Complete genome sequence of a novel foveavirus isolated from Allium sativum L. in China.

The complete genome sequence of a novel foveavirus identified in garlic (Allium sativum L.) in China was determined using RNA-seq, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The entire genomic RNA (GenBank accession MT981417) is 8748 nucleotides long excluding the 3'-terminal poly(A) tail and contains five open reading frames (ORFs). These ORFs encode the viral replicase, a triple gene block, and a coat protein. The virus was tentatively named "garlic yellow stripe associated virus" (GarYSaV). Pairwise comparisons of protein sequences show that GarYSaV encodes proteins that share less than 47% identity with those of other foveaviruses, suggesting that it represents a new species in the genus. Phylogenetic analysis of amino acid sequences of the replicase and CP confirm that GarYSaV is a member of the genus Foveavirus. To our knowledge, this is the first report of a foveavirus in a monocot plant.

Questions surrounding the taxonomic validity of the species Garlic mite-borne filamentous virus (genus Allexivirus).

Garlic mite-borne filamentous virus is one of the oldest recognized allexivirus species but, paradoxically, one with the least well studied member viruses. In this paper, we review the history of this taxon and highlight problems in designating a holotype (exemplar isolate). Analyses are presented that suggest that GarMbFV is conspecific with Garlic virus A, and therefore the former taxon should be abolished.

Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

Allexiviruses may have acquired inserted sequences between the CP and CRP genes to change the translation reinitiation strategy of CRP.

Allexiviruses are economically important garlic viruses that are involved in garlic mosaic diseases. In this study, we characterized the allexivirus cysteine-rich protein (CRP) gene located just downstream of the coat protein (CP) gene in the viral genome. We determined the nucleotide sequences of the CP and CRP genes from numerous allexivirus isolates and performed a phylogenetic analysis. According to the resulting phylogenetic tree, we found that allexiviruses were clearly divided into two major groups (group I and group II) based on the sequences of the CP and CRP genes. In addition, the allexiviruses in group II had distinct sequences just before the CRP gene, while group I isolates did not. The inserted sequence between the CP and CRP genes was partially complementary to garlic 18S rRNA. Using a potato virus X vector, we showed that the CRPs affected viral accumulation and symptom induction in Nicotiana benthamiana, suggesting that the allexivirus CRP is a pathogenicity determinant. We assume that the inserted sequences before the CRP gene may have been generated during viral evolution to alter the termination-reinitiation mechanism for coupled translation of CP and CRP.

Application of nucleic acid aptamers for detection of Apple stem pitting virus isolates.

DNA aptamers (PSA-H and MT32) were applied for the detection of Apple stem pitting virus (ASPV) isolates using an Enzyme-Linked Oligonucleotide Assay (ELONA) and Western blot analysis. The specificity and effectiveness of aptamers were verified in comparison to a conventional Enzyme Linked Immunosorbent Assay (ELISA). A genetically diverse group of ASPV isolates was tested. The results showed that aptamer MT32 detected a wider range of ASPV isolates than an aptamer PSA-H and proved to be superior to commercially available monoclonal antibodies. Aptamer MT32 produced higher signal intensity in ELONA with a virus-infected plant extracts than antibodies in ELISA. Moreover, the ELISA method failed to detect ASPV in six samples. The results presented in this study indicated that aptamer MT32 can be used as a receptor molecule of various immunoassay protocols for ASPV detection.

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.

Divergent molecular variants of Grapevine virus B (GVB) from corky bark (CB)-affected and CB-negative LN33 hybrid grapevines.

Analysis of two Grapevine virus B (GVB)-infected LN33 hybrid grapevines revealed that a plant exhibiting clear symptoms of corky bark (CB) disease was infected with two molecular variants of the virus, whereas a plant exhibiting no disease symptoms was infected with only one variant. Sequence results indicated that the single variant in the CB-negative grapevine was also one of the two present in the CB-affected hybrid. Plant extracts from these two grapevines were used to successfully transmit the virus to N. benthamiana. After further cloning and sequencing, two clearly divergent variants were identified. Comparative molecular analysis of the variants, named here GVB 953-1 and GVB-H1, respectively, transmitted from CB-affected and consistently CB-negative plants, revealed short genomic regions, most of them highly divergent, that encoded amino acid sequences, containing significant amino acid substitutions altering the net charges of their respective proteins. Interestingly, a comparison of these variants to genome sequence data of GVB variants GVB Italy and GVB 94/971 available from the GenBank, revealed that these significant amino acid substitutions were the same for, and unique to, the variant pairs GVB 953-1/GVB Italy and GVB-H1/GVB 94/971. This despite the variants of each pair being otherwise clearly different at nucleotide and amino acid levels. In addition, both sets of variants differed substantially in their respective 3'-non-translated (3'NTR) regions. The relevance of these findings is discussed.

