Serological detection of close and distant relationships of an isolated Tobamovirus

A virus causing mosaic and leaf yellowing symptoms on Mucuna pruriens was isolated and purified through its susceptible host Vigna unguiculata var. TVu 76. Yield of the purified virus obtained was 7.2 mg/kg. It had an ultraviolet (UV) absorption spectrum at 260 nm as 1.53±0.00 and at 280 nm as 1.45±0.00, which translated into nucleic acid content of 72.9 %. Determination of the molecular weight of the virus coat protein from a plotted graph gave 27 kiloDaltons. When the purified virus was electrophoresed in agarose gel, no secondary band was observed. Absorbance curve at 260 nm was obtained when the purified virus was measured spectrophotometrically. A volume of 16 000μL antiserum was obtained against the virus with concentration of 0.3 mg/ml. The titre value determined for the antiserum raised against the virus was 1:2000 as there was a wider difference between the readings obtained for the healthy and diseased controls. At this dilution, the titre value was two and a half times greater than that of the healthy control. The heterologous: homologous percentage (%) obtained for the isolated virus and TMV (Muguga strain) was 50.0 % when Protein-A sandwich enzyme linked immunosorbent assay (PAS ELISA) was used to determine its serological relationship with the virus and other Tobamovirus strains. Keyword: PAS ELISA, Tobamovirus, coat protein, molecular weight Corresponding author: OR odedaraoo@funaab.edu.ng ____________________________________________________________________ sdara@hotmail.com Department of Microbiology, Federal University of Agriculture, Abeokuta. International Rice Research Institute, Philliphines. Department of Botany, Faculty of Science, University of Ibadan, Ibadan, Nigeria. Departmant of Zoology, Faculty of Science, University of Ibadan, Ibadan, Nigeria. Virology Unit, International Institute of Tropical Agriculture, Ibadan, Nigeria. Introduction 2014) and Citrus (Kareem et al., 2014). Enzyme linked immunosorbent assay Protein A-sandwich (PAS) ELISA is an described by Clark and Adams (1977) showed indirect ELISA (Hughes & Thomas, 1988) and it that the microplate method of ELISA could be makes use of protein-A, a molecule isolated from very effectively applied to the detection and the cell wall of Staphylococcus aureus. The assay of plant viruses (Matthew, 1991). Since binding of Protein-A is used in several serological that time, the method has been more widely procedures (Matthews, 1991). For instance, an used (Matthews, 1991). The method is a indirect Protein-A sandwich (PAS-ELISA) which serological procedure and has been used to was more successful than double antibody detect various viruses in yams (Dioscorca alata) sandwich (DAS) ELISA in detecting heterologous (Odu et al., 1999; Eni et al., 2008), legumes isolates of cherry leaf roll virus was described by (Odedara et al., 2007; Odedara et al., 2012), Edwards & Cooper (1985). It was hypothesized that a het:hom PAS ELISA could be used to Solanaceous plants, cucurbits (Ayo-John et al., 114 Nig. J. Biotech. Vol. 35 (2018) 114-122 ISSN: 0189 1731 Available online at http://www.ajol.info/index.php/njb/index and www.biotechsocietynigeria.org DOI: https://dx.doi.org/10.4314/njb.v35i1.14 Serological detection of close and distant relationships of an isolated Tobamovirus 1,5 2,5 3 4,5 Odedara, O.O., Hughes, J.d'A., Odebode, A.C. and Ala, A. NIGERIAN JOURNAL OF BIOTECHNOLOGY Copyright resides with the authors in terms of the Creative Commons License 4.0. (See http://creativecommons.org/licenses/by/4.0/). Condition of use: The user may copy, distribute, transmit and adapt the work, but must recognize the authors and the Nigerian Journal of Biotechnology. centrifugation at 9,500 rpm for 30 min. The detect both close and distant serological supernatant was discarded into a clean beaker relationships of Tobamovirus (Hughes and and 4.0 g polyethylene glycol (PEG mol. wt. Thomas, 1988). 