Spatio-temporal clustering of African swine fever virus (Asfarviridae: Asfivirus) circulating in the Kaliningrad region based on three genome markers.

INTRODUCTION: The rapid spread of African swine fever in the Kaliningrad region makes it necessary to use the methods of molecular epidemiology to determine the dynamics and direction of ASF spread in this region of Russia. The aim of the study was to determine single nucleotide polymorphisms within molecular markers K145R, O174L and MGF 505-5R of ASFVs isolated in Kaliningrad region and to study the circulating of the pathogen in European countries by subgenotyping and spatio-temporal clustering analysis. MATERIALS AND METHODS: Blood samples from living domestic pigs and organs from dead domestic pigs and wild boars, collected in the Kaliningrad region between 2017 and 2022 were used. Virus isolation was carried out in porcine bone-marrow primary cell culture. Amplicons of genome markers were amplified by PCR with electrophoretic detection and subsequent extraction of fragments from agarose gel. Sequencing was performed using the Sanger method. RESULTS: The circulation of two genetic clusters of ASFV isolates on the territory of the Kaliningrad has been established: epidemic (K145R-III, MGF 505-5R-II, O174L-I - 94.3% of the studied isolates) and sporadic (K145R-II, MGF 505-5R-II, O174L-I - 5.7%). CONCLUSION: The broaden molecular genetic surveillance of ASFV isolates based on sequencing of genome markers is necessary in the countries of the Eurasian continent to perform a more detailed analysis of ASF spread between countries and within regions.

The evolutionary and genetic patterns of African swine fever virus.

African swine fever (ASF) is a serious animal disease, and has spread to Africa, Europe and Asia, causing massive economic losses. African swine fever virus (ASFV) is transmitted from a reservoir host (warthog) to domestic pigs via a sylvatic cycle (transmission between warthogs and soft ticks) and a domestic cycle (transmission between domestic pigs) and survives by expressing a variety of genes related to virus-host interactions. We evaluated differences in codon usage patterns among ASFV genotypes and clades and explored the common and specific evolutionary and genetic characteristics of ASFV sequences. We analysed the evolutionary relationships, nucleotide compositions, codon usage patterns, selection pressures (mutational pressure and natural selection) and viral adaptation to host codon usage based on the coding sequences (CDS) of key functional genes of ASFV. AT bias was detected in the six genes analysed, irrespective of clade. The AT bias of genes (A224L, A179L, EP153R) encoding proteins involved in interaction with host cells after infection was high; among them, the AT bias of EP153R was the greatest at 78.3%. A large number of overrepresented codons were identified in EP153R, whereas there were no overrepresented codons with a relative synonymous codon usage (RSCU) value of ≥3 in B646L. In most genes, the pattern of selection pressure was similar for each clade, but in EP153R, diverse patterns of selection pressure were captured within the same clade and genotype. As a result of evaluating host adaptation based on the codon adaptation index (CAI), for B646L, E183L, CP204L and A179L, the codon usage patterns in all sequences were more similar to tick than domestic pig or wild boar. However, EP153R showed the lowest average CAI value of 0.52 when selecting tick as a reference set. The genes analysed in this study showed different magnitudes of selection pressure at the clade and genotype levels, which is likely to be related to the function of the encoded proteins and may determine key evolutionary traits of viruses, such as the level of genetic variation and host range. The diversity of codon adaptations at the genetic level in ASFV may account for differences in translational selection in ASFV hosts and provides insight into viral host adaptation and co-evolution.

African swine fever virus DNA is present in non-biting flies collected from outbreak farms in Romania.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA. METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA). RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12. CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.

A proposed update of African swine fever virus (genotype II) subgenotyping based on the central variable region (CVR) of Russian isolates.

