Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis
Aim. Identification of the widespread Ukrainian isolate(s) of PVY (Potato virus Y) in different potato cultivars and subsequent phylogenetic analysis of detected PVY isolates based on NA and AA sequences of coat protein. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. PVY...
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| Zitieren: | Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis / I.G. Budzanivska, L.P. Ovcharenko, A.V. Kharina, I.I. Boubriak, V.P. Polischuk // Вiopolymers and Cell. — 2014. — Т. 30, № 2. — С. 141-148. — Бібліогр.: 20 назв. — англ. |
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Budzanivska, I.G. Ovcharenko, L.P. Kharina, A.V. Boubriak, I.I. Polischuk, V.P. 2019-06-14T16:22:44Z 2019-06-14T16:22:44Z 2014 Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis / I.G. Budzanivska, L.P. Ovcharenko, A.V. Kharina, I.I. Boubriak, V.P. Polischuk // Вiopolymers and Cell. — 2014. — Т. 30, № 2. — С. 141-148. — Бібліогр.: 20 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00088D https://nasplib.isofts.kiev.ua/handle/123456789/153797 578.85/86 Aim. Identification of the widespread Ukrainian isolate(s) of PVY (Potato virus Y) in different potato cultivars and subsequent phylogenetic analysis of detected PVY isolates based on NA and AA sequences of coat protein. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. PVY has been identified serologically in potato cultivars of Ukrainian selection. In this work we have optimized a method for total RNA extraction from potato samples and offered a sensitive and specific PCR-based test system of own design for diagnostics of the Ukrainian PVY isolates. Part of the CP gene of the Ukrainian PVY isolate has been sequenced and analyzed phylogenetically. It is demonstrated that the Ukrainian isolate of Potato virus Y (CP gene) has a higher percentage of homology with the recombinant isolates (strains) of this pathogen (approx. 98.8– 99.8 % of homology for both nucleotide and translated amino acid sequences of the CP gene). The Ukrainian isolate of PVY is positioned in the separate cluster together with the isolates found in Syria, Japan and Iran; these isolates possibly have common origin. The Ukrainian PVY isolate is confirmed to be recombinant. Conclusions. This work underlines the need and provides the means for accurate monitoring of Potato virus Y in the agroecosystems of Ukraine. Most importantly, the phylogenetic analysis demonstrated the recombinant nature of this PVY isolate which has been attributed to the strain group O, subclade N:O. Мета. Виявлення українських ізолятів Y вірусу картоплі (PVY) у різних сортах картоплі і їхній наступний філогенетичного аналіз на основі нуклеотидних і амінокислотних послідовностей білка оболонки. Методи. ІФА, ЗТ-ПЛР, секвенування ДНК і філогенетичний аналіз. Результати. У зразках картоплі вітчизняної селекції методом ІФА ідентифіковано PVY. Оптимізовано процес виділення РНК та розроблено тест-систему на базі ПЛР для діагностики українських ізолятів PVY. Секвеновано ділянку гена капсидного білка українського ізоляту та здійснено філогенетичний аналіз. Встановлено, що зазначений ген демонструє вищий ступінь гомології з генами капсидних білків рекомбінантних ізолятів (штамів) (98,8–99,8 % гомології для нуклеотидних і амінокислотних послідовностей). Український ізолят перебуває в окремому кластері разом з рекомбінантними ізолятами із Сирії, Ірану та Японії і має з ними спільне походження. Висновки. В результаті роботи визначено засоби для точного моніторингу вірусу картоплі в агроекосистемах України. Філогенетичний аналіз продемонстрував рекомбінантну природу досліджуваного ізоляту PVY, який раніше був приписаний до групи штамів О, субклади N:О. Цель. Выявление украинских изолятов Y вируса картофеля (PVY) в различных сортах картофеля и их последующий филогенетический анализ на основе нуклеотидных и аминокислотных последовательностей белка оболочки. Методы. ИФА, ОТ-ПЦР, секвенирование ДНК и филогенетический анализ. Результаты. В образцах картофеля отечественной селекции методом ИФА идентифицирован PVY. Оптимизирован процесс выделения РНК и разработана тест-система на базе ПЦР для диагностики украинских изолятов PVY. Секвенирован участок гена капсидного белка украинского изолята и проведен филогенетический анализ. Показано, что указанный ген демонстрирует высокую степень гомологии с генами капсидных белков рекомбинантных изолятов (штаммов) (98,8–99,8 % гомологии для нуклеотидных и аминокислотных последовательностей). Украинский изолят находится в отдельном кластере вместе с рекомбинантными изолятами из Сирии, Ирана и Японии и имеет с ними общее происхождение. Выводы. В результате работы определены средства для точного мониторинга вирусов картофеля в агроэкосистемах Украины. Филогенетический анализ продемонстрировал рекомбинантную природу исследуемого изолята PVY, который ранее был приписан к группе штаммов О, субклады N:O. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Viruses and Cell Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis Нуклеотидні і амінокислотні послідовності білка оболонки українського ізоляту Y вірусу картоплі:порівняння з гомологічними послідовностями інших ізолятів та філогенетичний аналіз Нуклеотидные и аминокислотные последовательности белка оболочки украинского изолята Y вируса картофеля: сравнение с гомологичными последовательностями других изолятов и филогенетический анализ Article published earlier |
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Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis |
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Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis Budzanivska, I.G. Ovcharenko, L.P. Kharina, A.V. Boubriak, I.I. Polischuk, V.P. Viruses and Cell |
| title_short |
Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis |
| title_full |
Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis |
| title_fullStr |
Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis |
| title_full_unstemmed |
Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis |
