3.4. Comparison of ORFs including LSD_019 and LSD_144
As compared to LSDV-NI2490, 6 single nucleotide polymorphisms (SNPs), 5 small indels (2 single nt deletions, 1 single nt insertion, and 1 double nt deletion) were observed in LSDV-WB/IND/19, whereas as compared to LSDV-KSGO 240, 6 single nucleotide polymorphisms (SNPs), 6 small indels (3 single nt deletions, 1 single nt insertion and 1 double nt deletion) were observed (Table 2). Out of 156 ORFs, LSDV-WB/IND/19 showed 100% amino acid identity in 148 ORFs with Kenyan LSDV strains except for eight ORFs viz. LSD_019 (all Kenyan strains), LSD_049 (LSDV-KSGO 240, LSDV-KSGO 240/cattle), LSD_089 (LSDV-KSGO 240/cattle), LSD_094 (LSDV-NI2490, LSDV-KSGO 240, LSDV-Kenya/58), LSD_096 (LSDV-NI2490, LSDV-Kenya/58), LSD_134 (LSDV-KSGO 240), LSD_140 (all Kenyan strains) and LSD_144 (all Kenyan strains). Out of eight ORFs, five ORFs viz. LSD_049 (Q393P), LSD_089 (I24V), LSD_094 (P85H, H536N, I537N), LSD_096 (R23E), LSD_140 (N203K) showed 99.4%-99.9% aa identities owing to non-synonymous changes (Table 2). LSD_134 is 100% identical except LSDV-KSGO 240 in which it has a truncated version. LSD_019 and LSD_144 showed 51% aa identity to that of Kenyan LSDV strains due to truncation of both kelch-like proteins in LSDV-WB/IND/19 (Fig. 2, 3).
12 bp nt insertion present in GPCR gene (LSD_011) of LSDV vaccine strains, recombinant LSDV strains as well as Kenyan strains was also observed in LSDV-WB/IND/19. LSD_126 having 27 bp nt insertion in wild-type LSDV strains including Kenyan strains was also present in LSDV-WB/IND/19 whereas deletion is present in vaccine and recombinant LSDV strains (Erster et al., 2017). Similar findings were observed in the GPCR and LSD_126 genes of LSDV strains from Bangladesh (Badhy et al., 2021). LSDV-KSGO 240 genome isolated from the Kenyavac vaccine vial (JOVAC) shared 99.9% homology with LSDV-NI 2490 strain differing by single aa substitution in LSD_ 049 and single nt deletion causing frameshifting and further truncation of LSD_134 gene (Vandenbussche et al., 2016). LSDV-KSGO 240 isolated from cattle has 99.99% identity to the parent KSGO 240 strain isolated from the parent vaccine vial with seven variants out of which 3 genes viz. LSD_026, LSD_089, and LSD_094 are affected (Bamouh et al., 2021). Also, LSDV-KSGO 240 isolated from cattle demonstrated intact LSD_134 protein. LSDV-WB/IND/19 also has intact LSD_134 protein similar to LSDV-KSGO 240 from cattle, LSDV-NI2490, and LSDV-Kenya/58. Genomes of three vaccine strains isolated from Lumpyvax, Herbivac LS, and LSDV-OBP share 99.9% homology (Mathijs et al., 2016b). LSD_134 is also disrupted in LSDV-LW1959 and OBP vaccines by a+1 frameshifting whereas Herbivac LS vaccine shows restoration/merging of LSD_134a and LSD_134b into single LSD_134 (Mathijs et al., 2016b). Also, Herbivac LS 008 vaccine batch showed disruption of 134 into 134a, 134b along with AT deletion in LSD_131 (Douglas et al., 2019). Among SPPV strains, frameshifting has been demonstrated in LSD_ 134 in case of SPPV-SA and SPPV-AG vaccine strains, whereas GTPV-G20‐LKV vaccine strain also reveals a + 1 frameshift leading to disruption of LSD_134 (Biswas et al., 2020). LSD_134 belongs to the poxviral B22R family of proteins. Monkeypox B22R homolog MPXV197 is associated with inhibition of T‐cell responses and its deletion leads to attenuation (Alzhanova et al., 2014).
