Pathogenicity and stability of a truncated DNAbeta associated with Tomato yellow leaf curl China virus

DNAbeta molecules are single-stranded satellite DNA associated with monopartite begomoviruses (family Geminiviridae). DNAbeta possesses a C1 gene on the complementary strand, which has a conserved position and size. To better understand the function of C1 gene in virus infection, a C1 deletion DNAbeta associated with a Tomato yellow leaf curl China virus (TYLCCNV) isolate was constructed. Co-agroinoculation with TYLCCNV showed the truncated DNAbeta was infectious in Nicotiana benthamiana and N. glutinosa plants but not in N. tabacum Samsun, N. tabacum and Lycopersicon esculentum plants. The wild-type TYLCCNV DNAbeta co-agroinoculated with TYLCCNV caused systemic infection in all the above hosts. Results of Southern blot analysis indicate that C1 gene is not required for TYLCCNV and DNAbeta replication. However, the presence of C1 gene in DNAbeta can increase both TYLCCNV and DNAbeta accumulation in infected plants. The truncated TYLCCNV DNAbeta was stable in N. benthamiana and N. glutinosa plants.     

The begomoviruses Honeysuckle yellow vein mosaic virus and Tobacco leaf curl Japan virus with DNAbeta satellites cause yellow dwarf disease of tomato

The complete nucleotide sequences of two begomoviruses (Nara virus-1 and Nara virus-2), a satellite DNA (DNAbeta-Nara) and defective DNAs were obtained from honeysuckle (Lonicera japonica) showing characteristic yellow vein mosaic symptoms in Nara Prefecture, Japan. One begomovirus (Ibaraki virus) and a satellite DNA (DNAbeta-Ibaraki) was isolated and cloned from honeysuckle plants exhibited typical yellowing of veins and small elliptical shaped enations along veins on the under side of the leaves in Ibaraki Prefecture, Japan. The genome organization of the three viruses is the same as those of other Old World monopartite begomoviruses. Nara virus-1 had overall nucleotide sequence identity with Nara virus-2 of 94% and Ibaraki virus of 90%. DNAbeta-Nara had overall nucleotide sequence identity with DNAbeta-Ibaraki of 83%. Comparison of the nucleotide sequences with other begomoviruses revealed that Nara virus-1 and Nara virus-2 are strains of Honeysuckle yellow vein mosaic virus (HYVMV), hence named as HYVMV-Nara1 and HYVMV-Nara2, whereas Ibaraki virus was a strain of Tobacco leaf curl Japan virus (TbLCJV), designated as TbLCJV-Hs[Iba]. HYVMV-Nara1 and HYVMV-Nara2 have hybrid genomes, which are likely to have formed recombination between HYVMV and TbLCJV. TbLCJV-Hs[Iba] or HYVMV-Nara2 could infect and cause yellowing, leaf crinkling and stunting symptoms when partial tandem dimeric constructs were agroinoculated on tomato plants. However, in the presence of DNAbeta, both TbLCJV-Hs[Iba] or HYVMV-Nara2 produced more severe stunting symptoms in tomato plants. Therefore, these viruses along with their satellites are causal agents of tomato yellow dwarf disease in Japan, and honeysuckle acts as a potential reservoir host. Previously available evidence indicated that DNAbeta elements do not contain iteron sequences of their helper viruses; hence this is the first evidence that DNAbeta satellites have the iteron of their helper virus.     

Particles and associated inclusions in sugarbeet infected with the curly top virus

Electron microscopy of sugarbeet leaves of different ages infected with curly top virus revealed spherical viruslike particles approximately 16 nm in diameter. The particles tended to form clumps and were sometimes associated with fibrils resembling those formed by nucleic acids. The particles were found in nuclei of phloem parenchyma cells. They appeared to arise in the nucleoplasm with no obvious relation to the nucleolus. Nuclear chromatin disappeared concomitantly with the increase in the amount of virus. The nucleolus apparently disintegrated also. Some views indicated that the nuclear envelope breaks down, but, thus far, particles were not detected in the cytoplasm. When particles first appear in a nucleus, a spherical granular inclusion of unknown identity occurs nearby. An amorphous inclusion body is found in the cytoplasm of cells in which the nuclei contain virus.     