| title_sort |
nucleotide and amino acid sequences of a coat protein of an ukrainian isolate of potato virus y: comparison with homologous sequences of other isolates and phylogenetic analysis |
| author |
Budzanivska, I.G. Ovcharenko, L.P. Kharina, A.V. Boubriak, I.I. Polischuk, V.P. |
| author_facet |
Budzanivska, I.G. Ovcharenko, L.P. Kharina, A.V. Boubriak, I.I. Polischuk, V.P. |
| topic |
Viruses and Cell |
| topic_facet |
Viruses and Cell |
| publishDate |
2014 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Нуклеотидні і амінокислотні послідовності білка оболонки українського ізоляту Y вірусу картоплі:порівняння з гомологічними послідовностями інших ізолятів та філогенетичний аналіз Нуклеотидные и аминокислотные последовательности белка оболочки украинского изолята Y вируса картофеля: сравнение с гомологичными последовательностями других изолятов и филогенетический анализ |
| description |
Aim. Identification of the widespread Ukrainian isolate(s) of PVY (Potato virus Y) in different potato cultivars and subsequent phylogenetic analysis of detected PVY isolates based on NA and AA sequences of coat protein. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. PVY has been identified serologically in potato cultivars of Ukrainian selection. In this work we have optimized a method for total RNA extraction from potato samples and offered a sensitive and specific PCR-based test system of own design for diagnostics of the Ukrainian PVY isolates. Part of the CP gene of the Ukrainian PVY isolate has been sequenced and analyzed phylogenetically. It is demonstrated that the Ukrainian isolate of Potato virus Y (CP gene) has a higher percentage of homology with the recombinant isolates (strains) of this pathogen (approx. 98.8– 99.8 % of homology for both nucleotide and translated amino acid sequences of the CP gene). The Ukrainian isolate of PVY is positioned in the separate cluster together with the isolates found in Syria, Japan and Iran; these isolates possibly have common origin. The Ukrainian PVY isolate is confirmed to be recombinant. Conclusions. This work underlines the need and provides the means for accurate monitoring of Potato virus Y in the agroecosystems of Ukraine. Most importantly, the phylogenetic analysis demonstrated the recombinant nature of this PVY isolate which has been attributed to the strain group O, subclade N:O.
Мета. Виявлення українських ізолятів Y вірусу картоплі (PVY) у різних сортах картоплі і їхній наступний філогенетичного аналіз на основі нуклеотидних і амінокислотних послідовностей білка оболонки. Методи. ІФА, ЗТ-ПЛР, секвенування ДНК і філогенетичний аналіз. Результати. У зразках картоплі вітчизняної селекції методом ІФА ідентифіковано PVY. Оптимізовано процес виділення РНК та розроблено тест-систему на базі ПЛР для діагностики українських ізолятів PVY. Секвеновано ділянку гена капсидного білка українського ізоляту та здійснено філогенетичний аналіз. Встановлено, що зазначений ген демонструє вищий ступінь гомології з генами капсидних білків рекомбінантних ізолятів (штамів) (98,8–99,8 % гомології для нуклеотидних і амінокислотних послідовностей). Український ізолят перебуває в окремому кластері разом з рекомбінантними ізолятами із Сирії, Ірану та Японії і має з ними спільне походження. Висновки. В результаті роботи визначено засоби для точного моніторингу вірусу картоплі в агроекосистемах України. Філогенетичний аналіз продемонстрував рекомбінантну природу досліджуваного ізоляту PVY, який раніше був приписаний до групи штамів О, субклади N:О.
Цель. Выявление украинских изолятов Y вируса картофеля (PVY) в различных сортах картофеля и их последующий филогенетический анализ на основе нуклеотидных и аминокислотных последовательностей белка оболочки. Методы. ИФА, ОТ-ПЦР, секвенирование ДНК и филогенетический анализ. Результаты. В образцах картофеля отечественной селекции методом ИФА идентифицирован PVY. Оптимизирован процесс выделения РНК и разработана тест-система на базе ПЦР для диагностики украинских изолятов PVY. Секвенирован участок гена капсидного белка украинского изолята и проведен филогенетический анализ. Показано, что указанный ген демонстрирует высокую степень гомологии с генами капсидных белков рекомбинантных изолятов (штаммов) (98,8–99,8 % гомологии для нуклеотидных и аминокислотных последовательностей). Украинский изолят находится в отдельном кластере вместе с рекомбинантными изолятами из Сирии, Ирана и Японии и имеет с ними общее происхождение. Выводы. В результате работы определены средства для точного мониторинга вирусов картофеля в агроэкосистемах Украины. Филогенетический анализ продемонстрировал рекомбинантную природу исследуемого изолята PVY, который ранее был приписан к группе штаммов О, субклады N:O.
|
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0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/153797 |
| citation_txt |
Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis / I.G. Budzanivska, L.P. Ovcharenko, A.V. Kharina, I.I. Boubriak, V.P. Polischuk // Вiopolymers and Cell. — 2014. — Т. 30, № 2. — С. 141-148. — Бібліогр.: 20 назв. — англ. |
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VIRUSES AND CELL
UDC 578.85/86
Nucleotide and amino acid sequences of a coat protein
of an Ukrainian isolate of Potato virus Y:
comparison with homologous sequences of other isolates
and phylogenetic analysis
I. G. Budzanivska1, L. P. Ovcharenko2, A. V. Kharina1, I. I. Boubriak3, 4, V. P. Polischuk1
1Educational and Scientific Centre «Institute of Biology», Taras Shevchenko National University of Kyiv
64/13, Volodymyrska Str., Kyiv, Ukraine, 01601
2Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
3Department of Biochemistry, University of Oxford
South Parks Road, Oxford, OX1 3QU, UK
4Institute of Cell Biology and Genetic Engineering, NAS of Ukraine
148, Akademika. Zabolotnoho, Kyiv, Ukraine, 03680
fitovirus@yandex.ru
Aim. Identification of the widespread Ukrainian isolate(s) of PVY (Potato virus Y) in different potato cultivars
and subsequent phylogenetic analysis of detected PVY isolates based on NA and AA sequences of coat protein.
Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. PVY has been identified sero-
logically in potato cultivars of Ukrainian selection. In this work we have optimized a method for total RNA ex-
traction from potato samples and offered a sensitive and specific PCR-based test system of own design for
diagnostics of the Ukrainian PVY isolates. Part of the CP gene of the Ukrainian PVY isolate has been sequen-
ced and analyzed phylogenetically. It is demonstrated that the Ukrainian isolate of Potato virus Y (CP gene)
has a higher percentage of homology with the recombinant isolates (strains) of this pathogen (approx. 98.8–
99.8 % of homology for both nucleotide and translated amino acid sequences of the CP gene). The Ukrainian
isolate of PVY is positioned in the separate cluster together with the isolates found in Syria, Japan and Iran;
these isolates possibly have common origin. The Ukrainian PVY isolate is confirmed to be recombinant. Con-
clusions. This work underlines the need and provides the means for accurate monitoring of Potato virus Y in
the agroecosystems of Ukraine. Most importantly, the phylogenetic analysis demonstrated the recombinant
nature of this PVY isolate which has been attributed to the strain group O, subclade N:O.
Keywords: Potato virus Y, potyvirus, PCR, phylogenetic analysis, recombinant strain.
Introduction. Potyvirus genus is a classical example
of employing computer technologies and phylogenetic
analysis not only to study virus strain diversity and re-
lationships among the representatives of this genus, but
also for developing our understanding of origin and
evolution of both viruses and their respective hosts.
Potato virus Y (PVY) is a typical member of Poty-
virus genus, the largest genus of plant viruses. Potato
growers from different parts of the world agree that to-
day PVY is assumed to be the most economically harm-
ful plant virus [1] with the exception of Australia whe-
re PVY has never led to serious potato crop losses. At
the same time novel PVY strains arise or spread to the
new geographical regions [2]. The development and
use of molecular techniques for characterization of PVY
allowed the appearance of many new names for its iso-
lates and strains.
From the very beginning of plant virology as the fun-
damental science, visual symptoms of virus-induced
diseases of cultivated plants were a major motive for
141
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 2. P. 141–148 doi: http://dx.doi.org/10.7124/bc.00088D
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
142
naming various viruses. At the same time, virus proper-
ties were mostly unknown and hence could not be inclu-
ded (and reflected) in their respective names. For in-
stance, the most typical symptoms induced by potato vi-
ruses were divided into two groups: rugose (wrinkled)
mosaics following the infection by aphid-transmitted
viruses, and mottling which developed after infection
with sap-transmitted pathogens. In 1931, Smith [3]
isolated infectious agents and named these viruses as Y
and X, in accordance with the type of transmission and
the symptoms developing on infected tobacco (Nicotia-
na tabacum L.). The «X» component induced double
concentric circles on tobacco and was named as the «ring-
spot virus» whereas the «Y» component was aphid-
transmitted and led to the dark discoloration of green tis-
sues along the leaf veins («vein streak»). These X and
Y components were the prototypes of Potato virus X
(PVX) and Potato virus Y (PVY) – the names which are
still in use. With time these viruses became (and still
remain) typical species of two respective virus genera -
Potexvirus and Potyvirus [4]. During the first years of
«potato virology» PVY had many names (synonymous)
including: potato virus 20, potato virus C, potato acro-
petal necrosis virus, potato leaf streak abscission virus,
potato severe mosaics virus, potato streak virus, potato
striation virus, potato vein necrosis virus, Solanum virus
2, tobacco vein streak virus, tobacco vein necrosis virus.
PVY is the important pathogen infecting not only
potato but also other cultures including pepper (Capsi-
cum spp.), tomato (Lycopersicum esculentum L.) and to-
baccos. Seemingly, potato and pepper serve as selecti-
ve hosts for PVY strains. Despite the PVY variants iso-
lated from these two species are specific to them, toma-
to and tobacco plants may be infected by the majority
of both potato and pepper isolates of PVY. Phylogene-
tic analysis of the coat protein (CP) gene sequences sug-
gests that some PVY variants isolated from tomatoes
are closely related to PVY-C. Grouping virus isolates on
the base of phylogenetic analysis of the CP gene sequen-
ces shows good correlation with phenotypic appearan-
ces (symptoms) of necrotic and mosaic strains [5–7].
In 1980ies new PVY-N isolates have been descri-
bed, some of which were related to the necrotic (reason
for «N» in the virus name) disease of potato tubers – po-
tato tuber necrotic ringspot disease (PTNRD) [8, 9].
These isolates (formerly named PVY-NN) were given
a new acronym, PVY-NTN. Another group of PVY-N
isolates characterized by the loss of virulence on potato
has been registered in Poland and called PVY-N-Wi
(following its description on «Wilga» cultivar of potato)
[10].
Several PVY-NTN isolates have been characteri-
zed at the molecular level and shown to be recombinants
of PVY-O («ordinary» strain) and PVY-N («necrotic»
strain) in CP-coding region. Later on the additional re-
combinational events were described for HC-Pro and
nuclear inclusion genes. Furthermore, PTNRD isolates
lacking recombinational events in the CP gene have be-
en found in the Northern America, Denmark, New Zea-
land, Germany, Poland and Japan [11].
The PVY-NTN isolates undetectable using primers
developed for European PVY-NTN were named Nor-
thern American PVY-NTN, or NA-PVY-NTN. In Ca-
nada and Spain, additional variants of PVY-N were des-
cribed (I-136 and I-L56 isolates in Canada and isolate
17 in Spain); these were capable of serological reaction
with PVY-O-specific monoclonal antibodies, at the sa-
me time inducing veinal necrosis on tobacco (similarly
to PVY-N-Wi). As was demonstrated later, these isola-
tes were widely spread in most countries cultivating po-
tato. These isolates were considered recombinant;
another recombinational «node» has been confirmed by
RT-PCR and this group was named PVY-N:O (a «cross»
between the ordinary and necrotic strains) [12–13].
