Garlic Virus Research Articles

Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions

Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material.

N-terminal in coat protein of Garlic virus X is indispensible for its serological detection

Conserved coat protein region of plant viruses is often used as source of antigen for production of polyclonal antibodies for broad-based detection of closely related viruses. Antigenic region in coat protein is located either on N-terminal, and/or C-terminal or in the middle of coat protein. A study was undertaken to determine if antigenic region resides in N-terminal in Garlic virus X (GarV-X) of Allexivirus. In allexiviruses, N-terminal of coat protein region (1-57 amino acids) was highly variable. A complete coat protein of 27 kDa and a truncated protein without N-terminal (20 kDa) of GarV-X were expressed in pET expression vector and confirmed in western blotting using anti-His antisera. These expressed proteins were purified and used for antisera production. Specific and strong reaction was obtained for antisera generated against GarV-X full CP and GarV-X was detected in field-grown allium crops viz., onion, garlic, leek, and bunching onion and chives in ELISA. Antisera against GarV-X CPΔ1-61 (truncated CP) did not show reaction for GarV-X detection in immunoassay. Epitope mapping also indicated N-terminal as major antigenic determinant region with highest antigenic signal score. Our studies confirm that antigenic signals or epitopes reside in the N-terminal region of GarV-X which can be synthesized and used for production of monoclonal antibodies for specific detection purposes.

Występowanie wirusów z rodzaju Allexivirus na roślinach czosnku w Polsce

Garlic (Allium sativum L.) can be infected by numerous viruses that are spread by the seed bulbs during the vegetative propagation of the plants. Eight garlic viruses that belong to the Allexivirus genus are transmitted by mites. The aim of this study was to detect and identify Allexiviruses infecting garlic in Poland. Identification of Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and Shallot virus X (ShVX) was based on ELISA (Enzyme-Linked Immunosorbent Assay) and confirmed by RT-PCR (Reverse Transcription Polymerase Chain Reaction). Garlic virus D (GarV-D), Garlic virus E (GarV-E) and Garlic virus X (GarV-X) were detected using RT-PCR. The samples of garlic plants were collected in September 2011 and July 2012 from garlic production field located in different part of Poland. Garlic virus D, Garlic virus X and Garlic virus B were the most abundant garlic viruses in all examined regions and were identified in 84, 67 and 54% of all samples, respectively. Garlic virus A and Garlic virus C were detected in all studied regions with low frequency. None of the tested garlic samples were infected with Shallot virus X. Allexiviruses that were present in garlic plants occurred always in mixed infections. Rośliny czosnku mogą być porażane przez wiele gatunków wirusów. Wirusy infekujące czosnek rozprzestrzeniają się wraz z materiałem rozmnożeniowym. Ponadto osiem gatunków należących do rodzaju Allexivirus jest przenoszonych przez szpeciele. Celem przeprowadzonych badań było wykrywanie i identyfikacja allexiwirusów infekujących rośliny czosnku w Polsce. Obecność wirusa A czosnku, wirusa B czosnku, wirusa C czosnku i wirusa X szalotki stwierdzano przy pomocy testu serologicznego ELISA (Enzyme-Linked Immunosorbent Assay) i potwierdzano techniką RT-PCR (Reverse Transcription Polymerase Chain Reaction). Wirus D czosnku, wirus E czosnku i wirus X czosnku były wykrywane przy użyciu techniki RT-PCR. Próbki materiału roślinnego pobierano w dwóch terminach, we wrześniu 2011 roku oraz w lipcu 2012 roku, z pól produkcyjnych, zlokalizowanych w różnych rejonach Polski. Najczęściej występującymi wirusami w polskich uprawach czosnku był wirus D czosnku, wirus X czosnku i wirus B czosnku (84, 67 i 54%). Wirus A czosnku i wirus C czosnku były wykrywane we wszystkich badanych regionach, jednak ze znacznie niższą częstotliwością występowania. Żadna z badanych prób nie była porażona przez wirus X szalotki. Allexiwirusy w roślinach czosnku występowały zawsze w mieszanych infekcjach.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

BackgroundGarlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors.ResultsIn this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR.ConclusionsThe expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

