Cornelia Heinze

Detection and differentiation of serologically cross-reacting tobamoviruses of economical importance by RT-PCR and RT-PCR-RFLP

A procedure involving reverse transcription followed by the polymerase chain reaction (RT-PCR) using a single primer pair was developed for the detection of five tobamovirus species which are related serologically. Either with a subsequent restriction enzyme analysis (RT-PCR-RFLP) or with a RT-PCR using species specific primers the five species can be differentiated. To differentiate those species by serological means is time consuming and might give ambiguous results. With the example of the isolate OHIO V, which is known to break the resistance in a selection of Lycopersicon peruvianum, the suitability of the RT-PCR-RFLP technique to detect variability at the species level was shown. In sequence analysis 47 codons of the coat protein gene of this isolate were found to be mutated compared to a tobacco mosaic virus (TMV) coat protein gene sequence. Forty of these mutations were silent and did not change the amino acid sequence. Both procedures are suitable to detect mixed infections. In addition, the RT-PCR-RFLP give information on the relative amounts of the viruses that are present in a doubly infected plant. The RT-PCR-RFLP using general primers as well as the RT-PCR using species specific primers were proven to be useful for the diagnosis and control of the disease and will be helpful for resistance breeding, epidemiological investigations and plant virus collections.

The phylogenetic structure of the cluster of tobamovirus species serologically related to ribgrass mosaic virus (RMV) and the sequence of streptocarpus flower break virus (SFBV)

Summary.Ribgrass mosaic virus (RMV), turnip vein-clearing virus (TVCV) and Youcai mosaic virus (YoMV; formerly designated as oilseed rape mosaic virus; ORMV) belong to the genus Tobamovirus and are arranged in one out of three subgroups because of their common host range, serological cross-reactivity and amino acid composition of their coat proteins. The recently defined species Wasabi mottle virus (WMoV) is closely related to the same subgroup. The distinction of the four species is difficult and the lack of sequence information of a wide range of isolates has led to an unclear nomenclature. To clarify this situation we sequenced the coat protein genes from 18 isolates which were serologically related to members of the species of this cluster. The size of the coat protein was conserved with the exception of one isolate which revealed an N-terminal extension due to the mutation of three stop-codons. Phylogenetic analysis of these CP ORFs resulted in a tree with three clusters each containing at least one of the approved species RMV, TVCV and 1ptYoMV/WMoV in which our isolates were distributed. The tree was congruent and did support the present taxonomic status of species within this subgroup.For practical purpose we developed a subgroup 3 specific primer pair and a species differentiating restriction fragment length polymorphism (RFLP). Sequencing of the genome of Streptocarpus flower break virus (SFBV) which is serologically distantly related to the subgroup 3 viruses revealed a distinct genome organization. Therefore we propose that this virus should be regarded as a member of a species not belonging to any of the subgroups so far established.

Peptide-derived broad-reacting antisera against tospovirus NSs-protein.

Sequence alignments of tospovirus species of serogroup I to IV revealed a stretch of 24 amino acids at the C terminus of the non-structural protein NSs with a highly conserved sequence. Based on this sequence the 24 amino acids peptide YFLSKTLEVLPKNLQTMSYLDSIQC was synthesized and used to raise antisera in two rabbits. The specificity of the antisera against NSs from infected plants was confirmed with Western blots and by immunogold labelling and electron microscopy. These antisera detected tospovirus isolates of serogroup I to III in antigen-coated plate ELISA and Western blots but failed to detect isolates of serogroup IV. Epitope scanning using overlapping octopeptides composing the peptide suggested that the antisera contained antibodies against two different epitopes. Strongly reacting peptides were found at the C-terminus of the original peptide sequence when probing with one of the antisera. In this part the sequence was homologous to serogroup I, II and III, with all deviations from serogroup IV located here. Additional octopeptides, based on this region, synthesized with sequence modifications back to the serogroup IV sequence in all possible combinations, had low reactivity. However two of the modified peptides with partly restored serogroup IV sequences revealed promising reactivity and could be suitable to raise an antiserum with broader reactivity, including serogroup IV.

