Laboratory confirmation of ASF
All collected tissue samples belonging to 13 different domestic pigs from Karonga district included in the present study were positive for ASFV after conducting diagnostic PCR using ASFV-specific primers.
Phylogenetic reconstruction of ASFV targeting B646L (p72) gene, TRS and CVR
In order to classify viruses characterized in this study among the 24 ASFV p72 known genotypes, the c-terminal end of B646L (p72) gene was amplified and sequenced. All sequences obtained in this study have been deposited to the GenBank and given accession numbers (Accession numbers MN755863-MN755874). The ASFV from domestic pigs in Karonga district named MAL/19/Karonga/1-4 had 100% nucleotide identity. The BLASTn of B646L (p72) nucleotide sequences of MAL/19/Karonga/1-4 against other ASFV strains available at GenBank showed 100% nucleotide identity with ASFV strains previously described in Tanzania, Zambia, Georgia, China, Vietnam, Estonia, Moldova, Czech Republic, Belgium and Poland. After phylogenetic reconstruction using ASFV strains indicated in Table 1, the MAL/19/Karonga/1-4 clustered together with ASFV belonging to genotype II (Figure 1).
Table 1. African swine fever virus (ASFV) strains circulating in Malawi together with genotype II ASFV previously described in Tanzania, Zambia, Mozambique,Zimbabwe, Georgia and China, sharing high nucleotide identity with strains that caused outbreak in Malawi during September 2019.
Isolate
|
Host species
|
Year of isolation
|
Town/district
|
Country
|
Accession number
|
P72 genotype
|
Reference
|
MAL 2011/5
|
Domestic pig
|
2011
|
NK[1]
|
Malawi
|
KC835275
|
II
|
Unpublished
|
MAL2011 4
|
Domestic pig
|
2011
|
NK
|
Malawi
|
JX524217
|
II
|
Unpublished
|
MAL/2011/3
|
Domestic pig
|
2011
|
NK
|
Malawi
|
KC662378
|
II
|
Unpublished
|
Mal 2011/01
|
Domestic pig
|
2011
|
NK
|
Malawi
|
JX294724
|
II
|
Unpublished
|
MAL/19/Karonga_1
|
Domestic pig
|
2019
|
Karonga district
|
Malawi
|
MN755863
|
II
|
This study
|
MAL/19/Karonga_2
|
Domestic pig
|
2019
|
Karonga district
|
Malawi
|
MN755864
|
II
|
This study
|
MAL/19/Karonga_3
|
Domestic pig
|
2019
|
Karonga district
|
Malawi
|
MN755865
|
II
|
This study
|
MAL/19/Karonga_4
|
Domestic pig
|
2019
|
Karonga district
|
Malawi
|
MN755866
|
II
|
This study
|
TAN/12/Iringa
|
Domestic pig
|
2012
|
Iringa
|
Tanzania
|
KF834193
|
II
|
(39)
|
TAN/10/Kyela
|
Domestic pig
|
2010
|
Kyela
|
Tanzania
|
JX391987
|
II
|
(35)
|
ZAM/13/Mbala
|
Domestic pig
|
2013
|
Mbala
|
Zambia
|
LC174750
|
II
|
(12)
|
ZAM/2017/Mbala/1
|
Domestic pig
|
2017
|
Mbala
|
Zambia
|
LC322016
|
II
|
(36)
|
ZIM/2015/01
|
Domestic pig
|
2015
|
Mashonaland
|
Zimbabwe
|
KX090923
|
II
|
(54)
|
MOZ_5/2006
|
Soft tick
|
2006
|
Gorongosa National Park
|
Mozambique
|
KY353984
|
II
|
(51)
|