IN VITRO EXPRESSION AND PURIFICATION OF THE COAT PROTEIN GENE OF GARLIC COMMON LATENT VIRUS AND ITS APPLICATION IN ELISA-BASED DIAGNOSTICS

The coat protein (CP) gene of Garlic common latent virus (GarCLV) was expressed in Escherichia coli strain BL21 expression system as a 40 kDa fusion protein with histidine tag (6His) at its N and C terminals. The purified protein reacted positively in Western blot with a commercial GarCLV polyclonal antiserum and hence, used as immunogen for production of polyclonal antisera in two New Zealand white rabbits. Antisera to GarCLV (titer 1:2000) detected the virus by direct antigen-coated ELISA in GarCLV positive garlic samples. Further, the specific reactivity of the antisera was confirmed through Western blotting and immunosorbent electron microscopy. Antisera developed were successfully utilized for detection of GarCLV in 46 out of 48 different garlic accessions. The immunoreagents developed will be useful for virus indexing in garlic tissue culture programs as well as in quarantine and/or certification programs.

Occurrence and Phylogenetic Analysis of Potyviruses, Carlaviruses and Allexiviruses in Garlic in Sudan

AbstractAllium sativum L. samples of three cultivars grown in Sudan were shown to contain several of the common garlic viruses of the genera Potyvirus, Carlavirus and Allexivirus. In particular, Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GarCLV), Garlic virus A (GVA), Garlic virus B (GVB), Garlic virus C (GVC) and Garlic virus X (GVX) were detected by reverse transcription polymerase chain reaction (RT‐PCR) assay using degenerate genus‐specific and virus‐specific primer sets. Multiple viral infections with members of all three genera were found in all but one sample. Further, molecular and phylogenetic analysis of nucleotide and deduced amino acid sequences revealed that Sudanese isolates of LYSV, GarCLV and of Allexivirus genus were significantly divergent in partial coat protein genomic region from isolates of the same species retrieved from GenBank. This is the first report and study on viruses infecting garlic in Sudan.

Molecular characterization of coat protein gene of Garlic common latent virus isolates from India: an evidence for distinct phylogeny and recombination

The coat protein (CP) gene of five Indian Garlic common latent virus (GarCLV) isolates was sequenced and it was 960 bp long in all the five isolates, encoding a protein of 319 amino acids. Comparative nucleotide sequence analysis revealed diversity of 4.3% among the Indian isolates and of 11.9% among all isolates worldwide. Amino acid sequence comparison showed a significant variability in the N-terminal of CP of GarCLV. Various protein analysis tools identified thirteen conserved domains and motifs including Carlavirus and Potexvirus-specific Flexi CP and Flexi N CP. Phylogenetic analysis clustered GarCLV isolates in the subgroup II with isolates from Australia, Brazil, Japan, and South Korea. Intraspecies recombination study revealed that only one of the Indian isolates was a recombinant. Interspecies recombination study suggested the absence of genetic exchange from Carlavirus species to GarCLV; conversely, GarCLV was identified as a putative donor for at least two other Carlavirus species. This is the first report of molecular variability and recombination in GarCLV isolates.

First Report of Garlic common latent virus Infecting Garlic in Sudan.

