Search Results (23)

Background/Objectives: African Swine Fever (ASF) represents one of the most serious threats to animal health and global food security. The causative agent of ASF is the African swine fever virus (ASFV), a DNA virus belonging to the Asfarviridae family. Here, we describe ex vivo results for an original anti-ASFV vaccine approach based on the cellular immune response induced by extracellular vesicles (EVs) engineered to express four ASFV proteins. EV engineering was achieved by expressing a DNA vector encoding a biologically inactive HIV-1 Nef protein (Nef^mut), which exhibits unusually high efficiency of incorporation into EVs, even when fused to foreign proteins. Previous studies have demonstrated that intramuscular injection of Nef^mut-based vectors leads to the engineering of Evs, spontaneously released by muscle cells, and induction of antigen-specific CD8⁺ T cell immunity. Methods: We designed DNA vectors expressing the fusion products between Nef^mut and each of the four ASFV structural proteins p30, p54, pp62, and p72. Engineered EVs were molecularly characterized by Western blot and nanotrack analysis, and their potential immunogenicity was assessed by priming and cross-presentation assays. Results: We assessed that the four fusion proteins were successfully expressed in transfected mammalian cells, with the release of valuable amounts of engineered EVs. When immature swine dendritic cells were challenged with the engineered EVs and then co-cultivated with autologous peripheral blood lymphocytes in priming assays, lymphocyte subpopulations specifically reacting against each ASFV antigen were elicited, as detected by an IFN-γ ELISpot assay. In addition, we provide evidence that the Nef^mut-based fusion products incorporated into the engineered EVs can be cross-presented by professional antigen-presenting cells, leading to cross-priming of autologous lymphocytes. Conclusions: These results represent the best premise to go forward with experiments examining immunogenicity and antiviral efficiency in pigs. Full article

African swine fever virus (ASFV) is the only member of the family Asfarviridae and can cause African swine fever, a disease with a consistently high mortality rate. The pE248R protein, a myristoylated integral membrane protein of ASFV, is required for virus infectivity and some early postentry event, making it a key target for studying the prevention and treatment of ASFV. In this study, BALB/c mice were immunized with purified recombinant pE248R protein, leading to the generation of five monoclonal antibodies (mAbs). Selected mAbs were subsequently subjected to further characterization. By identifying the reactivity of different pE248R protein peptide segments with these monoclonal antibodies, we screened and identified a linear B cell epitope (⁸⁷QEVALTQWMDAG⁹⁸) on the pE248R protein. These results provide a new theoretical basis for analyzing the structure and function of pE248R protein, particularly contributing to the construction of a comprehensive B-cell epitope map for ASFV immunogens. Full article

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170–190+ kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with a genome size of approximately 12 kB, belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS) allow for the establishment of NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution, facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10 kB and 6 kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV, providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the three known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with two different genotype 1 CSF viruses. ASFV and CSFV amplicons from optimized PCR were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1,000X for ASFV, with 99% of the genome covered, and 10,000X–20,000X for CSFV, with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation, as well as genetic characterization of these high-consequence swine viruses, globally and within the United States, should an outbreak occur. Full article

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time. Full article

African swine fever (ASF) is a deadly hemorrhagic disease of domestic and wild swine that was first described in the early 20th century after the introduction of European pigs to Kenya. The etiological agent, the African swine fever virus (ASFV), is a large DNA virus within the Asfarviridae family that is broadly categorized epidemiologically into genotypes based on the nucleotide sequence of B646L, the gene encoding the major capsid protein p72. ASF outbreaks in Africa have been linked historically to 25 genotypes by p72 nucleotide analysis and, recently, to 6 genotypes by amino acid comparison, whereas global outbreaks of ASF outside of Africa have only been linked to 2 genotypes: genotype I, which led to an outbreak in Europe during the 1960s that later spread to South America, and genotype II, responsible for the current pandemic that began in Georgia in 2007 and has since spread to Europe, Asia, and Hispaniola. Here, we present an analysis of the genome of ASFV Spencer, an isolate that was collected in 1951 near Johannesburg, South Africa. While nucleotide analysis of Spencer indicates the p72 coding sequence is unique, differentiating from the closest reference by five nucleotides, the predicted amino acid sequence indicates that it is 100% homologous to contemporary genotype 1. Full genome analysis reveals it is more similar to Mkuzi1979 and encodes genes that share similarity with either genotype 1 or genotype 2 outbreak strains. Full article