Genetic diversity in curtoviruses: a highly divergent strain of Beet mild curly top virus associated with an outbreak of curly top disease in pepper in Mexico

A full-length curtovirus genome was PCR-amplified and cloned from peppers in Mexico with symptoms of curly top disease. The cloned DNA of this isolate, MX-P24, replicated in Nicotiana tabacum protoplasts and was infectious in N. benthamiana plants. Sequence analysis revealed that the MX-P24 isolate had a typical curtovirus genome organization and was most similar to beet mild curly top virus (BMCTV). However, sequence identities were at the threshold value for establishment of a new curtovirus species. To further investigate the biological properties of MX-P24, an agroinoculation system was generated. Agroinoculated shepherd's purse plants developed typical curly top symptoms, and virus from these plants was transmissible by the beet leafhopper (Circulifer tenellus). The host range of MX-P24 was similar to that of BMCTV, with curly top symptoms induced in common bean, pepper, pumpkin, shepherd's purse and tomato plants and mild or no symptoms induced in sugar beet plants. Together, these results indicate that MX-P24 is a highly divergent strain of BMCTV associated with an outbreak of curly top disease in peppers in Mexico.     

Molecular characterization of a satellite RNA associated with blackcurrant reversion nepovirus

Summary.  A satellite RNA (satRNA) associated with blackcurrant reversion nepovirus (BRV) was isolated and its nucleotide sequence was determined from cDNA clones. BRV satRNA was 1432 nucleotides (nt) in length excluding the poly(A)-tail, and contained one open reading frame which encodes a polypeptide of 402 amino acids, with a calculated M_r of 44 220. The coding region was bordered by a 5′ leader sequence of 25 nt and a 3′-nontranslated region of 201 nt. Two in vitro translation products of approximately 45 kDa and 40 kDa were detected, indicating that two in-frame AUG codons at positions 26 and 134 may both be functional. Nucleotide sequence comparisons revealed a stretch of 865 nt that was 63% identical between BRV satRNA and the large satRNA of chicory yellow mottle nepovirus. A 5′-terminal consensus sequence and a 40 nt motif (located at positions 264–303 of BRV satRNA) were conserved between BRV satRNA and other nepoviral large satRNAs. Received March 22, 1999/Accepted August 30, 1999     

Mixed infection of black currant (Ribes nigrum L.) plants with Blackcurrant reversion associated virus and rhabdovirus-like particles with symptoms of black currant reversion disease

Black currant plants of cvs. Black Smith and Karlstejnsky dlouhohrozen showing symptoms of severe Russian (R) form of black currant reversion disease (BCRD) were found in 1999-2000 in the Czech Republic. Five selected plants of both cultivars originating from two distant loci were tested by polymerase chain reaction (PCR) for presence of the Blackcurrant reversion associated virus (BRAV), the causal agent of BCRD. In all plants, virus-specific 215 nt cDNA fragments proving the presence of BRAV were obtained. Moreover, in two of those five black currant plants, rhabdovirus-like particles were found in ultrathin sections by electron microscopical examinations. The particles measured 200-347 nm by 64-90 nm. They occurred mostly within nuclei of parenchyma cells of vascular bundles as single particles, rafts of particles, but also in aggregates. They were found also in the perinuclear space and occasionally directly in the cytoplasm. Clusters of particles either within the nucleus or in the perinuclear space were membrane-bound. We bring evidence on the occurrence of the severe (R) form of BCRD and the first evidence of BRAV in the Czech Republic.     