Garlic (Allium sativum L.) is one of the most important vegetable field crops in Sudan, cultivated on an area of more than 6,000 ha with a total yield of 27,000 t in 2010 (faostat.fao.org). As part of a project which started in 2010 to improve the garlic production in Sudan, samples from local varieties showing severe mosaic and/or mottling were collected in winter 2011 from the main production areas in River Nile State, Northern State, and Darfur State. The plant material used for garlic production came from Sudan and was not imported. Because no reliable data were available on which viruses occur in garlic in Sudan, specific tests were initially omitted. In order to get an overview of the viruses present, dsRNA was prepared of a mixed leaf sample (12 leaves of different samples). This resulted in a high molecular weight dsRNA of approximately 9 kbp that served as template for a random RT-PCR followed by cloning and sequencing (3). Three identical clones originating from one PCR product covering the C-terminal part of the coat protein to the N-terminal part of the nucleic acid binding protein showed the highest sequence similarity to Garlic common latent virus (GarCLV). The nucleotide sequence identities of the 554-bp insert range from 85% to an isolate from India (Accession No. FJ154841) up to 97% to a GarCLV isolate from The Netherlands (AB004804), identifying the virus as a Sudanese isolate of GarCLV, one of the most common garlic infecting viruses. GarCLV belongs to the genus Carlavirus (1) and has previously been reported from Asia, Europe, and South America ( http://sdb.im.ac.cn/vide/descr352.htm ). In order to confirm these results, a double antibody sandwich (DAS)-ELISA was performed with six individual garlic samples in which five samples showed a clear reaction with a GarCLV specific antiserum (AS-0230, DSMZ, Germany). The occurrence of GarCLV could be further confirmed for the ELISA positive samples by a specific RT-PCR using the primers published by Majumder and Baranwal (2). Fragments of the expected size were obtained for all five samples. In addition, one of the positive samples was examined by electron microscopy (Dr. K. Richert-Pöggeler, JKI Braunschweig); filamentous flexous particles typical for carlaviruses could be observed. The random RT-PCR sequence obtained in this study has been submitted to GenBank (KC013030). To our knowledge, this is the first report of GarCLV in garlic in Sudan and Africa. The impact of GarCLV on garlic production in Sudan needs to be evaluated, but the awareness of the occurrence of the virus and the availability of a reliable diagnostic tool will help to select virus-free propagation material. This will form the basis for a sustainable garlic production.

The Mechanisms Responsible for Garlic - Drug Interactions and their In Vivo Relevance

Garlic phytochemicals and garlic supplements influence the pharmacokinetic and pharmacodynamic behavior of concomitantly ingested drugs. In this paper we have summarized the mechanisms responsible for first-pass intestinal pharmacokinetic interactions by investigating the intestinal permeability of some cardiovascular, antiviral drugs, their transport with hepatic transporters and CYP3A4 metabolism. Transporter-enzyme interplay was studied with several in vitro models of varying complexity: rat small intestine and Caco-2 cell monolayers were used in studies of intestinal processes, and hepatic pharmacokinetics was monitored in HepG2 cells, isolated rat hepatocytes and rat liver slices. Garlic phytochemicals from aged garlic extract modified the activities of secretory and absorptive transporters in both intestine and liver and competitively inhibited CYP3A4 enzyme. The increased activities of the most important intestinal efflux (P-glycoprotein - Pgp, Multidrug Resistance Associated Protein 2 - MRP-2, Breast Cancer Resistance Protein - BCRP) and uptake (MonoCarboxylate Transporter 1 - MCT1, Organic Anion Transporting Polypeptide - OATP, Peptide transporter 1 - PepT1) transporters were caused by changes in electrophysiological membrane properties and by allosteric modifications. Because clinical studies investigating interactions between garlic and human immunodeficiency virus protease inhibitors saquinavir and ritonavir have already been performed, we used these in vivo data to evaluate the in vitro results and the reliability of the models employed as screening tools for forecasting the potential of first-pass intestinal metabolism changes. We also assessed the probability of pharmacokinetic interactions with garlic of the novel drug darunavir and other cardiovascular drugs. Finally, selected garlic phytochemicals were tested for their ability to influence P-glycoprotein and CYP3A4 activities.

Multiple Important Plant Viruses Are Present On Vegetable Crops in Kuwait

Plant virus diseases are wide-spread in Kuwait and cause significant economic losses in many crops. Four plant families, including Cucurbitaceae (cucumber, squash, melon, zucchini), Solanaceae (tomato, potato, pepper, eggplant), Liliaceae (onion) and Leguminosaea (bean) were surveyed for viruses by ELISA from December 2007 through May 2008. The survey was conducted on six farms located in the two main production areas, Wafra and Abdally agricultural districts. Each farm was visited four times during the production season. Eighteen plant viruses were detected, including Cucumber mosaic virus, Garlic common latent virus, Iris yellow spot virus, Onion yellow dwarf virus, Melon necrotic spot virus, Papaya ringspot virus, Pepino mosaic virus, Pepper mild mottle virus, Potato moptop virus, Potato virus M, Potato virus X, Potato virus Y, Squash mosaic virus, Tomato mosaic virus, Tomato spotted wilt virus, Tomato yellow leaf curl virus, Watermelon mosaic virus, and Zucchini yellow mosaic virus. Virus incidence was close to 100% on some crops, including cucurbit and onions and double or triple infections were common. Several important viruses were not found, including Potato virus A, Potato virus S, Potato leafroll virus, and Potato virus V.