Georgia 2007/1
|
Domestic pig
|
2007
|
Caucasus Region
|
Georgia
|
NC_044959
|
II
|
(59)
|
China 2018/1
|
Domestic pig
|
2018
|
Shenbei
|
China
|
MH722357
|
II
|
(20)
|
Tengani
|
Warthog
|
1960
|
Tengani
|
Malawi
|
AF301541
|
V
|
(16)
|
MAL/2002/1
|
Domestic pig
|
2002
|
Mpemba Camp
|
Malawi
|
AY494553
|
V
|
(27)
|
Malawi/1978
|
Domestic pig
|
1978
|
NK
|
Malawi
|
AF270707
|
VIII
|
(16)
|
ZAM/2/84
|
Domestic pig
|
1984
|
NK
|
Malawi
|
AF449471
|
VIII
|
(16)
|
Dezda
|
Domestic pig
|
1986
|
Chilikum-Wera, Dedza
|
Malawi
|
AF449479
|
VIII
|
(16)
|
NDA/1/90
|
Domestic pig
|
1990
|
Nadula
|
Malawi
|
AF449473
|
VIII
|
(16)
|
BAN/911
|
Domestic pig
|
1991
|
Bangula, Lower Shire
|
Malawi
|
AY351501
|
VIII
|
(27)
|
DED/891
|
Domestic pig
|
1989
|
Dedza District
|
Malawi
|
AY351502
|
VIII
|
(27)
|
DED/911
|
Domestic pig
|
1991
|
Mtenden Campus, Dedza
|
Malawi
|
AY351503
|
VIII
|
(27)
|
DOWA
|
Domestic pig
|
1986
|
Moya, Dowa
|
Malawi
|
AY351509
|
VIII
|
(27)
|
KAC/912
|
Domestic pig
|
1991
|
Kachendere Seminary
|
Malawi
|
AY351504
|
VIII
|
(27)
|
LIL/891
|
Domestic pig
|
1989
|
Lilongwe District
|
Malawi
|
AY351505
|
VIII
|
(27)
|
LIL/901
|
Domestic pig
|
1990
|
Kafere diptank, Lilongwe
|
Malawi
|
AY351510
|
VIII
|
(27)
|
MCH/891
|
Domestic pig
|
1989
|
Kachebere Seminary
|
Malawi
|
AY351506
|
VIII
|
(27)
|
MCH/893
|
Domestic pig
|
1989
|
Lilongwe District
|
Malawi
|
AY351507
|
VIII
|
(27)
|
MCHINJI/075
|
Domestic pig
|
1987
|
Mchinji
|
Malawi
|
AY351508
|
VIII
|
(27)
|
NGE/921
|
Domestic pig
|
1992
|
Karonga District
|
Malawi
|
AY351544
|
VIII
|
(27)
|
SAL/921
|
Domestic pig
|
1992
|
Salima District
|
Malawi
|
AY351546
|
VIII
|
(27)
|
SIY91/2
|
Domestic pig
|
1991
|
Sinyala diptank, Lilongwe
|
Malawi
|
AY351566
|
VIII
|
(27)
|
THY/901
|
Domestic pig
|
1990
|
Comforzi farm, Thyolo District
|
Malawi
|
AY351545
|
VIII
|
(27)
|
Malawi Lil-20/1 (1983)
|
Tick (pig)
|
1983
|
Chalaswa
|
Malawi
|
AY261361
|
VIII
|
(30)
|
MZI/921
|
Domestic pig
|
1992
|
Euthini, Mzinda District,
|
Malawi
|
AY351543
|
XII
|
(27)
|
The analysis of the intergenic region (IGR) between I73R and I329L genes of the strains that caused ASF outbreak in Karonga district in northern Malawi in 2019 showed 99.41% nucleotide identity with ASFV genotype II strains responsible for the 2017 outbreaks in Morogoro and Pwani regions of Tanzania and 99.16% nucleotide identity with some isolates circulating in Europe and Asia, including the Georgia 2007/1 isolate. The viruses characterized in this study lacked a GAATATATAG fragment between the I173R and the I329L genes (Figure 2) and were classified as IGR I variants as previously described (20,34). In addition, a similar G to A replacement were observed in ASFV described in this study and those previously described in Tanzania (Figure 2).