African swine fever virus (ASFV) belongs to the family of Asfarviridae, part of the group of nucleocytoplasmic large DNA viruses (NCLDV). Little is known about the internalization of ASFV in the host cell and the fusion membrane events that take place at early stages of the infection. Poxviruses, also members of the NCLDV and represented by vaccinia virus (VACV), are large, enveloped, double-stranded DNA viruses. Poxviruses were considered unique in having an elaborate entry-fusion complex (EFC) composed of 11 highly conserved proteins integrated into the membrane of mature virions. Recent advances in methodological techniques have again revealed several connections between VACV EFC proteins. In this study, we explored the possibility of an analogous ASFV EFC by identifying ten candidate proteins exhibiting structural similarities with VACV EFC proteins. This could reveal key functions of these ASFV proteins, drawing attention to shared features between the two virus families, suggesting the potential existence of an ASFV entry-fusion complex. Full article

The African Swine Fever Virus (ASFV) is a DNA virus of the Asfarviridae family, Asfivirus genus. It is responsible for massive losses in pig populations and drastic direct and indirect economic impacts. The ever-growing handling of ASFV pathological material in laboratories, necessary for either diagnostic or research activities, requires particular attention to avoid accidental virus release from laboratories and its detrimental economic and environmental effects. Recently, the Commission Delegated Regulation (EU) 2020/689 of 17 December 2019 repealed the Commission Decision of 26 May 2003 reporting an ASF diagnostic manual (2003/422/EC) with the minimum and supplementary requirements for ASF laboratories. This decision generated a regulatory gap that has not been addressed yet. This paper aims to describe the Italian National Reference Laboratory (NRL) efforts to develop an effective and reliable biological containment tool for ASF laboratories and animal facilities. The tool consists of comprehensive and harmonized structural and procedural requirements for ASF laboratories and animal facilities that have been developed based on both current and repealed legislation, further entailing a risk assessment and internal audit as indispensable tools to design, adjust, and improve biological containment measures. Full article

Arboviruses are a diverse family of vector-borne pathogens that include members of the Flaviviridae, Togaviridae, Phenuviridae, Peribunyaviridae, Reoviridae, Asfarviridae, Rhabdoviridae, Orthomyxoviridae and Poxviridae families. It is thought that new world arboviruses such as yellow fever virus emerged in the 16th century due to the slave trade from Africa to America. Severe disease-causing viruses in humans include Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Crimean–Congo hemorrhagic fever virus (CCHFV), severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV). Numerous methods have been developed to detect the presence of these pathogens in clinical samples, including enzyme-linked immunosorbent assays (ELISAs), lateral flow assays (LFAs) and reverse transcriptase–polymerase chain reaction (RT-PCR). Most of these assays are performed in centralized laboratories due to the need for specialized equipment, such as PCR thermal cyclers and dedicated infrastructure. More recently, molecular methods have been developed which can be performed at a constant temperature, termed isothermal amplification, negating the need for expensive thermal cycling equipment. In most cases, isothermal amplification can now be carried out in as little as 5–20 min. These methods can potentially be used as inexpensive point of care (POC) tests and in-field deployable applications, thus decentralizing the molecular diagnosis of arboviral disease. This review focuses on the latest developments in isothermal amplification technology and detection techniques that have been applied to arboviral diagnostics and highlights future applications of these new technologies. Full article

Acanthamoeba polyphaga mimivirus, so called because of its “mimicking microbe”, was discovered in 2003 and was the founding member of the first family of giant viruses isolated from amoeba. These giant viruses, present in various environments, have opened up a previously unexplored field of virology. Since 2003, many other giant viruses have been isolated, founding new families and taxonomical groups. These include a new giant virus which was isolated in 2015, the result of the first co-culture on Vermamoeba vermiformis. This new giant virus was named “Faustovirus”. Its closest known relative at that time was African Swine Fever Virus. Pacmanvirus and Kaumoebavirus were subsequently discovered, exhibiting phylogenetic clustering with the two previous viruses and forming a new group with a putative common ancestor. In this study, we aimed to summarise the main features of the members of this group of giant viruses, including Abalone Asfarvirus, African Swine Fever Virus, Faustovirus, Pacmanvirus, and Kaumoebavirus. Full article

African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars with a mortality of up to 100%. The causative agent, African swine fever virus (ASFV), is a member of the Asfarviridae family of the nucleocytoplasmic large DNA viruses. The genome size of ASFV ranges from 170 to 194 kb, encoding more than 50 structural and 100 nonstructural proteins. ASFV virions are 260–300 nm in diameter and composed of complex multilayered structures, leading to an intricate internalization pathway to enter host cells. Currently, no commercial vaccines or antivirals are available, due to the insufficient knowledge of the viral receptor(s), the molecular events of ASFV entry into host cells, and the functions of virulence-associated genes. During the early stage of ASFV infection, the fundamental aspects of virus-host interactions, including virus internalization, intracellular transport through the endolysosomal system, and membrane fusion with endosome, are precisely regulated and orchestrated via a series of molecular events. In this review, we summarize the currently available knowledge on the pathways of ASFV entry into host cells and the functions of viral proteins involved in virus entry. Furthermore, we conclude with future perspectives and highlight areas that require further investigation. This review is expected to provide unique insights for further understanding ASFV entry and facilitate the development of vaccines and antivirals. Full article

