Grapevine Fanleaf Virus Monoclonal Antibodies: their Use to Distinguish Different Isolates

Monoclonal antibodies (MCAs) specific for Grapevine Fanleaf Virus (GFV) were obtained by fusion of a non-secreting myeloma cell line with the spleen cells from Balb/c mice immunized with GFV isolate F 13. The MCAs were tested with apanel of GFV isolates, using both crude extracts and purified virus particles. Some of the MCAs recognize all 41 isolates tested and are suitable as a diagnostic reagent for GFV detection. Certain MCAs do not bind to some of the isolates, while others show a lower reactivity for certain isolates than for the immunogen F 13. MCAs make it possible to distinguish GFV isolates which until now could not be distinguished by using polyclonal antiserum.     

African Cassava Mosaic Virus (ACMV): Stability of Purified Virus and Improved Conditions for its Detection in Cassava Leaves by ELISA

African Cassava Mosaic Virus (ACMV) was purified by a method which allowed the separation of monomer from dimcr virus particles. Optimal conditions for storing purified virus to be used for immunization were determined by ELISA and inoculation on Nicotiana benthamiana. Purified virus could be stored without loss of infectivity and serological activity for more than 145 days at 4 °C or frozen at –20 °C, but not longer than 40 days in the presence of 50 % redistilled glycerol. Rabbit and chicken immunoglobulins were used to detect ACMV in cassava leaves by direct and indirect ELISA. To obtain the same absorbance values, it was necessary to use longer incubation times with the indirect method, but the virus detection end-point m sap from infected plants was the same for the two methods (1/512). Conditions for improving virus detection tn cassava samples were determined. The virus was better detected when leaves from diseased plants were ground in 100 mM Tris-HCl containing 1 % polyvinylpyrrolidone at pH 8.5 than in phosphate buffer. Plant inhibitors were the restricting factor in the detection of virus by ELISA, but this difficulty was avoided when leaves to be tested were harvested from the top of the cassava plants.     

The detection by serological methods of viruses infecting the rose

Homogenates of herbaceous test plants infected with arabis mosaic virus (AMV), prunus necrotic ringspot virus (PNRSV), or strawberry latent ringspot virus (SLRV), and purified virus preparations were used to assess the sensitivities of four serological methods (the enzyme-linked immunosorbent assay - ELISA, immunodiffusion in gels, the latex flocculation assay, and serologically specific electron microscopy -SSEM) for the detection of these viruses. The latex test was up to 250 times more sensitive than gel immunodiffusion, but SSEM and ELISA were respectively up to 1000 and 200 times more sensitive than the latex test. Gel immunodiffusion and latex tests failed to detect any of the viruses in infected roses. Although ELISA reliably detected PNRSV and SLRV when leaves from infected roses were homogenised in a leaf: buffer ratio of 1 g:10 ml, AMV was occasionally undetected. However, when a modified ELISA technique, which reduced non-specific reactions, was used some PNRSV-infected roses were also not detected. Detection by SSEM was c. twice as sensitive as ELISA for all three viruses in rose extracts. The relative advantages of ELISA and SSEM for the detection of plant viruses are discussed.     

Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3'-untranslated regions (UTRs) of both RNAs deleted (R1Delta/2Delta construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Delta/2Delta construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Delta/2 (3'-UTR of RNA 1 deleted), R1/2Delta (3'-UTR of RNA 2 deleted), or R1Delta/2Delta plus R3. This demonstrates that the 3'-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3'-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3'-termini of AMV RNAs is not required for assembly of virus particles.     

Alfalfa mosaic and cucumber mosaic virus infection in chickpea and lentil: incidence and seed transmission

