Production of Monoclonal and PolyclonalAntibodies for Detection of Geminiviruses and       the Virus –Vector Relationships...
Outline1.   Introduction2.   Justification & Work plan3.   Production of antibodies4.   Immunization effects on animals5. ...
1. Introduction                                                                                    GeminivirusesFamily: Ge...
BEGOMOVIRUSES:                                                  CASSAVA MOSAIC DISEASE (CMD)• CMD is the single most impor...
Vector of CMDBemisia tabaci (Gennadius) (Homoptera: Aleyrodidae)                         • Most economically important    ...
Mastrevirus: Maize streak virus        •   Most important virus disease of maize in            Africa and the neighbouring...
2. Justification & Work Plan1   Specific monoclonal antibodies are needed to differentiate EACMV    from other begomovirus...
Aims:• To produce antibodies for the detection and differentiation of  geminiviruses• Determine the virus-vector interacti...
3. Production polyclonal antibodies (PABS)1. PURIFICATION OF ACMV: Method adapted from Thottappilly, 1986)   Density gradi...
Fig: 1HAT IN MEDIA,Only HGPRT +ve cells survive;- salvage pathway         TAS/ACP-ELISA                                   ...
Table 1; Experimental animals used in immunisations                                                               Conc. Of...
Results                       Table 2 ACMV/EACMV AND MSV RABBIT AND MOUSE POLYCLONAL ANTISERA PRODUCED                    ...
(1 hr incubation)               1.4                                                                                       ...
(O/N incubation)                        4                                                                                 ...
All the polyclonal antisera raised against ACMV, EACMV and MSV are useful for virus detection by ACP-ELISA “Compared to TA...
BA    Plate 1; Growing hybridoma cells.      (a) Lower left corner: dividing cells      (b) Top right: cells forming a col...
Table 31st screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mousehybridomas          WELLS WITH                WELLS +VE ...
TABLE 4; 2nd screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mousehybridomas   CELL            ACMV R.                 A...
Table 5CLONING: EACMV/ cell lines in TAS-ELISA (O/N incubation)Cell lines    1st Cloning                               2nd...
Table 6; MSV/M/A1 AND MSV/M/A2 BALB/C (1st Screening)          WELLS WITH                WELLS +VE                        ...
Table 7; CLONING: MSV cell lines in ACP-ELISACell    Ist                                                      2NDlines   C...
Plate 2Fusions with Swiss Albino strain of mice yielded no Hybridomas                    International Institute of Tropic...
Plate 3  MAbs 6B3 and 3F1 differentiated EACMV from ACMV  and they are useful for specific detection of EACMV.“This is the...
4. Effects of Immunisation on Rabbits           International Institute of Tropical Agriculture – Institut international d...
Fig 4; Bi-monthly Temperature Differences In Immunized                            Animals Before And After Immunisationsof...
TABLE 8 : PERCENTAGE CHANGE OF PHYSICAL AND HAEMATOLOGICAL PARAMETERSBETWEEN GROUPS OF RABBITS IN 12 WEEKS GIVEN DIFFERENT...
5. TRANSMISSION EFFICIENCY OF MSV in     C. dabrowski and C. triangular          International Institute of Tropical Agric...
Fig 5: Percent transmission of MSV by C. triangular and C.                                    dabrowski given different vi...
Fig 6: Percent transmission of MSV by C. triangular and                                   C. dabrowski given different vir...
C. triangular is an efficient vector of MSVC. dabrowiski is relatively poor vector of MSV             International Instit...
6. SURVEY FOR GEMINIVIRUSES IN NIGERIA           International Institute of Tropical Agriculture – Institut international ...
Fig 7: MAP OF NIGERIA SHOWING STUDY SITES FOR GEMINIVIRUSESSurveyed during Oct-Nov 2002Cassava, maize, tomato, pepper, okr...
Methods for virus detection1. Serological tests (ELISA):    ACMV poly; MSV poly; SCRI Mabs; DMSZ MAbs2. PCR:• DNA Extracti...
Primers used for PCR amplification of geminiviruses                                                                       ...
Table 9: Occurrence of Geminiviruses in Nigeria        COWPEA               TOMATO                               PEPPER   ...
Table 9 (Continued) : Occurrence of Geminiviruses in Nigeria                       COWPEA                TOMATO           ...
Plate 4: PCR Indexing for Other Geminiviruses in Survey Samples Using                           Universal Primer Pair (Bro...
Total positive samples detected by PCR usinguniversal primers for geminiviruses (Primer A/F;Primer B/R)93 samples analysed...
% incidence of ACMV/EACMV using MAbs              80                                                                      ...
% incidence of MSV using MSV polyclonal antibody              120                                                         ...
This survey demonstrated usefulness of        antibodies produced in this study:MAbs 6B3 and 3F1 MAbs were very specific a...
7. CONCLUSIONS•   Eliminating the density gradient step in purifications of antigens is not    detrimental in the producti...
CONCLUSIONS   C. dabrowski is inefficient as a vector of MSV and is unlikely to contribute to    disease epidemics.   Th...
ACKNOWLEDGEMENTS                           IITA, Ibadan Scottish Crop Research Institute (SCRI), Scotland, UKDeushe Sammlu...
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Production of Monoclonal and Polyclonal Antibodies for Detection of Geminiviruses and the Virus –Vector Relationships