Viruses occurring in onion crop in amasya province, the major onion producing region in Turkey.

Amasya is the greatest onion producing area in Turkey. Onion fields from Amasya region were surveyed for virus diseases in 2009-2011 and tested for the presence of the most important onion viruses such as Onion yellow dwarf virus (OYDV), Iris yellow spot virus (IYSV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and Garlic common latent virus (GCLV). The presence of virus diseases and their identification was ascertained through symptom observation in the fields, sap transmission to hosts, and DAS-ELISA. Based on the ELSA results, 57 out of 332 samples (17.16%) were infected with viruses. The results showed that the highest infection was caused by OYDV (12.33%) followed by LYSV (3.60%). Only 1.19% of the samples were infected with SLV, but none of the samples were found to be infected for GCLV and IYSV.

The Mechanisms Responsible for Garlic - Drug Interactions and their In Vivo Relevance

Garlic phytochemicals and garlic supplements influence the pharmacokinetic and pharmacodynamic behavior of concomitantly ingested drugs. In this paper we have summarized the mechanisms responsible for first-pass intestinal pharmacokinetic interactions by investigating the intestinal permeability of some cardiovascular, antiviral drugs, their transport with hepatic transporters and CYP3A4 metabolism. Transporter-enzyme interplay was studied with several in vitro models of varying complexity: rat small intestine and Caco-2 cell monolayers were used in studies of intestinal processes, and hepatic pharmacokinetics was monitored in HepG2 cells, isolated rat hepatocytes and rat liver slices. Garlic phytochemicals from aged garlic extract modified the activities of secretory and absorptive transporters in both intestine and liver and competitively inhibited CYP3A4 enzyme. The increased activities of the most important intestinal efflux (P-glycoprotein - Pgp, Multidrug Resistance Associated Protein 2 – MRP-2, Breast Cancer Resistance Protein - BCRP) and uptake (MonoCarboxylate Transporter 1 – MCT1, Organic Anion Transporting Polypeptide - OATP, Peptide transporter 1 – PepT1) transporters were caused by changes in electrophysiological membrane properties and by allosteric modifications. Because clinical studies investigating interactions between garlic and human immunodeficiency virus protease inhibitors saquinavir and ritonavir have already been performed, we used these in vivo data to evaluate the in vitro results and the reliability of the models employed as screening tools for forecasting the potential of first-pass intestinal metabolism changes. We also assessed the probability of pharmacokinetic interactions with garlic of the novel drug darunavir and other cardiovascular drugs. Finally, selected garlic phytochemicals were tested for their ability to influence P-glycoprotein and CYP3A4 activities. Keywords: CYP3A4, flavonoids, garlic supplement, sulfur containing compounds, permeability, pharmacokinetic interactions, transporters

Characterization of viruses associated with garlic plants propagated from different reproductive tissues from Italy and other geographic regions

Garlic is an important crop cultivated worldwide and several different viruses have been associated with propagative material. Garlic is propagated from bulbs and/or from vegetative topsets of the inflorescences known as bulbils. The effects of the geographic origin and the type of the propagative material on the phylogenetic relationships and genetic variability of the coat protein genes of four allium viruses are presented here. Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic virus X (GVX), and Garlic common latent virus (GCLV) were detected in single and mixed infections in plants grown either from bulbils and/or bulbs originating from Italy, China, Argentina, and the U.S.A. OYDV and LYSV fell into five and three well supported clades respectively whereas isolates of GVX and GCLV all clustered into one well-supported clade each. Some of the OYDV and LYSV clades presented evidence of host tissue selection while some phylogenetic structuring based on the geographic origin or host was also observed for some virus clades. Unique haplotypes and novel coat protein amino acid sequence patterns were identified for all viruses. An OYDV coat protein amino acid signature unique to Chenopodium quinoa , an uncommon host of the virus, was of particular interest. The type of propagative material affected the population dynamics of all of the viruses. The virus populations in plants propagated from bulbs were more diverse than in plants propagated from bulbils.