Samples collected in 1994 and 1995 from commercial crops of chickpeas and lentils growing in the agricultural region of south-west Western Australia were tested for infection with alfalfa mosaic (AMV) and cucumber mosaic (CMV) viruses, and for members of the family Potyviridae using enzyme-linked immunosorbent assay (ELISA). In 1994 no virus was detected in the 21 chickpea crops tested but in 1995, out of 42 crops, AMV was found in two and CMV in seven. With lentils, AMV and/or CMV was found in three out of 14 crops in 1994 and 4 out of 13 in 1995, both viruses being detected in two crops in each year. Similar tests on samples from chickpea and lentil crops and plots growing at experimental sites, revealed more frequent infection with both viruses. No potyvirus infection was found in chickpeas or lentils in agricultural areas either in commercial crops or at experimental sites. However, bean yellow mosaic virus (BYMV) was detected along with AMV and CMV in irrigated plots of chickpeas and lentils at a site in Perth. When samples of seed from infected crops or plots of chickpeas and lentils were germinated and leaves or roots of seedlings tested for virus infection by ELISA, AMV and CMV were found to be seed-borne in both while BYMV was seed-borne in lentils. The rates of transmission found through seed of chickpea to seedlings were 0.1–1% with AMV and 0.1–2% with CMV. Seed transmission rates with lentil were 0.1–5% for AMV, 0.1–1% for CMV and 0.8% for BYMV. Individual seed samples of lentil and chickpea sometimes contained both AMV and CMV. With both species, infection with AMV and CMV was sometimes found in commercial seed stocks or seed stocks from multiplication crops of advanced selections nearing release as new cultivars. Seed-borne virus infection has important practical implications, as virus sources can be re-introduced every year to chickpea and lentil crops or plots through sowing infected seed stocks leading to spread of infection by aphid vectors, losses in grain yield and further contamination of seed stocks.     

Production and use of cDNA clones from arabis mosaic virus

Arabis mosaic virus (AMV) genomic RNAs were converted to dsDNA and cloned into bacterial plasmids. Insert sizes of cDNA clones ranged from 0·2 to 3·2 kbp. Restriction enzyme mapping identified clones representing at least 90% of the RNA-2 genome. A 0·9 kbp clone specific to RNA-1 was also identified. Northern blot hybridisations of AMV RNAs with clones from either RNA-1 or RNA-2 showed no cross reactions. The sensitivity of virus detection in dot hybridisation was 15 pg of purified genomic RNA and 40 pg of purified virus particles. The possibility of using cDNA clones for the detection of AMV in strawberry sap was demonstrated. Two AMV dsRNAs corresponding to genomic RNAs in size were isolated from infected cucumber plants and reacted in hybridisation experiments.     

Evaluation of Different Polymeric Organic Materials for Creating Conditions That Favor Reductive Processes in Groundwater

The ability of three different polymeric organic materials (POMs) to create redox conditions favorable for reductive processes such as dechlorination was evaluated over a period of 70 days. Corn crop residue, unrefined chitin, and wood shavings were mixed with sand, packed into columns, and flushed with fresh groundwater once per day. Extracted groundwater was evaluated for pH, hydrogen, methane, volatile fatty acids (VFAs), alcohols, dissolved organic carbon (DOC), and chemical oxygen demand (COD). The pH measured for all POMs was between 5 and 8.5, with values after 30 days generally between 6 and 8. Hydrogen and methane concentrations indicated that all columns remained anaerobic during the entire experiment. Acetate was the dominant VFA in all columns, although propionate, butyrate, isobutyrate, and isovalerate were detected at much lower concentrations. Also, acetate concentrations were greater at all times in columns containing unrefined chitin (4 to 43 mM) than in those containing either corn crop residue (1 to 7 mM) or wood shavings (0 to 3 mM). Total electron donating capacities for dechlorination (expressed as total chloride removal capacities) were estimated from total COD and hydrogen concentrations. This capacity was greater in the column containing unrefined chitin than in those containing either corn crop residue or wood shavings. Overall the electron donating capacities remaining after 70 days were 40%, 11%, and 44% of the initial values for the corn crop residue, the unrefined chitin, and the wood shavings, respectively. The results indicate that over a 70-day period, anaerobic and nutrient-rich conditions were sustained for all POMs, but that the amount of electron donor available for reductive terminal electron accepting processes varies between POMs and is greatest for unrefined chitin.     

Effect of Aliette on AMV Infection of Bean Leaves and on the Resultant Alterations in the Patterns of Proteins and Peroxidases

Increased peroxidase activity as well as changes in the patterns of soluble proteins, and peroxidases were observed following infection of bean leaves with AMV.
Foliar sprays with Aliette® (phosethyl Al) at 2000 ppm a. i. delayed the appearance of necrotic local lesions and reduced their final number on primary bean leaves following inoculation with alfalfa mosaic virus (AMV).
The delay in the appearance of symptoms observed on Aliette-treated inoculated leaves was correlative with a delay in all the above cited alterations, while reduction of final symptoms was correlative with a decrease of these alterations.     

