A variant of the satellite RNA of groundnut rosette virus that induces brilliant yellow blotch mosaic symptoms in Nicotiana benthamiana

Some Malawian cultures of groundnut rosette virus (GRV) give rise to variants that, although still causing symptoms of the chlorotic type of rosette in groundnut, induce brilliant yellow blotch mosaic symptoms, instead of the usual veinal chlorosis and mild mottle, in Nicotiana benthamiana. One such isolate (YB) induced the formation in infected plants of a 0.9 kbp dsRNA having extensive sequence homology with molecules of similar size in other naturally occurring isolates of GRV. These dsRNA molecules were shown to be double-stranded forms of single-stranded satellite RNA molecules. Experiments in which the satellite was removed from and restored to isolate YB, or exchanged with those from other GRV isolates, showed that it carries the determinant for yellow blotch mosaic symptoms. Plants inoculated with the 0.9 kbp dsRNA (denatured or undenatured) developed yellow blotch mosaic even when the satellite-free GRV helper was not inoculated until 11 days later. The satellite RNA is therefore a very stable molecule. Prior infection of N. benthamiana with a GRV isolate containing a normal form of the satellite protected against expression of yellow blotch mosaic symptoms when the plants were later inoculated with isolate YB, whereas prior infection with satellite-free isolates did not. This provides a simple method of determining whether a GRV isolate has an associated satellite RNA. The YB satellite seems to be a newly recognised variant additional to those known to cause the chlorotic, green and other forms of groundnut rosette disease.     

Different variants of the satellite RNA of groundnut rosette virus are responsible for the chlorotic and green forms of groundnut rosette disease*

Groundnut plants with symptoms of rosette disease contain groundnut rosette virus (GRV), but GRV is transmitted by Aphis craccivora only from plants that also contain groundnut rosette assistor virus (GRAV). Two main forms of rosette disease are recognised, ‘chlorotic rosette’ and ‘green rosette’. GRV cultures invariably possess a satellite RNA and this is the major cause of rosette symptoms: satellite-free isolates derived from GRV cultures from Nigerian plants with chlorotic or green rosette, or from Malawian plants with chlorotic rosette, induced no symptoms, or only transient mild mottle or interveinal yellowing, in groundnut. When the satellite RNA species from GRV cultures from Nigerian green or Malawian chlorotic rosette were reintroduced into the three satellite-free isolates in homologous and heterologous combinations, the ability to induce rosette symptoms was restored and the type of rosette induced was that of the cultures from which the satellite RNA was derived. Thus different forms of the satellite are responsible for the different forms of rosette disease. Other forms of the satellite induce only mild chlorosis or mottle symptoms in groundnut. Individual plants may contain more than one form of the satellite, and variations in their relative predominance are suggested to account for the variable symptoms (ranging from overall yellowing to mosaic) seen in some plants graft-inoculated with chlorotic rosette.     

Groundnut rosette and its assistor virus

Chlorotic rosette from Malawi (isolate CR1), passed through Stylosanthes gracilis and S. juncea, was not subsequently transmissible from groundnuts (Arachis hypogaea) by Aphis craccivora or A. gossypii, but with S. mucronata transmissibility was occasionally regained after a period of time. Aphid transmissibility was similarly lost after passage of two isolates (a chlorotic rosette from Rhodesia, CR2, and a green rosette from Nigeria, GR) through soybean (Soja max) and after manual inoculation to groundnuts. Groundnut plants that remained symptomless after exposure to rosette infection by aphids often contained a virus that restored aphid transmissibility when introduced into groundnuts containing the vectorless virus from that isolate. Groundnut rosette disease therefore consists of a symptom-inducing virus that we call groundnut rosette virus (GRV) and a symptomless assistor virus (GRAV) that must be present for aphid transmission. The interactions between the GRV and GRAV of chlorotic and green rosette, and their transmission by different vector races, are described.     

The nature of the resistance in groundnut to rosette disease

Groundnut rosette disease is caused by a complex of three agents, groundnut rosette virus (GRV) and its satellite RNA, and groundnut rosette assistor virus (GRAV); the satellite RNA is mainly responsible for the disease symptoms. Groundnut genotypes possessing resistance to rosette disease were shown to be highly resistant (though not immune) to GRV and therefore to its satellite RNA, but were fully susceptible to GRAV.     

