Prospects for development of African swine fever virus vaccines.

African swine fever virus is a large DNA virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. No vaccine is available, limiting options for control. The virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. Pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. CD8+ cells are required for protection induced by attenuated strain OURT88/3. Passive transfer of antibodies from immune to naïve pigs can also induce protection. Knowledge of the genome sequences of attenuated and virulent strains and targeted gene deletions from virulent strains have identified a number of virus genes involved in virulence and immune evasion. This information can be used to produce rationally attenuated vaccine strains. Virus antigens that are targets for neutralising antibodies have been identified and immunisation with these recombinant proteins has been shown to induce partial protection. However knowledge of antigens which encode the dominant protective epitopes recognised by CD8+ T cells is lacking.

Phytoplankton communities of the west coast of Florida - multiyear and seasonal responses to nutrient enrichment.

Blooms of the harmful algae species Karenia brevis are frequent off the southwest coast of Florida despite having relatively slow growth rates. The regional frequency of these harmful algal blooms led to the examination of the dominant estuarine outflows for effects on both K. brevis and the phytoplankton community in general. There is comparatively little information on the growth rates of non-Karenia taxonomic groups other than diatoms. A seasonally based series (Fall, Winter, and Spring) of bioassay experiments were conducted to determine the nutrient response of the coastal phytoplankton community. Treatments included estuarine waters (Tampa Bay, Charlotte Harbor, and the Caloosahatchee River) applied in a 1:25 dilution added to coastal water to mimic the influence of estuarine water in a coastal environment. Other treatments were 5-15 µM additions of nitrogen (N), phosphorus (P), and silica (Si) species, amino acids, and N (urea) + P added to coastal water. Incubations were conducted under ambient conditions with shading for 48 h. Analyses of dissolved and particulate nutrients were coupled with HPLC analysis of characteristic photopigments and taxonomic assignments of biomass via CHEMTAX. The coastal phytoplankton community, dominated by diatoms, cyanophytes and prasinophytes, was significantly different both by bioassay and by season, indicating little seasonal fidelity in composition. Specific growth rates of chlorophyll a indicated no significant difference between any controls, any estuarine treatment, P, or Si treatments. Conditions were uniformly N-limited with the highest growth rates in diatom biomass. Despite differing initial communities, however, there were seasonally reproducible changes in community due to the persistent growth or decline of the various taxa, including haptophytes, cyanophytes, and cryptophytes. For the one bioassay in which K. brevis was present, the slow growth of K. brevis relative to diatoms in a mixed community was evident, indicating that identifying the seasonally based behavior of other taxa in response to nutrients is critical for the simulation of phytoplankton competition and the successful prediction of the region's harmful algal blooms.

Surgical site infection prevention bundle in elective colorectal surgery.

OBJECTIVE: Surgical Site Infections (SSIs) are responsible for a significant economic burden as well as intangible costs suffered by the patient, with up to 60% deemed preventable. Colorectal patients are disproportionally affected by SSI due the risk of wound contamination with bowel content. We aimed to reduce the rate of superficial SSI after elective colorectal surgery using a bundle of evidence-based interventions. METHODS: An SSI prevention bundle was implemented in elective colorectal surgery, comprised of triclosan-coated sutures, 2% chlorhexidine skin preparation and use of warmed carbon dioxide (CO2) during laparoscopic procedures. The SSI reduction strategy was prospectively implemented and compared with historical controls. Our primary outcome measure was the overall rate of superficial SSI. Centres for Disease Control and Prevention criteria, which use microbiological evidence in conjunction with clinical features were used as the definition of SSI. RESULTS: The overall SSI rate was 27.4% in the pre-bundle group (N = 208) and 12.5% in the patients who received the SSI prevention bundle (N = 184) (adjusted odds ratio 0.38; confidence interval 0.21-0.67; P<0.001). The median time to SSI diagnosis was postoperative day 8. Overall patient length of stay (LOS) was unchanged from six days at baseline following implementation of the bundle. CONCLUSIONS: We have shown successful implementation of an SSI prevention bundle which has reduced superficial SSI rate. We recommend this SSI prevention bundle becomes standard practice in elective colorectal surgery and plan to extend the bundle to emergency general surgery.

