Kundu J K
Department of Virology, Crop Research Institute, Prague 6, 161 06 Czech Republic. Research sponsored by project No. MZE 0002700603 and QH81269.
Plant Dis. 2008 Nov;92(11):1587. doi: 10.1094/PDIS-92-11-1587B.
Barley yellow dwarf disease, an important, ubiquitous virus disease of cereal crops worldwide, is caused by a group of related single-stranded RNA viruses assigned to luteovirus (Barley yellow dwarf virus (BYDV) spp. PAV, PAS, MAV, and GAV) or polerovirus (Cereal yellow dwarf virus-RPV) genera or unassigned to a genera (BYDV-SGV, BYDV-RMV, and BYDV-GPV) in the family Luteoviridae (2). Incidence of BYDV in cereal crops (e.g., barley, wheat, and oats) was high and reached epidemic levels in recent years in many regions of the Czech Republic. Previously, only PAV isolates have been identified here on the basis of serological detection (4), although antibodies to differentiate between PAV, PAS, and MAV are not widely available. Field samples of cereal crops were routinely tested in 2006 and 2007 and BYDVs were detected by ELISA. One-step-reverse transcription (RT)-PCR (Qiagen, Hilden, Germany) was adapted for BYDV detection using primer pairs BYcpF (5'-CCACTAGAGAGGTGGTGAATG-3') and BYcpR (5'-CCGGTGTTGAGGAGTCTACC-3') designed from conserved sequences identified by aligning multiple BYDV sequences available in public databases. These primers amplify a 641-bp fragment spanning nucleotides 2839-3479 from PAV (GenBank Accession No. EF043235) or PAS (GenBank Accession No. NC_002160) that includes a region of the coat protein gene and the intergenic region. RT-PCR amplicons were generated from two field isolates, PS-RuJK (spring wheat isolate, cv. Granny, collected in July 2007 from experimental plots at the CRI in Prague) and JE-120JK (winter barley isolate, cv. Merlot, collected in January 2008 from a barley field in Rychnov), both of which induced severe BYD symptoms. Amplicons were sequenced in both directions in a CEQ2000XL sequencer (Beckman Coulter, Fullerton, CA). The partial coat protein gene sequence of 483 nt of PS-RuJK and JE-120JK was analyzed and compared with available sequences of 26 PAV, 17 PAS, and 13 MAV isolates by MEGA4 (3). PS-RuJK (GenBank Accession No. EU863652) nucleotide and amino acid sequence identities ranged from 96.3 to 99.2% and 93.7 to 98.7%, respectively, for available PAS isolates, and 89.9 to 90.5% and 85.5 to 86.9%, respectively, for available PAV isolates, and 78.3 to 79.5% and 70.0 to 72.5%, respectively, for available MAV isolates. Similarly, nucleotide and amino acid sequence identities JE-120JK (GenBank Accession No. EU863653) ranged from 95.2 to 98.6% and 90.6 to 96.9%, respectively, for PAS isolates, 88.8 to 90.1% and 83.1 to 84.4%, respectively, for PAV isolates, and 77.6 to 78.7% and 67.5 to 70.0%, respectively, for MAV isolates. Also, both of these isolates have the conserved amino acid motif "SIPGS" that is usually present in a variable region of the coat protein gene on the surface of virion (1) at position 52 to 56 of amino acid sequences of all published PAS-like isolates, including Vd29:AY167109, FH1:AJ223588, MA9516:AJ007926, FL2:AJ223586, ASL-1:AJ810418, and WS6603:DQ285680, contrary to "PVFRP" or "LISGP" motif in PAV or MAV, respectively. Therefore, the sequence data clearly confirm that these two isolates belong to the PAS species. To our knowledge, this is the first record of PAS detected in the Czech Republic. References: (1) C. A. Chay et al. Phytopathology 86:370, 1996. (2) C. J. D'Arcy and L. L. Domier. Page 891 in: Virus Taxonomy-8th Report of the ICTV. C. M. Fauquet et al., eds. Springer-Verlag, NY, 2005. (3) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007. (4) J. Vacke. Page 100 in: Sbornik Referatu z Odborneho Seminare, Aktualni Problemy Ochrany Polnich Plodin. Praha, 1991.
