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African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a disease with detrimental effects on the health, welfare, and production of domestic and wild pigs. The ASF laboratory confirmation is based on the analysis of blood, serum and organ samples. However, testing these samples could not be always convenient, economically feasible or possible. This study describes the validation process of a PCR-based assay targeting a portion of p72 gene, used for the molecular detection of ASFV, from meat juice samples obtained from pigs succumbed to ASFV. More specifically, we investigated the capability of a real-time PCR assay to detect ASFV DNA in meat juices obtained from the diaphragmatic muscle along with the correspondent spleens of 55 ASFV-positive pigs and wild boars sampled from confirmed outbreaks in Romania and from 73 ASFV-negative and regularly slaughtered healthy pigs collected in the Abruzzo region (Italy). The test was able to detect viral DNA in both types of samples, with lower Ct values in spleens (mean=21.11, median=20.61) than meat juices (mean=23.08, median=22.40). However, distributions of Ct values were strongly correlated each other (R 2 = 0.83, P<0.001). Considering the distribution of the observed Ct values in the 55 positive meat juice samples, a 1:10 dilution would be able to detect 90 % of positive samples, whereas a 1:100 dilution would reduce the detectability to 78 % of more contaminated samples. As meat juice could be obtained easily from muscles and considering the potential use of this test on pooled samples, it could represent a tool to aid the investigation of ASFV spread., Competing Interests: Declaration of Competing Interest Authors declare that no competing interests exist. Mention of trade names or commercial products in this article is solely for providing specific information and does not imply recommendation or endorsement by the IZSAM., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

African swine fever virus (ASFV) is a complex DNA virus causing severe hemorrhagic disease in domestic pigs and wild boar. The disease has spread worldwide, with important socio-economic consequences. Early virus detection and control measures are crucial as there are no effective vaccines nor antivirals on the market. While the diagnosis of ASFV is fast and based primarily on qPCR, the detection of infectious ASFV is a labor-intensive process requiring susceptible macrophages and subsequent antibody-based staining or hemadsorption. The latter cannot detect ASFV isolates devoid of functional CD2v (EP402R) expression. Here, we report the development of a plasmid-based reporter assay (RA) for the sensitive detection and titration of infectious ASFV. To this end, we constructed a plasmid for secreted NanoLuc luciferase (secNluc) expression driven by the ASFV DNA polymerase gene G1211R promoter. Infection of plasmid-transfected immortalized porcine kidney macrophages (IPKM) followed by measurement of secNluc from cell culture supernatants allowed reliable automated quantification of infectious ASFV. The RA-based titers matched the titers determined by conventional p72-staining or hemadsorption protocols. The novel assay is specific for ASFV as it does not detect classical swine fever virus nor porcine reproductive and respiratory syndrome virus. It is applicable to ASFV of different genotypes, virulence, and sources, including ASFV from sera and whole blood from infected pigs as well as non-hemadsorbing ASFV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

African swine fever (ASF) is a highly fatal viral disease affecting pigs. It is caused by the ASF virus (ASFV), and causes serious economic losses to the swine industry worldwide, including in Korea. Commercially available enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-ASFV antibodies are used for the diagnosis and surveillance of ASF. In this study, an ELISA was developed to detect anti-ASFV antibodies using two recombinant proteins, p22 and p30, from genotype II ASFV. Recombinant transmembrane domain-deleted p22 (p22∆TM) and p30 were expressed in E.coli vector system pET32a and mixed for use as antigens in indirect ELISA. The p22∆TM/p30-based indirect ELISA was validated using 31 sera from genotype I ASFV-infected pigs and 1133 sera from uninfected pigs. Area under the curve of this test was 0.999 [95 % concentration interval 0.992-1.000], and sensitivity and specificity were 93.5 % and 99.8 %, respectively. The between run coefficient of variation for internal quality control serum was 6.61 %. In the seroconversion analysis, the p22∆TM/p30-based indirect ELISA showed equal or better ability to detect antibodies in pigs experimentally challenged with ASFV p72 genotypes I and II (p < 0.05). In conclusion, the p22∆TM/p30-based indirect ELISA is a reliable diagnostic method for detecting anti-ASFV antibodies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 10 2 copies/μL for ASFV and 6.34 × 10 1 copies/μL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018-2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