African swine fever virus (ASFV) isolates are grouped and tracked through analysis of their central variable region (CVR) sequences. In this study, sequences of 70 ASFV isolates collected from different regions of Russia between 2018 and 2022 were analyzed. The analysis based on the CVR sequences indicated that the isolates belonged to three distinct groups. Group 1 shared 100% sequence identity to the isolate Georgia 2007/1. Group 5 had a C > A single-nucleotide polymorphism (SNP) at position 601, while group 13 is new and unique to the Far East of Russia, with five isolates from the Amur, Khabarovsk, and Primorsky regions. These findings demonstrate a new approach to phylogenomics and cladistics of ASFV isolates within genotype II on the basis of the CVR.

Porcine Circovirus Type 3 (PCV3) in Poland: Prevalence in Wild Boar Population in Connection with African Swine Fever (ASF).

Human health is dependent on food safety and, therefore, on the health of farm animals. One of the most significant threats in regard to swine diseases is African swine fever (ASF). Infections caused by porcine circoviruses (PCVs) represent another important swine disease. Due to the ubiquitous nature of PCV2, it is not surprising that this virus has been detected in ASFV-affected pigs. However, recent data indicate that coinfection of PCV3 and ASFV also occurs. It is still unclear whether PCV infection plays a role in ASFV infection, and that subject requires further analysis. The aim of this study was to assess whether PCV3 and PCV4 are present in the wild boar population in Poland (real-time PCR). The analysis was performed on wild boar samples collected for routine ASF surveillance in Poland, between 2018 and 2021. By extension, the obtained data were compared in regard to ASFV presence in these samples, thus investigating the odds of ASFV infection on the grounds of the PCV carrier state in free-ranging Suidae in Poland. In addition, sequencing of PCV3 and phylogenetic analysis were performed, based on a full genome and a capsid gene. In the current study, we demonstrated the high prevalence of PCV3 in the wild boar population in Poland; meanwhile, PCV4 was not detected. The odds of ASFV infection on the grounds of the PCV3 carrier state in free-ranging Suidae in Poland was more than twice as high. Ten full genome sequences of PCV3 were obtained, all of them belonging to clade 3a. The similarity between them was in the range of 98.78-99.80%.

Detection of Recombinant African Swine Fever Virus Strains of p72 Genotypes I and II in Domestic Pigs, Vietnam, 2023.

African swine fever virus (ASFV) genotype II is endemic to Vietnam. We detected recombinant ASFV genotypes I and II (rASFV I/II) strains in domestic pigs from 6 northern provinces in Vietnam. The introduction of rASFV I/II strains could complicate ongoing ASFV control measures in the region.

Characterization of an African Swine Fever Virus Field Isolate from Vietnam with Deletions in the Left Variable Multigene Family Region.

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 105 TCID50/pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 103 TCID50/pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

Development of a new effective African swine fever virus vaccine candidate by deletion of the H240R and MGF505-7R genes results in protective immunity against the Eurasia strain.

IMPORTANCE: African swine fever (ASF) caused by ASF virus (ASFV) is a highly contagious and acute hemorrhagic viral disease in domestic pigs. Until now, no effective commercial vaccine and antiviral drugs are available for ASF control. Here, we generated a new live-attenuated vaccine candidate (ASFV-ΔH240R-Δ7R) by deleting H240R and MGF505-7R genes from the highly pathogenic ASFV HLJ/18 genome. Piglets immunized with ASFV-ΔH240R-Δ7R were safe without any ASF-related signs and produced specific antibodies against p30. Challenged with a virulent ASFV HLJ/18, the piglets immunized with high-dose group (105 HAD50) exhibited 100% protection without clinical symptoms, showing that low levels of virus replication with no observed pathogenicity by postmortem and histological analysis. Overall, our results provided a new strategy by designing live-attenuated vaccine candidate, resulting in protection against ASFV infection.

Performance Characteristics of Real-Time PCRs for African Swine Fever Virus Genome Detection-Comparison of Twelve Kits to an OIE-Recommended Method.

