Резюме
Рудь Ю.П., Майстренко М.І., Бучацький Л.П. Ампліфікація та аналіз нуклеотидної послідовності генів VP2 та NS вірусу інфекційного панкреатичного некрозу, виділеного в Західній Україні.
Розроблено метод експрес діагностики українського ізоляту “Карпати” вірусу інфекційного панкреатичного некрозу (IPNV) на основі методу полімеразної ланцюгової реакції (ПЛР). Підібрано олігонуклеотидні праймери, специфічні до фрагментів генів VP2 та NS, та проведено оптимізацію постановки ПЛР. Аналіз нуклеотидних послідовностей ампліфікованих фрагментів IPNV “Карпати” свідчить, що український ізолят належить до штаму Sp. Ампліфіковані фрагменти кДНК на 95-99% ідентичні з послідовностями генів NS та VP2 інших ізолятів штаму Sp. Серед ізолятів штаму Sp найбільш спорідненими до українського ізоляту IPNV виявились віруси, виділені у Великобританії, Норвегії, Франції, Турції та Ірані.
Ключові слова: вірус інфекційного панкреатичного некрозу (IPNV), Західна Україна.
Резюме
Рудь Ю.П., Майстренко М.І., Бучацкий Л.П. Амплификация и анализ нуклеотидной последовательности генов VP2 та NS вируса инфекционного панкреатического некроза, выделенного в Западной Украине.
Разработан метод экспресс диагностики украинского изолята "Карпаты" вируса инфекционного панкреатического некроза (IPNV) на основе метода полимеразной цепной реакции (ПЛР). Подобраны олигонуклеотидные праймеры, специфические к фрагментам генов VP2 и NS, и проведена оптимизация постановки ПЛР. Анализ последовательностей нуклеотидов амплификованых фрагментов IPNV "Карпаты" свидетельствует, что украинский  изолят принадлежит к штамму Sp. Амплификованные фрагменты кДНК на 95-99%% идентичны  последовательностям генов NS и VP2 других изолятов штамма Sp. Среди изолятов штамма Sp наиболее родственными к украинскому изоляту IPNV оказались вирусы, выделенные в Великобритании, Норвегии, Франции, Турции и Иране.
Ключевые слова: вирус инфекционного панкреатического некроза (IPNV), Западная Украина.
Summary
Rud Yu., Maistrenko M., Buchatsky L. Amplification and analysis of the nucleotide sequences of the genes NS and VP2 of infectious pancreatic necrosis virus isolated in Western Ukraine.
The method of polymerase chain reaction for Infectious pancreatic necrosis virus (IPNV), isolated in Western Ukraine, was developed. Three sets of primers targeting NS and VP2 genes were used for virus identification and the parameters of PCR were optimized. It was shown that WB primers are the most efficient for virus diagnostic. The nucleotide sequences of amplified fragments were analysed and the prevalance of Ukrainian isolate of IPNV “Karpaty” to Sp strain was revealed.
Key words: infectious pancreatic necrosis virus (IPNV), Western Ukraine.
Рецензент: д.біол.н., проф. В.К. Рибальченко

УДК 576.858

Інститут рибного господарства НААН України

Институт рыбного хозяйства Национальной академии аграрных наук Украины (Киев)

Institute of Fisheries, National Academy of Agrarian Sciences of Ukraine

Київський національний університет імені Тараса Шевченка

Киевский национальный университет имени Тараса Шевченко

Украина, 01601, г. Киев, ул. Владимирская, 64/13 T

aras Shevchenko National University of Kyiv

64/13, Volodymyrska Street, City of Kyiv, Ukraine, 01601

irido1@bigmir.net

In present research the molecular approaches of polymerase chain reaction  (PCR) assay and nucleotide sequencing were used for investigation of Infectious pancreatic necrosis virus (IPNV), isolated from rainbow trout O. mykiss in Siret river, Chernivtsi region, Western Ukraine.

Water birnaviruses (Аquabirnavirus family) belong to the biggest group of viruses, which cause diseases in different fish species and invertebrates. Atlantic salmon S. salar, rainbow trout and char S. fontinalis  are the most sensitive species for this virus in world aquaculture and theirs mortality can reach up to 70% and more. IPNV is widely distributed in Europe and there are several new reports about virus isolation in Ukraine neighboring countries. Since salmonids breeding is mainly located in the west region of Ukraine the IPNV is an economically important fish pathogen for national trout farms. Consequently the water  birnavirus was isolated  from Siret river, Western Ukraine, in 2011. Preliminary investigations based on cell culture and electron microscopy methods revealed relatedness of isolated virus to IPNV, which was named strain "Karpaty". But for more consistent statement of virus relationship, further establishing of possible origin and also developing rapid diagnostics the sequences analysis of viral RNA is needed. Therefore the goals of the present study were to select valid oligonucleotide primers and test it in PCR assay; to provide sequencing of amplified products in way of verification of target amplification; and to accomplish the phylogenetic analysis of Ukrainian IPNV strain.

Genomic viral RNA was extracted from cell culture supernatant using GeneJET^TM RNA Purification Kit. The cDNA synthesis was conducted using RevertAid^TM Premium First Strand cDNA Synthesis Kit following the manufacturer’s instructions. Then cDNA was subjected for PCR amplification. Amplified DNA was analysed by agarose gel electrophoresis. For extraction of DNA from agarose gel the Silica Bead DNA Extraction Kit was used. Sequencing was performed on a 3130 Genetic Analyzer and analyzed using BLASTN, Vector NTI 10 and MEGA version 5.2 software.

  For rapid diagnostic of Ukrainian IPNV strain the method of PCR was developed. Three sets of primers targeting NS and VP2 genes were used for virus identification and the parameters of PCR cycling were optimized.  It was shown that WB primers are the most efficient for virus diagnostic, however the IPN and PrD primers also can be used. Amplified fragments were in size of 200 base pairs (bp) for WB primers, 620 and 175 bp for IPN and PrD primers respectively. In case of low concentration of target RNA only WB primers were enabled to identify the virus. It was noted that annealing temperature of 60⁰C was the most suitable for all primer sets.

The nucleotide sequences of amplified fragments were analysed and the prevalance of Ukrainian isolate of IPNV “Karpaty” to Sp strain was revealed. The comparison of sequences of  IPNVs VP2 and NS genes from NCBI and amplified fragments of IPNV strain “Karpaty” confirmed the high identity of  95-99% with Sp strain, firstly isolated in Denmark. Among the isolates of Sp strain the most related to IPNV “Karpaty” were viruses found in Great Britain, Norway, France, Turkey and Iran.

Thus the selected primers and developed PCR assay can be used for IPNV diagnostic in salmonids cultivated in fish-farming or native ponds of Ukraine. For rapid virus identification in PCR method the WB primers should be used. The complete monitoring of IPNV in Ukraine has to result in total data of virus distribution in Ukraine and also to identify another strains which are widespread in Europe. It will be the subject of our future research.

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