African Swine Fever Virus (ASFV) causes a transmissible and fatal disease in pigs that is currently devastating global swine production. Efficient and economical collection of genetic data from ASFV field isolates is essential for bio-surveillance, to limit and control its spread, and to better understand ASF disease ecology. Standard genotyping and subtyping of ASFV field isolates is currently limited to a few variable regions within the ASFV genome. However, more extensive sequencing is necessary to better understand ASFV molecular evolution and identify regions relevant to genetic diversity. In this study, we developed a method for rapid and efficient next generation sequencing of approximately 40% of the ASFV genome using long PCR amplification of six different genomic regions. The amplified regions contain all segments currently used for genotyping and additional genes predicted to contribute to ASFV diversity. The primers used for amplification are broadly compatible with published ASFV genomes, permitting their use on relevant ASFV isolates. This methodology provides the enhanced depth of coverage of amplicon-based sequencing while mitigating complications associated with ASFV whole-genome sequencing. Implementation of this methodology could substantially increase the scale of ASFV genetic data collection, which is necessary to effectively monitor and combat this critical agricultural disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

African swine fever (ASF) is an infectious disease of domestic and wild pigs, caused by ASF virus (ASFV). In this study, a triplex real-time PCR assay was developed to detect and differentiate the gene-deleted and wild-type ASFV strains. Three pairs of primers and probes were designed to target the conserved region of B646L gene (p72), MGF&#95;360-14L gene (located in the middle of MGF360-505R gene) and CD2v gene, respectively. Gene-deleted (with MGF360-505R and / or CD2v genes deletion) and wild-type ASFV strains were detected specifically and simultaneously by the assay developed without cross-reactions with other nucleic acids of PCV-2, CSFV, PRRSV, FMDV or SVA. The detection limits of the triplex rPCR were 7.9 copies, 9.7 copies, and 9.6 copies of standard plasmid DNA containing B646L gene, MGF&#95;360-14L gene and CD2v gene, respectively. A total of 1215 field samples were tested in parallel by the triplex rPCR and real-time PCR recommended by OIE, and the B646L gene detection results were completely consistent between these two assays. The triplex rPCR assay was successfully developed to identify pigs infected with wild-type ASFV strains or immunized with the ASFV gene-deleted vaccine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

African swine fever virus (ASFV) is a complex double stranded DNA virus, responsible for a highly infectious and fatal disease in pigs and boars and for important deterioration of animal welfare. Over the last decade, the disease spread to several European and Asian countries causing unprecedented dramatic economic losses in pig industry. In the absence of a vaccine, affected countries rely on trustful diagnostic tests and adapted testing policies to set up control programs to fight against the disease. In this study, we evaluated the sensitivity and specificity of seven commercially available ASFV real-time PCR detection kits and three Taq polymerases on 300 well-characterized wild boar samples collected in Belgium during the 2018-2019 outbreak. This study confirms that all commercial kits and two Taq polymerases are suitable for ASFV detection in diagnostic laboratories. Furthermore, the use of endogenous controls is emphasized when testing field samples harvested on carcasses in an advanced stage of decomposition, in order to avoid false negative results., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

African swine fever (ASF) is a fatal disease caused by a virus in domestic pigs. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay were developed for the detection of African swine fever virus (ASFV). LAMP primers targeting the p10 gene of ASFV were designed, the LAMP reaction system was optimized with plasmid pUC57 containing the p10 gene sequence, and the specificities of the real-time LAMP and the visual assays were tested with the DNA or cDNA of pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV) and ASFV, as well as the plasmid pUC57 containing the p10 gene sequence. The detection limits were determined using a serial dilution of plasmid pUC57 containing the p10 gene sequence. Our results showed that the LAMP assays could accurately and specifically detect ASFV with a detection limit of 30 copies per μl -1 of pUC57 containing p10 gene sequence. In addition, the LAMP assays were further evaluated using various genotypes of ASFV strains. Furthermore, the LAMP assays are comparable with the well-established real-time PCR assay. This study provides promising solutions for facilitating preliminary and cost-effective surveillance for prevention and control of ASFV., (Published by Elsevier B.V.)