Studies on Variability of Peanut Clump Virus: Symptomatology and Serology

Intensive survcys of groundnut virus diseases were carried out in Senegal from 1986 to 1990. Peanut clump virus (PCV; furovirus group) was detected in several regions in groundnuts (Arachis hypogaea), showing typical symptoms namely, small dark green leaves, short petioles and internodes, and reduced shoot size resulting in a dwarfed and bushy appearance (clumping) of the infected plants. PCV was also detected in groundnuts exhibiting variable symptoms like chlorotic leaf spots, specking, chlorotic rings or ringspots, line patterns, vein yellowing, mottle or light mosaic etc. with or without clumping. Symptoms induced by these different isolates on the test plant Chenopodium amaranticolor also showed considerable variability. Serological studies of 41 isolates of PCV (collected from Senegal, Burkina Faso, Niger and India), using seven monoclonal antibodies in Triple Antibody Sandwich ELISA (TAS-ELISA), permitted us to distinguish five different serogroups based on their reaction profiles. However, these did not correspond to the five groups formed in an arbitrary classification based on the symptomatology of C. amaranticolor. Serogroups do not correlate with the geographic origin.     

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3.     

An umbraviral protein,involved in long-distance RNA movement,binds viral RNA and forms unique,protective ribonucleoprotein complexes

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.     

Viruses associated with chlorotic rosette and green rosette diseases of groundnut in Nigeria*

Groundnut (Arachis hypogaea) plants from Nigeria with chlorotic rosette disease contained a manually transmissible virus, considered to be a strain of groundnut rosette virus (GRV(C)). GRV(C) infected nine out of 32 species in three out of nine families. It caused local lesions without systemic infection in Chenopodium amaranticolor, C. murale and C. quinoa, and systemic symptoms in Glycine max, Nicotiana benthamiana, N. clevelandii and Phaseolus vulgaris as well as in groundnut. Some ‘rosette-resistant’ groundnut lines were also infected. GRV(C) was transmitted by Aphis craccivora, but only from groundnut plants that were also infected with an aphid-transmissible second virus, which was not manually transmissible and was considered to be groundnut rosette assistor virus (GRAV). Plants infected with GRAV contained isometric particles c. 25 nm in diameter which were detectable by immunosorbent electron microscopy on grids coated with antisera to several luteoviruses, especially with antisera to bean leaf roll, potato leafroll and beet western yellows viruses. No virus-like particles were observed in extracts from plants infected with GRV(C) alone. A single groundnut plant obtained from Nigeria with symptoms of green rosette contained luteovirus particles, presumed to be of GRAV, and yielded a manually transmissible virus that induced symptoms similar to those of GRV(C) in C. amaranticolor but gave only mild or symptomless infection of N. benthamiana and N. clevelandii. It was considered to be a strain of GRV and designated GRV(G).     

应用DNA-RNA斑点杂交法检测登革病毒核酸

新近制备了大量纯化的pEH920 DNA,该质粒DNA插入了登革病毒2型核酸片段的互补DNA。以[a-~(32)P]dCTP按缺口转译法标记pEH 920 DNA作为探针,以感染病毒的蚊细胞c_6/36培养上清作标本,应用DNA-RNA斑点杂交法检测了登革病毒核酸。结果显示同位素标记探针(pEH 920)与登革病毒2型标本反应最强,具有一定的型特异性。但与其它血清型登革病毒也呈一定交叉反应。初步探讨了探针的敏感性,至少可检出TCID_(50)625的登革病毒2型核酸。     

不同CMV分离物侵染寄主的超微结构变化

应用电镜观察了黄瓜花叶病毒ＣＭＶ不同分离物侵染寄主的细胞超微结构变化。来自一患红（Ｓａｌｖｉａｓｐｌｅｎｄｅｎｓ）的不含卫星ＲＮＡ分离物Ｍ－２２侵染心叶烟,病毒粒子散布于细胞质,在液泡中形成大片病毒粒子结果,液泡膜边缘产生小泡结构,完整的病毒粒子穿过胞间连丝在细胞间运转,胞间连丝中央部分有扩张现象。     

A Novel Satellite RNA of Cucumber Mosaic Virus Induces Unique Line-pattern Mosaic Symptoms in Tobacco

The CMV(YW) isolate of cucumber mosaic virus (CMV) induced unique line‐pattern mosaic symptoms in systemically infected leaves of tobacco (Nicotiana tabacum cv. Ky57). By northern hybridization analysis using cDNA to CMV(Y) satellite RNA as a probe, it was confirmed that CMV(YW) contained a satellite RNA. which was designated sat‐YW RNA; this was 388 nucleotides in length and did not have either a conserved domain that induces necrosis in tomato or chlorosis in tobacco. CMV(YW) free of sat‐YW RNA. which was isolated by the single lesion isolation method using Chenopodium amaranticolor, did not induce the unique line‐pattern mosaic symptom. Furthermore, the sat‐YW RNA‐mediated line‐pattern mosaic symptom was also induced by in vitro transcribed infectious sat‐YW RNA in tobaccos infected with either CMV(YW) or CMV(Y) genomic RNA. These results clearly demonstrated that sat‐YW RNA induces the unique line‐pattern mosaic symptom on CMV‐infected tobaccos.     

