonstrate that HDACis control both PHB transcription and alternative splicing. The effect of HDACis on PHB alternative splicing was not due to the modification of the expression of the ASF/SF2 splicing factor.

We evaluated the relationship between apical surface fluid (ASF) and protein secretion in Calu-3 cells grown at an air-liquid interface. Calu-3 monolayers responded to forskolin, a cystic fibrosis transmembrane regulator (CFTR) channel agonist, by secreting a significant amount of ASF. Such a response from Calu-3 monolayers was not observed with CFTR channel blockers glybenclamide and DPC. Other ion channel mediators, PGF-2alpha, PMA, DNDS, and DIDS, had no effect on Calu-3 ASF secretion. Forskolin decreased Calu-3 protein secretion and glybenclamide increased protein secretion. Similarly, forskolin decreased Calu-3 lysozyme secretion, whereas glybenclamide and DPC increased lysozyme secretion. We observed significant changes in Calu-3 fluid and protein secretions with ion channel mediators known to alter CFTR activity. Our results demonstrate a functional link between fluid and protein secretions in Calu-3 apical surface and suggested a possible involvement of CFTR in these processes.

It has previously been suggested that auditory event related potentials (AEPs) are a potential marker of central serotonergic (5-HT) activity in man, with the slope of the AEP amplitude stimulus intensity function (ASF-slope) inversely correlating with 5-HT activity. However, two recent studies investigating this hypothesis in healthy subjects by rapidly lowering central 5-HT concentrations using the acute tryptophan depletion paradigm have found no effect on ASF-slope [Biological Psychology, 59 (2002) 121; Psychopharmacology (Berl), 146 (1999) 101]. These studies employed a 50g tryptophan depletion drink, which has been argued may not lower central 5-HT concentrations sufficiently. We here report the effect of tryptophan depletion on the AEP ASF-slope using 100g amino acid drinks with and without tryptophan in 14 healthy male subjects, employing a within subject, double blind, random, balanced order, cross-over design. No significant effect of tryptophan depletion was found on ASF-slope. These negative findings cast further doubt on the hypothesis that the ASF-slope is an indicator of central 5-HT function.

African swine fever (ASF) is caused by African swine fever virus (ASFV), which can cause substantial morbidity and mortality events in swine. The virus can be transmitted via direct and indirect contacts with infected swine, their products, or competent vector species, especially Ornithodoros ticks. Africa and much of Eastern Europe are endemic for ASF; a viral introduction to countries that are currently ASF free could have severe economic consequences due to the loss of production from infected animals and the trade restrictions that would likely be imposed as a result of an outbreak. We identified vulnerabilities that could lead to ASFV introduction or persistence in the United States or other ASF-free regions. Both legal and illegal movements of live animals, as well as the importation of animal products, byproducts, and animal feed, pose a risk of virus introduction. Each route is described, and current regulations designed to prevent ASFV and other pathogens from entering the United States are outlined. Furthermore, existing ASFV research gaps are highlighted. Laboratory experiments to evaluate multiple species of Ornithodoros ticks that have yet to be characterized would be useful to understand vector competence, host preferences, and distribution of competent soft tick vectors in relation to high pig production areas as well as regions with high feral swine (wild boar or similar) densities. Knowledge relative to antigenic viral proteins that contribute to host response and determination of immune mechanisms that lead to protection are foundational in the quest for a vaccine. Finally, sampling of illegally imported and confiscated wild suid products for ASFV could shed light on the types of products being imported and provide a more informed perspective relative to the risk of ASFV importation.

BACKGROUND

Fetal hemoglobin has been implicated in the modulation of sickle cell crisis though it is functional during infancy.

OBJECTIVE

The purpose of this study was to determine the waning time of fetal hemoglobin (HbF) and its persistence in later life.

METHODS

Ninety infants aged 0-12 months, admitted at hospital, were tested for their HbF levels. Adult patients numbering 690 were also examined for their sickle cell status and a sickle positive patient of SS type with HbF had her family members recruited and their sickle cell types determined.

RESULTS

The results revealed that HbF was highest (98%) at birth, decreasing at 5% per week till 6 months when it wane off. Ten infants aged 6-12 months had HbF persisting at a level of 10% or more. Adult patients examined showed proportions of their sickle cell types as AS forming 51%, AC 20%, SS 19%, and SC 10%. An SS adult patient with mild sickle cell crisis had an ASF father who had no crisis and a mother and brother with AS each who had severe crisis.