Some rare PVY-N-Wi isolates may have had four
recombinational events. Two Spanish isolates serologi-
cally reacted with PVY-O-specific antibodies and did
not induce veinal necrosis on tobacco. These viruses
were put into the group PVY-ZE (variants of PVY-Z).
It has been suggested to attribute them to a separate
group of PVY strains, PVY-E.
Molecular analysis of the PVY genomes witnesses
to an increase in the number of the recombinant PVY
isolates and strains belonging to various strain groups
defined on the base of host response or serological crite-
ria. However, the serological criteria for determination
of the PVY strain groups using monoclonal antibodies
have not been fully elaborated. Hence, at present we ha-
ve no clear understanding of molecular base for diffe-
rentiation of the PVY isolates and strains. Moreover,
different viewpoints are expressed and suggested by va-
rious authors.
BUDZANIVSKA I. G. ET AL.
This work has been aimed at the identification of
widespread (typical) Ukrainian isolate(s) of PVY in dif-
ferent potato cultivars and subsequent phylogenetic ana-
lysis of the detected PVY isolates based on the gene and
amino acid sequences of coat protein which was suc-
cessfully used by many researchers and proved to serve
as a reliable molecular marker for virus evolution.
Materials and methods. PVY (Potyvirus, Potyviri-
dae) was the object of this research. Many potato culti-
vars of Ukrainian selection (listed further in the «Re-
sults and Discussion» section) were employed in this
work and screened for PVY infection. Plant samples
were collected based on visual virus-like symptoms:
vein clearing, mottling leaf tips, streak necrotization
along the veins on the underside of the leaves, necrotic
and dry leaves. Mild virus-like symptoms typically pre-
vailed in the field.
The virus identification has been carried out via
ELISA using the PVY-specific polyclonal commercial
antisera («Loewe», Germany) according to the manu-
facturer’s recommendations. The results were measured
using automated ELISA microplate reader Stat Fax 2100
(«Awareness Technology», USA) at the wavelength of
405 nm. The results were considered positive if they ex-
ceeded the negative control optical density value by 3 ti-
mes or more. Each sample was replicated three times.
Simple average values were used [14].
In parallel to the classical method for RNA extrac-
tion [15], we have also used commercially available
E.Z.N.A. Plant RNA Mini Kit (USA) was applied for
comparing RNA yield and purity.
In order to select optimal primers’ set, three pairs of
primers of own design (widely recognized Entry and
Oligo 6 software packages) were used:
1st pair:
1 (As) 5'-CAAACCATAAGCCCATTCATC-3' ;
2 (S) 5'-GCACCAAATCAGGAGATTCTACT-3';
Expected product size: 569 bp;
2nd pair:
PVY-S 5'-CACGATTGCTCAAGCAAGAA-3';
PVY-As 5'-GGCGGAGTATGCACCAAGTA-3';
Expected product size: 412 bp;
3rd pair:
PVY1-S5'-TGCACGATTGCTCAAGCAAGAAT-3';
PVY1-As 5’GGCGGAGTATGCACCAAGTA-3';
Expected product size: 480 bp.
All these three primer pairs were specific to the res-
pective conserved regions of the CP gene of PVY (see
«Results and Discussion» section for details).
For the virus detection via RT-PCR we employed
one-step-RT-PCR kit («Qiagen», UK). Software packa-
ges Entry and Oligo 6 were used for design of own pri-
mers; optimal reaction conditions were selected.
The amplicons were analyzed using 1 % agarose
gel electrophoresis with markers Gene Ruller 100 bp
DNA Ladder plus («Fermentas Inc.», USA).
The purified amplicons were sequenced using Ap-
plied Biosystems 3730x1 DNA Analyzer with Big Dye
terminators, version 3.1 («Applied Biosystems», USA).
The identification and comparison of obtained sequ-
ences were carried out with BLAST analysis (http://
www.ncbi.nlm.him.gov). For phylogenetic analysis the
software package MEGA 5 was used [16]. To check the
validity of constructed trees, the boostrap test was emp-
loyed with 1000 replications [17]. Phylogenenic trees
were constructed using neighbour-joining (NJ) [18]
and maximum likelihood (ML) [19] approaches. Statis-
tic evaluation of results was done with the help of Mic-
rosoft Excel 2003 using Student’s test.
Results and discussion. Numerous potato cultivars
of Ukrainian selection kindly provided by the Institute of
potato research of NAASU have been screened for PVY
detection: Povin, Ariel, Dovira, Promin, Mandrivnytsya,
Serpanok, Bylyna, Presto, Zelenyy Gay, Okolytsya, Bro-
za, Fantaziya, Podolyanka, Maxam rosa. These are po-
pular cultivars selected and bred at the Institute faciliti-
es and then marketed in virtually every region of Ukrai-
ne for commercial production. This fact allows sugges-
ting that, in general, the fields of this Institute may serve
as a major «hotbed» of PVY origination/microevolu-
tion/recombination/spread and hence may represent ty-
pical Ukrainian population mix for this virus.
Further diagnostics using DAS-ELISA with the
PVY-specific commercial test system («Loewe») de-
monstrated high content of PVY in the cultivars Povin,
Dovira, Promin, Mandrivnytsya, Serpanok, Bylyna, Ze-
lenyy Gay, Okolytsya (ELISA data not shown). These
PVY-positive samples were then used for total RNA ex-
traction and subsequent amplification.
