Characterization of vaccine and field IBDV strains in Ukraine for proper vaccine selection for disease prevention

A. S. Pastyria, I. G. Budzanivska, V. P. Polischuk © 2018 A. S. Pastyria et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDС 578

Virus replicates in immature IgM+ B-cells residing in the bursa of Fabricius of young chickens and causes an immunosuppressive infectious bursal disease (IBD) [2].Primary viral antigen and structural protein is VP2.VP2 gene contains special hypervariable region (VP2 HRV).Mutations in this region lead to emergence of antigenically different IBDV strains [3].The most effective way of IBD prevention is vaccination with the live attenuated vaccines.These vaccines are divided into three groups: "mild", "intermediate" and intermediate plus or "hot" according to a degree of their residual virulence [4].Mild vaccines are safe for specific pathogen free (SPF) chickens but are not very effective in the presence of high levels of maternal antibodies or against very virulent strains of IBDV.Intermediate and 'hot' vaccines are much more effective but may induce moderate to severe lesions in the bursa of Fabricius [5].Choosing the appropriate vaccine is critical for controlling IBD because of the antigenic divergence observed among the serotype 1 viruses.Point mutations and recombination events have contributed to the antigenic drift and antigenic variation among IBDV strains.In order to choose the most effective vaccine it is necessary to analyze what kind of field virus circulates in the farm.Vaccine strain should be genetically and antigenically close to the field virus [5,6].
This study was aimed at comparing VP2 HRV of different vaccine strains and field isolates detected in Ukraine to find the most antigenically close vaccine strains for prevention IBD in each particular farm.
RNA was extracted with the use of magnetic separation method following supplier`s recommendations (MagVet TM , LSI) from bursa tissues samples and vaccine samples.Reverse transcription was performed using a commercial test kit (Reverta-L, Amplisens).Obtained cDNA was used for nested PCR.PCR amplification of hypervariable region of VP2 gene was carried out with specific primers [7].The oligonucleotide primers used in this work designated Bur1F (5'-TCACCGTCC TCAGCTTAC-3' nucleotide position 587-604) and Bur1R (5'-TCAGGATTTGGGATCAGC-3' nucleotide position 1212-1229) designed to amplify the hypervariable region of VP2 gene amplicon size -643 bp.To increase specificity and sensitivity of the reaction the second set of primers Bur2F (5'-CGCTATAGCGCT TGACCCAAAAA-3', nucleotide position 651 -673) and Bur2R (5'-CTCACCCCAGCG ACCGTAACGACG-3', nucleotide position 1179-1202) designed by Kataria et al [8] were used which allows the amplification of the inner region of the first amplicon obtained after the first round of the amplification using Bur1F and Bur1R primers.The resulting product had the length of 552 bp.[The] First round of amplification was carried out for 1 cycle at 95 °C for 2 min, 36 cycles at 95°C for 30 s, 52 °C for 30 s, 72 °C for 30 s, and 1 cycle at 72 °C for 2 min.Amplicons obtained from the first reaction were diluted by 20 times and used for the second reaction.Thermal profile for the second reaction was similar except the primer annealing temperature, which was 63 °C.PCR products were visualized in 1,5 agarosegel.Amplicons were separated from reaction components using the Thermo Scientific GeneJET Gel Extraction Kit.Purified amplicons were sequenced using forward primer (Bur2F) by Institute of Molecular Biology and Genetics NAS, Ukraine (3130 Genetic Analyzer, Applied Biosystems, USA).Sequences were analyzed using Mega 6 software [9].Nucleotide alignment was performed using ClustalW instrument.Phylogenetic analysis was performed using neighbor-joining method [10].
To analyze phylogenetic relationship of vaccine and field strains we added previously described very virulent (Ukraine 1517, Ukrai- of consensus tree remained the same and contained the same 5 clusters (Fig. 2).8 very virulent field isolates clustered together with 'hot' and 8 classical virulent -with intermediate vaccine strains.There also were no field strains represented in cluster III (Fig. 2).
To determine antigenic difference among vaccine and field strains, the deduced amino acid substitutions in the variable VP2 (vVP2)  in sixteen field strains compared with reference IBDV strains: 52/70 -field strain for numbering, UK661 -reference VVIBDV, МВ -''hot'' vaccine strain, D78 -mild vaccine strain, 228E and V877 -intermediate vaccine strains.Sites with amino acids similar to strain 52/70 are marked with dots.
For the reference and numbering the strain 52/70 (GeneBank accession no.D00869) was chosen [13].The reference vvIBDV strain UK661 (GeneBank accession no.AJ878898) and 'hot' vaccine strain MB (GeneBank accession no.AY739669) were taken for comparison with Ukrainian vvIBDV isolates.The analyzed region included 142 amino acid residues, from position 206 to 347.It was found that none of the local examined isolates was of vaccine or attenuated origin due to absence of 253-His tidi ne and 284-Threonine mutations that are typically found in attenuated vaccine strains [14,15].Figure 3 demonstrated that 8 of the examined strains (Ukraine 1517, Ukraine 55, Ukraine 691_35_4, Ukraine 691_35_5, Ukrai ne 760_45_4, Ukraine 2065, Ukraine 934, Ukraine 964) showed the characteristic of vvIBDV amino acid substitutions at residues 222A, 242I (except Ukraine 760_45_4), 256I, 294I, and 299S (except Ukraine 691_35_5).8 strains had the serine-rich heptapeptide SWSASGS which was found next to the second hydrophilic region 326-332 that confirmed the highly virulence nature among the analyzed strains [14].In five strains [Ukraine 1517, Ukraine 55, Ukraine 691_35_4, Ukraine 691_35_5, Ukraine 760_45_4] D to N mutation in 212 aa position was found, which was not described before.The influence of this mutation on IBDV virulence should be analyzed in further studies.The comparison amino acid sequences of very virulent isolates and vaccine strain MB showed that in the discussed critical aa sites MB strain is similar to the analyzed field isolates.These results indicate that based on phylogenetic and the amino acid sequence analyses vaccine strains can be chosen for preven-tion IBD in farms.The most phylogenetically and antigenically close to vvIBDV isolates were MB and MB/3 strains.

Conclusion
As a result of phylogenetic analysis the vaccine strains studied in the research formed 5 genetic clusters.After addition of the field strains for analysing they clustered to the most similar vaccine strains.Deduced amino acid analysis revealed that 8 Ukrainian isolates had the amino acid sites common for very virulent strains.These strains were antigenically close to the 'hot' vaccine strains.Therefore, these data can be used for the vaccine selection for prevention IBD in each particular poultry farm.

Fig. 1 .
Fig.1.Phylogenetic tree of VP2 hypervariable region (HRV) nucleotide sequences of 11 vaccine strains com monly used in Ukraine.The Neighbor-Joining consensus tree is shown.Results of the bootstrap test (1000 replicates) are shown next to the branches[11].The bar represents 0.01 nucleotide substitutions per site.Vaccine strains grou ped phylogenetically into mild (II), intermediate (I, III, V) and "hot" (IV) strains.

Fig. 2 .
Fig. 2. Phylogenetic tree of hypervariable region (HRV) nucleotide sequen ces of 11 vaccine strains and 16 field IBDV isolates from Ukraine.The Neighbor-Joining consensus tree is shown.Re sults of the bootstrap test (1000 replicates) are shown next to the branches [11].The bar represents 0.01 nucleotide substitutions per site.IBDV strains formed 5 clus ters.Classical virulent IBDV strains are represented in clusters I, II and V. Very virulent IBDV strains are represented in cluster V. Cluster III contains only intermediate vaccine strains.