Phylogenetic analysis of allexiviruses identified on garlic from Australia

Partial genome sequences with identity to members of the genus Allexivirus were described in Australia from Allium sativum L. (garlic). Phylogenetic analysis of coat protein and nucleotide binding protein gene sequences with those of 82 other allexivirus isolates confirmed that the Australian isolates were members of Garlic virus A, Garlic virus B, Garlic virus C, and Garlic virus D.

Spread of viruses in garlic fields cultivated under different agricultural production systems in Brazil

Samples from four regions, representing 76% of the garlic growing area in Brazil, were tested by RT-PCR for the presence of Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV), Garlic virus C (GarV-C), Garlic virus D (GarV-D) and Garlic mite-borne filamentous virus (GarMbFV). The samples (352 bulbs) represented five agricultural systems: traditional common garlic (CG), virus-free common garlic (VFCG), non-vernalized noble garlic (NVNGSG) and vernalized noble garlic cultivated by small growers (VNGSG), and vernalized noble garlic adopted by major growers (VNGMG). Multiple infections were detected in 22% of the samples. Potyvirus species were present in all regions. LYSV prevailed over OYDV while the carlavirus GCLV was less prevalent. GarV-C and GarMbFV were the most prevalent among the allexivirus species. The NVNG production system had a higher prevalence of LYSV and GarV-C. The CG production system, that uses less technology, had the highest prevalence for all species, especially LYSV that prevailed in 94% of the samples. Overall, the regions with higher technological input employing better quality seeds had the lowest viral prevalence for all species. This monitoring provides information to establish a strategy to raise the phytosanitary quality and the national productivity of garlic.

First Report of Garlic virus X Infecting Garlic in India.

Garlic (Allium sativum) is an important crop in several states of India. Filamentous viruses such as Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), and Garlic common latent virus (GarCLV) have been reported previously in different garlic cultivars from India (4). These viruses are transmitted from generation to generation through cloves and cause severe reduction in yield and quality. During December 2010, garlic plants were observed with mosaic leaf symptoms and stunting in an experimental field at the Indian Agricultural Research Institute, Delhi. Cloves and leaves from 3-month-old symptomatic plants of five different cultivars (G-282, IC-375416, Ruag, Yamuna Safed, and ACC-40), originally from different regions of India, were collected from the field in Delhi and total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The presence of OYDV and GarCLV was confirmed by reverse transcription (RT)-PCR in all cultivars, while the presence of SLV was only confirmed in cv. G-282 by RT-PCR. Since Allexiviruses are common in garlic, their detection in cloves was confirmed by RT-PCR using primers ALLEX 1 and ALLEX 2 (2). An ~200-bp amplification product was observed in all five cultivars. To further characterize the Allexivirus in these cultivars, an amplicon of ~900 bp was amplified with Allex-CP (1) and ALLEX 2 (2) primers and cloned and sequenced. BLAST analysis of the nucleotide sequences from five garlic cultivars showed identity with different allexiviruses, Garlic virus A (GarV-A) (74 to 83%), Garlic virus E (GarV-E) (74 to 80%), Garlic virus D (GarV-D) (76 to 79%), and Garlic virus X (GarV-X) (75 to 78%). Since species demarcation in the genus Allexivirus is based on the coat protein (CP) gene (3), another set of primers, 5'-MYT KGA GTG GCT VAC ACA YAT-3' and 5'-ATT RAA GTC GTG RGG ATG CAT-3' was designed. These primers were derived from conserved regions of ORF4 and ORF5 (CP) sequences of allexiviruses available in the NCBI database and used in RT-PCR to obtain the complete CP. An ~1.5-kb amplicon was obtained only in cv. G-282 that originated from the southern part of India. A similar amplicon was obtained from Chenopodium amaranticolor mechanically inoculated with leaf sap from cv. G-282. Sequences (1,422 bp) obtained from three clones each from garlic cv. G-282 and C. amaranticolor were identical and BLAST analysis of the consensus nucleotide sequence showed maximum identity of 75 to 81% with isolates of GarV-X. The 1,422 nucleotide sequence was comprised of 690 bp of ORF4 (partial) and 732 bp of the CP. The coat protein sequence (GenBank Accession No. HQ822272) shared a 79.6 to 81.1% identity in nucleotide and 89.3 to 90.9% in amino acid sequence with different isolates of GarV-X (GenBank Accession Nos. AJ292229, U89243, and GQ475426). To our knowledge, this is the first report of GarV-X in a garlic cultivar from India. The characterization and identification of allexiviruses is important for production of virus-free garlic plants through tissue culture in India.