Genome characterization and genetic diversity of beet curly top Iran virus: a geminivirus with a novel nonanucleotide

Beet curly top Iran virus (BCTIV) was previously reported as a distinct curtovirus in Iran. Complete nucleotide sequences of three BCTIV isolates, one each from central, southern, and south eastern Iran were determined to be 2844, 2844, and 2845 nt long, respectively. BCTIV shared highest nucleotide sequence identity (52.3%) with Spinach curly top virus (SpCTV) and lowest identity (46.6%) with Horseradish curly top virus (HrCTV). The BCTIV genome comprises three virion-sense (V1, V2, and V3) and two complementary-sense (C1 and C2) ORFs. ORFs C3 and C4 were not found in BCTIV genome. Based on a comparison of nucleotide sequence identity of individual genes, the three virion-sense ORFs were 72.7–79.9% related to the corresponding ORFs of curtoviruses, whereas no significant relationship was found between the C1 and C2 ORFs of BCTIV and curtoviruses. These two ORFs, however, were only distantly related with those of mastreviruses. Similar to the latter viruses, the BCTIV genome comprises two intergenic regions. The BCTIV large intergenic region included a sequence capable of forming a stem loop structure and a novel nonanucleotide (TAAGATT/CC) with a unique nick site. Phylogenetic analysis using deduced amino acid sequence of individual ORFs revealed that the V2 and V3 ORFs are monophyletic and the V1 ORF is classified with the related ORF of curtoviruses. Whereas the two complementary-sense ORFs are grouped with those of mastreviruses. Computer-based prediction suggested that BCTIV has a chimeric genome which may have arisen by a recombination event involving curto- and mastrevirus ancestors. Percent nucleotide sequence identities of the coat protein gene of ten isolates of BCTIV, collected from a wide range of geographical regions in Iran, varied from 87.1 to 99.9, with the isolates being distributed between two subgroups. Based on biological and molecular properties, BCTIV is proposed as a new member of the genus Curtovirus.     

Mutational analysis of the monopartite geminivirus beet curly top virus.

Mutants of the monopartite geminivirus beet curly top virus have been screened for infectivity and symptom development in Nicotiana benthamiana and Beta vulgaris, for replication competence in N. benthamiana leaf discs, and for transmission by the leafhopper Circulifer tenellus. Disruption of open reading frame (ORF) V2 by the introduction of a termination codon resulted in symptomless infection of N. benthamiana associated with low levels of virus and reduced single-stranded (ss) DNA and prevented systemic infection of B. vulgaris. Reduced levels of ssDNA were produced by the mutant in N. benthamiana leaf discs, suggesting that V2 affects the synthesis or accumulation of this viral DNA form. Mutants in which ORF C2 had been truncated by the introduction of termination codons or by frame-shifting remained highly infectious and induced severe symptoms in both N. benthamiana and B. vulgaris. Similarly, a mutant containing a termination codon within ORF C3 was highly infectious and induced severe symptoms in N. benthamiana although infectivity in B. vulgaris was greatly reduced, symptoms were extremely mild, and virus levels were low. A synergistic effect of a double mutation in ORFs C2 and C3, manifested by the inability of mutants to systemically infect N. benthamiana and the production of reduced amounts of ssDNA in N. benthamiana leaf discs, suggests that both ORFs are functional in this host. A mutant containing a termination codon within the 5' terminus of ORF C4 produced severe symptoms in both N. benthamiana and B. vulgaris resembling those induced by wild-type virus. Comparison with the phenotypes of previously characterized ORF C4 mutants suggests that a conserved core sequence of this ORF is an important symptom determinant. ORF C2, C3, and C4 mutants produced virus particles and were transmitted by C. tenellus, eliminating agroinoculation as a contributory factor to the mutant phenotypes. Our results are compared with those derived from mutagenesis studies on related bipartite geminiviruses.     

