Mixed infection of Sida jamaicensis in Jamaica reveals the presence of three recombinant begomovirus DNA A components

Begomoviruses impose serious constraints on agriculture throughout the temperate, tropical and subtropical regions. Previously, we characterised a sida golden yellow vein virus isolate, SiGYVV-[JM:Lig2:08] (HQ009519-20) from a symptomatic Sida jamaicensis plant. With the aim of establishing whether it was hosting a mixed infection that could facilitate recombination, PCR-RFLP was done on DNA extracted from this plant, and the results suggested the presence of two additional genetically distinct DNA-A molecules. Sequence analysis of these two DNA-A molecules (relying on BLAST searches and the CLUSTAL V algorithm within the DNASTAR MegAlign module) revealed that they belonged to novel species, and we have tentatively named these viruses sida golden mosaic Braco virus-[Jamaica:Liguanea:2008] and sida golden mosaic Liguanea virus-[Jamaica:1:2008]. Using RDP4 (recombination detection program), we determined that all three viruses were recombinant, with bases ~10 to ~440 of both SiGMLigV-[JM:Lig:08] and SiGYVV-[JM:Lig2:08] having been derived from a relative of SiGMBV-[JM:Lig:08] (P < 2.070 × 10^?7 for all seven of the recombination detection methods). SiGMBV-[JM:Lig:08] was itself a product of recombination, deriving bases ~490–1195 from a virus that was ~92 % similar to malvastrum yellow mosaic Helshire virus. Phylogenetically, these DNA-A components are most closely related to those of malvaceous weed-infecting begomoviruses from Jamaica, Cuba, Florida and Mexico. The SiGMBV DNA-A was able to elicit symptomatic infection in N. benthamiana.     

A novel monopartite begomovirus infecting sweet potato in Brazil

The complete genome sequences of two monopartite begomovirus isolates (genus Begomovirus, family Geminiviridae) present in a single sweet potato (Ipomoea batatas) plant collected in S?o Paulo, Brazil, are presented. Based on the current taxonomic criteria for the genus Begomovirus, one of the isolates was shown to represent a novel species, tentatively named Sweet potato leaf curl Sao Paulo virus (SPLCSPV). The other isolate represented a new strain of sweet potato leaf curl virus, named sweet potato leaf curl virus-Sao Paulo (SPLCV-SP). The full genome sequence of the SPLCSPV isolate shared the highest nucleotide identity (87.6%) with isolates of sweet potato leaf curl Spain virus (SPLCESV). Phylogenetic and recombination analyses were used to investigate the relationships of these isolates to other monopartite Ipomoea-infecting begomoviruses.     

A novel cassava-infecting begomovirus from Madagascar: cassava mosaic Madagascar virus

Cassava mosaic geminiviruses (CMGs) are implicated in cassava mosaic disease (CMD), the main constraint to cassava production in Africa. Here, we report the complete nucleotide sequences of the DNA-A and DNA-B of a newly characterized CMG found infecting cassava in Madagascar, for which we propose the tentative name cassava mosaic Madagascar virus. With the exception of two recombinant regions that resembled a CMG, we determined that the non-recombinant part of the DNA-A component is distantly related to the other CMGs. Whereas the DNA-B component possesses one recombinant region originating from an unidentified virus, the rest of the genome was seen to be closely related to members of the species East African cassava mosaic Zanzibar virus (EACMZV). Phylogenetic analysis based on complete genome sequences demonstrated that DNA-A and DNA-B components are outliers related to the clade of EACMV-like viruses and that DNA-A is related to the monopartite tomato leaf curl begomoviruses described in islands in the south-west Indian Ocean.     