青森県のニンニク (品種福地ホワイト) におけるニンニク潜在ウイルスの発生

青森県のニンニク (品種福地ホワイト) におけるニンニク潜在ウイルスの発生

산마늘(Allium victorialis var. platyphyllum)에서 바이러스병의 최초보고

2005년 울릉도에 자생하고 있는 산마늘에 대하여 바이러스병을 조사하기 위하여 성인봉 부근에서 61점의 시료를 채집하였다. 채집한 시료를 동정하기 위해 전자현미경 검경과 종 특이적 프라이머(GCLV, LYSV, SLV, OYDV) 및 속 특이적 프라이머(Allexivirus)를 이용하여 RT-PCR을 각각 수행하였다. 전자현미경 검경을 실시한 결과 61점의 시료중 4점의 시료에서 600~900 nm의 긴 사상형 입자가 관찰되었으며, RT-PCR 진단결과 SLV가 4점, 이 중 하나는 Allexivirus가 복합감염되었다. 바이러스에 감염된 산마늘은 외관상으로 병징을 나타내지 않았다. RT-PCR 분석결과 SLV와 Allexivirus로 동정된 울릉도 산마늘 바이러스의 외피단백질 유전자 염기서열을 비교 분석하였다. 분석결과, SLV로 분류된 울릉도 산마늘 바이러스 개체간은 약 99%의 상동성을 가지고 있었으며, 이전에 보고된 다양한 SLV와 GLV의 strain과의 염기 서열의 비교에서는 75.7~83.7%의 상동성을 보였으며 아미노산 서열의 비교는 89.2~97.0%의 높은 상동성을 나타냈다. 또한 산마늘에서 검출된 GarV-A는 이전에 마늘에서 보고된 GarV-A와 99.2% 외피단백질 유전자 염기서열 상동성을 보였으며, 아미노산 서열은 98% 상동성을 보였다. 그러나 다른 Allexiviruses(GarV-C, GarV-E, GarV-X, GMbMV and Shal X)와 아미노산 서열을 비교한 결과 60.6%~81.5%의 상대적으로 낮은 상동성을 보였다. 이러한 자료들을 바탕으로 산마늘에서 분리한 SLV와 GarV-A를 각각 SLV-Ulleungdo와 GarV-A-Ulleungdo로 명명하였다. 본 논문은 산마늘에서 발생하는 바이러스에 대한 첫 번째 보고이다. In 2005, a survey was conducted to identify virus diseases on victory onion, Allium victorialis var. platyphyllum grown in Ulleung island located in the East Sea. A total of 61 samples were collected from victory onion in the neighborhood of Seonginbong. The identification of viruses from the samples were carried out by electron microscopy and RT-PCR using primers species specific to GCLV, LYSV, SLV, OYDV and genus specific to Allexivirus, respectively. From sixty-one samples, filamentous rod particles (600-900 nm) were detected from four victory onion samples in EM, three samples containing SLV and one sample containing both SLV and Allexivirus in RT-PCR analysis, respectively. Victory onions naturally infected by the viruses were asymptomatic apparently. The viruses detected by RT-PCR were further characterized by the nucleotide sequence analysis of the coat protein region. Three isolates of SLV showed approximately 99% identities in the nucleotide and amino acid sequences, suggesting that they were likely to be the same strain. On the other hand, they showed approximately 75.7~83.7% identities in the nucleotide and 89.2~97.0% in amino acid sequences compared with the previously reported SLV isolates in Allium. The CP gene of the Allexivirus showed approximately 99.2% nucleotide identities and 98.8% amino acid identities with Garlic virus A. However, there was relatively low homology ranging from 60.6% to 81.5% compared with other Allexiviruses (GarV-C, GarV-E, GarV-X, GMbMV, and Shal-X). These data suggested that two viruses, SLV and GarV-A identified from victory onion, are named SLV-Ulleungdo and GarV-A-Ulleungdo, respectively. This is the first report of viruses infecting victory onion.

