Diversity and emergence of new variants of African swine fever virus Genotype I circulating in domestic pigs in Nigeria (2016–2018)

Abstract Background African swine fever (ASF) is the most lethal disease of pigs caused by ASF virus (ASFV) with severe economic implications and threat to the swine industry in endemic countries. Between 2016 and 2018, several ASF outbreaks were reported throughout pig producing states in Nigeria. Objectives Thereafter, this study was designed to identify the ASFV genotypes responsible for these outbreaks within the study period (2016–2018). Methods Twenty‐two ASFV‐positive samples by polymerase chain reaction were selected. The samples were collected during passive surveillance in eight states of Nigeria were characterised using 3 partial genes sequences of the virus namely, p72 capsid protein of the B646L, p54 envelope protein of E183L and the central variable region (CVR) within B602L of ASFV. Results Phylogenetic and sequences analysis based on p72 and p54 revealed ASFV genotype I as the circulating virus. Sequence analysis of the CVR of B602L revealed genetic variations with six ASFV tandem repeat sequence (TRS) variants namely, Tet‐15, Tet‐20a, Tet‐21b, Tet‐27, Tet‐31 and Tet‐34, thus increasing the overall genetic diversity of ASFV in Nigeria. Three of the TRS variants, Tet‐21b, Tet‐31 and Tet‐34, were identified for the first time in Nigeria. The new TRS variants of ASFV genotype I were identified in Enugu, Imo, Plateau and Taraba states, while co‐circulation of multiple variants of ASFV genotype I was recorded in Plateau and Benue states. Conclusions The high genetic diversity, emergence and increasing recovery of new variants of genotype I in Nigeria should be a concern given that ASFV is a relatively stable DNA virus. The epidemiological implications of these findings require further investigation.


INTRODUCTION
African swine fever (ASF) is a highly fatal haemorrhagic disease of domestic and wild pigs caused by the ASF virus (ASFV), resulting in severe morbidity and mortality (Odemuyiwa et al., 2000;Penrith & Vosloo, 2009). ASFV is a double-stranded DNA virus, and the sole member of the genus Asfivirus and family Asfarviridae (Alonso et al., 2018). ASF was first reported in over a hundred years ago in Kenya in 1921, after which the disease was reported in other parts of Africa, and the last few years the disease has spread to Europe, Asia, Oceania and Caribbean countries (Dixon et al., 2020;Montgomery, 1921;Penrith & Kivaria, 2022). The ASFV is transmitted by direct and indirect contact between pigs in the ASFV domestic cycle, soft ticks of the genus: Ornithodoros spp. in the tick transmission cycle and via warthogs in the sylvatic cycle (Costard et al., 2013;Jori et al., 2013). Genetic typing of ASFVs is achieved by characterisation of the p72 capsid protein of the B646L gene, full-length of envelope protein p54 of the E183L gene, and further differentiation of ASFV genotypes can be done using the central variable region (CVR) of the B602L gene (Bastos et al., 2003;Lubisi et al., 2005;. Phylogenic studies of B646L gene of ASFV have so far identified 24 genotypes of the virus (Blome et al., 2020). In addition, the serogrouping-identification approach is also used to distinguish ASFV strains with 8 ASFV serogroups reported based on the EP402R gene (Malogolovkin et al., 2015). Molecular characterisation of B646L, E183L and B602L of the ASFV can be used for investigating the source and extent of outbreaks and possible genetic diversity of circulating viral strains (Lubisi et al., 2005;Malogolovkin et al., 2015). ASFV is a relatively stable DNA virus with low mutation rates and coupled with lack of closely related viruses which reduces the risk of high genetic variation (Dixon et al., 2020;Gaudreault et al., 2020). However, certain regions of the virus such as the CVR are prone to mutations leading to the creation of new ASFV variants (Luka et al., 2016). These new variants might have implications for tracing and tracking the rate of ASF infections across time and space. Pig production activities are carried out in parts in 30 states of the country either as commercial/backyard intensive farms or free-roaming extensive pig production systems (Fasina et al., 2012). Due to the high demand for live pigs, mobility of these animals is unregulated usually from the north-central to the Southern coastal parts of the country where farmers and traders can obtain higher prices for their animals (Adedeji et al., 2022). Following the introduction of ASF into Nigeria in 1997, the disease is now endemic in the country with frequent reports of outbreaks in pig producing areas of the country (Odemuyiwa et al., 2000;Owolodun et al., 2010 Nigeria (Adedeji et al., 2021). Despite several studies, the epidemiology and probable drivers of the disease in Nigeria are poorly understood (Awosanya et al., 2015;Fasina et al., 2012). Between 2016 and 2018, there was an upsurge in reported cases of ASF in Nigeria affecting eight states of the country. Epidemiological investigations revealed limited understanding of how ASFV spread into and within farming communi-ties. Therefore, this study carried out the genetic characterisation of circulating ASFV to shed light on possible insights on the course and characteristics of these outbreaks.

ASF outbreak investigations and sample collection
Between 2016 and 2018, 37 ASF outbreaks were reported in domestic pigs in 8 states of Nigeria ( Figure 1). One hundred ten outbreak samples were collected consisting of whole blood (47) and tissue (63) (liver, spleen and lymph nodes) from 147 pig farms. Samples were collected from intensive/cluster pig farms, and free-roaming pigs in the affected states of the country. Although, 147 pig farms reported suspected ASF cases, samples were collected in selected farms if they were clusters of pig farms. The affected states were Abia, Benue, Enugu, Kaduna, Imo, Lagos, Plateau and Taraba states ( Figure 1, Table 1).