8000) was added for every 100 ml of the virus o Other viruses belonging to this group suspension. This was stirred for 1h at 4 C. are: Tomato mosaic virus (ToMV), Pepper mild Centrifugation was done at 10,000 g for 15 min mottle virus (PMMV), Cucumber green mottle and the pellet resuspended in 20 ml 0.01M mosaic virus (CGMMV), Sunnhemp mosaic virus phosphate buffer (pH 7.2) (Gooding & Herbert, (SHMV), Ullucus mild mottle virus (UMMV) 1967). To obtain a pure virus suspension, 0.4 g (Hughes & Thomas, 1988). They are rod-shaped sodium chloride (NaCl) and 0.4 g PEG were rigid tube viruses. Hosts for TMV include added for every 10 ml of the suspension while tobacco, tomato and other Solanacoeus plants. stirring and this continued till the salt dissolved TMV had caused 20 % loss on tomato (Scholthof, and the suspension centrifuged at 9,500 rpm for 2000). 15 minutes. The supernatant was discarded and Growth of Mucuna pruriens had been the pellets resuspended in 2 ml 0.01M phosphate emphasized for enhancement of soil fertility, buffer for each 100 ml of the initial extract. prevention of soil erosion and maintenance of Centrifugation was done for the suspension at soil physical structures (Versteeg & Koudokpon, 10,000 g for 5 minutes in order to remove the 1993, Sanginga et al., 1996; Makau & Gachene, contaminants (Gooding and Herbert, 1967). 2001). Viral symptoms expression such as The absorbance of the virus at 260 and 280 nm mosaic, leaf yellowing and leaf distortion were estimated and virus stored in 0.01M sometimes accompanying growth of M. pruriens. o phosphate buffer (pH 7.2) at -20 C from where it Some previous reports had shown that this plant was withdrawn for antiserum production and is sometimes infected with TMV. Hence, the other tests. present study was conducted with the following objectives: to purify the Tobamovirus from the Spectrophotometry of purified preparations of naturally infected host, determine the molecular isolated Tobamovirus. weight of its coat protein, analyze its serological The absorption spectrum of the purified relationships with its other strains and produce preparations of the isolated Tobamovirus was antibodies against the virus. determined with a Beckman DU 520 Model Spectrophotometer (Beckman Instruments, Materials and Methods USA) in order to ascertain the purity of the Isolation of the Tobamovirus and Purification preparation and to obtain information on Virus from Mucuna pruriens was concentration and the nucleic acid content of the propagated in cowpea (Vigna unguiculata var virus (Hughes, 1986). A silica quartz cuvette TVu 76) by homogenizing symptomatic leaves of (Sigma, USA) containing 1 ml of resuspending Mucuna pruriens in sterilized mortars and pestles buffer solution was placed in the cuvette in 0.1M phosphate buffer (pH 7.7) containing chamber with the blank side positioned in the 0.01M ethylene diaminetetraacetic acid and path of the light beam and the scale set to zero rubbing the extracted sap on carborundum absorbance (100% transmittance) at 260 and dusted leaves of a week old cowpea seedlings 280 nm. The absorbance of the cuvette (TVu 76). At 2 weeks after inoculation 70 g of containing the virus preparation was also taken systemically infected cowpea leaves were at 260 and 280 nm after the resuspending buffer harvested and homogenized in a waring has been discarded from the cuvette. The ratio laboratory blender (Model No 35-59, Christison, of the absorbance of the virus preparations at USA) with 0.5M phosphate buffer (pH 7.2) 260 and 280 nm was then used to estimate the containing 1% 2-mercapto ethanol in the rate of amount of nucleic acid in the preparation using 1g tissue to 2ml buffer. After straining the the following formula (Gibbs & Harrison, 1976): homogenate through cheesecloth, 8ml NA /A = 0.932 + 0.045 (RNA%) – 0.0006 260 280 butanol was added to every 100ml of extract 2 (RNA%) while stirring for further 15 min before 115 Odedara et al./ Nig. J. Biotech. Vol. 35 (2018): 114-122


Enzyme linked immunosorbent assay
