Resistance to African swine fever virus among African domestic pigs appears to be associated with a distinct polymorphic signature in the RelA gene and upregulation of RelA transcription.

African swine fever virus (ASFV) is a highly contagious and fatal hemorrhagic disease of domestic pigs, which poses a major threat to the swine industry worldwide. Studies have shown that indigenous African pigs tolerate ASFV infection better than European pigs. The porcine v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) encoding a p65 kD protein, a major subunit of the NF-kB transcription factor, plays important roles in controlling both innate and adaptive immunity during infection with ASFV. In the present study, RelA genes from ASFV-surviving and symptomatic pigs were sequenced and found to contain polymorphisms revealing two discrete RelA amino acid sequences. One was found in the surviving pigs, and the other in symptomatic pigs. In total, 16 nonsynonymous SNPs (nsSNPs) resulting in codon changes were identified using bioinformatics software (SIFT and Polyphen v2) and web-based tools (MutPre and PredictSNP). Seven nsSNPs (P374-S, T448-S, P462-R, V464-P, Q478-H, L495-E, and P499-Q) were predicted to alter RelA protein function and stability, while 5 of these (P374-S, T448-S, P462-R, L495-E, and Q499-P) were predicted as disease-related SNPs.Additionally, the inflammatory cytokine levels of IFN-α, IL-10, and TNF-α at both the protein and the mRNA transcript levels were measured using ELISA and Real-Time PCR, respectively. The resulting data was used in correlation analysis to assess the association between cytokine levels and the RelA gene expression. Higher levels of IFN-α and detectable levels of IL-10 protein and RelA mRNA were observed in surviving pigs compared to healthy (non-infected). A positive correlation of IFN-α cytokine levels with RelA mRNA expression was also obtained. In conclusion, 7 polymorphic events in the coding region of the RelA gene may contribute to the tolerance of ASFV in pigs.

First investigation on the presence of porcine parvovirus type 3 in domestic pig farms without reproductive failure in the Democratic Republic of Congo.

Porcine Parvovirus (PPV) is one of the major pathogens responsible for reproductive failure in sows. However, the information on its frequency in the Democratic Republic of Congo (DRC) is largely unknown. Thus, the present study was carried out to detect and genetically characterize some of known Parvovirus namely porcine parvovirus 1, 2, 3, 4, porcine bocavirus (PBoV) 1, and porcine bocavirus-like virus (PBolikeV) in 80 randomly selected archive pig farm samples during an African swine fever (ASF) survey in South Kivu, eastern DRC by polymerase chain reaction (PCR). The majority of animals analyzed (82.5%) were local breeds, and most of them (87.5%) were adults (above one year old). The majority of the animals (65%) were from the free range farms. The PCR result indicated that only PPV3 was detected in 14/80 pigs. Seven swine herds (8.7%) were co-infected with PPV3 and ASFV. Morever, a significantly high PPV3 infection rate was observed in the spleen (66.7%, P<0.0001) compared to the others type of samples. Further, the phylogenetic analysis of partial PPV3 sequences revealed one clade of PPV3 clustered with PPV3 isolates reported in a previous study in Cameroun, China, Slovakia, Germany, and China. This study is the first to report the detection of PPV in DRC. Further studies are needed to assess the levels of PPV3 viremia and the impact in co-infections with other endemic pig viruses, including ASFV.

Risk factors of African swine fever virus in suspected infected pigs in smallholder farming systems in South-Kivu province, Democratic Republic of Congo.

BACKGROUND: African swine fever (ASF) is an infectious viral disease of domestic pigs that presents as a hemorrhagic fever, and for which no effective vaccine is available. The disease has a serious negative social and economic impact on pig keepers. There is limited information on the potential risk factors responsible for the spread of ASF in South Kivu. OBJECTIVE: The aim of this study was to determine the potential risk factors associated with ASF infection in suspected ASF virus (ASFV)-infected pigs. METHODS: We sampled whole blood from 391 pigs. Additionally, 300 pig farmers were interviewed using a structured questionnaire. Viral DNA was detected by using the real-time polymerase chain reaction technique. RESULTS: The majority of pigs sampled, 78% (95% confidence interval [CI], 74.4-82.6), were of local breeds. Over half, 60.4% (95% CI, 55.5-65.2), were female, and most of them, 90.5% (95% CI, 87.6-93.4), were adult pigs (> 1 year old). Viral DNA was detected in 72 of the 391 sampled pigs, indicating an overall infection rate of 18.4% (95% CI, 14.5-22.4). Multivariable logistic regression analysis revealed several risk factors positively associated with ASFV infection: feeding with swill in pen (odds ratio [OR], 3.8; 95% CI, 2.12-6.77); mixed ages of pigs in the same pen (OR, 3.3; 95% CI, 1.99-5.57); introduction of new animals to the farm (OR, 5.4; 95% CI, 1.91-15.28). The risk factors that were negatively (protective) correlated with ASFV positivity were the presence of male animals and the use of an in-pen breeding system. CONCLUSION: Local pig farmers should be encouraged to adopt proper husbandry and feeding practices in order to increase the number of ASF-free farms.

Detection and genetic characterization of African swine fever virus (ASFV) in clinically infected pigs in two districts in South Kivu province, Democratic Republic Congo.

African swine fever (ASF) is a notifiable contagious disease caused by the African swine fever virus (ASFV), leading to a serious socio-economic impact, constraining pig industry, and affecting food security worldwide. This study aimed to detect and characterize ASFV strains from suspected infected domestic pigs in two South-Kivu province districts of the Democratic Republic of the Congo (DRC). A total of 155 pig samples were screened for viral DNA and sequencing at multiple loci. An infection rate of 5.2% (8/155) was recorded from a total of 155 blood samples with the highest ASFV infection rate of 8% for Uvira (6/75) and mostly in female pigs 5 (7.6%). Most ASF associated clinical signs were redness on the skin and snout at 49% (95% CI: 21-34), followed by the unwillingness of pigs to stand at 29 % (95%, CI: 19-35). Phylogenetic analysis of partial B646L (p72) and the full-length E183 (p54) gene sequences revealed the circulation of genotypes IX and X, which clustered with previously reported viruses in the same region, Uganda, Kenya, and Tanzania. Intragenotypic resolution of the CVR region clustered the viruses into two subgroups: the genotype X strain subgroup (10 repeats, AAAABNAABA) and the genotype IX strain subgroup (11 repeats, AAAAAAAAAAF). This finding provides additional evidence that genetically similar ASFV strains may be circulating within South Kivu province and highlights the need for improved coordination to prevent the spread of the disease in non-infected areas.

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.

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.