十字花科蔬菜上黄瓜花叶病毒分离物的比较研究

对从杭州市郊青菜、萝卜、花椰菜上分离的6个CMV分离物进行了生物学、血清学及双链RNA比较研究。生物学测定结果表明,不同分离物在所测定的6种十字花科蔬菜上的致病力有差异。6种分离物颗粒形态、衣壳蛋白分子量和血清学方面无差异,且都属血清型Ⅰ。dsRNA分析结果表明,6个分离物的dsRNAl,2,3和4在PAGE中迁移率相似,但在1.5kb～0.4kb之间有多条量相对较少的条带,这些条带在不同分离物之间差异较大。6个分离物均含有卫星RNA。     

Inheritance of resistance to groundnut rosette virus in groundnut (Arachis hypogaea L.)

A method of field screening groundnut seedlings for resistance to groundnut rosette virus (GRV), by means of which over 97% incidence was induced in rows of susceptible test plants, was developed at Chitedze Research Station in Malawi. Two GRV-resistant Virginia cultivars (RG 1 and RMP 40) were crossed with three susceptible cultivars, one from each of the Spanish (JL 24), Valencia (ICGM 48) and Virginia (Mani Pintar) botanical groups. Twelve F₁ reciprocal crosses and their F₂ and backcross generations were produced and the material screened in nurseries in 1985/86 and 1986/87. Seedlings raised from plants which did not become infected in the field were inoculated in the glasshouse in order to eliminate susceptible escapees. The numbers of diseased and healthy individuals in each population were subjected to χ² tests. In the majority of the F₂ populations a good fit was obtained for a ratio of one resistant to 15 susceptible plants, a ratio to be expected if resistance to GRV were determined by a pair of independent complementary recessive genes. This was further supported by data from backcross generations.     

The Effect of Groundnut rosette assistor virus on the Agronomic Performance of Four Groundnut (Arachis hypogaea L.) Genotypes

The effect of Groundnut rosette assistor virus (GRAV), in the absence of the other two agents (Groundnut rosette virus and its satellite RNA) of the groundnut rosette disease virus complex, was evaluated on the agronomic performance of four groundnut (=peanut) genotypes (CG‐7, ICGV‐SM‐90704, JL‐24 and ICG‐12991) with different botanical characteristics. All genotypes infected with GRAV showed mild yellowing/chlorosis of leaves and the symptoms persisted throughout their growth period. ELISA absorbance values indicated lower amounts of GRAV antigen in ICGV‐SM‐90704 than in the other genotypes. The reduction in leaf area due to GRAV infection varied between 15.5% and 21.7%, whereas the plant height was decreased between 11.3% and 13.4% among the four genotypes. GRAV infection caused 28.4%, 16.9%, 21.7% and 25.5% reduction in the dry weight of haulms in CG‐7, ICGV‐SM‐90704, JL‐24 and ICG‐12991 respectively. Plants infected with GRAV showed greater reduction in seed weight in CG‐7 (52.2%), followed by JL‐24 (46.1%), ICG‐12991 (40.7%) and ICGV‐SM‐90704 (25.7%). These results provide evidence for the first time that GRAV infection, without GRV and sat RNA, affect plant growth and contribute to yield losses in groundnut.     

A ribonuclease protection assay for the direct detection and quantitation of hepatitis C virus RNA

A ribonuclease protection assay (RPA) was developed for the direct detection and quantitation of HCV RNA in infected patients' sera or plasma using HCV [(32)P]RNA from the conserved 5'-untranslated region (5'-UTR) as a probe. We were able to directly detect the presence of HCV RNA by RPA in several infected patients' samples. The viremic status of HCV infected patients with indeterminate recombinant immunoblot assay (RIBA II) was also determined by this assay. Polymerase chain reaction (PCR) was also performed on all these samples and were found to be positive with a concordance of 100% between the results of PCR and RPA. The RPA was able to detect approximately 1 pg of HCV RNA. A limited sequence heterogeneity among HCV isolates was also observed by this assay, suggesting that this may be a faster method of detecting heterogeneous HCV sequences in patients' samples. This simple and specific method could be used to quantitate HCV RNA in order to better determine viremia and follow the course of HCV infection especially when RIBA II results are indeterminate.     

