DNA Arbovirus Research Articles

Identification of a conserved G-quadruplex within the E165R of African swine fever virus (ASFV) as a potential antiviral target

African swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G-quadruplex (G4). In addition, the G4-stabilizers pyridostatin (PDS) and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand PDS substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

African swine fever virus B175L inhibits the type I interferon pathway by targeting STING and 2'3'-cGAMP.

African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines.

Identification of potential CD8+ epitopes in pp62 polyprotein of African swine fever virus using computational immunology.

The highly infectious African swine fever virus (ASFV) is currently the only known DNA arbovirus within the Asfarviridae family which primarily infects domestic pigs and wild boars. African swine fever (ASF) leads to a mortality rate of up to 100% which has caused massive socio-economic losses worldwide. Previous research indicates that ASFV's virulence can be attributed to polyprotein pp62, which plays a crucial role in viral assembly and core maturation. This particular study utilized in silico analysis to identify highly conserved cytotoxic T-cell epitopes in pp62 that can potentially serve as key components for future ASFV vaccines. To achieve this, the researchers retrieved, clustered, and aligned the peptide sequences of pp62. Subsequently, the aligned sequences were analyzed to identify epitopes that bind promiscuously to the swine major histocompatibility complex I (MHC I) alleles and exhibiting MHC IC50 values < 500 nM. Additionally, peptide sequences with positive proteasome and TAP scores were considered. Potential cross-reactivity was assessed by comparing the peptide sequences against available proteome sequences of Sus scrofa domesticus in various databases. Furthermore, molecular docking was conducted to evaluate the binding of candidate epitopes with swine leukocyte antigen-1*0401 (SLA-1*0401). The dissociation constants, binding energies, root mean square deviation, and root mean square fluctuation values for the SLA-epitope complexes were compared with a positive reference. In the course of the study, 21 highly conserved CD8+ epitopes were identified, out of which four were further assessed for their potential immunogenicity. The results demonstrated that the highly conserved CD8+ epitopes discovered in this study are promising for integration into future ASFV vaccine formulations. As preliminary data, it is anticipated that these findings will subsequently undergo in vitro and in vivo studies in the future.

Genetic Analysis of African Swine Fever Virus From the 2018 Outbreak in South-Eastern Burundi.

African swine fever (ASF) is a contagious viral disease that causes high mortality, approaching 100%, in domestic pigs and wild boars. The disease has neither a cure nor a vaccine, and it is caused by an ASF virus (ASFV), the only member of the family Asfarviridae, genus Asfivirus, and the only known DNA arbovirus. Twenty-four genotypes of ASFV have been described to date, and all of them have been described in Africa. ASF is endemic in Burundi, and several outbreaks have been reported in the country; the disease continues to economically impact on small-scale farmers. This study aimed at genetic characterization of ASFV that caused an ASF outbreak in the Rutana region, Burundi, in the year 2018. Tissue samples from domestic pigs that died as a result of a severe hemorrhagic disease were collected in order to confirm the disease using polymerase chain reaction (PCR) and to conduct partial genome sequencing. Nucleotide sequences were obtained for the B646L (p72) gene, the intergenic fragment between the I73R and I329L genes, and the central variable region (CVR) of the B602L gene. Phylogenetic analysis of the Burundian 2018 ASFV grouped the virus within B646L (p72) genotype X and clustered together with those reported during the 1984 and 1990 outbreaks in Burundi with high nucleotide identity to some ASFV strains previously reported in neighboring East African countries, indicating a regional distribution of this ASFV genotype. Analysis of the intergenic fragment between I73R and I329L genes showed that the Burundian 2018 ASFV described in this study lacked a 32–base pair (bp) fragment present in the reference genotype X strain, Kenya 1950. In addition, the strain described in this study had the signature AAABNAABA at the CVR (B602L) gene and showed 100% amino acid sequence identity to viruses responsible for recent ASF outbreaks in the region. The virus described in this study showed high genetic similarities with ASFV strains previously described in domestic pigs, wild suids, and soft ticks in East African countries, indicating a possible common wild source and continuous circulation in domestic pigs in the region.