Evidence of local evolution of tomato-infecting begomovirus species in West Africa: characterization of tomato leaf curl Mali virus and tomato yellow leaf crumple virus from Mali

Tomato yellow leaf curl (TYLC) and tomato leaf curl (ToLC) diseases are serious constraints to tomato production in Mali and other countries in West Africa. In 2003 and 2004, samples of tomato showing virus-like symptoms were collected during a survey of tomato virus diseases in Mali. Three predominant symptom phenotypes were observed: (1) TYLC/ToLC (stunted upright growth and upcurled leaves with interveinal yellowing and vein purpling), (2) yellow leaf crumple and (3) broccoli or bonsai (severe stunting and distorted growth). Squash blot (SB) hybridization with a general begomovirus probe and/or SB/PCR analyses revealed begomovirus infection in plants with each of these symptom phenotypes and no evidence of phytoplasma infection. Sequence analysis of PCR-amplified begomovirus fragments revealed two putative new begomovirus species associated with the TYLC/ToLC and yellow leaf crumple symptom phenotypes, respectively. Full-length clones of these begomoviruses were obtained using PCR and overlapping primers. When introduced into N. benthamiana and tomato plants, these clones induced upward leaf curling and crumpling (the TYLC/ToLC-associated begomovirus) or downward leaf curl/yellow mottle (yellow leaf crumple-associated begomovirus) symptoms. Thus, these begomoviruses were named tomato leaf curl Mali virus (ToLCMLV) and tomato yellow leaf crumple virus (ToYLCrV). The genome organization of both viruses was similar to those of other monopartite begomoviruses. ToLCMLV and ToYLCrV were most closely related to each other and to tobacco leaf curl Zimbabwe virus (TbLCZV-[ZW]) and tomato curly stunt virus from South Africa (ToCSV-ZA). Thus, these likely represent tomato-infecting begomoviruses that evolved from indigenous begomoviruses on the African continent. Mixed infections of ToLCMLV and ToYLCrV in N. benthamiana and tomato plants resulted in more severe symptoms than in plants infected with either virus alone, suggesting a synergistic interaction. Agroinoculation experiments indicated that both viruses induced symptomatic infections in tomato and tobacco, whereas neither virus induced disease symptoms in pepper, common bean, small sugar pumpkin, African eggplant, or Arabidopsis. Virus-specific PCR primers were developed for detection of ToLCMLV and ToYLCrV and will be used to further investigate the distribution and host range of these viruses.     

A novel drug vehicle capable of ultrasound-triggered release with MRI functions

A novel remotely triggered drug vehicle, magnetic hydroxyapatite (HA)-coated liposome (i.e. HA-coated liposome decorated with superparamagnetic iron oxide (SPIO) nanodots; HA/SPIO-coated liposome), was developed to exhibit ultrasound-triggered release behavior, magnetic resonance imaging (MRI) contrast and ultrasound-induced MRI contrast change. In this study, the effects of the HA/SPIO coating layer on the background leakage, response to ultrasound and MR signal were investigated. The background leakage of the liposome was significantly reduced due to HA/SPIO coating of the liposome. This coating layer also enhanced the sensitivity of the drug vehicle to ultrasound under sonication conditions at high frequencies (1 and 3 MHz) and low power densities (0.2-0.4 W cm(-2)). Moreover, the ultrasound-triggered vehicle exhibited a concentration-dependent T(2) (spin-spin relaxation time) contrast in MR images due to their decoration with SPIO nanodots. In addition, r(2) and r(2)(?) (transverse relaxivity) values increased with increasing amounts of SPIO decoration, suggesting that the MR images of HA/SPIO-coated liposomes could be probed by the T(2) signal. Most importantly, the r(2)(?)-r(2) value of HA/SPIO-coated liposomes decreased after sonication, which was more prominent for the sample with lower SPIO amounts. This suggests that this novel drug vehicle can be used not only as an MR image-guided drug vehicle capable of ultrasound-triggered release, but also as an MR reporter to probe the status of vehicles after ultrasonic triggering.     