LSD_019 and LSD_144 encode kelch-like proteins with BTB (broad-complex, tramtrack and bric a brac)/POZ domain, BTB/Kelch-associated (BACK) domain, and kelch-type beta-propeller with C‐terminal kelch repeats. Kenyan LSDV strains as well as vaccine-associated LSDV strains from South Africa have a single LSD_019 protein (569 aa), whereas some LSDV vaccine strains (LW1959, Herbivac, OBP, and Cro2016) show inactivation of LSD_019 into 019a (440 aa) and 019b (150 aa) due to +1 frameshift mutations (Kara et al., 2003; Mathijs et al., 2016b) (Fig. 2). GTPV G20-LKV vaccine strain has also demonstrated similar findings (Biswas et al., 2020). Some LSDV wild-type strains encode other mutant forms of LSD_019 viz. 019a (290 aa) and 019b (270 aa). The amino acid pattern at different positions is the same in Kenyan LSDV strains, vaccine-associated LSDV strains, wild-type LSDV strains as well as in LSDV-WB/IND/19. LSDV vaccine strains show amino acid changes at these positions (Fig. 2). However, unlike Kenyan and vaccine-associated LSDV strains, LSDV-WB/IND/19 strain possesses a pattern of LSD_019 similar to wild-type strains viz. 019a (290 aa) and 019b (269 aa) based on the C-terminal part of LSD_019b except for the deletion of lysine (K) at position 229. SPPV_019 gene is required for virulence after intranasal and intradermal inoculation (Balinsky et al., 2007).
The amino acid pattern at different positions is the same in Kenyan LSDV strains, wild-type LSDV strains, and recombinant LSDV strains, as well as in LSDV-WB/IND/19. LSDV vaccine and vaccine-associated strains show amino acid changes at these positions (Fig. 3). Kenyan LSDV strains have a single LSD_144 protein with 547 aa length, whereas wild-type, as well as recombinant LSDV strains, also possess a single LSD_144 protein with 550 aa length due to additional 3 amino acids (VKT) at extreme C-terminus of the protein. All of the related LSDV vaccines (LW1959, Herbivac, OBP, and Cro2016), as well as vaccine-associated LSDV strains, encode LSD_144a and LSD_144b proteins of 269/270 aa and 281 aa, respectively. LSDV vaccine strains have a deletion of phenylalanine (F) at position 252 as compared to vaccine-associated LSDV strains. LSDV-WB/IND/19 encodes two ORFs 144a and 144b of 270 aa and 281 aa, respectively. LSD_144a and LSD_144b proteins resemble that of Kenyan LSDV strains based on amino acid patterns, however, the C-terminal part of LSD_144a resembles that of vaccine-associated LSDV strains due to premature truncation. LSDV/Evros/GR/15, the first LSDV reported in Europe, showed 99.8% homology with LSDV field isolate NI-Warmbaths-LW with frameshift in LSD_144. (Agianniotaki et al., 2017). LSDV-Russia/Saratov/17 showed 8 aa changes in LSD_144 leading to the merging of LSD_144a and LSD_144b to single LSD_144 like wild-type LSDVs (Sprygin et al., 2018). GTPV Gorgon vaccine isolate has a 1.6 kbp deletion spanning the 3′ end of LSD_144 and LSD_145 and appears to inactivate both genes. China/GD01/2020 shows LSD_019 gene identical to Neethling vaccine LW 1959 and LSD_144 gene identical to Neethling 2490 strain. The unique variants in LSD_019 and LSD_144 have been confirmed by Sanger sequencing of the same sample as well as from original scab material to rule out nucleotide change arising due to passaging of virus for five passages. Interestingly, the same variants in both the genes were also present in another field sample, LSDV-UK/IND/21 in our study as well as other isolates from India, LSDV/Cattle/India/2019/Ranchi/P10, P30, and P50 (Acc. No. OK422492, OK422493, OK422494) (Fig. 4, 5). These findings suggest that LSDV strains circulating in India from 2019-2021 carry the same variants.