The comparison of two different techniques for to-
tal RNA extraction proved that commercial E.Z.N.A.
Plant RNA Mini Kit (USA) was by far more preferable
143
NUCLEOTIDE AND AMINO ACID SEQUENCES OF COAT PROTEIN OF POTATO VIRUS Y
for molecular work and provided a higher RNA yield
(data not shown). The classical method was also suitab-
le, but less reliable.
Further on, we went to select optimal primers’ set
for the PVY detection in Ukrainian potato cultivars and
to optimize the PCR conditions (annealing temperature
and MgCl2 concentration in the reaction mix). We have
demonstrated that only one pair of primers (the first one
from the three tested, see «Materials and Methods»
section) worked well in PCR with total RNA extracted
from collected potato samples (i. e., with «Ukrainian»
isolates of PVY) yielding the amplicon of 569 bp:
1 (As) 5'-CAAACCATAAGCCCATTCATC-3';
2 (S) 5'-GCACCAAATCAGGAGATTCTACT-3'.
These sets of primers were of such design that theo-
retically allowed the PCR detection of each PVY iso-
late (i. e., every set of primers could be used for the
detection of both ordinary virus strain and necrotic or C
strains). In general we should add that the use of the coat
protein gene sequence for primers’ design and subse-
quent virus detection and its phylogenetic analysis is
the common practice in plant virology [11].
According to the obtained results, the second and
the third pairs of primers (based on the sequences (Gen
Bank: DQ000989.1 and GenBank: DQ000990.1), were
not capable of detecting PVY in collected plant samp-
les (data not shown).
However, using the first set of primers we have suc-
cessfully amplified the PVY cDNA from the initial po-
tato leaf samples. This primers’ set was designed against
a part of CP gene of the ordinary strain of PVY (Gen
Bank: DQ000987.1).
Most probably, the ordinary strain of PVY domina-
tes in Ukraine which is indirectly confirmed by the ab-
sence of PVY-invoked epidemics and by the relatively
mild virus-like symptoms prevailing in the field (see
«Materials and Methods» section). Hence, it is logical to
assume that the primers designed using the CP gene se-
quence of the ordinary strain of PVY would be most sui-
table for the virus detection. However, as it was pointed
above, all three sets of primers were of universal design
allowing detection of all three PVY strains. As such, we
may propose several reasons for only one pair of pri-
mers allowing PVY detection in the Ukrainian potato
samples. These include tentative mutations in the speci-
fic regions of the PVY CP gene preventing the annea-
ling of primers’ sets N 2 and 3, and possibly non-opti-
mal RT-PCR conditions.
The first set of primers (capable of amplifying PCY
cDNA) was further employed for optimization of the
PCR parameters (determination of optimal MgCl2 con-
tent and annealing temperature).
In short, we have found that optimal primer annea-
ling temperature was 62 °C and optimal content of MgCl2
equaled 1.75 mM, providing more specific product.
The amplicon obtained using primers’ set N 1 has
been cloned for generation of a positive control for PCR.
The product has been detected in bacteria samples via
PCR using specific primers (Fig. 1).
Thus, we have optimized the PCR-based diagnos-
tics of Ukrainian isolate(s) of PVY consisting of: (1) se-
lected specific primers with yield up to 569 bp product
and the positive reaction control; (2) optimal parame-
ters of RT-PCR: annealing temperature and MgCl2 con-
centration were 62 °C and 1.75 mM, respectively. This
forms the base for future design of the test system for
routine PVY screening.
The RT-PCR yielded cDNA corresponding to the
part of CP gene of Ukrainian isolate of PVY. This
cDNA has been further sequenced and compared with
some of the known sequences of PVY isolates and
strains published in the GenBank. Though more than
600 of such sequences are available in public databa-
ses, we have used only 18 published PVY sequences
showing the highest percent identity to our RT-PCR
product as determined by BLAST feature (NCBI). Ad-
ditionally, we have also compared corresponding ami-
144
BUDZANIVSKA I. G. ET AL.
1 2 3 4
Fig. 1. Results of RT-PCR for PVY diagnostics: 1 – markers (Gene Ru-
ler DNA Ladder Mix, Fermentas); 2 – PCR product generated on the in-
sertion of a fragment of the capsid gene in plasmid pJET1.2. (positive
control); 3 – RNA of PVY-positive sample; 4 – negative control
no acid sequences. It follows that the CP gene of Ukrai-
nian isolate of PVY is more homologous to the recom-
binant isolates (strains) – for instance, Syria Rec, Japan
PVYNTN-NW, Iran N:O, USA N:O, Poland N-Wi
(98.8–99.8 % of homology for both nucleotide and
amino acid sequences) (Table).
These relationships are even more obvious when
analyzed using phylogenetic trees constructed for
nucleotide and amino acid sequences of CP employing
the Neighbor-Joining (NJ) method (Fig. 2, 3).
Both nucleotide sequence- and amino acid se-
quence-based phylogenetic trees for PVY CP demonst-
rate higly similar (though not 100 % identical) position
of Ukrainian isolate in relation to others. In both cases
we can see a separate cluster including recombinant iso-
lates (strains) from Syria, Iran and Japan; PVY N-Wi
Poland isolate is one of the most closely related to the
cluster with Ukrainian isolate of PVY.
To make a conclusion on the evolutionary history
of these isolates, the maximum likelihood (ML) appro-
ach has been used on the base of Tamura-Nei model. A
tree with high logarithm of likelihood has been const-
ructed for 1000 bootstrap replications. The branch length
corresponds to the number of changes per site.
As shown in Fig. 4, the phylogenetic tree construc-
ted using ML method underlines that the main tenden-
cies remain the same. We can say that Ukrainian PVY
isolates and PVY isolates from Syria, Iran and Japan ha-
ve descended from a single ancestor.