Partial genome sequence of a Potyvirus and of a virus in the order Tymovirales found in Senna macranthera in Brazil

Senna macranthera is a common tropical tree native to South- and Northeastern Brazil. In July 2006, symptoms of chlorotic spots in the young leaves and mild mosaic in the old leaves were observed in S. macranthera trees at Vicosa, State of Minas Gerais, Brazil. Electron microscopy examination of leaf dip preparations from the young leaves revealed the presence of flexuous rods. Viral RNA was extracted from purified virion preparations and used as a template for the RT-PCR-based cloning of viral genomic fragments. Two fragments (0.9 kb and 2.0 kb in length) were cloned. Sequence analysis of the 0.9 kb fragment indicated that it corresponded to the NIb coding region of a potyvirus, with nucleotide (nt) identity level of 72% for Pepper mottle virus (PepMoV) and Peru tomato mosaic virus (PTV), while analysis of the 2.0 kb fragment indicated that it corresponded to the putative replicase ORF, which displayed 74% nt identity with the ORF1 (replicase) of Garlic virus A (GarV-A). Based on molecular analysis and the isolates' host range, it is concluded that the viral fragments represent new viruses, for which we propose the names Senna virus Y (genus Potyvirus, family Potyviridae) and Senna virus X (unassigned genus, order Tymovirales).

First Report of Shallot latent virus in Garlic in Brazil.

Garlic (Allium sativum L.) can be infected by several viruses of the genera Allexivirus, Carlavirus, and Potyvirus (3). Garlic common latent virus (GarCLV) and Shallot latent virus (SLV) are the most important Carlavirus species infecting garlic, but only GarCLV has been described on garlic in Brazil. Seven hundred thirty-one samples of garlic showing mosaic symptoms and chlorotic streaking were collected from the states of São Paulo (São Manuel), Minas Gerais (Santa Juliana and São Gotardo), Goiás (Campo Alegre and Ipameri), and Paraná (Guarapuava and Piraquara) from April 2008 to July 2009 and analyzed by double-antibody sandwich (DAS)-ELISA for the presence of GarCLV and SLV using specific antiserum for SLV and GarCLV according to the manufacturer's protocol (Agdia Inc., Elkhart, IN). Cultivars sampled were Caçador, Chonan, Ito, Jonas, Quitéria, and Tropical. Fifty-five samples (7.5% of 731) tested positive for GarCLV, and none of the samples tested positive for SLV. Total RNA was extracted (2) from 15 samples that represented different states of production and used with primers SLV 7044 (5'-CTTTTGGTTCACTTTAGG-3') and SLV 8004 (5'-GCACGCAATAGTCTACGG-3'), designed in this study, to detect SLV in a one-step reverse transcription (RT)-PCR assay. Only 3 of the 15 samples, two from São Paulo and one from Paraná State, produced a 960-bp fragment covering the putative coat protein gene (ORF 5) (1) of SLV. The amplicons of the three isolates were sequenced. A nucleotide sequence identity of 91 to 92% was detected in comparison with two strains of SLV (GenBank Accession Nos. AB004567 and DQ520093), indicating the presence of two isolates of SLV in São Manuel (São Paulo State) and one in Piraquara, Paraná State (submitted to GenBank as Accession Nos. GU120176, HQ128602, and GU120175, respectively). To confirm identity of the virus, another pair of primers was constructed and tested (SLV 6737: 5'-YCCSGCCARGAAYTTCCC-3', and SLV 7060: 5'-TTAGAGCGCTGTWAACC-3'), from which a 340-bp fragment covering a portion of TGB2 (ORF 3) and TGB3 (ORF 4) (1) was amplified using the two isolates from São Paulo (GenBank Accession Nos. HQ123181 and HQ123182, respectively). The amplicon sequences shared 87% identity with that of an SLV isolate (Accession No. AJ292226), which confirmed the presence of SLV. The low titer of SLV in garlic might account for the false negative results by DAS-ELISA. The source of cultivated garlic bulbs in these regions of Brazil is unknown. Garlic cloves have been cultivated in São Manuel for approximately 15 years, indicating that SLV may have been present in Brazil for many years. To our knowledge, this is the first report of SLV in Brazil.