Laboratory analysis
Total genomic DNA was extracted from tissue and blood samples using QIAamp DNA mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. ASF diagnosis was carried out by detection of ASFV genome using conventional polymerase chain reaction (PCR) as previously described (Agüero et al., 2003).

Molecular characterisation of African swine fever virus
For ASFV genotyping and assessing the patterns of genetic variation among ASFV positive samples, three regions within the ASFV genome were amplified by PCR and sequenced. These were the C-terminal end of the B646L gene encoding the p72 protein (Bastos et al., 2003), the full length of E183L gene encoding p54 protein  and CVR within the B602L gene as previously described (Lubisi et al., 2005;Phologane et al., 2005;Nix et al., 2006) . The PCR products were purified using MinElute PCR purification kit (Qiagen) as described by the manufacturer's protocol and characterised commercially at Macrogen Inc. (Netherlands Europe) using Sanger sequencing method. The  assemblage of sequence reads was carried out using the Staden software package (http://staden.sourceforge.net/) and Bioedit (Hall, 1999) with default settings. Confirmation of sequences type was carried out using the BLAST tool, (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were constructed using MEGA X and inferred using neighbour Joining method for p72 and Minimum Evolution method for p54 (Kumar et al., 2018). ASFV genotypes were retrieved from the by the activities of butchers who also kept pigs (Table 2)

Phylogenetic analysis of B646L of ASFV
The p72 of B646L gene sequences generated from this study when compared with other sequences in the GenBank revealed a 99%-100% TA B L E 3 African swine fever viruses p72 and p54 sequences used for phylogenetic analysis

Country (Genbank Accession number) p54 References
Benin AF302816  and Ivory Coast (MG674296) using the BLAST search tool. Phylogenetic analysis of the Nigerian sequences with representatives of the 18 genotypes showed sequences generated in this study clustered with genotype 1 (Figure 2, Table 3).

Phylogenetic analysis of E183L (p54) of ASFV
The phylogenetic analysis of the p54 protein of ASFV revealed that all Nigerian sequences clustered with some sequences from West African countries belonging to genotype 1a (Figure 3). The phylogenetic tree was constructed using sequences of 16 genotypes retrieved from the Genbank (  Tet-21b recovered from pigs in Imo State which were reported to have been introduced from Cameroon but has not reported in Cameroon.

DISCUSSION
This study presents an update on molecular data of ASFV responsible for outbreaks in Nigeria between 2016 and 2018. Results of phylogeny of ASFV sequences obtained in this study identified genotype I Simi-lar previous studies in the country (Owolodun et al., 2010;Luka et al., 2016). However, sequence analysis of the hypervariable CVR encoded within B602L gene revealed six ASFV genotype I variants, three of which were new (Tet-21b, Tet-31 and Tet-34) and never reported in Nigeria. Although, Tet-31 has been recently reported in Burkina Faso (Minoungou et al., 2021). The TRS Tet-31 newly identified in Nigeria in this study was recovered in pigs reported to be imported from Cameroon; however, it is yet to be reported in that country. Earlier genetic studies had recovered 18 ASFV genotype I variants in Nigeria (Adedeji et al., 2022;Owolodun et al., 2010;Luka et al., 2016) (Table 4).
With 3 newly recovered variants (Tet-21b, Tet-31 and Tet-34) in this study, 21 ASFV genotype I TRS variants have now been recovered in Nigeria. In this study, Tet-15, Tet-27 and Tet-34 were the most widely distributed ASFV variants co-circulating in Benue and Plateau states.
(    (Brown et al., 2017;El-Hicheri 1998). ASF outbreaks frequently occur in Nigeria with severe clinical outcomes, thereby affecting pig farmers' financial income and threatening food security. In this study, ASF outbreaks were reported in eight out of 30 pig-producing states of Nigeria (Figure 1). Although the number of outbreaks may be higher but were underreported due to a lack of financial compensation to pig farmers. Rather, farmers rapidly sell-off or slaughter sick pigs leading to further spread of the disease in Nigeria (Fasina et al., 2010). Epidemiological data collected in this study showed that basic biosecurity measures such as proper quarantine before the introduction of new stock and traffic control were not observed, leading to the introduction and spread of ASF in the affected pig farms. Several risk factors have been identified as being responsible for the spread of ASFV in Nigeria, namely, poor husbandry system, live pigs trading and slaughtering of pigs on the farm and movement of ASF infected and recovered animals (Fasina et al., 2012). Other factors include external sourcing of replacement stock, presence of ASF-infected farms within the neighbourhood of other farms and exchange of feed and farm tools by farmers and their workers (Olugasa & Ijagbone, 2007;Awosanya et al., 2015). This study further confirms the importance of these risk factors in the spread and sustenance of the virus in the pig populations in Nigeria.

CONCLUSION
This study elucidates the continuous spread and emergence ASFV genotype I TRS variants in pig farming areas in Nigeria within 3 years of the study (2016)(2017)(2018). Although phylogeny revealed genotype I, however, six variants were recovered following sequence analysis of CVR of B602L gene. This study presents the first report of three of the variants that are co-circulating in four states in Nigeria. The increasing recovery of new variants of genotype I in Nigeria should be a source of serious concern, particularly for a stable DNA virus like ASFV. But the epidemiological implications of the findings are unknown and need further investigation.