A viral mechanism for inhibition of the cellular phosphatase calcineurin.

The transcription factor NFAT (nuclear factor of activated T cells) controls the expression of many immunomodulatory proteins. African swine fever virus inhibits proinflammatory cytokine expression in infected macrophages, and a viral protein A238L was found to display the activity of the immunosuppressive drug cyclosporin A by inhibiting NFAT-regulated gene transcription in vivo. This it does by binding the catalytic subunit of calcineurin and inhibiting calcineurin phosphatase activity.

African swine fever.

The continuing spread of African swine fever (ASF) outside Africa in Europe, the Russian Federation, China and most recently to Mongolia and Vietnam, has heightened awareness of the threat posed by this devastating disease to the global pig industry and food security. In this review we summarise what we know about the African swine fever virus (ASFV), the disease it causes, how it spreads and the current global situation. We discuss current control methods in domestic and wild pigs and prospects for development of vaccines and other tools for control.

African swine fever virus evasion of host defences.

African swine fever virus causes a haemorrhagic fever in domestic pigs and wild boar. The continuing spread in Africa, Europe and Asia threatens the global pig industry. The lack of a vaccine limits disease control. To underpin rational strategies for vaccine development improved knowledge is needed of how the virus interacts with and modulates the host's responses to infection. The virus long double-stranded DNA genome codes for more than 160 proteins of which many are non-essential for replication in cells but can have important roles in evading the host's defences. Here we review knowledge of the pathways targeted by ASFV and the mechanisms by which these are inhibited. The impact of deleting single or multiple ASFV genes on virus replication in cells and infection in pigs is summarised providing information on strategies for rational development of modified live vaccines.

African swine fever: A re-emerging viral disease threatening the global pig industry.

African swine fever (ASF) recently has spread beyond sub-Saharan Africa to the Trans-Caucasus region, parts of the Russian Federation and Eastern Europe. In this new epidemiological scenario, the disease has similarities, but also important differences, compared to the situation in Africa, including the substantial involvement of wild boar. A better understanding of this new situation will enable better control and prevent further spread of disease. In this article, these different scenarios are compared, and recent information on the pathogenesis of ASF virus strains, the immune response to infection and prospects for developing vaccines is presented. Knowledge gaps and the prospects for future control are discussed.

Survival of African Swine Fever Virus in Excretions from Pigs Experimentally Infected with the Georgia 2007/1 Isolate.

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of swine which can be transmitted through direct contact with infected animals and their excretions or indirect contact with contaminated fomites. The shedding of ASFV by infected pigs and the stability of ASFV in the environment will determine the extent of environmental contamination. The recent outbreaks of ASF in Europe make it essential to develop disease transmission models in order to design effective control strategies to prevent further spread of ASF. In this study, we assessed the shedding and stability of ASFV in faeces, urine and oral fluid from pigs infected with the Georgia 2007/1 ASFV isolate. The half-life of infectious ASFV in faeces was found to range from 0.65 days when stored at 4°C to 0.29 days when stored at 37°C, while in urine it was found to range from 2.19 days (4°C) to 0.41 days (37°C). Based on these half-lives and the estimated dose required for infection, faeces and urine would be estimated to remain infectious for 8.48 and 15.33 days at 4°C and 3.71 and 2.88 days at 37°C, respectively. The half-life of ASFV DNA was 8 to 9 days in faeces and 2 to 3 days in oral fluid at all temperatures. In urine, the half-life of ASFV DNA was found to be 32.54 days at 4°C decreasing to 19.48 days at 37°C. These results indicate that ASFV in excretions may be an important route of ASFV transmission.

Immunization of African Indigenous Pigs with Attenuated Genotype I African Swine Fever Virus OURT88/3 Induces Protection Against Challenge with Virulent Strains of Genotype I.