A betasatellite-dependent begomovirus infects ornamental rose: characterization of begomovirus infecting rose in Pakistan

The Begomovirus genus of the family Geminiviridae comprises the largest group of geminiviruses. The list of begomoviruses is continuously increasing as a result of improvement in the methods for identification. Ornamental rose plants (Rosa chinensis) with highly stunted growth and leaf curling were found in Faisalabad, Pakistan. Plants were analyzed for begomovirus infection, through rolling circle amplification and PCR methods. Based on complete genome sequence homologies with other begomoviruses, a new begomovirus species infecting the rose plants was discovered. In this paper, we propose a new species name, Rose leaf curl virus (RoLCuV), for the virus. RoLCuV showed close identity (83 %) with Tomato leaf curl Pakistan virus, while associated betasatellite showed 96 % identity with Digera arvensis yellow vein betasatellite (DiAYVB), justifying a new isolate for the betasatellite. Recombination analysis of newly identified begomovirus revealed it as a recombinant of tomato leaf curl Pakistan virus from its coat protein region. The infectious molecules for virus/satellite were prepared and inoculated through Agrobacterium tumefaciens to N. benthamiana plants. RoLCuV alone was unable to induce any level of symptoms on N. benthamiana plants, but co-inoculation with cognate betasatellite produced infection symptoms. Further investigation to understand the trans-replication ability of betasatellites revealed their flexibility to interact with Rose leaf curl virus.     

A novel membrane glycoprotein capable of inhibiting membrane attack by homologous complement

Neuraminidase-treated human erythrocytes become sensitive to haemolysis by heterologous serum via activation of the alternative complement pathway (ACP), while remaining insensitive to homologous serum because of the presence of inhibitors on the cell membrane. We obtained a monoclonal antibody which renders the neuraminidase-treated erythrocytes sensitive to haemolysis by homologous human serum via the ACP. This antibody reacts with a 20 KDa membrane glycoprotein which interferes with the terminal stage of complement action on cell membranes. The 20 KDa protein is anchored to the membrane via phosphatidylinositol.     

Functional analysis of the naturally recombinant DNA-A of the bipartite begomovirus Tomato chlorotic mottle virus

All geminiviruses found in Brazil belong to the Begomovirus genus with a bipartite genome that is split between two genomic components, DNA-A and DNA-B. The DNA-A of the bipartite begomovirus ToCMoV-[MG-Bt] (Tomato chlorotic mottle virus), however, possesses as a peculiar characteristic the capacity to systemically infect Nicotiana benthamiana. Here we further characterize this variant DNA-A and show that it also infects Solanum lycopersicum and other host plants, in the absence of DNA-B. The ToCMoV-[MG-Bt]-DNA-A encodes an additional ORF, designated AC5, but otherwise its genome organization is similar to other DNA-A from Western Hemisphere begomoviruses. We showed that this AC5 putative ORF is not essential for infection, as disruption of its coding capacity caused no effect on ToCMoV-[MG-Bt]-DNA-A-mediated infection process. Likewise, the ToCMoV-[MG-Bt]-DNA-A ac4 mutant was indistinguishable from its wild type counterpart in all hosts tested. In contrast, an av1 (coat protein) mutant was unable to infect systemically N. benthamiana and Chenopodium quinoa in the absence of DNA-B. However, inclusion of DNA-B in the infection assay fully rescued the movement defect of the ToCMoV-[MG-Bt]-DNA-A av1 mutant. These results suggest that at suboptimal conditions for infection the coat protein is required for ToCMoV-[MG-Bt] systemic movement.     

Molecular characterization of a novel monopartite begomovirus isolated from Pouzolzia zeylanica in China

The complete genome sequence of a monopartite begomovirus isolate TY01 was obtained from diseased Pouzolzia zeylanica plants exhibiting golden mosaic symptoms in Baise, Guangxi Province, China. It consisted of 2723 nucleotides (nt) and encoded two ORFs (CP and AV2) in the virion-sense DNA and five ORFs (AC1-AC5) in the complementary-sense DNA. Compared with the DNA-A sequences of other begomoviruses, it has the highest (78.5 %) nucleotide sequence identity with ageratum yellow vein virus (AYVV) isolate AFSP6D from Thailand, which is less than the 89 % identity in the complete genome that has been defined as the threshold value for demarcation of species in the genus Begomovirus, family Geminiviridae. Phylogenetic analysis showed that TY01 was grouped in a separate clade from the other 28 begomovirus isolates. These results indicate that isolate TY01 is a member of a novel Begomovirus species, for which the name “Pouzolzia golden mosaic virus” (PGMV) is proposed.     