Study of state-of-the-art (2006–2012 yy) and pre-
vious (1992–2005 yy) literature sources confirms the
absence of common opinion of the authors regarding
unified nomenclature for strains and isolates of PVY (in
particular, those found in potato). The comparison of
phylogenetic trees published by different researchers is
not an easy task as (most often) different «source» virus
145
NUCLEOTIDE AND AMINO ACID SEQUENCES OF COAT PROTEIN OF POTATO VIRUS Y
GenBank reference number Country Strain Homology of nucleotide sequences, % Homology of AA sequences, %
PVY_gi325053317_ Syria Rec 99.8 99.8
PVY_gi264160858_ Japan PVYNTN-NW 99.8 99.8
PVY_gi264160830_ Japan PVYNTN-NW 99.8 99.8
PVY_gi264160900_ Japan PVYNTN-NW 99.5 99.5
PVY_gi264160917_ Japan PVYNTN-NW 99.3 99.3
PVY_gi192763859_ Iran N:O 99.3 99.3
PVY_gi336318904_ USA N:O 99 99
PVY_gi347514591_ Poland N-Wi 98.8 98.8
PVY_gi336318948_ USA O 98.8 98.8
PVY_gi307094821_ France NW 98.8 98.8
PVY_strain_Wilga_gi90968475_ Germany Wigna 98.8 98.8
PVY_gi336318902_ USA O 98.6 98.6
PVY_strain_O_gi90968473_ Germany O 98.3 98.3
PVY_gi2808577_ Switzerland – 97.6 97.6
PVY_gi12005812_ Brazil O 97.4 97.4
PVY_gi2808576_ Switzerland O 97.4 97.4
PVY_gi156766622 China Isolate = «Laiwu1» 97.1 97.1
PVY_gi31338457_ China Isolate = «Hangzhou» 97.1 97.1
Evolutionary homology of Ukrainian isolate of PVY with selected PVY sequences available in the GenBank (%)
sequences (both nucleotide and amino acid) have been
used for the analysis. Apparently, the recombination
among the PVY genomes may lead to the development
of novel variants of the virus which may have differing
phenotype (without any significant correlation with co-
ding sequences). In turn, phenotypic appearances will
depend on the specific species and cultivar of the virus-
infected plant.
As soon as our work has not been aimed at studying
phenotypic variations of Ukrainian isolates of PVY,
our conclusions are based exclusively on the compara-
tive analysis of nucleotide sequences in the part of vi-
rus genome (the coat protein gene of PVY). The phylo-
genetic relationships based on the CP gene of PVY and
explored using ML method may be an indirect index of
the relationships for full-size genomes of potyviruses
146
BUDZANIVSKA I. G. ET AL.
Sequence 21 P VY Uk raine
Potato virus Y gi325053317 Syria
Potato virus Y gi264160858 PVYNTN-NW Japan
Potato virus Y gi264160830 PVYNTN-NW Japan
Potato virus Y gi192763859 N:0 Iran
Potato virus Y gi264160900 PVYNTN-NW Japan
Potato virus Y gi264160917 PVYNTN-NW Japan
Potato virus Y gi347514591 N W i Poland
Potato virus Y strain O gi90968473 Germany
P otato virus Y gi307094821 Straine NW Franse
Potato virus Y strain W ilga gi90968475 Germany
Potato virus Y gi336318948 O USA
Potato virus Y gi336318902 O USA
Potato virus Y gi12005812 O Brazil
Potato virus Y gi2808577 O S witzerland
Potato virus Y gi2808576 O Switzerland
Potato virus Y gi31338457 United Kingdom
Potato virus Y gi156766622 China
35
25
75
63
57
40
27
31
54
59
58
32
0.005
Fig. 2. Evolutionary relation-
ships of Ukrainian PVY iso-
lates with PVY isolates/
strains deposited in the Gen
Bank. Evolutionary history
has been reconstructed using
NJ method [17] for nucleoti-
de sequence of the CP gene.
Evolutionary distances were
calculated using p-distance
method [15] for 1000 boot-
strap replications (number of
base changes per site). ME-
GA5 has been employed for
evolutionary analysis
Sequence 21 PVY Ukraine
Potato virus Y gi325053317 Syria
Potato virus Y gi264160858 PVYNTN-NWJapan
Potato virus Y gi264160830 PVYNTN-NW Japan
Potato virus Y gi264160900 PVYNTN-NW Japan
Potato virus Y gi264160917 PVYNTN-NW Japan
Potato virus Y gi192763859 N:0 Iran
Potato virus Y gi347514591 N Wi Poland
Potato virus Y gi307094821 Straine NW Franse
Potato virus Y gi156766622 China
Potato virus Y gi336318948 O USA
Potato virus Y gi336318902 O USA
Potato virus Y strain Wilga gi90968475 Germany
Potato virus Y strain O gi90968473 Germany
Potato virus Y gi2808577 O Switzerland
Potato virus Y gi12005812 O Brazil
Potato virus Y gi2808576 O Switzerland
Potato virus Y gi31338457 United Kingdom77
61
26
47
44
27
12
39
8
66
0.005
Fig. 3. Evolutionary relation-
ships of Ukrainian PVY iso-
lates with PVY isolates/
strains deposited in the Gen
Bank. Evolutionary history
has been reconstructed using
NJ method [17] for amino
acid sequence of the CP ge-
ne. Evolutionary distances
were calculated using Pois-
son correction method [15]
for 1000 bootstrap replica-
tions (number of base chan-
ges per site). MEGA5 has be-
en employed for evolutiona-
ry analysis
[10] and for establishing the species of viruses, for which
only the CP gene sequences are available (which prevail
in the GenBank).