Transreplication of a Tomato yellow leaf curl Thailand virus DNA-B and replication of a DNAbeta component by Tomato leaf curl Vietnam virus and Tomato yellow leaf curl Vietnam virus

The genomes of two tomato-infecting begomoviruses from Vietnam were cloned and sequenced. A new variant of Tomato leaf curl Vietnam virus (ToLCVV) consisting of a DNA-A component and associated with a DNAbeta molecule as well as an additional begomovirus tentatively named Tomato yellow leaf curl Vietnam virus (TYLCVV) consisting also of a DNA-A component were identified. To verify if monopartite viruses occurring in Vietnam and Thailand are able to transreplicate the DNA-B component of Tomato yellow leaf curl Thailand virus-[Asian Institute of Technology] (TYLCTHV-[AIT]) infectivity assays were performed via agroinoculation and mechanically. As result, the DNA-B component of TYLCTHV-[AIT] was transreplicated by different DNA-A components of viruses from Vietnam and Thailand in Nicotiana benthamiana and Solanum lycopersicum. Moreover, the TYLCTHV-[AIT] DNA-B component facilitated the mechanical transmission of monopartite viruses by rub-inoculation as well as by particle bombardment in N. benthamiana and tomato plants. Finally, defective DNAs ranging from 735 to 1457 nucleotides were generated in N. benthamiana from those combinations containing TYLCTHV-[AIT] DNA-B component.     

Genetic diversity and host range studies of turnip curly top virus

Turnip curly top virus (TCTV) is a unique geminivirus that has recently been characterised as infecting turnips in Iran. The genome of TCTV shares <68 % pairwise identity with other geminiviruses and has a genome organisation similar to that of curtoviruses and topocuvirus. The replication-associated protein (Rep) bears the highest similarity to curtovirus Reps (48.5–69.0 %); however, in the case of the capsid protein (CP), the extent of similarity is only 39.5–44.5 %. We constructed an agroinfectious clone of TCTV and undertook host range studies on ten plant species; in three species (turnip, sugar beet and cowpea), we detected infection which presents curly top symptoms in turnip and sugar beet. The efficiency of TCTV infection in agroinoculated turnip plants was 71.7 %, and the infection was successfully transmitted to 80 % of the healthy turnip plants used in the insect transmission studies by Circulifer haematoceps under greenhouse conditions. We also determined the genome sequence of 14 new TCTV isolates from southern Iran isolated from turnips. We observed ~13 % diversity amongst all the TCTV isolates and found evidence of recombination in the CP- and Rep-coding regions of the genomes.     

Detection of a hepatitis B virus variant with a truncated X gene and enhancer II

Summary Approximately 15% of the viral genomes in the serum of a hepatitis B carrier were found to exhibit an 8 base-pair deletion within the enhancer II element, which would truncate the X protein by 26 amino acids at the carboxy-terminus.     

The coat protein of beet curly top virus is essential for infectivity

We have applied the procedure of Agrobacterium-mediated inoculation to develop a simple, efficient, and reproducible assay for the infectivity of the leafhopper-transmitted geminivirus, beet curly top virus (BCTV). This assay system was used to show that a coat protein mutant of BCTV is not infectious, but could be complemented by coagroinoculation with a second mutant bearing a lethal mutation in the complementary-sense open reading frame, C1. Furthermore, the coat protein mutant retained the ability to replicate and to produce both ssDNA and dsDNA when electroporated into Nicotiana tabacum protoplasts. We conclude that the coat protein of BCTV is essential for spread of the virus. The results are discussed in the light of results with coat protein mutants of other geminiviruses.     

Molecular characterization of Tobacco leaf curl Pusa virus, a new monopartite Begomovirus associated with tobacco leaf curl disease in India

Leaf curl disease of tobacco (TbLCD) is endemic in India. A monopartite Begomovirus, a betasatellite and an alphasatellite were found associated with the disease in Pusa, Bihar. The DNA-A of the Begomovirus associated with TbLCD in Pusa, Bihar was found to comprise of 2707 nt with a typical Old World begomovirus-like genome organization. The full-length sequence of DNA-A [HQ180391] showed that the Pusa isolate is a newly described member of the genus Begomovirus, as it had <89% sequence homology with DNA-A of all the known begomoviruses. The isolate is tentatively named as Tobacco leaf curl Pusa virus [India:Pusa:2010]. The betasatellite (HQ180395) associated with TbLCD in Pusa was identified as a variant of Tomato leaf curl Bangladesh betasatellite [IN:Raj:03], with which it shared 90.4% sequence identity. The alphasatellite (HQ180392) associated with the disease had highest 87% nucleotide sequence identity with Tomato leaf curl alphasatellite. The Begomovirus, betasatellite, and alphasatellite associated with TbLCD in Pusa, Bihar, India were found to be recombinants of extant begomoviruses, betasatellites and alphasatellites spreading in the Indian sub-continent and South-East Asia.     