Simultaneous Detection of Several Nepoviruses Infecting Grapevine in a Single DAS-ELISA Test Using Mixed Antisera

Single and mixed antisera have been compared in DAS-ELISA for the routine diagnosis of nepoviruses infecting grapevine. The use of mixed polyclonal antibodies allowed in a single test the simultaneous detection of several nepoviruses (ArMV + GFLV) or serotypes of nepoviruses (TBRV serotypes G + S and RRV serotypes E + G) whatever the nature of the antigens, e.g. purified virions, diseased grapevine leaves or grapevine wood shavings. The detection was as reliable and efficient as with simple antibodies. The plant samples which were positively diagnosed by mixed antisera often showed an increase of their absorbance values, in comparison with the detection using simple antisera, while the background level was unchanged. The origin of this enhancement remains unclear, but it seems to be closely related to the mixing of the conjugated antibodies.     

Incidence and Control of Necrotic Leaf Mosaic Caused by Arabis Mosaic Virus in Lilium tigrinum splendens in the Netherlands

Necrotic mosaic on leaves and ring spots on bulb scales of Lilium tigrinum splendens, can be caused by arabis mosaic virus (AMV). Primarily infected bulbs can show spongy roots and large necrotic areas on creamy coloured bulb scales. Consecutive series of plants replanted for monthly periods in infested soil were mostly infected by AMV at a high rate (70%) throughout the year. Very low or undetectable numbers of Xiphinema diversicandatum, nematodes in soil dilution experiments infected lilies very efficiently (50–70 %). In general, soil disinfestation with dichloropropene, dazomet, methylbromide, and other disinfectants were variably fairly effective, particularly when yellow crocus among which couch was abundant, was previously grown for two years. The influx of AMV infected material into the soil was assumed to increase the number of AMV-carrying nematodes, and may be one cause of the failure of soil disinfestation. A survey of AMV infested soil in lily-growing regions in The Netherlands indicated its occurrence in a few fields only. Complex control measures applicable under growers' conditions, in addition to the variably effective soil disinfestation, are indicated.     

Studies on rose mosaic disease in field-grown roses produced in the United Kingdom

Arabis mosaic virus (AMV) and prunus necrotic ringspot virus (PNRSV), separately or together, caused in field-grown roses the range of symptoms recognised as rose mosaic disease. PNRSV infection alone generally induced chlorotic line patterns, ring-spots or mottles in the leaves at some time during the growing season; AMV plus PNRSV normally caused chlorotic vein-banding. However, during prolonged periods of high temperatures (c. 21 °C or more) vein banding occurred in some roses infected only with PNRSV. Isolates of PNRSV from rose had particles which were similar in shape, protein mol. wt, density and sedimentation coefficients to previously described isolates of PNRSV from cherry, plum and rose; all were cherry serotypes. In graft-inoculated roses, apple serotypes of PNRSV induced stunting and chlorosis, puckering and distortion of leaves, which closely resembled symptoms associated with rose mosaic in the USA and chlorotic mottle rose mosaic in New Zealand. To avoid possible confusion in using the name rose mosaic it is suggested that the virus(es) present in roses should be named.     

VSV Pseudotype Particles with the Coat of Avian Myeloblastosis Virus

PSEUDOTYPES of vesicular stomatitis virus (VSV) with the coat of avian myeloblastosis (AMV) or murine leukaemia viruses—VSV(AMV) and VSV(MLV)—can be produced by growing VSV in chick cells preinfected with AMV or in mouse cells preinfected with MLV¹. The VSV particles carrying their own neutralization antigen and double-neutralizable particles may be inactivated with antiserum against VSV. The surviving pseudotypes possess neutralization, host-range and interference specificities corresponding to the tumour virus donating their coat. It has also been shown that a conditional lethal mutant of VSV in which a structural protein is affected is complemented under restrictive conditions with AMV. This mutant, ts-45, when complemented with AMV again predominantly produces the pseudotype VSV(AMV).     

The effect of crystallinity of cellulose on the rate of reducing sugars production by heterogeneous enzymatic hydrolysis

A kinetic model is devised, from the reaction mechanism steps, to predict the rate of reducing sugar production by hydrolysis of two types of cellulose, namely, amorphous carboxymethylcellulose (CMC) and highly crystalline wood shavings, using Aspergillus niger cellulase. Experimental results in a stirred batch reactor at 40 degrees C show that the production of reducing sugar reduced at much shorter times for wood shavings in comparison to CMC at the same initial substrate concentration. The experimental results are used to determine the kinetic parameters of the model equations. The significance of crystallinity was determined using inert fraction coefficient, which is assumed to be constant and equals 0.05 and 0.98 for CMC and wood shavings, respectively. It is shown there is a good agreement between the experimental results and proposed kinetic model predictions. The effect of the inert fraction coefficient on the production of reducing sugar by the enzymatic hydrolysis of cellulose is also determined. It is found that the cellulase used extracted from A. niger is much more sensitive towards the substrate structure in comparison to that extracted from Trichoderma reesei.     