African swine fever (ASF) is an important disease of domestic pigs and wild boar. The disease is caused by African swine fever virus (ASFV). In 2014, ASFV was introduced into Eastern Europe, and it has since then continued to spread within various Eastern European countries. Investigating differences in sequences between ASFV isolates may be a valuable tool to understand differences in virulence among them, however currently, no complete genome sequences of the viruses responsible for the Eastern European outbreaks have been reported. In this study, the complete genome sequence of a highly virulent ASFV was determined directly from erythrocyte-associated nucleic acids obtained from a pig experimentally infected with an isolate from Poland (ASFV POL/2015/Podlaskie). The sequence (ca. 189 kb) of this recent European ASFV showed 95 nt differences (99.95% identity) from the ASFV Georgia 2007/1 genome. The complete sequence of ASFV POL/2015/Podlaskie should assist further studies on the genetic diversity and evolution of the European ASFVs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Generation of African swine fever virus (ASFV) recombinants has so far relied mainly on the manipulation of virus strains which had been adapted to growth in cell culture, since field isolates do not usually replicate efficiently in established cell lines. Using wild boar lung cells (WSL) which allow for propagation of ASFV field isolates, a novel approach for the generation of recombinant ASFV directly from field isolates was developed which includes the integration into the viral thymidine kinase (TK) locus of an ASFV p72-promoter driven expression cassette for enhanced green fluorescent protein (EGFP) embedded in a 16 kbp mini F-plasmid into the genome of the ASFV field strain NHV. This procedure enabled the monitoring of recombinant virus replication by EGFP autofluorescence. Selection for the TK-negative (TK(-)) phenotype of the recombinants on TK(-) Vero (VeroTK(-)) cells in the presence of 5-bromo-2'-deoxyuridine (BrdU) led to efficient isolation of recombinant virus due to the elimination of TK(+) wild type virus by BrdU-phosporylation in infected VeroTK(-) cells. The recombinant NHV-dTK-GFP produced titres of both cell-associated and secreted viral progeny in WSL cells similar to parental NHV indicating that insertion of large heterologous sequences into the viral TK locus and EGFP expression do not impair viral replication in these cells. In summary, a novel method has been developed for generation of ASFV recombinants directly from field isolates, providing an efficacious method for further manipulations of wild-type virus genomes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 10⁹ to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10⁻¹ to 10⁻⁵. The detection limit was 10⁻⁵ dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In this study, a new in situ hybridisation (ISH) protocol has been developed to identify African swine fever virus (ASFV) genome in formalin-fixed, paraffin-embedded tissues. Different digoxigenin labelled ASFV-probes were tested, including single ASFV-specific oligonucleotides, an 18.5kb restriction fragment from the viral genome and the entire ASFV genome. The latter showed the highest sensitivity in all tissues tested, independently of the virus used for challenge: E75L or Ba71L. Although a similar ASFV genome distribution was observed, the number of ISH-positive cells was higher for Ba71L compared to E75L infected tissues. As expected, the monocyte-macrophage cell lineage was the main target cell for ASFV infection. Corresponding with the last stages of infection, ISH-positive signals were also found in other cell types, including endothelial cells, hepatocytes and neutrophils. Furthermore, two unexpected findings were also noticed: the detection of a specific ISH-signal in lymphocytes and a tendency to find the signal in the nucleus of infected cells. In summary, the present findings demonstrate the utility of this new ISH protocol to study ASFV pathogenesis and its potential use as a diagnostic tool., (Copyright 2010 Elsevier B.V. All rights reserved.)

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study. The assay can detect ASFV DNA in a range of clinical samples. Sensitivity was equivalent to the Office International des Epizooties (OIE) recommended TaqMan assay. In addition the assay was found to have a detection limit 10-fold more sensitive than the conventional PCR recommended by the OIE. Linear range was ten logs from 2x10(1) to 2x10(10). The assay is rapid with an amplification time just over 2h. The development of this assay provides a useful tool for the specific diagnosis of ASF in statutory or emergency testing programs or for the detection of ASFV DNA in research applications., (Copyright 2010 Elsevier B.V. All rights reserved.)

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of African swine fever virus (ASFV). This assay targets the topoisomerase II gene of ASFV and its specificity was confirmed by restriction enzyme digestion of the reaction products. The analytical sensitivity of this ASFV LAMP assay was at least 330 genome copies, and the test was able to detect representative isolates of ASFV (n=38) without cross-reacting with classical swine fever virus. The performance of the LAMP assay was compared with other laboratory tests used for ASF diagnosis. Using blood and tissue samples collected from pigs experimentally infected with ASFV (Malawi isolate), there was good concordance between the LAMP assay and real-time PCR. In addition to detecting the reaction products using either agarose gels or real-time PCR machines, it was possible to visualise dual-labelled biotin and fluorescein ASFV LAMP amplicons using novel lateral flow devices. This assay and detection format represents the first step towards developing a practical, simple-to-use and inexpensive molecular assay format for ASF diagnosis in the field which is especially relevant to Africa where the disease is endemic in many countries., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