The CVR sequences obtained in this study were translated into amino acids and coded to obtain corresponding signature. The CVR tetrameric repeats of ASFV that caused the outbreak in Karonga district in 2019 included CADT, NVDT, CASM, CAST and CSTS, corresponding to B, N, D and A codes, respectively. The ASFV characterized in this study showed 10 amino acid tetramers (BNDBNDBNAA) that were 100% identical to each other. A similarity search against other ASFV amino acids sequences performed by BLASTp showed 100% amino acids identity to ASFV that caused previous ASF outbreaks in Tanzania, Madagascar, Zambia, Mozambique, Mauritius, Russia and China (Table 2).
Table 2. Selected African swine fever viruses belonging to p72 genotype II with high amino acids sequences identity with viruses collected in Malawi in September 2019 at the Central variable region (CVR) of the B604L gene.
Virus name
|
Year
|
Country of origin
|
Host species
|
CVR Accession number
|
CVR signature
|
reference
|
MAL/19/Karonga_1
|
2019
|
Malawi
|
Domestic pig
|
MN755871
|
BNDBNDBNAA
|
This study
|
MAL/19/Karonga_2
|
2019
|
Malawi
|
Domestic pig
|
MN755872
|
BNDBNDBNAA
|
This study
|
MAL/19/Karonga_3
|
2019
|
Malawi
|
Domestic pig
|
MN755873
|
BNDBNDBNAA
|
This study
|
MAL/19/Karonga_4
|
2019
|
Malawi
|
Domestic pig
|
MN755874
|
BNDBNDBNAA
|
This study
|
ASFV_Tan_17_PTF2
|
2017
|
Tanzania
|
Domestic pig
|
MK276893
|
BNDBNDBNAA
|
(38)
|
ASFV_Tan_17_PTF1
|
2017
|
Tanzania
|
Domestic pig
|
MK276892
|
BNDBNDBNAA
|
(38)
|
ASFV_Tan_17_01
|
2017
|
Tanzania
|
Domestic pig
|
MK276887
|
BNDBNDBNAA
|
(38)
|
ASFV_Tan_15_4
|
2015
|
Tanzania
|
Domestic pig
|
MK276894
|
BNDBNDBNAA
|
(38)
|
ZAM/13/Mbala
|
2013
|
Zambia
|
Domestic pig
|
BAW94569
|
BNDBNDBNAA
|
(12)
|
ZAM/2017/Mbala/1
|
2017
|
Zambia
|
Domestic pig
|
LC322013
|
BNDBNDBNAA
|
(36)
|
Antani03
|
NK[1]
|
Madagascar
|
Domestic pig
|
EU649696
|
NK
|
Unpublished
|
Arm07
|
2007
|
Armenia
|
Domestic pig
|
JX857522
|
NK
|
(34)
|
MOZ_2/2006
|
2006
|
Mozambique
|
Tick
|
ATD84005
|
BNDBNDBNAA
|
(51)
|
MOZ/1/2002
|
2002
|
Mozambique
|
Domestic pig
|
QBG64414
|
NK
|
Unpublished
|
CN201801
|
2018
|
China
|
Domestic pig
|
AYD60223
|
NK
|
Unpublished
|
ASFV-wbBS01
|
2018
|
China
|
Wild boar
|
QAU54736
|
NK
|
Unpublished
|
MAD/1998
|
1998
|
Madagascar
|
Domestic pig
|
AAQ18412
|
NK
|
(16)
|
Tver0511/Torjo
|
2011
|
Russia
|
Domestic pig
|
AII03124
|
NK
|
(34)
|
Irkutsk2017
|
2017
|
Russia
|
Domestic pig
|
AUC64211
|
NK
|
Unpublished
|
MAU/1/2008
|
2008
|
Mauritius
|
Domestic pig
|
QBG64413
|
NK
|
Unpublished
|
Key: (CAST, CVST, CTST, CASI=A), (CADT, CADI, CTDT, CAGT, CVDT=B), (NVDT, NVGT, NVDI=N) and (CASM=D).