African Swine Fever Virus: An Emerging DNA Arbovirus.

African swine fever virus (ASFV) is the sole member of the family Asfarviridae, and the only known DNA arbovirus. Since its identification in Kenya in 1921, ASFV has remained endemic in Africa, maintained in a sylvatic cycle between Ornithodoros soft ticks and warthogs (Phacochoerus africanus) which do not develop clinical disease with ASFV infection. However, ASFV causes a devastating and economically significant disease of domestic (Sus scrofa domesticus) and feral (Sus scrofa ferus) swine. There is no ASFV vaccine available, and current control measures consist of strict animal quarantine and culling procedures. The virus is highly stable and easily spreads by infected swine, contaminated pork products and fomites, or via transmission by the Ornithodoros vector. Competent Ornithodoros argasid soft tick vectors are known to exist not only in Africa, but also in parts of Europe and the Americas. Once ASFV is established in the argasid soft tick vector, eradication can be difficult due to the long lifespan of Ornithodoros ticks and their proclivity to inhabit the burrows of warthogs or pens and shelters of domestic pigs. Establishment of endemic ASFV infections in wild boar populations further complicates the control of ASF. Between the late 1950s and early 1980s, ASFV emerged in Europe, Russia and South America, but was mostly eradicated by the mid-1990s. In 2007, a highly virulent genotype II ASFV strain emerged in the Caucasus region and subsequently spread into the Russian Federation and Europe, where it has continued to circulate and spread. Most recently, ASFV emerged in China and has now spread to several neighboring countries in Southeast Asia. The high morbidity and mortality associated with ASFV, the lack of an efficacious vaccine, and the complex makeup of the ASFV virion and genome as well as its lifecycle, make this pathogen a serious threat to the global swine industry and national economies. Topics covered by this review include factors important for ASFV infection, replication, maintenance, and transmission, with attention to the role of the argasid tick vector and the sylvatic transmission cycle, current and future control strategies for ASF, and knowledge gaps regarding the virus itself, its vector and host species.

Host genome depletion to determine the evolution, genetic diversity and transmission patterns of full genome sequences of African swine fever genotype IX from Uganda

African swine fever (ASF) is a highly contagious viral haemorrhagic disease of domestic pigs, wild boar and feral swine. It is caused by the only DNA arbovirus, African swine fever virus (ASFV), which is the only member of the family Asfarviridae and is transmitted by the soft ticks in the genus Ornithodoros. ASF results in mortalities of up to 100% and exhibits different epidemiological patterns in Africa, Europe and Asia. This ASFV has recently spread to other countries in Europe and is also now present in Asia where it has spread like wildfire, resulting into high economic losses. The lack of a treatment or vaccine against this disease makes its control and prevention extremely difficult. This is partly due to the large size of the ASFV genome (170-190 kbp) and the many genes (187–190) that it encodes. There are currently 24 genotypes, with genotype IX predominating in Uganda and Kenya. Understanding the genetic characteristics of ASFV have focused mainly on partial fragments of selected genes, thus missing the opportunity to investigate the evolution, genetic diversity and patterns of transmission at the whole genomic level. Using the host-genome depletion approach we show an increase in the amount of ASFV DNA recovered directly from clinical material and generate genotype IX complete genomes on an NGS platform. We further show the inter-epidemic relationships of ASFV outbreaks in Uganda. The data contribute towards understanding the patterns and dynamics of transmission and maintenance of ASFV in a country in which ASF is endemic.

