Virus isolation
Out of 21 samples collected 13 representative samples from each outbreak were subjected to virus isolation using Baby Hamster Kidney (BHK-21) monolayer cell culture. From 13 samples cultured, cytopathic effect (CPE) was observed in 10 (76.9%) samples (Table 1), while virus did not grow on the rest of the three samples even when blind passaged three times. The CPE observed was fast destruction of mono-layer cells and the infected cell appeared as singly and round in shape. Additionally, the CPE was characterized by complete destruction of the cell and cell detachment which was mostly seen within 48 hrs of inoculation.
All the 21 samples were subjected to serotype differentiation using antigen detection sandwich ELISA and only serotype O was detected (Table 1), indicating that this serotype was the predominant in the study area. The same serotype was found in both Shewarobit and South Wollo areas of Amhara regional state.
Table 1: Result of FMDV serotyping by antigen detection ELISA and virus isolation by BHK-21 cell
Site of outbreak
|
Kebele
|
No. of sample inoculated
|
Type of samples inoculated
|
Virus isolated
|
Serotype detected
|
Shewarobit
|
Yelen
|
4
|
3Tissue and 1OP
|
ETH/17/18
ETH/20/18
ETH/23/18
|
O
|
Kewet
|
3
|
Tissue
|
ETH/24/18
ETH/27/17
|
O
|
South
Wollo
|
Albeko
|
2
|
Tissue
|
ETH/35/18
ETH/36/18
|
O
|
Werebabo
|
4
|
Tissue
|
ETH/37/18 ETH/39/18
ETH/41/18
|
O
|
Phylogenetic analysis
From the FMD viruses isolated, amplicons corresponding to the complete VP1 coding region were generated by reverse transcription (RT-PCR) and the sequence data were generated for 9 of the serotype O FMD viruses in WRLFMD, Pirbright, UK. The sequence data were used for comparison with other available viral sequences downloaded from NCBI, GenBank to reconstruct phylogenetic relationships of the isolates. List of FMD serotype O isolates in this study and some selected serotype O sequences of different countries downloaded from NCBI which are used for comparison are summarized in Table 2 (Additional file 1).
Assessment of phylogenetic analysis of VP1 gene of FMDV serotype O isolates (Figure 1) showed that both Shewarobit and South Wollo strains were clustered with other strains belonging to the East African topotype‑3(EA3).TheisolatesfromYelen (O/ETH/23/2018, O/ETH/17/2018, and O/ETH/20/201) and Kewet (O/ETH/24/2018 and O/ETH/27/2017) kebele of Shewarobit shared 99.5% nucleotide identity. Similarly, isolates from Albeko (O/ETH/36/2018 and O/ETH/35/2018) and Werebabo (O/ETH/37/2018 and O/ETH/39/2018) kebeles of South Wollo had only 99.4% nucleotide similarity. Furthermore, isolate from both outbreak sites (Shewarobit and South Wollo) shared more than 90% nucleotide similarity with each other. The VP1 sequences FMDV serotype O isolates in the present study were also compared with other Ethiopian and African isolates available in the GenBank database (Figure 1). Accordingly, the isolate O/ETH/17/2018 and O/ETH/24/2018 from Yelen and Kewet kebeles respectively of Shewarobit had 7.8% nucleotide divergence with Ethiopian isolates of O/ETH/3/96 and O/ETH/30/94. However, only 3.4% nucleotide diversities were recorded when compared to Sudan isolates (O/SUD/3/2008, O/SUD/5/2008 and O/SUD/4/2008 with accession numbers KR149728, GU566061 and KJ831704, respectively) which are genetically homologs on their VP1 gene nucleotide sequence and this is supported by 99% bootstrap value. The phylogenetic analysis of isolates in this study also showed that, they are differing by 15.7% with EA-1 (O/UGA/5/96, O/KEN/83/79), 17.1% with EA-2 (O/KEN/5/2002, O/UGA/3/2002), 21.5% with EA-4 (O/ETH/58/2005, O/ETH/59/2005) and 22.1% with WA (O/BKF/1/92, O/BKF/2/92) topotype of FMD serotype O virus group.
Figure 1: Minimum-Evolution tree showing the relation between the serotype O isolates from Ethiopia and reference viruses. The red rectangle ( ) indicates the new isolates obtained from the outbreak cases of Shewarobit and South Wollo. Bootstrap values<70% were not shown in the picture. EA, East Africa; WA; West Africa.