African swine fever (ASF) is a major threat to pig production, and real-time PCR (qPCR) protocols are an integral part of ASF laboratory diagnosis. With the pandemic spread of ASF, commercial kits have risen on the market. In Germany, the kits have to go through an approval process and thus, general validation can be assumed. However, they have never been compared to each other. In this study, 12 commercial PCR kits were compared to an OIE-recommended method. Samples representing different matrices, genome loads, and genotypes were included in a panel that was tested under diagnostic conditions. The comparison included user-friendliness, internal controls, and the time required. All qPCRs were able to detect ASFV genome in different matrices across all genotypes and disease courses. With one exception, there were no significant differences when comparing the overall mean. The overall specificity was 100% (95% CI 87.66-100), and the sensitivity was between 95% and 100% (95% CI 91.11-100). As can be expected, variability concerned samples with low genome load. To conclude, all tests were fit for purpose. The test system can therefore be chosen based on compatibility and prioritization of the internal control system.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus.

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION: A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Deletion of E184L, a Putative DIVA Target from the Pandemic Strain of African Swine Fever Virus, Produces a Reduction in Virulence and Protection against Virulent Challenge.

African swine fever (ASF) is currently causing a major pandemic affecting the swine industry and protein availability from Central Europe to East and South Asia. No commercial vaccines are available, making disease control dependent on the elimination of affected animals. Here, we show that the deletion of the African swine fever virus (ASFV) E184L gene from the highly virulent ASFV Georgia 2010 (ASFV-G) isolate produces a reduction in virus virulence during the infection in swine. Of domestic pigs intramuscularly inoculated with a recombinant virus lacking the E184L gene (ASFV-G-ΔE184L), 40% experienced a significantly (5 days) delayed presentation of clinical disease and, overall, had a 60% rate of survival compared to animals inoculated with the virulent parental ASFV-G. Importantly, all animals surviving ASFV-G-ΔE184L infection developed a strong antibody response and were protected when challenged with ASFV-G. As expected, a pool of sera from ASFV-G-ΔE184L-inoculated animals lacked any detectable antibody response to peptides partially representing the E184L protein, while sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognize the same set of peptides. These results support the potential use of the E184L deletion for the development of vaccines able to differentiate infected from vaccinated animals (DIVA). Therefore, it is shown here that the E184L gene is a novel ASFV determinant of virulence that can potentially be used to increase safety in preexisting vaccine candidates, as well as to provide them with DIVA capabilities. To our knowledge, E184L is the first ASFV gene product experimentally shown to be a functional DIVA antigenic marker. IMPORTANCE No commercial vaccines are available to prevent African swine fever (ASF). The ASF pandemic caused by the ASF virus Georgia 2010 (ASFV-G) strain is seriously affecting pork production in a contiguous geographical area from Central Europe to East Asia. The only effective experimental vaccines are viruses attenuated by deleting ASFV genes associated with virus virulence. Therefore, identification of such genes is of critical importance for vaccine development. Here, we report the discovery of a novel determinant of ASFV virulence, the E184L gene. Deletion of the E184L gene from the ASFV-G genome (ASFV-G-ΔE184L) produced a reduction in virus virulence, and importantly, animals surviving infection with ASFV-G-ΔE184L were protected from developing ASF after challenge with the virulent parental virus ASFV-G. Importantly, the virus protein encoded by E184L is highly immunogenic, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that unlike what is observed in animals inoculated with the vaccine candidate ASFV-G-ΔMGF, ASFV-G-ΔE184L-inoculated animals do not mount a E184L-specific antibody response, indicating the feasibility of using the E184L deletion as the antigenic marker for the development of a DIVA vaccine in ASFV.

First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia.

African swine fever virus (ASFV) is one of the pathogens of highest concern worldwide. Despite different virus lineages co-circulating in several areas, dual infections in the same animal have been rarely observed, suggesting that ASF superinfections are infrequent events. Here we present the first genome-wide detection and analysis of two intragenotype dual ASFV infections. The dual infections have been detected in a hunted wild boar and in a pig carcass, both infected by ASFV genotype I in Sardinia in 1984 and 2018, respectively. We characterize the genetic differences between the two sequences, their intra-host frequency, and their phylogenetic relationship among fully sequenced ASFV strains from Sardinia. Both dual infections involve pairs of closely related but different viruses that were circulating in Sardinia in the same period. The results imply that dual ASFV infections or similar ASFV strains are more common than expected, especially in ASF endemic areas, albeit difficult to detect.

Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection.

The Georgia-07-like genotype II African swine fever virus (ASFV) with high virulence has been prevalent in China since 2018. Here, we report that genotype I ASFVs have now also emerged in China. Two non-haemadsorbing genotype I ASFVs, HeN/ZZ-P1/21 and SD/DY-I/21, were isolated from pig farms in Henan and Shandong province, respectively. Phylogenetic analysis of the whole genome sequences suggested that both isolates share high similarity with NH/P68 and OURT88/3, two genotype I ASFVs isolated in Portugal in the last century. Animal challenge testing revealed that SD/DY-I/21 shows low virulence and efficient transmissibility in pigs, and causes mild onset of infection and chronic disease. SD/DY-I/21 was found to cause necrotic skin lesions and joint swelling. The emergence of genotype I ASFVs will present more problems and challenges for the control and prevention of African swine fever in China.

ASFV-G-∆I177L as an Effective Oral Nasal Vaccine against the Eurasia Strain of Africa Swine Fever.

The African swine fever virus (ASFV) is currently causing a pandemic affecting wild and domestic swine from Western Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in overwhelming economic losses to the swine industry. We recently developed a recombinant vaccine candidate, ASFVG-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV strain Georgia (ASFV-G). ASFV-G-ΔI177L has been proven safe and highly efficacious in challenge studies using parental ASFV-G. Here, we present data demonstrating that ASFV-G-ΔI177L can be administered by the oronasal (ON) route to achieve a similar efficacy to that of intramuscular (IM) administration. Animals receiving ON ASFV-G-ΔI177L were completely protected against virulent ASFV-G challenge. As previously described, similar results were obtained when ASFV-G-ΔI177L was given intramuscularly. Interestingly, viremias induced in animals inoculated oronasally were lower than those measured in IM-inoculated animals. ASFV-specific antibody responses, mediated by IgG1, IgG2 and IgM, do not differ in animals inoculated by the ON route from that had IM inoculations. Therefore, the ASFV-G-ΔI177L vaccine candidate can be administered oronasally, a critical attribute for potential vaccination of wild swine populations.

The first complete genome sequence of the African swine fever virus genotype X and serogroup 7 isolated in domestic pigs from the Democratic Republic of Congo.