The attenuated African swine fever virus genotype I strain OURT88/3 has previously been shown to induce protection of European breeds of domestic pigs against challenge with virulent isolates. To determine whether protective immune responses could also be induced in indigenous breeds of pigs from the Kinshassa region in Democratic Republic of Congo, we immunized a group of eight pigs with OURT88/3 strain and challenged the pigs 3 weeks later with virulent genotype I strain OURT88/1. Four of the pigs were protected against challenge. Three of the eight pigs died from African swine fever virus and a fourth from an unknown cause. The remaining four pigs all survived challenge with a recent virulent genotype I strain from the Democratic Republic of Congo, DRC 085/10. Control groups of non-immune pigs challenged with OURT88/1 or DRC 085/10 developed signs of acute ASFV as expected and had high levels of virus genome in blood.

African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases.

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.

Identification of a variable region of the African swine fever virus genome that has undergone separate DNA rearrangements leading to expansion of minisatellite-like sequences.

Nucleotide sequencing identified a tandemly repeated sequence array 22 x 10(3) base-pairs from the right-hand DNA terminus of the African swine fever virus (ASFV) genome. The sequence of the repeat array and sequences closely flanking it were compared in the genomes of four groups of ASFV isolates that had very different restriction enzyme site maps. Arrays present in one group of ASFV isolates from East Zambia/Malawi varied in length and contained between 8 and 38 copies of a 17-nucleotide repeat unit. Repeat arrays in a second group of ASFV isolates from Europe were less variable in length but consisted of different types of repeat unit that were divergent in sequence. A third genetically diverse ASFV isolate. LIV 13 from a South Zambia Game Park, contained repeat unit types that were similar to those of European viruses. MFUE6 isolate from an East Zambia Game Park contained a shorter version of the European repeat unit. An eight-base-pair core sequence was conserved between the East Zambia/Malawi and European and LIV 13 repeat units. These tandemly repeated sequence arrays share a number of properties with chromosomal minisatellite DNA. Similar tandem repeat arrays have not been described in poxviruses.

Mutagenesis of cauliflower mosaic virus.

A series of insertion mutants of cauliflower mosaic virus (CaMV) DNA has been constructed in vitro. These insertions consist of a short DNA sequence (10 or 22 bp) containing a restriction endonuclease site (SmaI) not represented on the viral DNA. Viral infectivity was analyzed by inoculating plants with the mutated cloned viral DNA and observing symptoms. Insertions within ORFVII, and in one site within the large intergenic region, did not interfere with viral infectivity, whilst insertions within ORFII and at the end of ORFIV retarded the development of viral symptoms. All other insertion mutants analyzed were lethal. CaMV with a deletion of 105 bp within ORFVII was viable. Such viable mutants can be used to construct additional deletions or to insert foreign DNA into the viral genome.

An ARID family protein binds to the African swine fever virus encoded ubiquitin conjugating enzyme, UBCv1.

The NH(2)-terminal end of a protein, named SMCp, which contains an ARID (A/T rich interaction domain) DNA binding domain and is similar to the mammalian SMCY/SMCX proteins and retinoblastoma binding protein 2, was shown to bind the African swine fever virus encoded ubiquitin conjugating enzyme (UBCv1) using the yeast two hybrid system and in in vitro binding assays. Antisera raised against the SMCp protein were used to show that the protein is present in the cell nucleus. Immunofluorescence showed that although UBCv1 is present in the nucleus in most cells, in some cells it is in the cytoplasm, suggesting that it shuttles between the nucleus and cytoplasm. The interaction and co-localisation of UBCv1 with SMCp suggest that SMCp may be a substrate in vivo for the enzyme.

A lipid modified ubiquitin is packaged into particles of several enveloped viruses.

An anti-ubiquitin cross-reactive protein which migrates more slowly (6.5 kDa) by SDS-PAGE than ubiquitin was identified in African swine fever virus particles. This protein was extracted into the detergent phase in Triton X-114 phase separations, showing that it is hydrophobic, and was radiolabelled with both [3H]palmitic acid and [32P]orthophosphate. This indicates that the protein has a similar structure to the membrane associated phosphatidyl ubiquitin described in baculovirus particles. A similar molecule was found in vaccinia virus and herpes simplex virus particles, suggesting that it may be a component of uninfected cell membranes, which is incorporated into membrane layers in virions during morphogenesis.

The cellular immune recognition of proteins expressed by an African swine fever virus random genomic library.