CONCLUSIONS

These findings suggest that HbF wanes off during infancy but persist in some adults and may modulate crisis in these adults. This has implications in sickle cell management.

Coronavirus Disease 2019 (COVID-19) has now become a global pandemic. This has led the United States to declare a national emergency and a ban on all elective diagnostic and therapeutic procedures, as well as elective surgery in inpatient and outpatient settings. Ambulatory surgery facilities that perform only elective procedures are thus likely to be closed. However, these facilities may be able assist acute care hospitals, as essential (urgent and emergent) surgeries and diagnostic and therapeutic procedures will still need to be performed. The aim of this article is explore the potential contribution of ASFs in the current healthcare crisis. It is important to understand that COVID-19-related information is continually evolving, and thus, the discussion provided here is subject to change.

There have been successful interventions fortifying staple foods to mobilize micronutrients as well as agricultural efforts to raise yields of staple foods to increase food availability. Zambia serves as an interesting case study because since 1961 there has been a notable decline in the availability of animal source foods (ASFs) and pulses and a significant increase in the supply of cassava and vegetable oils. The shift in food availability was partly attributed to the agricultural success in high-yielding and drought-resistant varieties that made cassava and oil crops more affordable and readily available. In this research, we explore another policy strategy that involves ASF as a mechanism to help remedy micronutrient inadequacies in a population. A scenario modeling analysis compares the changes in the nutrient profile of the Zambian diet through adding either staple plant source foods (PSFs) or ASFs. The scenarios under study involve the addition of (1) 18 fl oz of whole cow's milk; (2) 60 g of beef, 30 g of chicken, and 5 g of beef liver; (3) milk plus meat; or (4) 83 g of maize flour, 123 g of cassava, and other staple PSF, that is, isocaloric to the "milk + meat" group. The findings alert program planners and policy makers to the value of increasing the availability, accessibility, and utilization of ASF to simultaneously address multiple nutrient deficiencies, as well as the nutrition challenges that remain when expanding the availability of plant-based staples.

Post-splicing activities have been described for a subset of shuttling serine/arginine-rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras-PI 3-kinase-Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co-regulated levels of isoform-specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5'-terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap-independent manner by the action of cis-acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap-dependent manner while the other by internal ribosome entry site-dependent initiation, to show that in vivo over-expression of SF2/ASF increases the ratio between cap-dependent and internal ribosome entry site-dependent translation. Consistently, knocking-down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor-2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field.

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.

CONCLUSIONS

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.

Global shifts toward an increasingly Western diet and rises in nutrition-related noncommunicable diseases necessitate systematic examination of dietary change in adults and children. This study longitudinally examined mother and child dietary intakes and their relationship with socioeconomic factors across 4 mutually exclusive cohorts followed over 6- to 7-y time periods (cohort A: 1991-1997, cohort B: 1993-2000, cohort C: 1997-2004, cohort D: 2000-2006). The cohorts included 966 mother-child pairs (children 3-5 y at baseline) from the China Health and Nutrition Survey. Dietary intake was assessed using 24-h recall and household food consumption data; dietary variables were the percentage of total energy from animal-source foods (ASF), fats/oils, and grains. Mother-child comparison of dietary variables used average annual change measures, Spearman partial correlations, random effects models, and seemingly unrelated regression models and estimation. Whereas children were earlier adopters and maintainers of a less traditional Chinese diet, mothers experienced greater shifts away from the traditional Chinese diet with increasing child age. Mother-child correlations for the dietary variables ranged from 0.46 to 0.89 (P < 0.001). Similar increased intake of ASF and decreased intake of grains were reported for mothers and children of urban (vs. rural) residence and with higher levels of maternal education (P < 0.001). A comparable cohort effect was shown, with mothers and children consuming a less traditional Chinese diet in the later (C and D) compared to earlier (A and B) cohorts (P < 0.05). Our findings provide insight into dietary changes in mothers and children within the context of a rapidly changing nutrition and socioeconomic environment.