BACKGROUND: African swine fever (ASF), a highly contagious hemorrhagic disease, affects domestic pigs in the Democratic Republic of Congo (DRC) where regular outbreaks are reported leading to high mortality rates approaching 100% in the affected regions. No study on the characteristics of the complete genome of strains responsible for ASF outbreaks in the South Kivu province of DRC is available, limited a better understanding of molecular evolution and spread of this virus within the country. The present study aimed at determining the complete genome sequence of ASFV strains genotype X involved in 2018-2019 ASF disease outbreaks in South Kivu province of DRC. MATERIALS AND METHODS: Genomic DNA of a spleen sample from an ASFV genotype X-positive domestic pig in Uvira, during the 2018-2019 outbreaks in South Kivu, was sequenced using the Illumina HiSeq X platform. Obtained trimmed reads using Geneious Prime 2020.0.4 were blasted against a pig reference genome then contigs were generated from the unmapped reads enriched in ASFV DNA using Spades implemented in Geneious 2020.0.4. The assembly of the complete genome sequence of ASFV was achieved from the longest overlapping contigs. The new genome was annotated with the genome annotation transfer utility (GATU) software and the CLC Genomics Workbench 8 software was further used to search for any ORFs that failed to be identified by GATU. Subsequent analyses of the newly determined Uvira ASFV genotype X genome were done using BLAST for databases search, CLUSTAL W for multiple sequences alignments and MEGA X for phylogeny. RESULTS: 42 Gbp paired-end reads of 150 bp long were obtained containing about 0.1% of ASFV DNA. The assembled Uvira ASFV genome, termed Uvira B53, was 180,916 bp long that could be assembled in 2 contigs. The Uvira B53genome had a GC content of 38.5%, encoded 168 open reading frames (ORFs) and had 98.8% nucleotide identity with the reference ASFV genotype X Kenya 1950. The phylogenetic relationship with selected representative genomes clustered the Uvira B53 strain together with ASFV genotype X reported to date (Kenya 1950 and Ken05/Tk1). Multiple genome sequences comparison with the two reference ASFV genotype X strains showed that 130 of the 168 ORFs were fully conserved in the Uvira B53. The other 38 ORFs were divergent mainly due to SNPs and indels (deletions and insertions). Most of 46 multigene family (MGF) genes identified were affected by various genetic variations. However, 8 MGF ORFs present in Kenya 1950 and Ken05/Tk1 were absent from the Uvira B53 genome including three members of MGF 360, four of MGF 110 and one of MGF 100 while one MGF ORF (MGF 360-1L) at the left end of the genome was truncated in Uvira B53. Moreover, ORFs DP96R and p285L were also absent in the Uvira B53 genome. In contrast, the ORF MGF 110-5L present in Uvira B53 and Ken05/Tk1 was missing in Kenya 1950. The analysis of the intergenic region between the I73R and I329L genes also revealed sequence variations between the three genotype X strains mainly characterized by a deletion of 69 bp in Uvira B53 and 36 bp in Kenya 1950, compared to Ken05/Tk1. Assessment of the CD2v (EP402R) antigen unveiled the presence of SNPs and indels particularly in the PPPKPY tandem repeat region between selected variants representing the eight serogroups reported to date. Uvira B53 had identical CD2v variable region to the Uganda (KM609361) strain, the only other ASFV serogroup 7 reported to date. CONCLUSION: We report the first complete genome sequence of an African swine fever virus (ASFV) p72 genotype X and CD2v serogroup 7, termed Uvira B53. This study provides additional insights on genetic characteristics and evolution of ASFV useful for tracing the geographical spread of ASF and essential for improved design of control and management strategies against ASF.

Laboratory diagnosis of a new outbreak of acute African swine fever in smallholder pig farms in Jos, Nigeria.

African swine fever (ASF) is a highly contagious fatal infectious disease of pigs and wild suids. The disease has a worldwide occurrence and significant impact on pig production. Two adult intensively raised large white boars from two farms in Jos with a history of sudden death were diagnosed of ASF between July and August 2019. Post-mortem examination of carcasses grossly showed splenomegaly, haemorrhagic lymphadenitis and hepatomegaly with severe congestion. The kidneys were enlarged and had generalized petechiae and blood clot in the pelvis. The heart was moderately enlarged. Microscopic examination of the spleen and lymph nodes revealed severe lymphocytic depletion, haemorrhage and severe haemosiderosis. The liver was severely congested with focal coagulative necrosis of the hepatocytes. The kidneys were severely congested and showed renal tubular necrosis with few tubular protein casts. Tissue samples were confirmed to be positive for African swine fever virus (ASFV) by polymerase chain reaction (PCR) assay, and phylogenetic analysis revealed that the isolate belonged to genotype I.

Genetic characterization of African swine fever viruses circulating in North Central region of Vietnam.

Since the first outbreak of African swine fever virus (ASFV) in China in 2018, the disease has spread to Mongolia, Vietnam, Cambodia, Korea, Laos, Myanmar, Philippines, Timor-Leste, Indonesia and Papua New Guinea. ASFV was officially reported in Vietnam on 19 February 2019. The continued spread of ASFV has occurred in the whole country within 7 months. The phylogenetic analysis showed that ASFVs isolated in the North Central region of Vietnam belong to genotype II and serotype 8. Additionally, tandem repeat sequence (TRS) studies indicated that these ASFVs are very close to ASFV strains detected in China and Belgium, 2018, and differ from ASFV isolated in Georgia in 2007.

Isolation and Genetic Characterization of African Swine Fever Virus from Domestic Pig Farms in South Korea, 2019.

