Genetic characterisation of African swine fever virus from 2017 outbreaks in Zambia: Identification of p72 genotype II variants in domestic pigs

African swine fever (ASF) is a contagious haemorrhagic disease associated with causing heavy economic losses to the swine industry in many African countries. In 2017, Zambia experienced ASF outbreaks in Mbala District (Northern province) and for the first time in Isoka and Chinsali districts (Muchinga province). Meanwhile, another outbreak was observed in Chipata District (Eastern province). Genetic analysis of part of the B646L gene, E183L gene, CP204L gene and the central variable region of the B602L gene of ASF virus (ASFV) associated with the outbreaks in Mbala and Chipata districts was conducted. The results revealed that the ASFV detected in Mbala District was highly similar to that of the Georgia 2007/1 isolate across all the genome regions analysed. In contrast, while showing close relationship with the Georgia 2007/1 virus in the B646L gene, the ASFV detected in Chipata District showed remarkable genetic variation in the rest of the genes analysed. These results suggest that the Georgia 2007/1-like virus could be more diverse than what was previously thought, underscoring the need of continued surveillance and monitoring of ASFVs within the south-eastern African region to better understand their epidemiology and the relationships between outbreaks and their possible origin.


Introduction
African swine fever virus (ASFV) causes African swine fever (ASF), an acute, contagious and devastating haemorrhagic disease that affects swine. Because of its high mortality rates (up to ≈100%), serious socio-economic impact, high capacity for transboundary dissemination and lack of an effective vaccine or treatment, ASF is considered to be one of the most challenging animal diseases to contend with (Costard et al. 2009).
Historically, ASF was first described in Kenya in 1921 and is considered to be endemic in many African countries (Penrith et al. 2013). Indeed, reports of outbreaks in at least 26 African countries during 2009-2011 testify to the disease's continued eminent presence and the threat it poses to the pig industry in sub-Saharan Africa (Penrith et al. 2013). Based on sequence analysis of the variable C-terminus of the B646L gene encoding the p72 capsid protein, ASFV field strains are genetically categorised into 24 genotypes (Achenbach et al. 2016;Bastos et al. 2003;Quembo et al. 2018). Traditionally, while all the genotypes have been circulating within south-eastern Africa, only genotype I has been reported from and predominates in West Africa (Brown et al. 2017). Moreover, genotype I ASFV was introduced into Europe, South America and the Caribbean, but has only remained endemic in Sardinia (Italy) (Costard et al. 2009). However, the global ASF situation changed dramatically following the introduction of a highly virulent genotype II ASFV, which was circulating in south-eastern Africa, into Georgia in 2007 (Costard et al. 2009;Rowlands et al. 2008). The virus spread rapidly and eventually advanced into eastern Europe, a situation that has raised serious concerns over the global pig industry (Vergne et al. 2017).
In recent years, Zambia has suffered frequent sporadic ASF outbreaks in almost all its provinces, which have been associated with multiple genotypes (Simulundu et al. 2017(Simulundu et al. , 2018Thoromo et al. 2015). In 2017, ASF outbreaks occurred in Mbala (Northern province), Isoka and Chinsali (Muchinga Province) districts. Furthermore, an outbreak was also observed in May 2017 in Chipata District (Eastern province). With the aim of increasing knowledge about the epidemiology African swine fever (ASF) is a contagious haemorrhagic disease associated with causing heavy economic losses to the swine industry in many African countries. In 2017, Zambia experienced ASF outbreaks in Mbala District (Northern province) and for the first time in Isoka and Chinsali districts (Muchinga province). Meanwhile, another outbreak was observed in Chipata District (Eastern province). Genetic analysis of part of the B646L gene, E183L gene, CP204L gene and the central variable region of the B602L gene of ASF virus (ASFV) associated with the outbreaks in Mbala and Chipata districts was conducted. The results revealed that the ASFV detected in Mbala District was highly similar to that of the Georgia 2007/1 isolate across all the genome regions analysed. In contrast, while showing close relationship with the Georgia 2007/1 virus in the B646L gene, the ASFV detected in Chipata District showed remarkable genetic variation in the rest of the genes analysed. These results suggest that the Georgia 2007/1-like virus could be more diverse than what was previously thought, underscoring the need of continued surveillance and monitoring of ASFVs within the south-eastern African region to better understand their epidemiology and the relationships between outbreaks and their possible origin.
http://www.ojvr.org Open Access of ASF, findings on genetic analysis of distinct genome regions of ASFVs associated with outbreaks in Mbala and Chipata districts are reported.  Phylogenetic analysis showed that both ZAM/2017/Mbala/1 and ZAM/2017/Chipata belonged to p72 genotype II (Figure 1). In contrast, the p54 phylogeny revealed that ZAM/2017/Mbala/1 belonged to genotype IIa while ZAM/2017/Chipata/1 grouped under genotype VIIIa (Figure 2a). Thus, the p54 genetic tree showed that p72 genotype II viruses were separated into distantly related genotypes IIa, IIb (solely comprising ZAM/14/Chipata) and IIc (composed of ASFVs associated with outbreaks in Tanzania and Malawi in 2011) and the Zambian virus that fell into genotype VIIIa (Figure 2a). Consistent with the p72 and p54 evolutionary relationships, ZAM/2017/Mbala/1 was closely related to ZAM/13/Mbala and Georgia 2007/1 viruses in the p30 tree (Figure 2b). In contrast, ZAM/2017/Chipata/1 and ZAM/14/Chipata formed a distinct group that was distantly related to the Georgia 2007/1-like viruses (Figure 2b).  (Figure 2a), these findings suggest that in the south-eastern African region, these viruses could be diverse, probably because of the presence of a sylvatic cycle and long-term endemicity. The p30 phylogeny provided further evidence on the genetic diversity of p72 genotype II viruses as ZAM/2017/Chipata/1 and ZAM/14/Chipata belonged to Open Access

Conclusion
The present study investigated the molecular epidemiology of ASF outbreaks that occurred in 2017 in Northern, Muchinga and Eastern provinces of Zambia. Genetic analysis showed that the outbreaks in the Northern region were caused by genotype II ASFV, which was highly similar to the Georgia 2007/1 isolate. Although belonging to genotype II, the ASFV associated with the outbreak in Eastern province was genetically diverse, suggesting that these outbreaks were not related. Overall, this study supports the idea that genotype II ASFV circulating in south-eastern Africa is genetically diverse, probably because of the long-term endemicity in domestic pigs and a possible sylvatic origin.