Investigating the impact of environmental factors on the African Swine Fever epidemic in Belgium

Investigating the impact of environmental factors on the African Swine Fever epidemic in Belgium

Comprehensive Phylogenetic Reconstructions of African Swine Fever Virus: Proposal for a New Classification and Molecular Dating of the Virus

African swine fever (ASF) is a highly lethal disease of domestic pigs caused by the only known DNA arbovirus. It was first described in Kenya in 1921 and since then many isolates have been collected worldwide. However, although several phylogenetic studies have been carried out to understand the relationships between the isolates, no molecular dating analyses have been achieved so far. In this paper, comprehensive phylogenetic reconstructions were made using newly generated, publicly available sequences of hundreds of ASFV isolates from the past 70 years. Analyses focused on B646L, CP204L, and E183L genes from 356, 251, and 123 isolates, respectively. Phylogenetic analyses were achieved using maximum likelihood and Bayesian coalescence methods. A new lineage-based nomenclature is proposed to designate 35 different clusters. In addition, dating of ASFV origin was carried out from the molecular data sets. To avoid bias, diversity due to positive selection or recombination events was neutralized. The molecular clock analyses revealed that ASFV strains currently circulating have evolved over 300 years, with a time to the most recent common ancestor (TMRCA) in the early 18th century.

Control of African swine fever virus replication by small interfering RNA targeting the A151R and VP72 genes

African swine fever virus (ASFV) is the unique member of the Asfarviridae family and Asfivirus genus. It is an enveloped double-stranded DNA arbovirus that replicates in the cell cytoplasm, similar to poxviruses. There is no vaccine and no treatment available to control this virus. We describe the use of small interfering RNA (siRNA) targeting the A151R and VP72 (B646L) genes to control the ASFV replication in vitro. Results suggest that siRNA targeting the A151R and VP72 genes can reduce both the virus replication and its levels of messenger RNA transcripts. The reduction was up to 4 log(10) copies on the virus titre and up to 3 log(10) copies on virus RNA transcripts levels. The combination of multiple siRNA did not improve the antiviral effect significantly, compared with use of individual siRNAs. The function of the A151R gene product in the virus replication cycle is yet unclear, but is essential. We also demonstrate that it is possible to inhibit, using small interfering RNA, a virus that replicates exclusively in the cell cytoplasm in specific viral factories.

African swine fever : transboundary diseases

African swine fever (ASF) is a devastating haemorrhagic fever of pigs that causes up to 100 % mortality, for which there is no vaccine. It is caused by a unique DNA virus that is maintained in an ancient cycle between warthogs and argasid ticks, making it the only known DNA arbovirus. ASF has a high potential for transboundary spread, and has twice been transported from Africa to other continents - Europe and subsequently the Caribbean and Brazil (1957, 1959) and the Caucasus (2007). It is also a devastating constraint for pig production in Africa. Research at Onderstepoort Veterinary Institute has made and is making important contributions to knowledge of this disease, focusing on the cycle in warthogs and tampans and transmission from that cycle to domestic pigs, resistance to its effects in domestic pigs, and the molecular genetic characterisation and epidemiology of the virus.

Polymerase Chain Reaction (PCR) Detection Of The Genome Of African Swine Fever Virus (ASFV) From Natural Infection In A Nigerian Baby Warthog (&lt;i&gt;Phacochoereus Aethiopicus&lt;/i&gt;)