African swine fever (ASF) is a highly infectious and fatal haemorrhagic disease of pigs that is caused by a complex DNA virus of the genus Asfivirus and Asfarviridae African suids family. The disease is among the most devastating pig diseases worldwide including Africa. Although the disease was first reported in the 19th century, it has continued to spread in Africa and other parts of the world. Globally, the rising demand for pork and concomitant increase in transboundary movements of pigs and pork products is likely to increase the risk of transmission and spread of ASF and pose a major challenge to the pig industry. Different genotypes of the ASF virus (ASFV) with varying virulence have been associated with different outbreaks in several countries in sub-Saharan Africa (SSA) and worldwide, and understanding genotype circulation will be important for ASF prevention and control strategies. ASFV genotypes unique to Africa have also been reported in SSA. This review briefly recounts the biology, genomics and genotyping of ASFV and provides an account of the different genotypes circulating in SSA. The review also highlights prevention, control and progress on vaccine development and identifies gaps in knowledge of ASFV genotype circulation in SSA that need to be addressed. Full article

African swine fever (ASF), caused by a DNA virus (ASFV) belonging to genus Asfivirus of the Asfarviridae family, is one of the most threatening diseases of suids. During last few years, it has spread among populations of wild boars and pigs in countries of Eastern and Central Europe, causing huge economical losses. While local ASF occurrence is positively correlated with wild boar density, ecology of this species (social structure, movement behavior) constrains long-range disease transmission. Thus, it has been speculated that carnivores known for high daily movement and long-range dispersal ability, such as the wolf (Canis lupus), may be indirect ASFV vectors. To test this, we analyzed 62 wolf fecal samples for the presence of ASFV DNA, collected mostly in parts of Poland declared as ASF zones. This dataset included 20 samples confirmed to contain wild boar remains, 13 of which were collected near places where GPS-collared wolves fed on dead wild boars. All analyzed fecal samples were ASFV-negative. On the other hand, eight out of nine wild boar carcasses that were fed on by telemetrically studied wolves were positive. Thus, our results suggest that when wolves consume meat of ASFV-positive wild boars, the virus does not survive the passage through intestinal tract. Additionally, wolves may limit ASFV transmission by removing infectious carrion. We speculate that in areas where telemetric studies on large carnivores are performed, data from GPS collars could be used to enhance efficiency of carcass search, which is one of the main preventive measures to constrain ASF spread. Full article

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus), dengue virus (Flavivirus), African swine fever virus (Asfarviridae) and poliovirus (Picornaviridae). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR^(−/−) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed. Full article

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Short-read sequencing (SRS) can produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for comprehensively annotating transcriptomes. Long-read sequencing (LRS) can overcome some of SRS’s limitations, but it also has drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the ASFV transcriptomic atlas. This work identified many novel transcripts and transcript isoforms and annotated the precise termini of previously described RNAs. This study identified a novel species of ASFV transcripts, the replication origin-associated RNAs. Additionally, we discovered several nested genes embedded into larger canonical genes. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism, although a large proportion of these transcripts are expressed in low abundance. A multifaceted meshwork of transcriptional overlaps was also discovered. Full article

Kaumoebavirus infects the amoeba Vermamoeba vermiformis and has recently been described as a distant relative of the African swine fever virus. To characterize the diversity and evolution of this novel viral genus, we report here on the isolation and genome sequencing of a second strain of Kaumoebavirus, namely LCC10. Detailed analysis of the sequencing data suggested that its 362-Kb genome is linear with covalently closed hairpin termini, so that DNA forms a single continuous polynucleotide chain. Comparative genomic analysis indicated that although the two sequenced Kaumoebavirus strains share extensive gene collinearity, 180 predicted genes were either gained or lost in only one genome. As already observed in another distant relative, i.e., Faustovirus, which infects the same host, the center and extremities of the Kaumoebavirus genome exhibited a higher rate of sequence divergence and the major capsid protein gene was colonized by type-I introns. A possible role of the Vermamoeba host in the genesis of these evolutionary traits is hypothesized. The Kaumoebavirus genome exhibited a significant gene strand bias over the two-third of genome length, a feature not seen in the other members of the “extended Asfarviridae” clade. We suggest that this gene strand bias was induced by a putative single origin of DNA replication located near the genome extremity that imparted a selective force favoring the genes positioned on the leading strand. Full article