Outbreaks of African swine fever (ASF) have been reported from many countries, particularly in Sub-Saharan Africa, but until 2007 the disease had never been reported from the Republic of Mauritius. This is the first report describing field epidemiological and laboratory investigations into the outbreak of the lethal pig disease on the island. The official index case displayed clinical and necropsy signs suggestive of ASF. Serological and agent identification methods used to confirm and investigate the outbreak yielded negative and a few positive results respectively. Phylogenetic analysis based on DNA sequencing clustered the outbreak strain within genotype II viruses. The outbreak was controlled by modified stamping out and risk assessment revealed the possibility of disease endemicity in the country.

African swine fever (ASF) is a severe viral disease that is currently spreading among domestic pigs and wild boar (Sus scrofa) in large areas of Eurasia. Wild boar play a key role in the spread of ASF, yet despite their significance, little is known about the key mechanisms that drive infection transmission and disease persistence. A mathematical model of the wild boar ASF system is developed that captures the observed drop in population density, the peak in infected density and the persistence of the virus observed in ASF outbreaks. The model results provide insight into the key processes that drive the ASF dynamics and show that environmental transmission is a key mechanism determining the severity of an infectious outbreak and that direct frequency dependent transmission and transmission from individuals that survive initial ASF infection but eventually succumb to the disease are key for the long-term persistence of the virus. By considering scenarios representative of Estonia and Spain we show that faster degradation of carcasses in Spain, due to elevated temperature and abundant obligate scavengers, may reduce the severity of the infectious outbreak. Our results also suggest that the higher underlying host density and longer breeding season associated with supplementary feeding leads to a more pronounced epidemic outbreak and persistence of the disease in the long-term. The model is used to assess disease control measures and suggests that a combination of culling and infected carcass removal is the most effective method to eradicate the virus without also eradicating the host population, and that early implementation of these control measures will reduce infection levels whilst maintaining a higher host population density and in some situations prevent ASF from establishing in a population.

African swine fever (ASF) is a lethal hemorrhagic disease that affects wild and domestic swine. The etiological agent of ASF is African swine fever virus (ASFV). Since the first case was described in Kenya in 1921, the disease has spread to many other countries. No commercial vaccines are available to prevent ASF. In this study, we generated a recombinant Newcastle disease virus (rNDV) expressing ASFV protein 72 (p72) by reverse genetics and evaluated its humoral and cellular immunogenicity in a mouse model. The recombinant virus, rNDV/p72, replicated well in embryonated chicken eggs and was safe to use in chicks and mice. The p72 gene in rNDV/p72 was stably maintained through ten passages. Mice immunized with rNDV/p72 developed high titers of ASFV p72 specific IgG antibody, and had higher levels of IgG1 than IgG2a. Immunization also elicited T-cell proliferation and secretion of IFN-γ and IL-4. Taken together, these results indicate that rNDV expressing ASFV p72 might be a potential vaccine candidate for preventing ASF.

African swine fever (ASF) is a major pig health problem, and the causative virus is moving closer to Western European regions where pig density is high. Stopping or slowing down the spread of ASF requires mitigation strategies that are both effective and practical. Based on the elicitation of ASF expert opinion, this study identified surveillance and intervention strategies for ASF that are perceived as the most effective by providing the best combination between effectiveness and practicality. Among the 20 surveillance strategies that were identified, passive surveillance of wild boar and syndromic surveillance of pig mortality were considered to be the most effective surveillance strategies for controlling ASF virus spread. Among the 22 intervention strategies that were identified, culling of all infected herds and movement bans for neighbouring herds were regarded as the most effective intervention strategies. Active surveillance and carcase removal in wild boar populations were rated as the most effective surveillance and intervention strategies, but were also considered to be the least practical, suggesting that more research is needed to develop more effective methods for controlling ASF in wild boar populations.

Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions.

A Western blot technique using a recombinant protein has been developed to confirm positive results obtained in African swine fever (ASF)-specific antibody detection by ELISA. The new confirmatory Western blot is based on the use of protein p54, one of the most antigenic ASF virus structural proteins, expressed in Escherichia coli fused to the N-terminus of MS2 polymerase. The recombinant Western blot assay was highly specific and equally sensitive for ASF virus-infected pigs detection as the conventional Western blot, which uses virus-induced proteins ranging in molecular weight between 23 and 35 kDa. The novel Western blot assay provides a simpler interpretation of the test, eliminates the possibility of false-positive reactions produced by cellular compounds that contaminate the antigen employed in the conventional technique, and avoids the use of live virus in antigen production.