Properties and partial purification of infective material from plants containing groundnut rosette virus*

Groundnut plants with chlorotic rosette disease contain a manually transmissible virus, groundnut rosette (GRV), which is also transmitted in the persistent (circulative) manner by aphids (Aphis craccivora), but only from plants that are co-infected with a manually non-transmissible luteovirus, groundnut rosette assistor virus (GRAV). Strains of GRV from plants with chlorotic or green forms of rosette are called GRV(C) and GRV(G) respectively. An isolate of GRV(C) from Nigeria remained infective in Nicotiana clevelandii leaf extracts for 1 day at room temperature and for 15 days at 4d?C, but lost infectivity after 1 day at -20d?C or after dilution to 10^--⁴. Its infectivity and longevity in vitro were not altered by addition of 1 mg/litre bentonite to the extraction buffer. Infectivity in leaf extracts was abolished by treatment with 50% (v/v) ether, 10% (v/v) chloroform or 8% (v/v) n-butanol, but not by treatment for 30 min with RNase A at up to 100 ng/ml. In attempts to purify GRV(C), nearly all the infectivity from N. clevelandii extracts was found in the pellets from centrifugation at 65 000 g for 1. 5 h; infectivity also occurred in a cell membrane fraction that collected at the top of a 30% sucrose ‘cushion’ containing 4% polyethylene glycol and 0.2 M NaCI. However, no virus-like particles were found in either type of preparation by electron microscopy. Nucleic acid preparations made directly from GRV(C)-infected N. clevelandii leaves were very infective; this infectivity was totally inactivated by treatment for 30 min with RNase A at 10 ng/ml in buffers of both low and high ionic strength and was therefore attributed to ssRNA. When nucleic acid preparations were electrophoresed in gels no virus-specific bands were visible but the position of the infectivity indicated that the infective ssRNA has an apparent mol. wt of c. 1.55 × 10⁶. A similar mol. wt was indicated by the rate of sedimentation of the infective ssRNA in sucrose gradients. Preparations of dsRNA made from GRV(C)-infected N. clevelandii leaves contained a species of mol. wt c. 3.0 × 10⁶; in addition some dsRNA preparations contained an abundant component of mol. wt c. 0.6 × 106 together with several other components of intermediate mol. wt. Similar patterns of bands were observed in dsRNA preparations made from Nigerian-grown groundnut material infected with GRV(C) alone, or with GRV(C) + GRAV, or with GRV(G) + GRAV. The properties of GRV closely resemble those of two other viruses that depend on luteoviruses for transmission by aphids, carrot mottle virus and lettuce speckles mottle virus.     

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.     

生物素标记贾第虫全基因组DNA探针的制备及其特异性敏感性测定

用生物素标记的贾第虫全基因组ＤＮＡ探针,在斑点杂交试验中显示高度的敏感性和特异性。用它可检出１０ｎｇ贾第虫ＤＡ,１０＾３个贾第虫滋养体或包囊,且不与阴道毛滴虫、溶组织内阿米巴、弓形虫和ＢＡＢＬ／ｃ小鼠肝细胞ＤＮＡ,以及贾第虫患者粪便上清液发生交叉反应。本探针可用于贾第虫病病原体检测和虫株鉴定研究。     

Cucumber-mosaic-virus-associated RNA-5: XII. — Symptom-modulating effect is codetermined by the helper virus satellite replication support function

In tomato, the disease-modulating effects of a cucumber mosaic virus (CMV) satellite isolate from Belgium, here designated T-CARNA-5 (CARNA-5 = CMV-associated RNA-5), were found to be different depending on the supporting helper virus strain. With two CMV strains, T-CARNA-5 induced lethal necrosis, but with a third strain from Ixora spp. (CMV-Ix), aggravated stunting was observed. However, the primary structure of the T-CARNA-5 contained within virus isolated from tobacco or tomato infected with each of these three CMV strains, conformed to the conserved sequence profile of CARNA-5 isolates which are necrogenic in tomato. Dilution end-point bioassay of T-CARNA-5 established a direct cause-effect relationship between it and tomato necrosis or stunting, depending on the helper virus. Total nucleic acid extracts taken at different times from tomato plants infected with the above CMV strains and T- or S-CARNA-5 (used as non-necrogenic control) showed viral RNA, ssCARNA-5 and dsCARNA-5 to be present in significant amounts, but in sometimes dissimilar proportions depending on the combination; except in CMV-Ix/S-CARNA-5 infection where neither ss-nor dsCARNA-5 was found.The experiments established that CARNA-5 biological expression studies in CMV-infected tomato have to take into account the helper virus satellite replication support function, which may be a primary codeterminant of quantitative or qualitative differences in the symptom modulation observed.     

Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus

The nucleotide sequence of the satellite of arabis mosaic virus was determined using the satellite RNA encapsidated in virions. The 300-nucleotide long sequence showed extensive homology (50%) with that of the 359-nucleotide satellite RNA of tobacco ringspot virus, which occurs both in a linear and a circular form. This homology also revealed the presence of conceived sequences believed to mediate self-cleavage of the latter as well as other viral satellite RNAs. A circular form of the arabis mosaic virus satellite can be isolated from infected tissues and partially converts to the linear form upon elution from denaturing gels.