Retrospective multi-locus sequence analysis of African swine fever viruses by “PACT” confirms co-circulation of multiple outbreak strains in Uganda
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Date
Authors
Kabuuka, T.
Mulindwa, Henry
Bastos, Armanda D.S.
Van Heerden, Juanita
Heath, Livio
Fasina, Folorunso Oludayo
Journal Title
Journal ISSN
Volume Title
Publisher
MDPI
Abstract
African swine fever (ASF) is a haemorrhagic fever of swine that severely constrains pig
production, globally. In Uganda, at least 388 outbreaks of ASF were documented from 2001 to
2012. We undertook a retrospective serological and molecular survey of ASF virus (ASFV) using
banked samples collected from seven districts (Pallisa, Lira, Abim, Nebbi, Kabarole, Kibaale, and
Mukono) of Uganda. Six assays (ELISA for antibody detection, diagnostic p72 gene PCR and genomic
amplification, and sequencing of four gene regions (p72 [P], p54 [A], CVR of the 9RL-ORF [C], and TK
[T]), hereinafter referred to as P-A-C-T (PACT)) were evaluated. Antibodies to ASFV were detected
in the Abim district (6/25; 24.0%), and the remainder of the serum samples were negative (187/193;
96.9%). For the tissue samples, ASFV detection by assay was 8.47% for P, 6.78% for A, 8.47% for C,
and 16.95% for T. The diagnostic PCR (p72 gene) detected seven positive animals from four districts,
whereas the TK assay detected ten positives from all seven districts. In addition to the superior
detection capability of TK, two virus variants were discernible, whereas CVR recovered three variants,
and p72 and p54 sequencing each identified a single variant belonging to genotype IX. Our results
indicate that dependence on serology alone underestimates ASF positivity in any infected region,
that multi-locus sequence analysis provides better estimates of outbreak strain diversity, and that the
TK assay is superior to the WOAH-prescribed conventional p72 diagnostic PCR, and warrants further
investigation.
Description
This article belongs to the Special Issue titled 'African Swine Fever Virus Transmission and Control: The Role of Wild and Domestic Suids'.
SUPPORTING INFORMATION: FILE S1: Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani14010071/s1, Figures S1–S4: Figure S1: Agarose gel electrophoresis of p72-PCR products amplified with P1 and P2 OIE p72 screening primers (Lane m: 100 bp ladder; lane N: Negative control; lane P: Positive control; lanes 10, 36, 46, 48, 52, 58 and 59; Positive amplicons (n = 7)); Figure S2: Agarose gel electrophoresis of p54 [A], CVR-ORF [C], p72 [P] and Tk [T] gene cycle sequenced products using modified reaction conditions and demonstrating improved TK gene amplification (Lanes L = 100 bp ladder; N = Negative control; A1, P1, P6, T1, T2, T3, T4, T6 and T7 are positive amplicons from purified DNA products. The figure showed that agarose gel bands for positive cycle sequenced products are evident in lanes in A1, P1, P6, T1, T2, T3, T4, T6 and T7. Figure 3 shows the second PCR results in lanes A2, A6, A7, C1, C2, C5, C6, C7, P1, P2, P5, P6, P7, T2, T5, T6 and T7); Figure S3: Agarose gel electrophoresis of p54 [A], CVR-ORF [C], p72 [P] and Tk [T] gene products (Lanes L = 100 bp ladder; N = Negative control; A2, A6, A7, C1, C2, C5, C6, C7, P1, P2, P5, P6, P7, T2,T5, T6 and T7 are positive amplicons from purified DNA products); Figure S4: Agarose gel electrophoresis of TK gene-PCR products amplified with TK-1 + TK-Rev primers (Lane L = 100 bp ladder; N = Negative control; P1= Positive control; 2, 15 and 17 are new Tk gene positive amplicons).
DATA AVAILABILITY STATEMENT : All supporting data used in this research are freely available as Supplementary Materials or at the UPeTD (https://repository.up.ac.za/handle/2263/31741, accessed on 15 December 2023). All sequence data are available in the manuscript with their Accession numbers.
SUPPORTING INFORMATION: FILE S1: Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani14010071/s1, Figures S1–S4: Figure S1: Agarose gel electrophoresis of p72-PCR products amplified with P1 and P2 OIE p72 screening primers (Lane m: 100 bp ladder; lane N: Negative control; lane P: Positive control; lanes 10, 36, 46, 48, 52, 58 and 59; Positive amplicons (n = 7)); Figure S2: Agarose gel electrophoresis of p54 [A], CVR-ORF [C], p72 [P] and Tk [T] gene cycle sequenced products using modified reaction conditions and demonstrating improved TK gene amplification (Lanes L = 100 bp ladder; N = Negative control; A1, P1, P6, T1, T2, T3, T4, T6 and T7 are positive amplicons from purified DNA products. The figure showed that agarose gel bands for positive cycle sequenced products are evident in lanes in A1, P1, P6, T1, T2, T3, T4, T6 and T7. Figure 3 shows the second PCR results in lanes A2, A6, A7, C1, C2, C5, C6, C7, P1, P2, P5, P6, P7, T2, T5, T6 and T7); Figure S3: Agarose gel electrophoresis of p54 [A], CVR-ORF [C], p72 [P] and Tk [T] gene products (Lanes L = 100 bp ladder; N = Negative control; A2, A6, A7, C1, C2, C5, C6, C7, P1, P2, P5, P6, P7, T2,T5, T6 and T7 are positive amplicons from purified DNA products); Figure S4: Agarose gel electrophoresis of TK gene-PCR products amplified with TK-1 + TK-Rev primers (Lane L = 100 bp ladder; N = Negative control; P1= Positive control; 2, 15 and 17 are new Tk gene positive amplicons).
DATA AVAILABILITY STATEMENT : All supporting data used in this research are freely available as Supplementary Materials or at the UPeTD (https://repository.up.ac.za/handle/2263/31741, accessed on 15 December 2023). All sequence data are available in the manuscript with their Accession numbers.
Keywords
Conventional PCR, Diagnosis, p54 gene, p72 gene, Central variable region (CVR), 9RL ORF, TK gene, African swine fever (ASF), Polymerase chain reaction (PCR), Enzyme-linked immunosorbent assay (ELISA), SDG-03: Good health and well-being
Sustainable Development Goals
SDG-03:Good heatlh and well-being
Citation
Kabuuka, T.; Mulindwa, H.;
Bastos, A.D.S.; van Heerden, J.; Heath,
L.; Fasina, F.O. Retrospective
Multi-Locus Sequence Analysis of
African Swine Fever Viruses by
“PACT” Confirms Co-Circulation of
Multiple Outbreak Strains in Uganda.
Animals 2024, 14, 71. https://doi.org/10.3390/ani14010071.