Protein A-sandwich (PAS) ELISA is an described by Clark and Adams (1977) showed indirect ELISA (Hughes & Thomas, 1988) and it that the microplate method of ELISA could be makes use of protein-A, a molecule isolated from very effectively applied to the detection and the cell wall of Staphylococcus aureus.The assay of plant viruses (Matthew, 1991).Since binding of Protein-A is used in several serological that time, the method has been more widely procedures (Matthews, 1991).For instance, an used (Matthews, 1991).The method is a indirect Protein-A sandwich (PAS-ELISA) which serological procedure and has been used to was more successful than double antibody detect various viruses in yams (Dioscorca alata) sandwich (DAS) ELISA in detecting heterologous (Odu et al., 1999;Eni et al., 2008), legumes isolates of cherry leaf roll virus was described by (Odedara et al., 2007;Odedara et al., 2012), Edwards & Cooper (1985).It was hypothesized that a het:hom PAS ELISA could be used to Solanaceous plants, cucurbits (Ayo-John et al., centrifugation at 9,500 rpm for 30 min.The detect both close and distant serological supernatant was discarded into a clean beaker relationships of Tobamovirus (Hughes and and 4.0 g polyethylene glycol (PEG mol. wt. Thomas, 1988).8000) was added for every 100 ml of the virus o Other viruses belonging to this group suspension.This was stirred for 1h at 4 C. are: Tomato mosaic virus (ToMV), Pepper mild Centrifugation was done at 10,000 g for 15 min mottle virus (PMMV), Cucumber green mottle and the pellet resuspended in 20 ml 0.01M mosaic virus (CGMMV), Sunnhemp mosaic virus phosphate buffer (pH 7.2) (Gooding & Herbert, (SHMV), Ullucus mild mottle virus (UMMV) 1967).
To obtain a pure virus suspension, 0.4 g (Hughes & Thomas, 1988).They are rod-shaped sodium chloride (NaCl) and 0.4 g PEG were rigid tube viruses.Hosts for TMV include added for every 10 ml of the suspension while tobacco, tomato and other Solanacoeus plants.
stirring and this continued till the salt dissolved TMV had caused 20 % loss on tomato (Scholthof, and the suspension centrifuged at 9,500 rpm for 2000).
15 minutes.The supernatant was discarded and Growth of Mucuna pruriens had been the pellets resuspended in 2 ml 0.01M phosphate emphasized for enhancement of soil fertility, buffer for each 100 ml of the initial extract.prevention of soil erosion and maintenance of Centrifugation was done for the suspension at soil physical structures (Versteeg & Koudokpon, 10,000 g for 5 minutes in order to remove the 1993, Sanginga et al., 1996;Makau & Gachene, contaminants (Gooding and Herbert, 1967).

2001). Viral symptoms expression such as
The absorbance of the virus at 260 and 280 nm mosaic, leaf yellowing and leaf distortion were estimated and virus stored in 0.01M sometimes accompanying growth of M. pruriens.o phosphate buffer (pH 7.2) at -20 C from where it Some previous reports had shown that this plant was withdrawn for antiserum production and is sometimes infected with TMV.Hence, the other tests.present study was conducted with the following objectives: to purify the Tobamovirus from the Spectrophotometry of purified preparations of naturally infected host, determine the molecular isolated Tobamovirus.weight of its coat protein, analyze its serological The absorption spectrum of the purified relationships with its other strains and produce preparations of the isolated Tobamovirus was antibodies against the virus.
determined with a Beckman DU 520 Model Spectrophotometer (Beckman Instruments, Materials and Methods USA) in order to ascertain the purity of the Isolation of the Tobamovirus and Purification preparation and to obtain information on Virus from Mucuna pruriens was concentration and the nucleic acid content of the propagated in cowpea (Vigna unguiculata var virus (Hughes, 1986).A silica quartz cuvette TVu 76) by homogenizing symptomatic leaves of (Sigma, USA) containing 1 ml of resuspending Mucuna pruriens in sterilized mortars and pestles buffer solution was placed in the cuvette in 0.1M phosphate buffer (pH 7.7) containing chamber with the blank side positioned in the 0.01M ethylene diaminetetraacetic acid and path of the light beam and the scale set to zero rubbing the extracted sap on carborundum absorbance (100% transmittance) at 260 and dusted leaves of a week old cowpea seedlings 280 nm.
