王林发) 186 • Guoping Wang (王国平) 85 • Yanxiang Wang (王雁翔) 85 • Yaqin Wang (王亚琴) 38 • Muhammad Waqas 187 • Tàiyún Wèi (魏太云) 188 • Shaohua Wen (温少华) 85 • Anna E. Whitfield 189 • John V. Williams 190 • Yuri I. Wolf 99 • Jiangxiang Wu (吴建祥) 38 • Lei Xu (徐雷) 138 • Hironobu Yanagisawa (栁澤広 宣) 191 • Caixia Yang (杨彩霞) 69 • Zuokun Yang (杨作坤) 85 • F. Murilo Zerbini 192 • Lifeng Zhai (翟立峰) 193 • Yong-Zhen Zhang (张永振) 220,221 • Song Zhang (张松) 34 • Jinguo Zhang (张靖国) 194 • Zhe Zhang (张哲) 85 • Xueping Zhou (周雪平) 195

BackgroundThere is a lack of published evidence on the importance of methotrexate (MTX) dose and route of administration on both its efficacy and adverse events in children with Juvenile Idiopathic Arthritis (JIA). We aimed to document our clinical practice based on the treat-to-target approach in order to support the concept that better therapeutic effect achieved with an optimal dose of parenteral MTX is associated with clinically acceptable adverse effects comparable to those reported for oral treatment.MethodsStudy inclusion criteria were indication of new MTX therapy for active arthritis in confirmed JIA patients younger than 18 years. Eligible patients were evaluated prospectively every 3 months for 1 year using standardized instruments for treatment response (American College of Rheumatology Pediatric (ACRPedi) response, Juvenile Arthritis Disease Activity Score (JADAS) 71, Clinically Inactive Disease (CID)) and adverse events (laboratory monitoring, Methotrexate Intolerance Severity Score (MISS)). MTX responders had to achieve at least ACRPedi 70 response. MTX intolerance was defined by MISS ≥ 6.ResultsIn 45/55 patients (81.8 %) MTX was started as subcutaneous injection. The initial median weekly dose was 14.4 mg/m2 in parenteral and 11.7 mg/m2 in oral administration. MTX therapy was effective in the level of ACRpedi70 and CID in 50.9 % and 30.9 % of patients at month 6 and in 70.9 % and 56.4 % after 12 months of the treatment, respectively. MTX intolerance at 6 and 12 months was noted in 25.5 % and 30.6 %, respectively. Management of intolerance included change in the dose and/or route of administration, education and councelling. Adverse events led to MTX withdrawal in 5 patients (9 %) due to toxicity (n = 3) and intolerance (n = 2). We did not find any significant predictive factors for either MTX therapeutic response or intolerance.ConclusionSubcutaneous MTX weekly dose around 15 mg/m2 is associated not only with a high response rate within the first 12 months of treatment, but also with a relatively low rate of significant adverse effects that would lead to the treatment termination. It allows early recognition of MTX non-responders and addition of biologic therapy. Sustainability of therapeutic effect and longer-term evolution of adverse events will be addressed by an ongoing extension of the study.

During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.

Winter oilseed rape grown in several areas in South Bohemia showed symptoms of stunting, leaf reddening and extensive malformation of floral parts. Phytoplasmas were consistently observed by using electron microscopy only in phloem tissue of symptomatic plants. DNA isolated from infected and healthy control plants was used in PCR experiments. Primer pairs R16F2/R2, P1/P7 and rpF2/R2, amplifying, respectively, 16S rDNA, 16S rDNA plus spacer region and the beginning of the 23S and ribosomal protein gene L22 specific for phytoplasmas, were used. According to RFLP and sequence analyses of PCR products, the phytoplasma from rape was classified in the aster yellows phytoplasma group, subgroup 16SrI-B. The PCR products from the Czech phytoplasma-infected rape also had RFLP profiles identical to those of phytoplasma strains from Italian Brassica. This first molecular characterization of phytoplasmas infecting rape compared with strains from Brassica does not, however, clearly indicate differences among isolates of the same 16SrI-B subgroup. Further studies on other chromosomal DNA portions could help the research on host specificity or on geographical distribution of these phytoplasmas.

Virus diseases of strawberry present several complex problems. More than 25 viruses have been described in the genus Fragaria thus far. Here, we describe a novel rhabdovirus, tentatively named strawberry virus 1 (StrV-1), that infects F. ananassa and F. vesca plants. Genomic sequences of three distinct StrV-1 genotypes co-infecting a single F. ananassa host were obtained using combined Illumina and Ion Proton high-throughput sequencing. StrV-1 was transmitted to herbaceous plants via Aphis fabae and A. ruborum, further mechanically transmitted to Nicotiana occidentalis 37B and sub-inoculated to N. benthamiana, N. benthamiana DCL2/4i, N. occidentalis 37B, and Physalis floridana plants. Irregular chlorotic sectors on leaf blades and the multiplication of calyx leaves seem to be the diagnostic symptoms for StrV-1 on indexed F. vesca clones. StrV-1 was detected in asymptomatic grafted plants and in 49 out of 159 field strawberry samples via RT-PCR followed by Sanger sequencing. The bacilliform shape of the virions, which have a cytoplasm-limited distribution, their size, and phylogenetic relationships support the assignment of StrV-1 to a distinct species of the genus Cytorhabdovirus. Acyrthosiphon malvae, A. fabae, and A. ruborum were shown to transmit StrV-1 under experimental conditions.

Strawberry crinkle disease is one of the major diseases that threatens strawberry production. Although the biological properties of the agent, strawberry crinkle virus (SCV), have been thoroughly investigated, its complete genome sequence has never been published. Existing RT-PCR-based detection relies on a partial sequence of the L protein gene, presumably the least expressed viral gene. Here, we present complete sequences of two divergent SCV isolates co-infecting a single plant, Fragaria x ananassa cv. Čačanská raná.

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.

Visual inspections of elm trees in south Moravia in 1997-2007 revealed a rare occurrence of plants with smaller and cowl-forming leaves on some twigs, i.e. a feature resembling witches'-broom disease observed on the end of twigs. The presence of phytoplasma-like bodies was observed by transmission electron microscopy of phloem tissue. On the other hand, no phytoplasmas were found in asymptomatic trees. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specific for phytoplasmas. Sequence analyses of the 16S-23S ribosomal operon (1852 bp) allowed for the classification of the detected phytoplasmas in the elm yellows group, but its position remained on the boundary of the 16SrV-A and 16SrV-C ribosomal subgroups. Sequence analyses of the ribosomal protein of the rpl22-rps3 and secY genes lead to further classification and revealed the phytoplasmas' affiliations to the 'Candidates Phytoplasma ulmi'. Some exceptions in unique oligonucleotide sequences defined for 'Ca. Phytoplasma ulmi' were found in the Czech isolate. This is the northernmost confirmed occurrence of phytoplasma on elm trees within Europe.