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

Abstract

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 isolates. 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 isolates, 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 isolates, 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 isolates in two subgroups: the Uvira subgroup (10 TRS repeats; AAAABNAABA) and another subgroup from all other isolates (8 TRS repeats; AABNAABA). The phylogenetic analysis of the EP402L gene clustered all the isolates into CD2v serogroup 7. Analyzing the intergenic region between I73R and I329L genes revealed that the isolates were identical but contained a deletion of a 33-nucleotide internal repeat sequence compared to ASFV isolate Kenya 1950. Conclusion The ASF disease in the South Kivu province of DRC in 2018–2019 was caused by ASFV genotype X and serogroup 7. This represents the first report of ASFV genotype X in DRC. CVR tetrameric repeat sequences clustered the ASFV isolates studied in two subgroups. Our finding emphasizes the need for improved coordination of the control of ASF.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus

“…However, after the reaction conditions were optimized, the two reaction systems showed the same detection sensitivity with different reaction temperatures. The detection limit for both the fluoresceinand biotin-labeled CRISPR/Cas12a reaction systems in this study was 6.8 copies per microliter, which is approach to the detection limits (sensitivities) of 6 copies per microliter as reported previously (27,32). This result suggests that the detection sensitivity of RPA-assisted RT-qPCR was higher than that of LAMP-assisted CRISPR/Cas12a.…”

“…For example, some researchers have used the CRISPR/Cas12a system combined with RPA isothermal amplification technology to detect human papillomavirus (HPV) and encephalitis B virus, which has simplified the detection process and shortened the detection time, while greatly improving detection accuracy (25,26). Several studies have reported the molecular detection techniques for ASFV, including RPA (27)(28)(29), RPA combined with RT qPCR (30), RPA combined with CRISPR/Cas (31), and LAMP combined with CRISPR/Cas12a (32). Some of them only use a single RPA or LAMP amplification technique to establish detection, while others establish joint methods with RT-qPCR or CRISPR/Cas, but these joint methods also only use fluorescence staining or colorimetric analysis without using test strip staining.…”

“…Some of them only use a single RPA or LAMP amplification technique to establish detection, while others establish joint methods with RT-qPCR or CRISPR/Cas, but these joint methods also only use fluorescence staining or colorimetric analysis without using test strip staining. The detection limit of viral DNA for these assays' ranges from as low as three point five copies per microliter (27) to as high as five hundred and eighty copies per microliter (32).…”