大麦黄矮病是一种重要的、在全球谷类作物中普遍存在的病毒病，由一组相关的单链RNA病毒引起，这些病毒被归类于黄症病毒属（大麦黄矮病毒（BYDV）的PAV、PAS、MAV和GAV株系）、马铃薯卷叶病毒属（谷类黄矮病毒-RPV）或未归类于任何属（BYDV-SGV、BYDV-RMV和BYDV-GPV），属于黄症病毒科（2）。近年来，在捷克共和国的许多地区，BYDV在谷类作物（如大麦、小麦和燕麦）中的发病率很高，并达到了流行水平。此前，尽管用于区分PAV、PAS和MAV的抗体并不广泛可用，但在此仅基于血清学检测鉴定出了PAV分离株（4）。2006年和2007年对谷类作物的田间样本进行了常规检测，并通过ELISA检测到了BYDV。采用一步逆转录（RT）-PCR（德国希尔德市的Qiagen公司），使用根据公共数据库中多个BYDV序列比对确定的保守序列设计的引物对BYcpF（5'-CCACTAGAGAGGTGGTGAATG-3'）和BYcpR（5'-CCGGTGTTGAGGAGTCTACC-3'）来检测BYDV。这些引物从PAV（GenBank登录号EF043235）或PAS（GenBank登录号NC_002160）中扩增出一个641 bp的片段，该片段跨越核苷酸2839 - 3479，包括衣壳蛋白基因的一个区域和基因间隔区。从两个田间分离株PS-RuJK（春小麦分离株，品种为Granny，2007年7月从布拉格CRI的试验田中采集）和JE-120JK（冬大麦分离株，品种为Merlot，2008年1月从Rychnov的一个大麦田中采集）产生了RT-PCR扩增产物，这两个分离株均引发了严重的BYD症状。扩增产物在CEQ2000XL测序仪（美国加利福尼亚州富勒顿市的贝克曼库尔特公司）中进行双向测序。对PS-RuJK和JE-120JK的483 nt的部分衣壳蛋白基因序列进行了分析，并通过MEGA4（3）与26个PAV、17个PAS和13个MAV分离株的可用序列进行了比较。对于可用的PAS分离株，PS-RuJK（GenBank登录号EU863652）的核苷酸和氨基酸序列同一性分别为96.3%至99.2%和93.7%至98.7%；对于可用的PAV分离株，分别为89.9%至90.5%和85.5%至86.9%；对于可用的MAV分离株，分别为78.3%至79.5%和70.0%至:72.5%。同样，对于PAS分离株，JE-120JK（GenBank登录号EU863653）的核苷酸和氨基酸序列同一性分别为95.2%至98.6%和90.6%至96.9%；对于PAV分离株，分别为88.8%至90.1%和83.1%至84.4%；对于MAV分离株，分别为77.6%至78.7%和67.5%至70.0%。此外，这两个分离株在所有已发表的PAS样分离株（包括Vd29:AY167109、FH1:AJ223588、MA9516:AJ007926、FL2:AJ223586、ASL-1:AJ810418和WS`6603:DQ285680）的氨基酸序列的第52至56位都具有保守的氨基酸基序“SIPGS”，该基序通常存在于病毒粒子表面衣壳蛋白基因的可变区域（1），与PAV或MAV中的“PVFRP”或“LISGP”基序相反。因此，序列数据清楚地证实这两个分离株属于PAS种。据我们所知，这是在捷克共和国检测到PAS的首次记录。参考文献：（1）C. A. Chay等人，《植物病理学》86:370，1996年。（2）C. J. D'Arcy和L. L. Domier。载于：《病毒分类学 - ICTV第8次报告》。C. M. Fauquet等人编。施普林格出版社，纽约，2005年，第891页。（3）K. Tamura等人，《分子生物学与进化》:24:1596，2007年。（4）J. Vacke。载于：《保护植物的当前问题 - 防御研讨会论文集》第100页。布拉格，1991年。