On 17 September 2019, the first outbreak of African swine fever in a pig farm was confirmed in South Korea. By 9 October, 14 outbreaks of ASF in domestic pigs had been diagnosed in 4 cities/counties. We isolated viruses from all infected farms and performed genetic characterization. The phylogenetic analysis showed that all of fourteen ASFV isolates in South Korea belong to genotype II and serogroup 8. Additionally, all isolates had an intergenic region (IGR) II variant with additional tandem repeat sequences (TRSs) between the I73R and I329L genes and showed characteristics of central variable region (CVR) 1 of the B602L gene and IGR 1 of MGF 505 9R/10R genes. These are identical to the genetic characteristics of some European isolates and Chinese isolates.

Recombination shapes African swine fever virus serotype-specific locus evolution.

The recombination is one of the most frequently identified drivers of double-stranded DNA viruses evolution. However, the recombination events in African swine fever virus (ASFV) genomes have been poorly annotated. We hypothesize that the genetic determinants of ASFV variability are potential hot-spots for recombination. Here, we analyzed ASFV serotype-specific locus (C-type lectin (EP153R) and CD2v (EP402R)) in order to allocate the recombination breakpoints in these immunologically important proteins and reveal driving forces of virus evolution. The recombinations were found in both proteins, mostly among ASFV strains from East Africa, where multiple virus transmission cycles are notified. The recombination events were essentially associated with the domain organization of proteins. The phylogenetic analysis demonstrated the lack of clonal evolution for African strains which conclusively support the significance of recombinations in the serotype-specific locus. In addition, the signature of adaptive evolution of these two genes, pN/pS > 1, was demonstrated. These results have implications for the interpretation of cross-protection potential between evolutionary distant ASFV strains and strongly suggest that C-type lectin and CD2v may experience substantial selective pressure than previously thought.

First detection of African swine fever (ASF) virus genotype X and serogroup 7 in symptomatic pigs in the Democratic Republic of Congo.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe hemorrhagic viral disease of domestic pigs. The analysis of variable regions of African swine fever virus (ASFV) genome led to more genotypic and serotypic information about circulating strains. The present study aimed at investigating the genetic diversity of ASFV strains in symptomatic pigs in South Kivu province of the Democratic Republic of Congo (DRC). MATERIALS AND METHODS: Blood samples collected from 391 ASF symptomatic domestic pigs in 6 of 8 districts in South Kivu were screened for the presence of ASFV, using a VP73 gene-specific polymerase chain reaction (PCR) with the universal primer set PPA1-PPA2. To genotype the strains, we sequenced and compared the nucleotide sequences of PPA-positive samples at three loci: the C-terminus of B646L gene encoding the p72 protein, the E183L gene encoding the p54 protein, and the central hypervariable region (CVR) of the B602L gene encoding the J9L protein. In addition, to serotype and discriminate between closely related strains, the EP402L (CD2v) gene and the intergenic region between the I73R and I329L genes were analyzed. RESULTS: ASFV was confirmed in 26 of 391 pigs tested. However, only 19 and 15 PPA-positive samples, respectively, were successfully sequenced and phylogenetically analyzed for p72 (B646L) and p54 (E183L). All the ASFV studied were of genotype X. The CVR tetrameric repeat clustered the ASFV strains in two subgroups: the Uvira subgroup (10 TRS repeats, AAAABNAABA) and another subgroup from all other strains (8 TRS repeats, AABNAABA). The phylogenetic analysis of the EP402L gene clustered all the strains into CD2v serogroup 7. Analyzing the intergenic region between I73R and I329L genes revealed that the strains were identical but contained a deletion of a 33-nucleotide internal repeat sequence compared to ASFV strain Kenya 1950. CONCLUSION: ASFV genotype X and serogroup 7 was identified in the ASF disease outbreaks in South Kivu province of DRC in 2018-2019. This represents the first report of ASFV genotype X in DRC. CVR tetrameric repeat sequences clustered the ASFV strains studied in two subgroups. Our finding emphasizes the need for improved coordination of the control of ASF.