The absorbance of the cuvette (TVu 76).At 2 weeks after inoculation 70 g of containing the virus preparation was also taken systemically infected cowpea leaves were at 260 and 280 nm after the resuspending buffer harvested and homogenized in a waring has been discarded from the cuvette.The ratio laboratory blender Christison, of the absorbance of the virus preparations at USA) with 0.5M phosphate buffer (pH 7.2) 260 and 280 nm was then used to estimate the containing 1% 2-mercapto ethanol in the rate of amount of nucleic acid in the preparation using 1g tissue to 2ml buffer.After straining the the following formula (Gibbs & Harrison, 1976): homogenate through cheesecloth, 8ml N-A /A = 0.932 + 0.045 (RNA%) -0.0006 260 280 butanol was added to every 100ml of extract 2 (RNA%) while stirring for further 15 min before Concentration of the purified virus was intramuscularly into the second rear thigh of the calculated based on the extinction co-efficient of rabbit.The third and fourth injections were the virus from the formula described by Hughes given as previously described for second (1986).injection.
Fourth injection, which was the booster injection was done 4 weeks after the third injection.Blood was collected after the last where; injection from the ear vein of the rabbit caged in C = concentration of the virus in mg/ml; OD = a rabbit cage (Nalgene, Italy, Model No COD 16 optical density at 260 nm; D = dilution of the 0012).virus preparations; E = Extinction co-efficient of The tip of one of the ear was shaved with the virus new razor blade, a cotton wool soaked with The specific extinction coefficient of the xylene was placed just at the edge of the ear virus used was as described by Hollings & Brunt, opposite the place where a cut was made to (1981).
It could also be estimated by allow vein dilation.
The shaved area was substituting the estimated percentage RNA in cleansed using a cotton wool soaked with the following equation according to Gibbs & absolute ethanol and an ear vein that runs Harrison, (1976).
through the area was cut with a clear razor blade.from the blood by incubating the blood at 37 C virus is the optical density of 1 cm layer of for 6 h during which the clot was ringed with a 1mg/ml (0.1%) virus preparation at a needle to allow shrinkage (Hughes, 1986).The wavelength of 260 nm.
From the former serum was collected at 6,000 rpm for 10 minutes formula, the concentration of the virus, was then and an equal volume of glycerol added before adjusted to 1mg/ml by using a normal saline o storing at 4 C. solution (0.95 % NaCl) in sterile distilled water to dilute out the purified virus which was kept in o Eppendorf tubes and stored at -20 C from where Titre value determination for the antiserum each was withdrawn for rabbit injection in the produced against the isolated Tobamovirus.production of polyclonal antibody.To find the The titre of the antiserum produced yield of the virus from a given weight of plant against the isolated Tobamovirus was tissue, the formula (Hughes, 1986) was used as determined using the PAS-ELISA described by below.
Hughes (1986).A two-fold serial dilutions of antiserum to the isolated Tobamovirus was made in phosphate buffer saline with Tween 20 PBS-Twhere; containing 0.15M NaCl, 0.003M KCl, pH 7.4 with Y = yield of virus (mg/kg fresh weight); V = 0.05% tween 20).For a particular dilution of an volume of the purified virus (ul;) C = antiserum, two duplicate wells were used for concentration of the virus (mg/ml); W = fresh both the infected sap (antigen) and the healthy weight of plant tissue (kg) (negative control) sap.During the addition of antibodies, wells in the same row were coated Production of antiserum against the isolated with different dilutions of crude antibody while Tobamovirus.