In tropical countries the diagnosis of viral infections of humans or animals is often hampered by the lack of suitable clinical material and the necessity to maintain a cold chain for sample preservation up to the laboratory. This study describes the use of filter papers for rapid sample collection, and the molecular detection and genotyping of viruses when stored over long periods at elevated temperatures. Infected blood was collected on filter papers, dried and stored at different temperatures (22, 32 and 37 degrees C) for various periods (up to 9 months). Two animal viruses, African swine fever, a large double-stranded DNA virus and Peste des Petits Ruminants, a negative single-stranded RNA virus, were used to validate the method. Filter papers with dried blood containing virus or control plasmid DNA were cut in small 5mm(2) pieces and added directly to the PCR tube for conventional PCR. Nucleic acid from both viruses could still be detected after 3 months at 32 degrees C. Moreover, the DNA virus could be detected at least 9 months after conservation at 37 degrees C. PCR products obtained from the filter papers were sequenced and phylogenetic analysis carried out. The results were consistent with published sequences, demonstrating that this method can be used for virus genotyping.

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 x 10(1) copies of target and are linear between 2 x 10(9) and 2 x 10(2) copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.

A closed tube isothermal Invader assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader assays were designed successfully and tested on a real-time PCR instrument (iCycler, BioRad). The assay exhibiting the lowest signal/noise ratio (VP73 ASFV Invader Assay) was analysed further using serial 10-fold dilutions of Lisbon 60 ASFV viral genome. The assay sensitivity was determined to be in the order of 2500 copies of ASFV DNA and showed a dynamic range of 4 logs, from 2.5x10(6) to 2500 copies. The high specificity of the test was demonstrated by the lack of cross-reactivity to the clinically similar but heterologous virus, classical swine fever virus. The sensitivity of the Invader assay is sufficient for the testing of acutely infected viremic animals in which the viral load will be high. The robustness and ease of use of the ASFV Invader assay, combined with the possibility to run and read the assay using simple and relatively inexpensive equipment, makes it suitable for laboratories lacking containment facilities and/or real-time PCR instrumentation or on a regional basis for on-site diagnosis close to putative sites of ASFV outbreaks.

African swine fever (ASF) has a substantial economic impact in many African developing countries and its eradication is based only on an efficient diagnosis program because of the absence of an available vaccine. Previous data suggested the convenience of using the highly antigenic virus protein p30 as ELISA antigen for serological diagnosis of this disease. A simple and efficient method is described for producing the recombinant protein p30 from ASF virus in Trichoplusia ni larvae (cabbage looper) in order to facilitate the large-scale production of this recombinant protein in the absence of fermentation procedures. A baculovirus encoding the virus protein p30 was used to infect insect larvae, showing that recombinant protein production had a sharp optimal peak with a time of occurrence dependent on the initial virus dose inoculated to the larvae. Crude lysates of infected larvae were used without further purification as coating antigen in ELISA to analyse a limited number of sera from natural or experimentally ASF virus infected pigs. Remarkably, the recombinant protein obtained from a single infected larva was sufficient for serological diagnosis of at least 3750 serum samples. Recombinant p30 obtained by this procedure was also used in a confirmatory immunoblotting, reacting with all positive sera tested previously by ELISA. In conclusion, production of the recombinant ASF virus protein p30 in larvae should be applicable to large-scale production of diagnostic reagents for this disease in developing countries, eliminating the need for specialised facilities for tissue culture.

The techniques for determining cellular sites of establishment and persistence of African swine fever virus (ASFV) were established in susceptible domestic pigs and the resistant African reservoir hosts, the warthog and bushpig. Detection, both in vitro and in vivo, was achieved by in situ hybridisation and immunocytochemistry, focusing principally on specific probes for vp73, a major capsid protein. Hybridisation of radio-labelled probes for DNA and RNA was relatively insensitive and time consuming whereas hybridisation of non-radioactive DNA probes was quicker and more sensitive. Detection of vp73 protein by immunocytochemistry was as sensitive as non-radioactive DNA hybridisation, offering in addition improved speed, ease and morphology. Both non-radioactive DNA hybridisation and immunocytochemistry were therefore used to detect ASFV DNA and protein in a range of porcine cells infected in vitro with different ASFV isolates. Malta and Malawi isolates infected both alveolar and bone marrow macrophages, but infected negligible numbers of endothelial (< 1%) and kidney cells (IBRS2 cells). Attenuated Uganda isolate, however, infected a high percentage of endothelial cells and IBRS2 cells as well as alveolar and bone marrow macrophages. When used to investigate the cell tropism of ASFV in tissues from pigs infected with the highly virulent Malawi isolate of ASFV, both techniques identified virus principally in cells of the mononuclear phagocytic system. In the lung, double staining revealed that pulmonary intravascular macrophages, but not alveolar macrophages, were infected.

