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.