Restriction analysis and differentiation of Ukrainian strains of infectious bursal disease virus

© 2017 A. 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


Introduction
Since the first outbreak of infectious bursal disease (IBD) it became one of the main problems in poultry industry.The infectious bursal disease virus (IBDV) has been discovered in the area of Gumboro, Delaware, USA.Since 1962 the classical strains of the virus have been prevalent in North and South America.In 1986 in central Europe new virus strains appeared [1].They induced 70 % mortality of affected birds.During next 10 years such very virulent strains (vvIBDV) have been spread in Europe, Asia, Africa and South America.In ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2017. Vol. 33.N 1. P 58-63 doi: http://dx.doi.org/10.7124/bc.000945

Viruses and Cell
Ukraine, the emergence of vvIBDV was registered in 2000 [2,3,4].Molecular mechanisms of emergence of new IBDV strains include mutations in hypervariable region of VP2 gene.The virus replicates in immature B-lymphocytes that develop in bursa of Fabricius, which leads to immunosuppression of infected animals [5,6,7].
The infectious bursal disease occurs primarily in poultry-producing areas thus leads to significant economic losses.IBDV is highly variable, that's why new virus strains with different level of virulence appear.Therefore, to control IBDV many different types of vaccines have been developed.They include live attenuated, inactivated, immune complex, vectored, DNA vaccine and ex.[1,8].Live vaccines provide the best level of defense against IBD, so they are used in most of poultry farms in Ukraine [4,8].About 15 different live attenuated vaccines have been registered in Ukraine.They can be classified as mild, intermediate and intermediate-plus vaccines based on the level of attenuation and residual virulence for SPF chickens [8,9].The intermediate-plus vaccines are regularly applied to protect chickens against vvIBDV challenges, while mild and intermediate vaccines are used for protection against classical strains of the virus.The choice of vaccine depends on type of virus that circulates in particular farm.Therefore, characterization of field isolates is required for proper vaccination.
Several approaches have been developed to the classify IBDV strains.Most of them are based on the nucleotide sequence analyses of VP2 gene, which encodes the main virus capsid protein [5,9,10].The most efficient methods for diagnostics, molecular characterization and differentiation of IBDV field isolates include RT-PCR and restriction fragment length polymorphism (RFLP), nucleotide sequence analysis, and quantitative real time RT-PCR (qRT-PCR) [4,5,10].
The aim of the study was to characterize restriction profile of IBDV vaccine strains and field isolates, detected in Ukraine and to differentiate vaccine and field strains using restriction analysis.

Materials and Methods
During the study 120 samples of bursa tissues taken from chickens from 16 farms in 10 regions of Ukraine, which include Kyiv, Cherkasy, Lviv, Vinnytsia, Volyn, Dnipropetrovsk, Luhansk, Ternopil, Kharkiv and Crimea have been analyzed.

Results and Discussion
During the study RNA of IBDV has been detected in 75 of total 120 analyzed samples of organs taken from infected birds.
Most of the current research of IBDV aimed to determine the nucleotide sequence of the VP2 gene of different strains [5,10].Comparing the sequences of vaccine and field isolates makes it possible to determine virus strain, but such research takes long time and requires big expenses.In terms of speed of diagnosis, we considered it appropriate to use restriction analysis as a method of differencing strains because it allows to quickly and accurately detect the difference between the vaccine and field virus strains by specific restriction sites.
Results of restriction analysis of strains 228E, GM97, V877 and MB correlate with previously described data by Borodavka et.al [4].
Restriction sites for strains MB/20, MB/5, MB/3 and Winterfield-2512 have been described for the first time.
Restriction profile of strain MB/20 was similar to that of strains 228E and GM97, and characterized by the presence of sites SacI, MvaI and MboI.
Strains V877 and MB/5 belonging to the group of intermediate vaccine strains, characterized by the presence of restriction sites for enzymes BstEII, MboI, SacI and MvaI.
For strain MB/3 5 restriction sites were identified for all restriction endonucleases used in the study, except for SacI.This restriction profile was similar to the previously described strain MB [4].Cleavage of obtained amplicons of strains MB/3 and MB by SspI indicates that they have been attenuated from vvIBDV strains.Amplicons of MB/3 and MB strains have been also cleaved by BspMI enzyme.This restriction site have not been shown for other vaccine strains.Detection of BstEII restriction site in amplicons of vaccine strains MB and MB/3 can be used for differentiation from field vvIBDV.
Strain Winterfield-2512 have been characterized by a unique set of restriction sites, different from all other vaccine strained used in the study.Restriction map for this strain have not been previously described.MboI, SacI, MvaI BstNI and SspI restriction sites have been detected (Fig. 1).Simultaneous presence of restriction sites SacI and SspI have been shown.These restriction sites are considered to be markers of classical and vvIBDV strains respectively [4,5,9].
Vaccine strains belonging to one group by the level of attenuation have been characterized by similar restriction profile (tab.1).
Data obtained from restriction analyses of vaccine strains have been used for differentiation of detected virus isolates.
Thus, it was shown that 38 virus isolates had restriction profile similar to vaccine strains V877 and MB/. 25 identified isolates were similar to strains GM97, 228E and MB/20, while 8 have the same restriction sites as the strains of MB and MB/3 (Fig. 2).
For four analyzed virus isolates restricrion profile had been similar to reference vvIBDV strain UK661.These four isolates differ from intermediate plus vsccine strain in BstEII restriction site.Unlike the vaccine strains MB and MB/3 BstEII have not been shown (fig.3)

Conclusion
We have described restriction profile of eight vaccine strains used in Ukraine and found that strains with the same level of residual virulence have similar restriction sites.However, for strain Winter field-2512 unique restriction profile have been described that was different from all vaccine strains used in the study.
Based on restriction analyses for most of the analyzed virus isolates vaccine origin have been shown.These results show that tissue samples have been taken from vaccinated birds.
However for 4 isolates restriction sites similar to vvIDBV strain have been identified.This indicates that these isolates originated from field vvIBDV strains.Identification of

IBDV strain
Resriction enzyme and length of restriction fragments (nt) vvIBDV shows a disadvantaged situation on the farm concerning Gumboro disease.