duplicated wells were coated with similar dilutions of antisera.Description of protein-A Partially purified virus from M. pruriens at sandwich ELISA wells of microtitre plates were a concentration of 1mg/ml mixed with equal precoated with protein-A at a concentration of 1 volume of Freund's complete adjuvant was ug/ml in 0.05M Na CO coating buffer, pH 9.6.injected intramuscularly into a 3-4 months old 2 3 o rabbit (Hampton et al., 1990).Healthy blood Plates were incubated at 37 C for 2 h was collected from the rabbit before injection to after which they were emptied and washed by serve as a control during serological analysis.
flooding the wells with 0.02M phosphate Two weeks after the first injection, 1 ml of the buffered saline containing 0.015M NaCl and virus, at concentration of 1 mg/ml mixed with 0.003M KCl, pH 7.4 with 0.05% tween -20 (PBS-Freund's incomplete adjuvant was injected T).
After each wash, plates were allowed to filled with PBS-T.The homologous antiserum stay for 3 min before the wells were emptied.
produced against the isolated Tobamovirus from Plates were drained and tapped dry after two M. pruriens was diluted in PBS-T as 1:1000 more washings.The corresponding antiserum respectively and filled into the four wells per each against the isolated Tobamovirus that have been antiserum used to detect the virus isolate.serially diluted appropriately in PBS-T were The mean absorbance value at 405 nm (A nm) Denatured, purified preparations of the Plates were washed as above and the isolates were used to determine the molecular serially diluted antiserum in PBS-T was added weight of the isolated Tobamovirus, appropriately to each well as 100ul/well well and Discontinuous, denaturing polyacrylamide gel o then incubated at 37 C for 2 h before washing electrophoresis (SDS-PAGE) was carried out and drying.Plates were developed by addition according to Laemmli (1970).The denatured of 200 ul aliquots/well of enzyme substrate virus isolate with the extracted protein from (para-nitro phenyl phosphate) dissolved at a healthy (negative control) Vigna unguiculata var.concentration of 1mg/ml in substrate buffer TVu 76 were subjected to this process.The which consisted of 12% diethanolamine molecular weight of the viral coat protein was adjusted with concentrated HCl to pH 9.8.estimated from the plotted graph of logarithms of molecular weights of standard protein Incubation was done at room temperature for 1 markers against their Rf values (ratio of the hr and absorbance readings taken at 405 nm distance migrated by individual protein to total with Dynex MRX ELISA reader (UK).
distance migrated by the tracking dye from the Absorbance values obtained for each base line of the stacking gel).serially diluted antiserum were plotted against those values for healthy controls and the titre Results determined from the graph.
The purified preparations of the isolated Tobamovirus appeared milky in the resuspension buffer.The volume of the virus obtained was Determination of serological relationship 1000 ul The yield when calculated was 7.2 between the isolated Tobamovirus and other mg/kg of fresh leaves of Vigna unguiculata var.Tobamoviruses.
TVu 76 (Table 1).The ultraviolet absorption Serological relationship between virus spectrum of the purified isolated virus had a isolated from Mucuna pruriens and other viruses minimum absorbance at 260 nm of 1.53 ± 0.00 to which antisera were available was carried out while at 280 nm it was 1.45 ± 0.00.This by challenging each of the 5 antisera available for translated to a nucleic acid content of 72.9%.these Tobamoviruses against the virus isolated The antiserum raised against the isolated from Mucuna pruriens in extracted sap of Tobamovirus from Mucuna pruriens was 16,000 infected Cowpea (Vigna unguiculata var.TVu 76) ul in volume with concentration of 0.3 mg/mL.according Hughes and Thomas (1988).
