Wave 1 – English ‘background’ questionnaire (PAPI)

where a substitution mutation occurred and in two Ethiopian viruses used for comparison, the Nigerian viruses had an I at this position while the Ethiopia viruses had an A. At positions 145, 146 and 147 different amino acid motifs were seen, while the vvIBDVs had a TDN at these three positions, the Nigerian viruses displayed three different motifs; QED, HEG and HDG. The non-vvIBDVs had SEG, NEG, TDS, NED and TEG at these positions. L148 was conserved except in one Nigerian virus that had a P at this position. Likewise D150 was conserved except in the six Nigerian and Gx from China that had an E. A163 was also conserved except in 5 out of 6 of the Nigerian viruses and Gx that have a V. D219 was conserved in all the IBDV strains used for comparism but the Nigerian viruses had a E at this position. E242 was conserved in all the vvIBDV included for comparism while the Nigerian viruses and other non-vvIBDVs had a D at this position.

trees (NJ and UPGMA) formed their own distinct clusters. On the branch containing the vvIBDV where three Nigerian IBDV strains were found, they formed a cluster of their own within the vvIBDVs (bootstrap value 96 and 98 respectively). On the NJ and UPGMA trees, five significant genome segment B lineages were observed. Segment B sequences from IBD viruses in Nigeria were found in two of these five lineages. The first lineage with a bootstrap value of 99%

consisted of 64 out of the 67 Nigerian IBDV strains exclusively but found on the same branch with variant, vaccine and serotypes 2 IBDV strains. The second lineage with a bootstrap value of 98% and 96% respectively consists of the variant and vaccine IBDV (Del E and bursa 706). The third lineage consists of the serotype 2 IBD viruses, with the fourth lineage containing IBDV from China (Henan) and none of the Nigerian IBDVs were seen on the second, third and fourth lineage. The fifth lineage contains vvIBDV with the turkey isolate from Nigeria and within this lineage, Nigerian IBDV strains used in this study were also found here. Within the vvIBDV, two significant lineages (bootstrap value of 97%) were observed. IBDV strains from Nigeria used in this study were found exclusively clustered in one of those lineages while the vvIBDV from China, Japan and UK were in the other. All 64 Nigerian viruses that were seen here on the same branch with the variant, vaccine, serotype 2 and Chinese IBDV strains though forming a unique cluster of their own and distant from them, had a genome segment A that indicated that they could be vvIBDV. Their exclusive clustering may not be unconnected with the observations made on the triplet amino acids at positions 145-147 coupled with other substitution mutations seen on their genome segment B amino acid sequences where four different motifs of amino acids QEG, HEG, HDG and QDG was observed. The non-vvIBDVs have the motif NEG at the triplet position (145-147). All vvIBDV have TDN at amino acid 145, 146 and 147 respectively and the three Nigerian IBDV strains that clustered with the vvIBDV had these amino acids at these positions. When phylogenetic trees constructed using the neighbor joining and UPGMA methods with 1000 bootstrap replicates with the nucleotide sequences of segment B of the 67 Nigerian IBDV strains used in this study and 50 IBDV segment B sequences consisting of thirty-eight wild-type strains representing scIBDV, cvIBDV and vvIBDV, seven vaccine strains, three naturally reassorted IBDV strains from Venezuela and two serotype 2 strains from Europe submitted by Le Nouën et al, 2006 with two others; turkey IBDV isolate from Nigeria and OH from US obtained from GenBank were analyzed, two major branches were also produced. On

both trees, the first branch showed two sub branches with the Nigerian IBDV strains forming a cluster containing exclusively again of 64 out of the 67 of the IBDV strains used in this study (bootstrap value 100% for NJ and UPGMA trees respectively). The second sub branch with bootstrap values of 74% and 69% (for NJ and UPGMA trees respectively), a large cluster consisting of IBDV strains from Australia, Venezuela, China, serotype 2, variant, classic and vaccine was seen. The second major branch (bootstrap value 100% for NJ and UPGMA trees respectively) consists of vvIBDV from various countries of the world with three of the Nigerian IBDV strain used in this study. On both trees, seven IBDV genome segment B lineages were observed. On the both trees, the first lineage consists of the Nigerian IBDV strains alone with none of the other IBDV strains from other countries seen here (bootstrap value 100%). The second lineage consists of the Australian and Venezuela IBDV strains on the NJ tree but on the UPGMA tree it was Australia and China. The third lineage on the NJ tree is IBDV from China while on the UPGMA it is IBDV from Venezuela. The fourth lineage on both trees were the serotype 2 IBDVs, fifth lineage consists of the variant, classic and vaccine IBDV strains, sixth lineage consists of IBDV strains from Ivory Coast and the seventh lineage consists of all the vvIBDV from various countries including three Nigerian IBDV strains.