Sequence-based comparison of Serotype O isolates with the vaccine strain (O/ETH/38/2005)
The VP1 sequence of serotype O field isolates were compared with the VP1 sequence of vaccine strain currently used for vaccine production in Ethiopia to determine their genetic relationship. Out of the total 639 nucleotides compared, 107 (17%) variable sites were detected between VP1 sequence of the vaccine strain and serotype O isolate from Shewa Robit and South Wollo, while 532 (83%) were conserved over the region as shown in Figure 4 (Additional file 2). Most of these nucleotide variations (71.03%) were noticed at 3rd codon positions, while 18.69% and 10.28% nucleotide variations occurred in the 1st and 2nd codon positions, respectively. The deduced amino acid sequences were also aligned and investigated in an attempt to determine the amino acid variations. A total of 22 out of 213 (10.3%) amino acid variations were observed in different sites of the VP1 gene, while the remaining 89.7% amino acids (aa) were conserved with reference to the vaccine strain. The variations were predominantly observed in two distinct regions, comprising the G-H loop (aa positions 133–158) and the C-terminus (aa positions 194–213), reported to be the main immunogenic sites of VP1 [9,24]. The amino acid substitutions at various positions obseved in the vaccine strain and field isolates, respectively, were (Figure 2): 133 asparagine (N) → serine (S), 138 valine(V) → glutamate (E), 139 threonine(T) → alanine (A), 140 serine (S) → arginine (R), 141 valine(V) → alanine (A), 142 threonine (T) → alanine (A), 158 proline(P) → threonine (T), 197 serine (S) → alanine (A) and 198 glutamate (E) →glycine (G) amino acid. At the position 134, majority of the isolates including the vaccine strain in the present study, contained amino acid cysteine (C) except , isolates O/ETH/23/2018 and O/ETH/24/2018, which had glycine (G). However, an amino acid change was also observed in antigenic site three at position 45 [25] , wherein threonine (T) was replaced by glutamine (Q). Interestingly, the Arginine-Glycine-Aspartate (RGD) cell attachment sites within the G–H loop of the gene, at position 145-147 were conserved in all the isolates including the vaccine strain.
10 20 30 40 50
....|....|....|....|....|....|....|....|....|....|
O/ETH/38/2005 VP1/FJ798108 TTSPGESADPVTATVENYGGETQVQRRQHTDVSFILDRFVKVTPTDQINV
O/ETH/27/2017 ................D...............A...........QS.T..
O/ETH/17/2018 ................................A...........QS.T..
O/ETH/20/2018 ................................A...........QS.T..
O/ETH/23/2018 ................................A...........QS.T..
O/ETH/24/2018 ................................A...........QS.T..
O/ETH/35/2018 ................................A...........QS.T..
O/ETH/36/2018 ................................A...........QS.T..
O/ETH/37/2018 ................................A...........QS.T..
O/ETH/39/2018 ................................A...........QS.T..
60 70 80 90 100
....|....|....|....|....|....|....|....|....|....|
O/ETH/38/2005 VP1/FJ798108 LDLMQTPAHTLVGALLRAATYYFADLEVAVKHEGNLTWVPNGAPESALDN
O/ETH/27/2017 .....I.S.........S...........................T....
O/ETH/17/2018 .....I.S.........S...........................T....
O/ETH/20/2018 .....I.S.........S...........................TS...
O/ETH/23/2018 .....I.S.........S...........................T....
O/ETH/24/2018 .....I.S.........S...........................T....
O/ETH/35/2018 .....I.S.........S...........................T....
O/ETH/36/2018 .....I.S.........S...........................T....
O/ETH/37/2018 .....I.S.........S...........................T....
O/ETH/39/2018 .....I.S.........S...........................T....
110 120 130 140 150
....|....|....|....|....|....|....|....|....|....|
O/ETH/38/2005 VP1/FJ798108 TTNPTAYHKAPLTRLALPYTAPHRVLATVYNGNCKYGVTSVTNVRGDLQV
O/ETH/27/2017 ................................S....EARAA........
O/ETH/17/2018 ................................S....EARAA........
O/ETH/20/2018 ................................S....EARAA........
O/ETH/23/2018 ................................SG...EARAA........
O/ETH/24/2018 ................................SG...EARAA........
O/ETH/35/2018 ................................S....EARAA........
O/ETH/36/2018 ................................S....EARAA........
O/ETH/37/2018 ................................S....EARAA........
O/ETH/39/2018 ................................S....EARAA........
160 170 180 190 200
....|....|....|....|....|....|....|....|....|....|
O/ETH/38/2005 VP1/FJ798108 LAQKAARPLPTSFNYGAIKATQVTELLYRMKRAETYCPRPLLAIHPSEAR
O/ETH/27/2017 .....V.T......................................AG..
O/ETH/17/2018 .......T......................................AG..
O/ETH/20/2018 .......T......................................AG..
O/ETH/23/2018 .......T......................................AGT.
O/ETH/24/2018 .......T......................................AG..
O/ETH/35/2018 .......T......................................AG..
O/ETH/36/2018 .......T......................................AG..
O/ETH/37/2018 .......T......................................AG..
O/ETH/39/2018 .......T......................................AG..
210
....|....|...
O/ETH/38/2005 VP1/FJ798108 HKQKIVAPVKQLL
O/ETH/27/2017 .............
O/ETH/17/2018 .............
O/ETH/20/2018 .............
O/ETH/23/2018 .............
O/ETH/24/2018 .............
O/ETH/35/2018 .............
O/ETH/36/2018 .............
O/ETH/37/2018 .............
O/ETH/39/2018 .............
Figure 2: Amino acid sequence alignments of the VP1 gene of vaccine strain (O/ETH/38/2005) with serotype O FMD field isolates. The amino acid sequence of the vaccine strain is written on top row and the conserved amino acids are indicated by dots of different colors.