Restriction of Virus Movement into Axillary Buds is an Important Aspect of Resistance in Cassava to African cassava mosaic virus

Axillary buds and bark samples of resistant, moderately resistant and susceptible (control) cassava genotypes either naturally infected under field conditions or experimentally inoculated by grafting were indexed for African cassava mosaic virus (ACMV). Virus detection was carried out using enzyme‐linked immunosorbent assay and polymerase chain reactions to determine the distribution of the virus within the plant and elucidate the genotypes response to virus movement. Significantly more bud and bark samples were positive for virus on the susceptible genotype TME 117 than resistant genotypes TMS 30001 and TMS 91/02319, or the moderately resistant genotype TMS 30572. Detectable virus concentration was significantly lower in the buds of moderately resistant and resistant genotypes than the susceptible control. Under field conditions, it was significant that more primary stem buds were infected than the buds of secondary and tertiary stems but such a gradient was not obvious with bark samples. Shoots that had asymptomic new leaves after the initial symptomatic leaves had no virus in their buds, but some of the bark samples from the same plants tested positive. A significant interaction was observed between year and stem type, and among year, genotype and stem type with respect to virus detection in bud and bark samples. Restriction of virus movement into axillary buds occurred in all the resistant and moderately resistant genotypes. This may explain ACMV‐infected stem cuttings of resistant genotypes producing healthy plants in subsequent generation.     

Virus-coded origin of a 32,000-dalton protein from avian retrovirus cores: structural relatedness of p32 and the beta polypeptide of the avian retrovirus DNA polymerase.

A 32,000-dalton protein (p32) located in avian retrovirus cores was immunoprecipitated from [35S]methionine-labeled avian myeloblastosis virus (AMV) propagated in cultured chicken embryo fibroblast cells by an antiserum preparation (sarc III) derived from tumor-bearing hamsters injected with cloned and passaged cells from an avian sarcoma virus-induced primary hamster tumor. Since sarc III serum apparently contained antibodies only to virus-coded proteins and not to chicken cellular proteins, the immunoprecipitation of p32 from AMV by sarc III serum strongly suggested that p32 is virus coded. The origin of p32 was more definitively established by demonstrating the existence of a structural relationship between p32 and the AMV DNA polymerase. AMV p32 cross-reacted with the beta polypeptide of AMV alphabeta DNA polymerase in radioimmunoprecipitation and radioimmunoprecipitation inhibition assays, indicating that p32 and beta share common antigenic determinants. This relationship was clarified by sodium do-decyl sulfate-polyacrylamide gel electrophoretic analysis of the peptides generated by limited proteolysis of 125I-labeled AMV DNA polymerase polypeptides and of 125I-labeled AMV p32 by chymotrypsin or Staphylococcus aureus V-8 protease. The peptides which appeared during proteolytic digestion of p32 were a subset of those produced by digestion of the beta polypeptide; however, p32 had no discernible peptides in common with the alpha polypeptide. Further, all of the peptides produced by limited proteolysis of beta were present in the digests of either p32 or alpha. Our findings suggest that p32 is apparently derived by cleavage of the beta polypeptide of AMV DNA polymerase, presumably at a site near or identical to that at which alpha is generated from beta by proteolytic cleavage.     

Virus content of leaves of cassava infected by African cassava mosaic virus

African cassava mosaic virus (ACMV) was detected in cassava leaves by ELISA. Some normal constituents of cassava leaves interfered with virus detection but leaf extracts of Nicotiana benthamiana did not. The symptom pattern was determined early in the growth of a leaf and subsequently changed little. ACMV was found only in the yellow or yellow green areas of the mosaic pattern. Virus content of the leaves increased with increasing symptom intensity, but decreased with leaf age and ACMV was not detected in mature leaves. Most whiteflies were found on young growing cassava leaves and the number decreased progressively with leaf age. This distribution will aid both the acquisition and inoculation of the virus.     