Our conclusions regarding close relationships bet-
ween Ukrainian PVY isolate and isolates from Syria,
Iran and Japan, as well as their putative common origin
are strongly supported by the fact that these isolates are
closely positioned at the other phylogenetic trees. Fol-
lowing this lead and according to the latest trends in
classification of PVY isolates/strains, it is viable to sug-
gest that Ukrainian isolate of PVY belongs to the strain
group O, subclade N:O (or to the subclade O:N-Wilga
N:O) [11, 12, 20].
Based on these comparative data we may conclude
that Ukrainian isolate of PVY is a recombinant virus.
Considering the experience of other research groups [1,
12, 13, 20], we would assume that such recombinant
isolate of PVY might be capable of phenotypic appea-
rances typical for both ordinary (O) and necrotic (N)
strains, depending on the specific conditions of the
plant virus infection development.
The fact that the recombinations more likely occur
in the limits of the strain is soothing; however, PVY
strain diversity in wild nature (which may serve as a
rich additional source of recombination material) is
completely obscure and requires accurate monitoring
for forecasting the appearance of new highly patho-
genic virus variants and prevention of the disease de
v-elopment.
This has been brightly examplified in 1980ies in
Europe by the appearance of necrotic strain invading
potato tubers, PVY-NTN.
Conclusions. This work underlines the need and
provides means for accurate monitoring of Potato virus
Y in the agroecosystems (and preferably – in wild-
growing plants) of Ukraine where potato remains one
of the most commercially important crop.
Aiming at designing proprietary PCR-based test
system for PVY, we have isolated PVY isolate, desig-
ned own specific primers and optimized the RT-PCR
conditions.
Most importantly, phylogenetic analysis demonstra-
ted the recombinant nature of this PVY isolate which
has been attributed to strain group O, subclade N:O.
Further work will focus on search of other wide-
spread PVY isolates and their molecular characteri-
zation with subsequent progress with the evaluation of
sensitivity and specificity of the test system.
Funding. The part of this research has been granted
by National Academy of Sciences of Ukraine (the grant
«System biotechnology of potato protection and mole-
cular diagnostics of potato pathogens»).
147
NUCLEOTIDE AND AMINO ACID SEQUENCES OF COAT PROTEIN OF POTATO VIRUS Y
Potato virus Y gi325053317 Syria
Potato virus Y gi264160858 PVYNTN-NWJapan
Potato virus Y gi264160900 PVYNTN-NW Japan
Potato virus Y gi264160917 PVYNTN-NW Japan
Sequence 21 PVY Ukraine
Potato virus Y gi192763859 N:0 Iran
Potato virus Y gi264160830 PVYNTN-NW Japan
Potato virus Y gi347514591 N Wi Poland
Potato virus Y strain O gi90968473 Germany
Potato virus Y gi307094821 Straine NW Franse
Potato virus Y strain Wilga gi90968475 Germany
Potato virus Y gi336318948 O USA
Potato virus Y gi336318902 O USA
Potato virus Y gi2808577 O Switzerland
Potato virus Y gi12005812 O Brazil
Potato virus Y gi2808576 O Switzerland
Potato virus Y gi31338457 United Kingdom
Potato virus Y gi156766622 China
37
70
36
58
36
30
33
56
63
0.005
Fig. 4. Molecular phyloge-
netic analysis of nucleotide
sequences of PVY CP gene
using maximum likelihood
(ML) method [16]
Íóêëåîòèäí³ ³ àì³íîêèñëîòí³ ïîñë³äîâíîñò³ á³ëêà îáîëîíêè
óêðà¿íñüêîãî ³çîëÿòó Y â³ðóñó êàðòîïë³:ïîð³âíÿííÿ ç
ãîìîëîã³÷íèìè ïîñë³äîâíîñòÿìè ³íøèõ ³çîëÿò³â òà
ô³ëîãåíåòè÷íèé àíàë³ç
². Ã. Áóäçàí³âñêà, Ë. Ï. Îâ÷àðåíêî, À. Â. Õàð³íà, ². ². Áóáðÿê,
Â. Ï. Ïîë³ùóê
Ðåçþìå
Ìåòà. Âèÿâëåííÿ óêðà¿íñüêèõ ³çîëÿò³â Y â³ðóñó êàðòîïë³ (PVY) ó
ð³çíèõ ñîðòàõ êàðòîïë³ ³ ¿õí³é íàñòóïíèé ô³ëîãåíåòè÷íîãî àíàë³ç
íà îñíîâ³ íóêëåîòèäíèõ ³ àì³íîêèñëîòíèõ ïîñë³äîâíîñòåé á³ëêà
îáîëîíêè. Ìåòîäè. ²ÔÀ, ÇÒ-ÏËÐ, ñåêâåíóâàííÿ ÄÍÊ ³ ô³ëîãåíå-
òè÷íèé àíàë³ç. Ðåçóëüòàòè. Ó çðàçêàõ êàðòîïë³ â³ò÷èçíÿíî¿ ñå-
ëåêö³¿ ìåòîäîì ²ÔÀ ³äåíòèô³êîâàíî PVY. Îïòèì³çîâàíî ïðîöåñ
âèä³ëåííÿ ÐÍÊ òà ðîçðîáëåíî òåñò-ñèñòåìó íà áàç³ ÏËÐ äëÿ ä³àã-
íîñòèêè óêðà¿íñüêèõ ³çîëÿò³â PVY. Ñåêâåíîâàíî ä³ëÿíêó ãåíà êàï-
ñèäíîãî á³ëêà óêðà¿íñüêîãî ³çîëÿòó òà çä³éñíåíî ô³ëîãåíåòè÷íèé
àíàë³ç. Âñòàíîâëåíî, ùî çàçíà÷åíèé ãåí äåìîíñòðóº âèùèé ñòó-
ï³íü ãîìîëî㳿 ç ãåíàìè êàïñèäíèõ á³ëê³â ðåêîìá³íàíòíèõ ³çîëÿò³â
(øòàì³â) (98,8–99,8 % ãîìîëî㳿 äëÿ íóêëåîòèäíèõ ³ àì³íîêèñëîò-
íèõ ïîñë³äîâíîñòåé). Óêðà¿íñüêèé ³çîëÿò ïåðåáóâຠâ îêðåìîìó
êëàñòåð³ ðàçîì ç ðåêîìá³íàíòíèìè ³çîëÿòàìè ³ç Ñèð³¿, ²ðàíó òà
ßïîí³¿ ³ ìຠç íèìè ñï³ëüíå ïîõîäæåííÿ. Âèñíîâêè.  ðåçóëüòàò³
ðîáîòè âèçíà÷åíî çàñîáè äëÿ òî÷íîãî ìîí³òîðèíãó â³ðóñó êàð-
òîïë³ â àãðîåêîñèñòåìàõ Óêðà¿íè. Ô³ëîãåíåòè÷íèé àíàë³ç ïðîäå-
ìîíñòðóâàâ ðåêîìá³íàíòíó ïðèðîäó äîñë³äæóâàíîãî ³çîëÿòó PVY,
ÿêèé ðàí³øå áóâ ïðèïèñàíèé äî ãðóïè øòàì³â Î, ñóáêëàäè N:Î.