At present, the eradication of African swine fever (ASF) in affected countries is based only on an efficient diagnosis program because of the absence of an available vaccine. The highly antigenic ASF virus proteins p54 and p30, encoded by genes E183L and CP204L respectively, were expressed in baculovirus for diagnostic purposes. A sequence comparison analysis of these genes from different field virus strains which are geographically diverse and isolated in different years, revealed that both genes are completely conserved among the isolates. Partially purified baculovirus-expressed proteins were used in ELISA and Western blot for ASF antibody detection in sera from experimentally inoculated pigs and field sera from ASF innaparent carriers. These comparative analyses showed that p54 presents better reactivity than p30 in Western blot. However, recombinant p30 was more efficient for antibody detection by ELISA, improving the discrimination between positive and negative sera by this technique. These data suggest the convenience of using p30 as ELISA antigen, while p54 should be the selected antigen for ASF virus antibody detection by Western blot. The combined use of both antigens for serodiagnosis of ASF disease will improve the sensitivity of innaparent carriers detection, facilitating also the interpretation of the tests, and eliminating the use of ASF virus in antigen production.

An efficient method for construction of random epitope libraries using filamentous phage is described. Random DNA fragments generated by DNase I digestion were blunt ended by T4 DNA polymerase and ligated with a 12-mer linker, followed by PCR amplification using the same oligonucleotide linker as primers. The results showed that only the ligated product containing linker sequences on both ends was specifically amplified. When the linker ligated-PCR (LL-PCR) product was used for the construction of phage display epitope libraries, the total number of independent clones in the libraries was increased by 100 to 1000 fold in comparison to that obtained for libraries constructed using other methods. In addition, the LL-PCR strategy also increased the probability of isolating recombined DNA fragments which are derived by random in-frame ligation of two or more discontinuous peptide-coding sequences before being inserted into the display vector. Such randomly recombined DNA fragments might be useful in defining conformational epitopes.

Antibody neutralization of African swine fever (ASF) virus measured by a plaque reduction assay presents frequent difficulties because of the absence or delay in plaque formation by many strains, especially low-passage viruses. To overcome this problem, a new ASF virus neutralization test has been developed. The new test consists of a conventional plaque reduction assay in which the viral plaques are detected by expression of marker genes. For the development of this neutralization assay 4 mutant viruses were generated by homologous recombination, containing beta-galactosidase or beta-glucuronidase reporter genes inserted into the thymidine kinase locus of the viral genome. These recombinant viruses have the following advantages with respect to parental viruses: (1) the neutralization assay takes less than a third of the time needed using non-recombinant viruses; (2) the small plaques can be detected more accurately by color contrast; and (3) the neutralization-resistant virus clones can be recovered easily post-plaque counting. Additionally, these recombinant viruses permit differentiation by chromogenic staining of individual infected pig macrophages, the natural host cell for ASF virus, facilitating neutralization assays in these primary cultures as described in cell lines.

ELISA, immunodot and DNA hybridization methods have been adapted to detect African swine fever virus (ASFV), and their sensitivities were compared using virus obtained from cell cultures. About 2.3 x 10(2) 50% hemadsorbing doses (HAD50) of virus were detected with ELISA sandwich using an anti-ASFV IgG biotinylated followed by avidin-peroxidase. The immunodot technique showed similar sensitivity, detecting about 4.6 x 10(2) HAD50 of virus. ASFV-DNA was detected using radioactive DNA probes and molecular hybridization. The maximal viral detection capacity of this technique was about 1.8 x 10(3) HAD50. The antigenic and DNA detection of ASFV during the infection of animals with virulent and attenuated viruses, was also studied. For this purpose, sera and red blood cells from several infected pigs were obtained at different days post-inoculation. The virus was detected at the third day after infection by the three methods. However, ASFV-DNA detection was more efficient than antigenic detection at nine days post-inoculation, when antigen detection failed, because immunocomplexes with circulating viruses were formed in the subacute infection.