Rapid reversion of a deletion mutation in Moloney murine leukemia virus by recombination with a closely related endogenous provirus

During abortive infection of mouse cells, defective retroviruses carrying deletions in essential functions can recombine with endogenous retroviral sequences to form viable, replication-competent viruses. We have examined the reversion of a mutant Moloney murine leukemia virus with a deletion in the protease domain of the pol gene after infection of NIH/3T3 cells. In this system revertants arise quickly, only 2 weeks after infection. Analysis of DNA clones of the revertant viral genomes showed that they were derived by recombination with a long sequence of gag and pol exhibiting 95% sequence identity to Moloney virus. One such cloned recombinant was fully infectious, indicating that the repertoire of viral sequences in the NIH/3T3 genome must include substantial stretches of functional viral genes. Examination of the viral DNAs very early in the infection revealed the presence of defective genomes, formed by nonhomologous crossovers between the two parental sequences. We suggest that these may serve as intermediates in the eventual formation of the viable revertant genomes.     

Diversity of Beet curly top Iran virus isolated from different hosts in Iran

Beet curly top Iran virus (BCTIV) is a major pathogen of sugar beet in Iran. In order to study diversity of BCTIV, we sampled 68 plants in Iran during the summer of 2010 with curly top disease symptoms on beans (Phaseolus vulgaris), cowpeas (Vigna unguiculata), tomatoes (Solanum lycopersicum L.), sea beets (Beta vulgaris subsp. maritima), and sugar beets (Beta vulgaris). Plant samples showing leaf curling, yellowing, and/or swelling of veins on the lower leaf surfaces were collected from various fields in Khorasan Razavi, Northern Khorasan (north-eastern Iran), East Azarbayejan, West Azarbayejan (north-western Iran), and Fars (southern Iran) provinces. Using rolling circle amplification coupled with restriction digests, cloning, and Sanger sequencing, we determined the genomes of nine new BCTIV isolates from bean, cowpea, tomato, sea beet, and sugar beet in Iran. Our analysis reveals ~11 % diversity amongst BCTIV isolates and we detect evidence of recombination within these genomes.     

Characterization of beet curly top virus subgenomic DNA localizes sequences required for replication.

Subgenomic viral DNA is accumulated in Nicotiana benthamiana and Beta vulgaris plants agroinoculated with the geminivirus beet curly top virus. The subgenomic DNA is more abundant in N. benthamiana and is distributed between two broad size groups in this host. Six unique examples, ranging in size from 887 to 1311 nucleotides, have been cloned from viral double-stranded DNA purified from N. benthamiana and analyzed by sequence determination. Deletions are distributed throughout most of the genome and only nucleotides 2946-410 are represented in all subgenomic DNAs. Comparison with a previously characterized subgenomic DNA suggests that cis-acting signals necessary for viral DNA replication are located in a predominantly intergenic region between nucleotides 2946-308.     

Transmission of cocoa swollen shoot virus by seeds

A study was undertaken to determine whether cocoa swollen shoot virus is transmitted by seeds, to improve the robustness of quarantine procedures for international exchange and long term conservation of cocoa germplasm. PCR/capillary electrophoresis, using cocoa swollen shoot virus primers designed from the most conserved regions of the six published cocoa genome sequences, allowed the detection of cocoa swollen shoot virus in all the component parts of cocoa seeds from cocoa swollen shoot virus-infected trees. PCR/capillary electrophoresis revealed the presence of cocoa swollen shoot virus in seedlings raised from seeds obtained from cocoa swollen shoot virus-infected trees. The high frequency with which the virus was transmitted through the seedlings suggested that cocoa swollen shoot virus is transmitted by seeds. This has serious implications for cocoa germplasm conservation and distribution.