A novel mycovirus from Clitocybe odora

The Ninth Report of the International Committee on Taxonomy of Viruses (ICTV) reports only a few species whose members replicate in fungi. Most of these mycoviruses are described to replicate in phytopathogenic and commercially cultivated fungi. A few reports describe virus-like symptoms and virus-like particles in non-cultivated basidiocarps such as Boletus edulis, Laccaria spp. and Cantharellus spp. However, viral sequences from non-cultivated Agaricomycotina are not available yet. In this report, I present a partial sequence of a virus found in Clitocybe odora (Bull.:Fr.) P. Kumm var. odora coding for a putative RNA-dependent RNA polymerase (RdRp) and a small 20-kDa ORF that may encode a coat protein (CP). The sequence of the putative RdRp (ORF 1) of C. odora clusters with those of the Tanathephorus cucumeris virus RdRp and the Tuber aestivum mitovirus RdRp. In addition to sequence homology, Tanathephorus cucumeris virus shows a similar codon usage and TA content in the 5′- and 3′ non-translated regions, but it does not encode a putative CP. A viral DNA form proposed for Tanathephorus cucumeris virus was not found in Clitocybe odora. This viral sequence does not fit into any of the existing virus taxa.

An unusual large intergenic region in the S-RNA of a Bulgarian tomato spotted wilt virus isolate

Summary. The complete S-RNA sequences of four Bulgarian and one German isolate of tomato spotted wilt tospovirus were determined. All isolates show a high conservation in their N proteins, while the NSs proteins and the intergenic regions (IGR) are more variable. The Bulgarian isolate 10HK96 has the largest S-RNA (3364 nucleotides) among tomato spotted wilt viruses reported so far. The enlargement is based on an insertion of 365 nts in the IGR that may have resulted from stuttering of the viral polymerase or non-homologous recombination. This insertion is present in the N protein gene subgenomic messenger, upstream of a proposed transcription termination signal.

Pelargonium necrotic spot virus: a new member of the genus Tombusvirus

Summary.A virus isolate from Pelargonium spp., provisionally designated UPEV (

Tomato spotted wilt virus (TSWV) infection of Physcomitrella patens gametophores

{\underline {\rm u}}{\rm nknown} \ {\underline {\rm pe}} {\rm largonium} \ {\underline {\rm v}}{\rm irus}

Duplications in the 3′ termini of three segments of Fusarium graminearum virus China 9

), had isometric particles 31–33 nm in diameter, with a granular surface structure similar to that of viruses in three genera of family Tombusviridae. Immunoelectron microscopy proved that UPEV was serologically distinct from all examined morphologically similar members of the family Tombusviridae. The induced cytopathology was characterized by large cytoplasmic virion aggregates and the formation of multivesicular bodies derived from mitochondria. Analysis of the complete ssRNA genome sequence revealed four open reading frames (ORFs) arranged like those of viruses in the genera Tombusvirus and Aureusvirus. Sequence comparisons indicated that three of the four ORFs had a high identity (52–97% identical amino acids) with the respective ORFs of tombusvirus species, especially with Carnation Italian ringspot virus, but not with those of viruses in other genera in Tombusviridae. On the contrary, UPEV coat protein had a low indentity (36–53% identical amino acids) with that of the aureusvirus Pothos latent virus. The data suggested that UPEV originated in a recombination event between a tombus- and an aureusvirus. According to its original host and symptom expression we proposed the new virus be named Pelargonium necrotic spot virus (PeNSV) and classified it as a distinct and new species in the genus Tombusvirus.

The complete sequence of tobacco mosaic virus isolate Ohio V reveals a high accumulation of silent mutations in all open reading frames

Following mechanical inoculation of the moss Physcomitrella patens (Hedw.) B.S.G. with Tomato spotted wilt virus (TSWV), the virus encoded N nucleocapsid protein was detected in gametophores harvested 11 and 29 dpi and the non-structural NSm movement protein was observed 29 dpi. The detection of both viral proteins presumes that P. patens could serve as a new lab–host for TSWV, allowing reverse genetics by gene targeting to elucidate the role of specified molecular virus–host interactions.

Production of antibodies against a synthetic peptide based on a sequence common to all tospovirus non-structural NsS protein

The hypovirulence-inducing Fusarium graminearum virus China 9 (FgV-ch9) was described recently and is closely related to the Fusarium graminearum mycovirus-2 (FgV2). Both viruses share common properties of viruses belonging to the family Chrysoviridae. Re-sequencing of FgV-ch9 revealed duplications of the 3′ non-coding regions of segments 2 and 3. Both duplications are arranged in a head-to-tail array, are attached to the complete terminus, and do not affect the encoded gene. An internal duplication was found in segment 5. This duplication resulted in an increase in the size of the encoded protein. In silico analysis showed similar duplications in segments 2 and 3 of FgV2.