An ultraviolet absorption spectrum curve was Two wells of Dynatech microtitre plate obtained at 260 nm for the purified isolated were filled per antiserum for the infected sap and Tobamovirus (Fig 1).other two wells which served as control were During electrophoresis of the purified for use.virus on the gel, a clear band was obtained for The reaction of isolated Tobamovirus to the purified virus in the undiluted form (Fig 2).
antiserum against TMV (Muguga), gave a strong There were no secondary bands.When the immunogenic reaction.This showed that they molecular weight of the coat protein of the virus were closely related with the het:hom % was determined from the plotted graph, a relationship of 50.0 (Table 2).The reactions of molecular weight of 27 kiloDalton was obtained the isolated Tobamovirus to other antisera raised (Fig. 3).
against other Tobamoviruses such as Tomato At 1:250 dilution, the antiserum when Mosaic virus (ToMV) genus Tobamovirus, Ullucus reacted against the virus-infected sap of V.
mild mottle virus genus Tobamovirus and Pepper unguiculata var.TVu 76, gave absorbance values mild mottle virus genus Tobamovirus were that were four times greater than that of the weakly immunogenic, thus suggesting that they healthy control (Fig. 4).Between 1:1000 and 1: are distantly related.
The het:hom % 4000 dilutions, the titre values were more or less relationships were 39.9 %; 35.6 % and 28.7 % stable, being about two and half times greater respectively (Table 2).than that of the healthy control.Therefore, a working dilution of 1:2000 was recommended    Antiserum against Tobacco mild green presence of a curve at 260 nm when absorbance value of the purified preparation was measured virus genus Tobamovirus however reacted spectophotometrically .This might be due to moderately with the isolated Tobamovirus with many factors ranging from the host species to het:hom % of 45.0.This showed that Tobacco the conditions of the extraction medium used mild green virus and the isolated Tobamovirus during purification procedure.As Matthew from M. pruriens were related to some extent but (1991) stated that some inhibitory substances not closely.
such as phenolic materials, organic acids, mucilage, gums, certain proteins and enzymes Discussion particularly ribonucleases do inhibit the isolation A Tobamovirus was isolated from of some viruses from their natural hosts.naturally infected Mucuna pruriens collected Examples are plant members of the Rosaceae from the campus of International Institute of which contain high concentration of tannins in Tropical Agriculture (IITA), Ibadan.Symptoms their leaves.For the isolated Tobamovirus to observed on the plant include mosaic, yellowing have a high yield as obtained in this study means and leaf distortion.
that the host on which this virus was maintained Purification of this virus was did not possess such inhibitory substances.carried out and it was observed that there was a The ultra violet absorption spectrum high yield of the purified virus as observed in the curve for the isolated Tobamovirus at 260 nm milky appearance of the preparation and the 405 added to the wells as 100 ul/well and plates obtained for each control was substracted from o incubated at 37 C for 2 h, washed and dried as the mean value obtained for virus-containing earlier described.Infected leaf samples of TVu wells.Corrected values were used to calculate Tobamovirus heterologous: homologous (het:hom) ratio 76 containing the isolated were expressed in percentage using the formula ground as 0.1g/ml in PBS-T containing 2% w/v (Hughes & Thomas, 1988) as below: poly vinyl pyrrolidone (PVP) and incubated as o 100 ul/well overnight at 4 C. Leaf extract of healthy TVu 76 were coated into the first duplicate wells of the inner sixty wells of the ELISA plate.Outer wells were not used to Determination of Molecular weight of the coat prevent errors.The third duplicate wells were protein of the isolated Tobamovirus coated with sap of the infected TVu 76.

Fig 2 :
Fig 2: Electrophoresed gel containing migrated coat protein of the purified preparation of the isolated Tobamovirus

Fig. 3 :
Fig.3: Determination of the molecular weight of coat protein of the isolated Tobamovirus in purified form from the plotted graph of logarithm of molecular weight of protein markers against their Rf values.

Fig 4 :
Fig 4: Mean absorbance values of antiserum against the isolated Tobamovirus at 405 nm during different dilutions

Table 1 :
Yield parameters of the purified isolated Tobamovirus preparation

Table 2 :
Serological relationship of the isolated Tobamovirus to other strain