Use of protein A to improve sensitisation of latex particles with antibodies to plant viruses

Protein A-coated latex (PAL) was compared with uncoated latex (L) for sensitisation with antibodies to five plant viruses: apple mosaic virus (ApMV), arabis mosaic virus (AMV), plum pox virus (PPV), potato virus Y ordinary strain (PVY°) and prunus necrotic ringspot virus (NRS V). A range of globulin concentrations was used with each antiserum and detection end points determined in serial dilutions of infective sap. When sensitised with antibodies to ApMV, PAL detected ApMV readily, whereas L did not. When sensitized with antibodies to PVY° and AMV, PAL gave higher detection end points than L. However, PAL gave little increase in sensitivity with the antisera to PPV and NRSV. Non-specific aggregation of latex, which sometimes occurred in very dilute sap with PAL, could be dispersed by adding 0.02% Tween-20 to the extraction buffer. Globulins of PVY° and AMV could be used at higher dilutions with PAL than with L, giving a saving in antiserum. Both types of latex sensitised with PVY° antibody globulins readily detected the tobacco veinal necrosis and C strains of this virus.     

Serological diagnosis and host range studies of important viral diseases of a few cucurbitaceous crops in Maharashtra,India

Around 39 well characterised viruses affect cucurbits crops in developing countries and their viral diversity may be the consequence for genetic and ecological diversity of their hosts. Indeed, cucurbits are grown in variety of climatic, environmental and agricultural conditions, and this may provide more or less favourable conditions for the specific viruses or their hosts. The presence of various viral diseases caused by different viruses in Maharashtra was studied from the infected samples collected from cucurbits and melons during the survey conducted in 2009–2010 in different locations. The virus isolates collected from various cucurbitaceous crops were established and their host ranges were studied by sap transmission. The study revealed Cucumber Mosaic Virus (CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and Cucumber green mottle mosaic virus infections predominately found in Nashik region, and Watermelon bud necrosis virus (WBNV), CMV, ZYMV, WMV and Watermelon silver mottle virus (WSMoV) infections in Aurangabad and Paithan regions. In Sangamner region, the crop was mostly affected by WBNV, ZYMV and WSMoV, and CMV was found only in Sillod region. The protocols for performing sap transmission tests in assay hosts were standardised for ZYMV, CMV and WBNV. Using direct antigen-coating enzyme-linked immunosorbent assay, of all the plant parts, young leaves were found to have high concentration of virus and suitable for virus detection in screening programmes. CMV and ZYMV was found to have high concentration of virus and suitable for virus detection in screening programmes.     

Transmission of the hop strain of arabis mosaic virus by Xiphinema diversicaudatum*

Hop plants became infected with the hop strain of arabis mosaic virus (AMV(H)) when grown in hopfield and woodland soil in which infected plants had been growing. Infection occurred in soil infested with the dagger nematode Xiphinema diversicaudatum, but neither in uninfested soil nor in soil previously heated to kill nematodes. X. diversicaudatum transferred direct from hop soils transmitted AMV(H) to young herbaceous plants and to hop seedlings; some of the hop seedlings developed nettlehead disease. A larger proportion of plants was infected using X. diversicaudatum obtained from a woodland soil and then given access to the roots of hop or herbaceous plants infected with AMV(H). AMV(H) was transmitted by adults and by larvae, in which the virus persisted for at least 36 and 29 wk, respectively. Difficulties were encountered in detecting AMV(H) in infected hop plants, due partly to the delay in virus movement from roots to shoots. Infection of hop shoots was seldom detected until the year after the roots were infested and sometimes nettlehead symptoms did not appear until the third year. Isolates of arabis mosiac virus from strawberry did not infect hop. The results are discussed in relation to the etiology and control of nettlehead and related diseases of hop.     

Transformation of chicken myelomonocytic cells by a retrovirus expressing the v-myb oncogene from the long terminal repeats of avian myeloblastosis virus but not Rous sarcoma virus.

To test the effect of long terminal repeat (LTR) regulatory sequences on the transforming capability of the v-myb oncogene from avian myeloblastosis virus (AMV), we have constructed replication-competent avian retroviral vectors with nearly identical structural genes that express v-myb from either AMV or Rous sarcoma virus (RSV) LTRs. After transfection into chicken embryo fibroblasts, virus-containing cell supernatants were used to infect chicken myelomonocytic target cells from preparations of 16-day-old embryonic spleen cells. Both wild-type AMV and the virus expressing v-myb from AMV LTRs (RCAMV-v-myb) were able to transform the splenocyte cultures into a population of immature myelomonocytic cells. The transformed cells expressed the p48v-Myb oncoprotein and formed compact foci when grown in soft agar. In contrast, the virus expressing v-myb from RSV LTRs (RCAS-v-myb) was repeatedly unable to transform the same splenocyte cells, despite being able to infect fibroblasts with high efficiency. This difference in the transforming activities of v-myb-expressing viruses with different LTRs most likely results from the presence of a factor (or factors) within the appropriate myelomonocytic target cell that promotes specific expression from the AMV but not from the RSV LTR.