Complete genome sequence of a novel monopartite begomovirus infecting sweet potato in China

The complete genome sequence of a new monopartite begomovirus isolate SC-1 was obtained from sweet potato samples in Sichuan province, China. The viral genome consists of 2,764 nucleotides (nt) and encodes two open reading frames (ORFs) called AV1 and AV2 genes in the viral-sense strand and four ORFs (AC1–AC4) in the complementary-sense strand. Sequence comparisons revealed that it shared the highest level of nt sequence identity (81.2 %) with Sweet potato leaf curl Georgia virus (AF326775). Phylogenetic analysis showed that the SC-1 genome was in a separate clade from other 29 begomovirus isolates. Thus, the SC-1 isolate is a novel species according to the demarcation criteria of species in the genus Begomovirus, for which the name “Sweet potato leaf curl China Sichuan Virus” (SPLCCSV) is proposed. Recombination analysis suggests that SPLCCSV has sequences derived from recombination between Sweet potato leaf curl virus (SPLCV) isolate GZ01 (JX286653) and SPLCV isolate Merremia N4 (DQ644563).     

Molecular characterization of tomato-infecting begomoviruses in Thailand

Bipartite geminiviruses infecting tomatoes in Thailand were detected by polymerase chain reaction (PCR) using CPA5/CPA2 primers. Products derived from PCR-amplified full-length DNA-A and DNA-B of TYLCV collected from Chiang Mai, Nong Khai, and Sakon Nakhon were cloned and sequenced. DNA-A from Chiang Mai was 2747 nts long; Nong Khai, 2744 nts; and Sakon Nakhon, 2747 nts, and those of DNA-B from Chiang Mai were 2750 nts long; Nong Khai, 2749 nts; and Sakon Nakhon, 2749 nts. The genomes of these virus isolates were organized like those of other begomoviruses. The DNA-A had two ORFs in the virion sense and four ORFs in the complementary sense. The DNA-B had two ORFs in the virion sense and one ORF in the complementary sense. Nucleotide sequences of DNA-A of TYLCV from Chiang Mai, Nong Khai, and Sakon Nakhon were closely related to those of Tomato yellow leaf curl Thailand virus (TYLCTHV) and Tomato yellow leaf curl Thailand virus-[Myanmar] (TYLCTHV-[MM]) with nucleotide sequence identity ranging from 89% to 95%. Based on sequence comparisons and phylogenetic analyses, these three virus isolates studied were identified as new strains of TYLCTHV and named Tomato yellow leaf curl Thailand virus-Chiang Mai (TYLCTHV-[CM]The GenBank accession codes for DNA-A of TYLCTHV-[CM], -[NK], and -[SK] are , and , respectively. The GenBank accession codes for DNA-B of TYLCTHV-[CM], -[NK], and -[SK] are , , and , respectively.), Nong Khai (TYLCTHV-[NK] and Sakon Nakhon (TYLCTHV-[SK]).     

Complete genome sequence of a novel monopartite begomovirus infecting sweet potato in China

The complete genome sequence of a novel monopartite begomovirus, isolate G-YU-12-10, was obtained from sweet potato samples exhibiting severe leaf curl symptoms in Xinxiang, Henan Province, China. The genome sequence consisted of 2766 nucleotides and encoded two open reading frames (ORFs) (AV1 and AV2) in the viral-sense strand and four ORFs (AC1-AC4) in the complementary-sense strand. The genome of isolate G-YU-12-10 was closely related to other sweet-potato-infecting begomoviruses (sweepoviruses) and shared the highest nucleotide sequence identity (89.0 %) with sweet potato leaf curl China Sichuan virus (SPLCCSV, KC488316). Thus, the G-YU-12-10 isolate represents a novel species according to the demarcation criteria of species in the genus Begomovirus, for which the name Sweet potato leaf curl Henan virus (SPLCHnV) is proposed. Interspecific recombination analysis supported the recombination hypothesis, indicating that recombination with other begomoviruses had taken place within AC2 and AC3 ORFs of SPLCHnV and also in the non-coding intergenic region (IR).     

