Complete genome sequence analysis:
Raw reads obtained from next generation sequencing of DNA extracted from enriched viral pellets were between 7,197,372 and 7,377,167 for the two samples. The size of assembled complete genome of IND/AS/SD-02/2020 and IND/AR/SD-61/2020 ASFV isolates was 190517 bp and 190572 bp, respectively. The average coverage depth of the assembled sequences of the two ASFV isolates were 801x and 978x.
The assembled sequences had GC content of around 38.4% including inverted terminal repeats (ITR) at 5’ (1547 & 1596 bp) and 3’ (1135&1144 bp)) ends. The complete genome sequences were annotated to 219 putative genes as compared to 194 genes in ASFV Georgia/2007. Global alignment of the complete genome sequences showed nucleotide identity of 99.96% among the two Indian isolates (IND/AS/SD-02/2020 and IND/AR/SD-61/2020). With other genotype-2 African swine fever viruses such as Georgia/2007 and Wuhan 2019-1, the nucleotide identity was 99.73%to 99.75% and 99.95%to99.98%, respectively.
On aligning the complete genome sequences of Indian isolates with ASFV Georgia/2007, IND/AS/SD-02/2020 had 43 single nucleotide insertions (19A, 18T, 2C, 4G), and 34 single nucleotide deletions (15A, 16T, 1C, 2G) at different genomic coordinates.IND/AR/SD-61/2020 had 33 single nucleotide insertions (13A, 14T, 3C, 3G), 26 single nucleotides deletions (11A, 14T, 1G) and one double nucleotide deletion (CC) at various positions (Fig. 2) (Supplementary table S1). Due to insertion/deletion, frame shifts were observed in the Indian viruses in the protein coding sequences of DP60R, ASFV-G_ACD 00190, MGF 110-10-L- MGF110-14L fusion, MGF 360-14L and I267L genes as compared to Georgia/2007. Both sequences of Indian ASFVs had insertion of one extra TRS (TATATAGGAA) in the intergenic region between I73R and I329L (Fig. 3), showing that Indian isolates belong to IGR II cluster of genotype II reported from China, Vietnam, Korea etc ., (Kim et al., 2019; Tran et al., 2021).
Single nucleotide variant analysis of sequences of Indian isolates with Georgia/2007 showed 70 nucleotide dissimilarity (29 in 5’ ITR, 26 in 3’ ITR, 1 to 2 in intergenic regions, 13 to 14 in genic region) (Supplementary table S2). SNP analysis among important Genotype II ASF viruses showed 19 non-synonymous SNP, 5 synonymous SNP in 14 different ORFs (Fig. 4). Synonymous SNP in 5 ORFs (MGF_110_1L, ASFVACD_320, MGF 505-4R, C717R, B263R) and non-Synonymous SNP in 14 ORFs (MGF110-1L, MGF110-4L, MGF360-10L, MGF360-11L, MGF505-4R, MGF 505-9R, K205R, C717R, B263R, O174L, NP419L, E199L, MGF 360-21R) were observed. Out of the 14 ORFs, MGF110-1L, MGF110-4L, MGF505-4R, C717R, O174L, B263R, E199L showed more than one mutation. Among 19 non-synonymous SNP, four were unique mutations present only in the Indian isolates. Two SNP in B263R and MGF505-4R genes were unique to SD-61/2020, one in MGF 360-11L gene was unique to SD-02/2020 isolate and one SNP in K205R gene was observed in both the isolates (Fig. 4).
Complete genome based phylogenetic analysis of Indian isolates with 33 additional ASFV sequences retrieved from Genbank showed their clustering under clade 2.2.2 with other p72-genotype-II ASFV reported from Georgia, Tanzania, China, Vietnam, Poland, Ukraine, East Timor lesteetc . between 2007 and 2020 (Fig. 5). Further phylogenetic analysis of only p72-genotype-II viruses showed the clustering of Indian isolates with ASFV/Wuhan/2019 in a separate clade (Fig. 6). Phylogenetic analysis of concatenated sequence of the 14 ORFs having SNP showed two distinct groups within genotype-II viruses, one with Tanzania/Rukwa/2017/1 and the other with Georgia/2007. Within the latter group, Indian isolates along with ASFV reported from other Asian countries reported during 2018-2020 formed a group distinct from Georgia/2007 (Fig. 7). Thus, our results showed the significance of the 14 ORFs in understanding the evolution of ASFV in Asian countries and their divergence from prototype Georgia/2007 ASFV. This might have implications on cross protectivity by the attenuated genotype-II vaccines, if derived from the Georgia/2007 isolate.