Êëþ÷îâ³ ñëîâà: Y â³ðóñ êàðòîïë³, ïîò³â³ðóñ, ÏËÐ, ô³ëîãåíåòè÷-
íèé àíàë³ç, ðåêîìá³íàíòíèé øòàì.
Íóêëåîòèäíûå è àìèíîêèñëîòíûå ïîñëåäîâàòåëüíîñòè áåëêà
îáîëî÷êè óêðàèíñêîãî èçîëÿòà Y âèðóñà êàðòîôåëÿ: ñðàâíåíèå
ñ ãîìîëîãè÷íûìè ïîñëåäîâàòåëüíîñòÿìè äðóãèõ èçîëÿòîâ
è ôèëîãåíåòè÷åñêèé àíàëèç
È. Ã. Áóäçàíèâñêàÿ, Ë. Ï. Îâ÷àðåíêî, À. Â. Õàðèíà, È. È. Áóáðÿê,
Â. Ï. Ïîëèùóê
Ðåçþìå
Öåëü. Âûÿâëåíèå óêðàèíñêèõ èçîëÿòîâ Y âèðóñà êàðòîôåëÿ (PVY)
â ðàçëè÷íûõ ñîðòàõ êàðòîôåëÿ è èõ ïîñëåäóþùèé ôèëîãåíåòè-
÷åñêèé àíàëèç íà îñíîâå íóêëåîòèäíûõ è àìèíîêèñëîòíûõ ïîñëå-
äîâàòåëüíîñòåé áåëêà îáîëî÷êè. Ìåòîäû. ÈÔÀ, ÎÒ-ÏÖÐ, ñåêâå-
íèðîâàíèå ÄÍÊ è ôèëîãåíåòè÷åñêèé àíàëèç. Ðåçóëüòàòû.  îá-
ðàçöàõ êàðòîôåëÿ îòå÷åñòâåííîé ñåëåêöèè ìåòîäîì ÈÔÀ èäåí-
òèôèöèðîâàí PVY. Îïòèìèçèðîâàí ïðîöåññ âûäåëåíèÿ ÐÍÊ è ðàç-
ðàáîòàíà òåñò-ñèñòåìà íà áàçå ÏÖÐ äëÿ äèàãíîñòèêè óêðàèí-
ñêèõ èçîëÿòîâ PVY. Ñåêâåíèðîâàí ó÷àñòîê ãåíà êàïñèäíîãî áåëêà
óêðàèíñêîãî èçîëÿòà è ïðîâåäåí ôèëîãåíåòè÷åñêèé àíàëèç. Ïîêà-
çàíî, ÷òî óêàçàííûé ãåí äåìîíñòðèðóåò âûñîêóþ ñòåïåíü ãîìî-
ëîãèè ñ ãåíàìè êàïñèäíûõ áåëêîâ ðåêîìáèíàíòíûõ èçîëÿòîâ
(øòàììîâ) (98,8–99,8 % ãîìîëîãèè äëÿ íóêëåîòèäíûõ è àìèíî-
êèñëîòíûõ ïîñëåäîâàòåëüíîñòåé). Óêðàèíñêèé èçîëÿò íàõîäèò-
ñÿ â îòäåëüíîì êëàñòåðå âìåñòå ñ ðåêîìáèíàíòíûìè èçîëÿòàìè
èç Ñèðèè, Èðàíà è ßïîíèè è èìååò ñ íèìè îáùåå ïðîèñõîæäåíèå.
Âûâîäû.  ðåçóëüòàòå ðàáîòû îïðåäåëåíû ñðåäñòâà äëÿ òî÷íî-
ãî ìîíèòîðèíãà âèðóñîâ êàðòîôåëÿ â àãðîýêîñèñòåìàõ Óêðàèíû.
Ôèëîãåíåòè÷åñêèé àíàëèç ïðîäåìîíñòðèðîâàë ðåêîìáèíàíòíóþ
ïðèðîäó èññëåäóåìîãî èçîëÿòà PVY, êîòîðûé ðàíåå áûë ïðèïèñàí
ê ãðóïïå øòàììîâ Î, ñóáêëàäû N:O.
Êëþ÷åâûå ñëîâà: Y âèðóñ êàðòîôåëÿ, ïîòèâèðóñ, ÏÖÐ, ôèëî-
ãåíåòè÷åñêèé àíàëèç, ðåêîìáèíàíòíûé øòàìì.
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Received 10.06.13
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