Deletion and recombination events between the DNA-A and DNA-B components of Indian cassava-infecting geminiviruses generate defective molecules in Nicotiana benthamiana

Cloned DNA-B components, belonging to the bipartite begomoviruses Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), family Geminiviridae, when co-inoculated along with previously cloned DNA-A components of the respective viruses onto the experimental host Nicotiana benthamiana, generated defective DNAs (def-DNA) ranging in size from 549 to 1555 nucleotides. All the cloned def-DNAs contained the common region (CR) as well as portions of either DNA-A or DNA-B and, in a few cases, both DNA-A and DNA-B, representing recombinant products, the junction points of which correspond to repeats of 2-11 bases found in the parental molecules. The DNA-B-derived def-DNAs were, in some cases, associated with a decrease in levels of DNA-B, with a concomitant change in the symptoms from downward leaf curling in the older leaves to upward leaf-rolling in newly emerging leaves, more typical of monopartite begomoviruses.     

Specificity of heterologous antisera to components solubilized from rat thymocyte membranes

Rat thymocyte membranes were solubilized by citraconic anhydride and the soluble preparation was fractionated by gel chromatography. Antithymocyte sera that were elicited by injection of either these soluble components or intact thymocytes or membranes of thymocytes, were compared for their cytotoxic effect on various cell populations and prolongation of skin grafts. The different antisera were equally effective in cytolysis and lysed 100% of thymus cells, 60% and 15% of lymph node and bone marrow cells, respectively. The antiserum raised by the soluble components contained significantly fewer antibodies against red blood cells or kidney and liver tissues and was also much less toxic when injected into rats. The different antisera were also comparably effective in the prolongation of skin grafts. The soluble components of rat thymocyte membranes inhibited the cytotoxic activity of the antisera raised in rabbits by either cells or the soluble preparation. However, the soluble component did not inhibit the cytotoxic activity of alloantisera, suggesting that the soluble component does not contain active histocompatibility antigens.     

Characterization of begomovirus components from a weed suggests that begomoviruses may associate with multiple distinct DNA satellites

A begomovirus disease complex associated with Sonchus arvensis, a common weed in Pakistan was studied using cloning, nucleic acid sequencing and phylogenetic analysis. The complex associated with this weed consists of a monopartite begomovirus and several distinct betasatellites and alphasatellites. The monopartite begomovirus associated with yellow vein disease of Sonchus arvensis showed 95–99% nucleotide sequence identity with Alternanthera yellow vein virus (AlYVV) reported from China, Vietnam and India. Two betasatellites were isolated from S. arvensis: one sharing between 91.4 and 95.3% nucleotide sequence identity with isolates of Ageratum yellow leaf curl betasatellite (AYLCB), and the other sharing between 78.2 and 99.9% identity with isolates of Cotton leaf curl Multan betasatellite (CLCuMB). Two alphasatellites were identified: one was homologous to Potato leaf curl alphasatellite (PotLCuA), while the other was closely related to Hibiscus leaf curl alphasatellite (HLCuA). Thus, AlYVV in S. arvensis is associated with satellites shown previously to be associated with other begomoviruses in Pakistan. Our results suggest that monopartite begomoviruses may associate with distinct satellites that are prevalent in the region.     

A novel human enterovirus recombinant from a child with diarrhea

Recombination is a well-known phenomenon for enteroviruses. In the present study, a novel recombinant HEV-B strain (ChZJ-1) was found in a 2-year-old child with diarrhea in Zhenjiang, China. The whole genome of ChZJ-1 was determined. Recombination and phylogenetic analysis revealed that ChZJ-1 might have been produced by recombination between coxsackievirus B5 and echovirus 18.