The ASFV used in this study belonged to genotype II (Fig. 1), and the nucleotide sequences encoding p72, p54 and p30 were completely similar to those of the first ASFV strain previously published in northern Vietnam (Vietnam/Hanoi/2019), which was identified as originating in China.
The inoculation of ASFV into the PAM, MARC-145, Vero, PK15, MA104 and BHK21 cell lines, as monitored through 3 passages, showed that MA104 and MARC-145 have potential for ASFV replication (Table 1). With infectious medium B, in MA104 cells, the Ct value of ASFV DNA was 23.55, 22.14 and 19.95 at passages 1, 2 and 3, respectively, while in MARC-145 cells, the Ct value of ASFV DNA was 25.51, 26.31 and 25.37 at passages 1, 2, and 3, respectively. With infectious medium A, in MA104 cells, the Ct value of ASFV DNA was 23.87, 23.06, and 19.24 at passages 1, 2 and 3, respectively; whereas in MARC-145 cells, the Ct value of ASFV DNA was 23.81, 24.71 and 22.84 at passages 1, 2, and 3, respectively. Therefore, the findings showed that infectious media A and B are more suitable and optimal than α-MEM for the growth and adaptation of ASFV in this study (Table 1).
Table 1
The susceptibility of available cell lines to ASFV in various passages (pass#)
Media/Passage
|
PAM
|
MARC-145
|
Vero
|
PK-15
|
MA104
|
BHK21
|
Infectious medium B
|
Pass#1
|
na
|
25.51
|
na
|
na
|
23.55
|
na
|
Pass#2
|
na
|
26.31
|
na
|
na
|
22.14
|
na
|
Pass#3
|
na
|
25.37
|
na
|
na
|
19.95
|
na
|
Mock
|
na
|
Neg
|
na
|
na
|
Neg
|
na
|
Infectious medium A
|
Pass#1
|
22.79
|
23.81
|
30.01
|
23.13
|
23.87
|
26.41
|
Pass#2
|
29.19
|
24.71
|
34.43
|
29.34
|
23.06
|
Neg
|
Pass#3
|
Neg
|
22.84
|
Neg
|
36.02
|
19.24
|
Neg
|
Mock
|
Neg
|
Neg
|
Neg
|
Neg
|
Neg
|
Neg
|
α-MEM
|
Pass#1
|
26.75
|
30.08
|
33.12
|
25.02
|
28.18
|
30.42
|
Pass#2
|
27.53
|
32.91
|
35.01
|
29.34
|
30.75
|
Neg
|
Pass#3
|
Neg
|
36.23
|
Neg
|
33.48
|
Neg
|
Neg
|
Mock
|
Neg
|
Neg
|
Neg
|
Neg
|
Neg
|
Neg
|
na, not available |
MA104 cells were used to compare ASFV growth and adaptation from tissue homogenate and blood sample on various infectious media (Fig. 2). At the 10th passage, the ASFV obtained from the blood sample had a significantly higher viral load than that obtained from the tissue sample (P = 0.000), exhibiting a Ct value = 20.39 ± 1.99 compared with 25.36 ± 2.11. With blood sample, ASFV grew on infectious medium B better than on infectious medium A (P = 0.006), corresponding to a Ct value = 19.58 ± 2.10 compared to 21.20 ± 1.47; ASFV originating from blood specimen continued to multiply gradually and peaked in the 15th passage with a Ct value = 14.36 ± 0.22 in infectious medium B and a Ct value = 15.42 ± 0.14 in infectious medium A. However, there was no difference (P = 0.062) in ASFV growth between infectious media A and B when cultured from tissue homogenate. The results showed that in MA104 cells, ASFV isolation from blood sample under infectious medium B condition promoted optimal growth of this virus (black line in Fig. 2; Fig. 3). The sequences of nucleotide/amino acid encoding the p72, p54 and p30 of the ASFV collected from MA104 cell supernatants through passages P0, P1, P5, P10 and P15 were 100% homologous when aligned. Moreover, antigenic regions in proteins p30 and p54 were highly conserved among the various ASFV culture passages (Table 2).
Table 2
Comparison of the p30 and p54 antigenic regions of the studied ASFVs with those of other reference strains
Virus strain
|
Genotype
|
p30
|
|
p54
|
61-DIVKSARIYAGQGYTEHQAQEEWNMILHVL-90
|
96-ESSASSENIH-105
|
Grp1 (65–75)
EDIQFINPYQD
|
Grp2a (93–107)
ATTASVGKPVTGRPA
|
Grp2b (98–113)
VGKPVTGRPATNRPAT
|
Grp3 (118–127)
TDNPVTDRLV
|
D/ASF/POT/Vietnam/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
D/ASF/POB/Vietnam/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
D/ASF/P1/Vietnam/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
D/ASF/P5/Vietnam/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
D/ASF/P10/Vietnam/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
D/ASF/P15/Vietnam/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
MK543947/Belgium/Etalle/wb/2018
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
MK628478/Lithuania/LT14/1490/2014
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
MT180393/Vietnam/NgheAn/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
MT166692/ Vietnam/Hanoi/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
MN172368/China/CAS19-01/2019
|
II
|
.. .. .. .. .. .. .. .. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
.. .. .. .. .. . .
|
.. .. .. .. .. .. .. .
|
.. .. .. .. .. .. .. . .
|
.. .. .. .. . .
|
AM712240/Portugal/OURT88/3
|
I
|
.. .. .. H. .. .. .. .. .. .. .. .. .. .. .
|
.. .. .. . S. .
|
.. .. .. .. .. . .
|
.. .. . A. .. .. .. . .
|
A.. .. .. .. .. .. .. .
|
.. .. .. .. . .
|
MH025916/Uganda/R8/2015
|
I
|
.. . RN.. .. .. .. .. .Q.. .. .. .. .. .. .
|
. .TS. TSLES
|
.. .. .. .. .. . .
|
.. .GN.. . .I. D.. .
|
.. .. I. D.. . .D. .V.
|
MNS.. .. .. I
|