Fig 10: NJ of Nigerian viruses with IBDV segment B from other countries, variant, classic, vaccine and serotype 2.

Fig 11: UPGMAof Nigerian viruses with IBDV segment B from other countries, variant, classic, vaccine and serotype 2.

4.10.2 Phylogenetic analysis of segment B of six Nigerian viruses and some segment B IBDV strains from other countries in GenBank

Phylogenetic trees constructed with the segment B nucleotide sequences (nt312 – 854) of the six Nigerian viruses and nucleotide sequence of some IBDV VP1 obtained from the GenBank consisting of IBDV strains from North and South America, Europe, Asia and Africa using the neighbor joining and UPGMA methods with 1000 bootstrap replicates produced two major branches on both the NJ and UPGMA trees with sub branches. On the NJ tree (Figure 12), the first major branch with sub branch (82% bootstrap value) showed the vvIBDV forming a cluster with 100% bootstrap value and HN04 from China on the same but separate branch. On the branch with bootstrap value of 84%, IBDVs from China, Brazil, Taiwan, Zambia, classic, variant and serotype 2 clustered on this branch. The first major branch on the UPGMA tree (Figure 13) consists of sub branches with the IBDVs from USA, China, Taiwan, Brazil, Zambia, classic, variant and serotype 2 forming a cluster (80% bootstrap value), the next sub branch consists of IBDV from Tanzania (100% bootstrap value). IBDV from China and USA formed the next cluster (98% bootstrap value) followed by IBDV from China and vvIBDV with bootstrap value of 48% although the IBDV strain from China formed its own cluster. The second major branch on the NJ tree consists of IBDV from Tanzania (100% bootstrap value), IBDV from China and USA (99% bootstrap value) and the six IBDV from Nigeria formed a separate cluster (100%

bootstrap value). Also the major second branch on the UPGMA tree with 100% bootstrap value consisted exclusively of the six IBDV from Nigeria, none of the other IBDV included in this study was found on this branch.

Fig 12: NJ of the segment B nucleotide sequences of six Nigerian viruses with IBDV from some countries.

AB368958_KZC-107 AB368959_KZC-103

AB368954_KARS-53 AJ318897_UK661

AF092944_HK46 D49707_OKYM GQ449693_HuB-1 AF362770_BD 3/99

FJ695139_YS07 AY099457_T09

vvIBDV

China KC109815_HN04

AF362774_23/82 JN982247_MG8 JQ619640_T1 HQ441154_USA06CA22 AB368961_KZC-104

AF133905_VARIANT E AY459321_9109

AY134875_SH95 EF576658_IL JQ619639_STC

Serotype 2, Brazil, USA, Cihna, Variant, Classic, Taiwan, Zambia IBDV

AB368955_KDSM-02

AB368956_KMRG-00 Tanzania IBDV AY705393_Gx

JQ403647_7741 EF517529_HARBIN-1

China and USA IBDV

BAUCHI58/NG/2012 BAUCHI37/NG/2012

PLATEAU19/NG/2009 PLATEAU7/NG/2009 KADUNA61/NG/2012 PLATEAU53/NG/2010

Nigerian IBDV 100

81

38 50 100 100

70 99

73 62 78 48 37 20 36 71 100 82

50 26

84 88

83

30 26 58 39

25 22

0.01

Fig 13: UPGMA of the segment B nucleotide sequences of six Nigerian viruses with IBDV from some countries AY459321_9109

JQ619639_STC AY134875_SH95 AB368961_KZC-104 HQ441154_USA06CA22 EF576658_IL JQ619640_T1 AF133905_VARIANT E JN982247_MG8 AF362774_23/82

USA, Classic, Variant, China, Taiwan, Brazil, Zambia and serotype 2 IBDV

AB368955_KDSM-02 AB368956_KMRG-00

Tanzania IBDV

EF517529_HARBIN-1 AY705393_Gx JQ403647_7741

China and USA IBDV

China KC109815_HN04 AY099457_T09 FJ695139_YS07 AF362770_BD 3/99 GQ449693_HuB-1 AF092944_HK46 D49707_OKYM AB368954_KARS-53 AJ318897_UK661 AB368958_KZC-107 AB368959_KZC-103

vvIBDV

BAUCHI58/NG/2012 PLATEAU19/NG/2009 PLATEAU7/NG/2009 KADUNA61/NG/2012 PLATEAU53/NG/2010 BAUCHI37/NG/2012

Nigerian IBDV 100

80 73 66

35 97

100 45 48 60

33 44 38

77 38 65

83 48

100 96

100 69 80

91

48 98 23

14

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07

CHAPTER 5