The National Veterinary Research Institute, Vom procured baby warthog (Phacochoreus aethiopicus) caught by hunters in the forest of Adamawa state of Nigeria to be screened for the presence of genome to African swine fever virus (ASFV). The Spleen, lymph nodes and liver tissues obtained from the warthog were accessed for the presence of African swine fever virus genomic DNA by the polymerase chain reaction (PCR). DNA extracted from these tissues were amplified for a region of ASFV DNA genome by the PCR using specific primers of 20bp (base pair) from a highly conserved region of the ASFV DNA genome. The amplified genome was visualized following gel electrophoresis of PCR products using 1.5% agarose gel in 1x tris-acetate buffer (TAE buffer). A single, discrete and specific band of expected size (278bp) when measured against 200bp (base pair) DNA molecular marker (Roche) was observed from all the three tissues tested. The detection of these bands confirms virus presence. This finding therefore, provides current evidence of the presence of ASFV in Nigerian warthogs. African swine fever virus (ASFV), the causative agent of African swine fever (ASF) is a unique, complex, icosahedral DNA arbovirus belonging to the genus asfivirus in the family asfaviridae (Dixon et al., 2000). The virus multiplies in the cytoplasm of the infected cells. In nature, maintenance and transmission of this virus involves the cycling of virus between argasid soft ticks (Ornithodoros moubata complex) and wild pig population (warthogs and bush pigs) in sub-Saharan Africa. This association is unlikely ever to be eliminated, hence making it potentially very difficult to eradicate the disease in Africa. And it appears that this continent will be the main reservoir of ASFV for the foreseeable future (Majiyagbe et al., 2004). The ASFV genome comprised of a linear double stranded DNA molecule which are covalently closed at both ends by a 37 nucleotide-long hair pin loop composed almost entirely of partially paired adenine (A) and thymine (T) residues (Dixon et al., 2000). Depending upon the virus isolate, the size of the DNA molecule oscillates between 170 and 190-kilo base pair (Kbp) (Murphy et al., 1995). ASF, a highly significant disease of domestic swine occurs in several disease forms ranging from per acute to sub-clinical or inapparent forms (Mebus and Dardiri, 1980 and Mebus, 1988). The disease entered Nigeria around 1997 through the southwestern region of the country most probably through cross border contacts with infected pigs/or pig products smuggled through the Nigerian border from neighboring Benin Republic who had earlier reported the outbreak of the disease (Anon, 1998a). Within a short period the disease became widely distributed through pig producing areas of Nigeria (Majiyagbe, 1999 and Luther, 2001) and has been causing a lot of devastation to the national pig herd. The present situations of ASF in Nigeria revealed that virtually all the pig producing centres are affected. Of the 36 states of the Federation, the disease has been confirmed through laboratory test in 18, covering the south-west, south-east and central states of Nigeria (Majiyagbe et al., 2004). Fast and accurate detection of ASF outbreaks is required to limit the spread of the disease because the disease resembles several other haemorrhagic diseases of pigs both clinically and pathologically. The application by PCR for the detection of regions of ASFV genomic DNA presents a sensitive and specific method of identifying the virus. The technique consists essentially of the enzymatic synthesis of millions of copies of a target DNA sequence (Saiki et al., 1985). Using a thermostable DNA polymerase (Saiki et al., 1988), and a succession of cycles that includes denaturation of the template DNA, hybridization of specific DNA primers to the template and extension of the primers, it becomes possible to enzymatically generate several copies of the target region. Using this highly sensitive technique, it became possible to detect the presence of ASFV genomic DNA in a Nigerian warthog. This communication herein reported is the first documented report of the detection of ASFV from a Nigerian warthog reported hitherto only in eastern and southern-African countries (Scott, 1965 and Plowright et al., 1969). Keywords: PCR, ASF, ASFV, Oligonucleotide primers, WarthogNigerian Veterinary Journal Vol. 28 (2) 2007 pp. 63-67

Pathogenesis of African swine fever virus inOrnithodorosticks

African swine fever virus (ASFV) is the only known DNA arbovirus and the sole member of the family Asfarviridae. It causes a lethal, hemorrhagic disease in domestic pigs. ASFV is enzootic in sub-Saharan Africa and is maintained in a sylvatic cycle by infecting both wild members of the Suidae (e.g. warthogs) and the argasid tick Ornithodoros porcinus porcinus. The pathogenesis of ASFV in O. porcinus porcinus ticks is characterized by a low infectious dose, lifelong infection, efficient transmission to both pigs and ticks, and low mortality until after the first oviposition. ASFV pathogenesis in warthogs is characterized by an inapparent infection with transient, low viremic titers. Thus O. porcinus porcinus ticks probably constitute the most important natural vector of ASFV, although both the mammalian and tick hosts are probably required for the maintenance of ASFV in the sylvatic cycle. The mechanism of ASFV transmission from the sylvatic cycle to domestic pigs is probably through infected ticks feeding on pigs. In addition to O. porcinus porcinus, a number of North American, Central American and Caribbean species of Ornithodoros have been shown to be potential vectors of ASFV.