The cellular immune recognition of peptides expressed by an African swine fever virus (ASFV) random genomic library has been studied. DNA from the Malawi (LIL20/1) ASFV isolate was randomly sheared by sonication, cloned into a plasmid vector downstream of a bacteriophage T7 promoter, and 72 recombinant plasmids were arbitrarily selected. These plasmids were transiently expressed following transfection into major histocompatibility complex (MHC) class I(+) class II(-) matched pig skin cells, which had been co-infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. Such cells served as antigen presenting cells and each recombinant plasmid was screened in a proliferation assay for recognition by CD8(+) lymphocytes from inbred pigs previously exposed to ASFV. This assay was demonstrated to measure CD8(+) T cell proliferation, as predicted by the phenotype of the antigen presenting cell. Of the 72 randomly selected clones, 14 were reproducibly recognised by immune pig lymphocytes and 10 corresponded to non-overlapping and distinct nucleic acid sequences. This high frequency of ASFV encoded antigenic epitopes supports the concept that cellular immunity to the virus may play an important role in resistance to ASF.

African swine fever virus genome content and variability.

A 55 kilobase pair (kb) region from the right end of the virulent African swine fever virus isolate, Malawi LIL20/1, has been sequenced. The 68 major open reading frames (ORFs) encoded are generally closely spaced and read from both DNA strands across the complete sequence. Comparison of the amino acid sequences of predicted ORFs with sequence databases identified 15 ORFs which encode proteins that are similar to proteins of known function. Two ORFs are homologous to copies of multigene family 360 (MGF360) and one ORF is homologous to copies of multigene family 110 (MGF110). Both of these multigene families have been described previously.

Familial transmission of alcohol consumption and the flushing response to alcohol in three Oriental groups.

Multivariate path analysis was used to examine the etiologies of variation and covariation of flushing after alcohol use in nuclear families of Korean, Taiwanese, Japanese and Caucasian ancestries. Phenotypic variances and covariances were partitioned into familial (additive genetic and common family environment) and environmental components. Although alcohol consumption and flushing varied greatly among the different groups, familialities, estimated from components of mother, father and at least one child, were remarkably similar. The familialities for flushing were 0.48 for Japanese, 0.56 for Koreans and 0.35 for Taiwanese; flushing is infrequent in Caucasians and thus was not analyzed. Familialities were lower for consumption, but like flushing, were consistent across ethnic groups (Japanese, 0.27; Koreans, 0.24; Taiwanese, 0.15; Caucasians, 0.28). The genetic correlation between flushing and alcohol consumption was high. Thus, to the extent that flushing influences alcohol consumption, the covariance is most likely genetic.

Molecular epidemiology of African swine fever virus studied by analysis of four variable genome regions.

Variable regions of the African swine fever virus genome, which contain arrays of tandem repeats, were compared in the genomes of isolates obtained over a 40-year period. Comparison of the size of products generated by polymerase chain reaction (PCR) from four different genome regions, within the B602L and KP86R genes and intergenic regions J286L and BtSj, placed 43 closely related isolated from Europe, the Caribbean, West and Central Africa into 17 different virus sub-groups. Sequence analysis of the most variable fragment, within the B602L gene, from 81 different isolates distinguished 31 sub-groups of virus isolates which varied in sequence and number of a tandem repeat encoding 4 amino acids. Thus, each of these analysis methods enabled isolates, which were previously grouped together by sequencing of a more conserved genome region, to be separated into multiple sub-groups. This provided additional information about strains of viruses circulating in different countries. The methods could be used in future to study the epidemiology and evolution of virus isolates and to trace the sources of disease outbreaks.

Development of a nested PCR and its internal control for the detection of African swine fever virus (ASFV) in Ornithodoros erraticus.

A nested PCR assay, with an internal control, was developed to detect African swine fever virus (ASFV) DNA in Ornithodoros erraticus. The assay revealed a better analytical sensitivity than virus isolation and the OIE PCR protocol. All ticks collected from the field, which were positive by virus isolation, were also positive by PCR. Viral DNA was detected in a further 19 out of 60 ticks from which no virus was isolated. Our results show that this assay is reliable and can easily be used to screen large tick populations collected in the field for the presence of ASFV.