Allexivirus transmitted by eriophyid mites in garlic plants.

Viruses in garlic plants (Allium sativum L.) have accumulated and evolved over generations, resulting in serious consequences for the garlic trade around the world. These viral epidemics are also known to be caused by aphids and eriophyid mites (Aceria tulipae) carrying Potyviruses, Carlaviruses, and Allexiviruses. However, little is known about viral epidemics in garlic plants caused by eriophyid mites. Therefore, this study investigated the infection of garlic plants with Allexiviruses by eriophyid mites. When healthy garlic plants were cocultured with eriophyid mites, the leaves of the garlic plants developed yellow mosaic strips and became distorted. In extracts from the eriophyid mites, Allexiviruses were observed using immunosorbent electron microscopy (ISEM). From an immunoblot analysis, coat proteins against an Allexivirus garlic-virus antiserum were clearly identified in purified extracts from collected viral-infected garlic plants, eriophyid mites, and garlic plants infected by eriophyid mites. A new strain of GarV-B was isolated and named GarV-B Korea isolate 1 (GarV-B1). The ORF1 and ORF2 in GarV-B1 contained a typical viral helicase, RNA-directed RNA polymerase (RdRp), and triple gene block protein (TGBp) for viral movement between cells. The newly identified GarV-B1 was phylogenetically grouped with GarV-C and GarV-X in the Allexivirus genus. All the results in this study demonstrated that eriophyid mites are a transmitter insect species for Allexiviruses.

Grapevine vitivirus A eradication in Vitis vinifera explants by antiviral drugs and thermotherapy.

Grapevine shoot cultures infected by Grapevine vitivirus A (GVA) were grown on Quorin-Lepoivre basic medium and submitted to in vitro chemotherapy and thermotherapy sanitation techniques. Ribavirin (Rb) at 20gml(-1), dihydroxypropyladenine (DHPA) at 60gml(-1) and their combination (RbDH) were added to the proliferating medium for three subsequent subcultures of 30 days each. Phytotoxicity was observed on drug-treated plantlets, which displayed a high percentage of mortality for each drug at doses higher than those aforementioned. Sequential ELISA were performed at the end of each subculture and ELISA-negative explants were submitted to RT-PCR. ELISA showed no antiviral activity following DHPA administration. Rb and RbDH treatment produced ELISA-negative explants which were assayed by RT-PCR and nested PCR. Biomolecular results showed no virus eradication in Rb treated explants but RbDH administration generated a percentage (40.0%) of GVA-free plantlets that permitted restoration of a new healthy generation of explants. Sixty percent (60%) of GVA eradication as confirmed by RT-PCR was obtained by in vitro thermotherapy at 36 degrees C for 57 days.

[Cloning and expression of two garlic virus coat protein genes].

The coat protein(CP) genes of garlic mosaic virus(GMVc) and garlic latent virus(GLVc) isolated from garlic(Allium) plants in Tianjin, China, were amplified from an established cDNA library by PCR method and subsequently expressed in E. coli. using the pET-30a expression system. The determined sequences of GMVc and GLVc CP genes show that the complete GMVc CP gene has 867 nucleotides encoding 289 amino acids. It has 88.5% and 97.2% homology, at the levels of nucleotide and amino acid, respectively, to a reported GMV, indicating that it belongs to Potyvirus. The complete GLVc CP gene has 885 nucleotides coding for 294 amino acids. It has 73.6% and 90.9% homologous percents, in nucleotide and amino acid, respectively, compared to a previously reported GLV, suggesting that it is a member of Carlavirus. The expressed products presented in inclusion body and were analyzed by SDS-PAGE. The molecular weights of GMVc and GLVc CPs appear in 32 kD and 34 kD size, respectively, which are consistent with the deduced sizes of these two CPs. These data will be virtually significant to the further investigation of viruses infecting parlic plant, the control of garlic virus diseases and the production of virus-freed garlic plants.