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Immunization effects on animals,Vector transmission studies,Surveys for geminiviruses in Nigeria,Production of antibodies for the detection and differentiation of geminiviruses

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Production of Monoclonal and Polyclonal Antibodies for Detection of Geminiviruses and the Virus –Vector Relationships

  1. 1. Production of Monoclonal and PolyclonalAntibodies for Detection of Geminiviruses and the Virus –Vector Relationships Adeola Ala Ph.D Student Virology Unit, IITA-Ibadan, Nigeria & Animal Physiology Unit, Department of Zoology University of Ibadan, Nigeria International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  2. 2. Outline1. Introduction2. Justification & Work plan3. Production of antibodies4. Immunization effects on animals5. Vector transmission studies6. Surveys for geminiviruses in Nigeria7. Conclusions International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  3. 3. 1. Introduction GeminivirusesFamily: Geminiviridae –Genome: circular single stranded DNA 2.5–3.0 kb in length, encapsidated in twinned (geminate) quasi-isometric particles. –Responsible for several devastating diseases in economically important crops of both monocotyledonous and dicotyledonous plants worldwide (Cassava, maize, wheat, tomato, pepper, bean, cotton, etc.)Genera:Begomovirus: ACMV, BGMVMastrevirus: MSVCurtovirus: BCTVTopocuvirus: TPCTV International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  4. 4. BEGOMOVIRUSES: CASSAVA MOSAIC DISEASE (CMD)• CMD is the single most important production constraint to cassava in sub- Saharan Africa, including Nigeria.• The disease results in 60-80% decrease in tuber yield, also effects the quality and impede germplasm movement (Bock, 1983)• In Africa, several species and strains of begomoviruses have been identified in the CMD etiology.• In Nigeria, African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and East African cassava mosaic Cameroon virus (EACMCV) are most prevalent (Ariyo et al. 2005). International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  5. 5. Vector of CMDBemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) • Most economically important virus vector (Geddes, 1990) • It is a pest on 350 plant species, including cassava, around the world International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  6. 6. Mastrevirus: Maize streak virus • Most important virus disease of maize in Africa and the neighbouring islands of Mauritus, La Reunion and Madagascar (Rybicki and pietersen, 1998). • Yield losses in maize due to MSV range from 0 to 100% ( Mzira 1984 and Barrow 1992) • The virus is transmitted by Cicadulina spp, (Homoptera: cicadellidae) C. storeyi, (= triangula) one of the main five species found In West Africa (Bosque-Perez et al., 1990)International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  7. 7. 2. Justification & Work Plan1 Specific monoclonal antibodies are needed to differentiate EACMV from other begomoviruses.2 Polyclonal and monoclonal antisera against MSV are needed for virus surveys and screening germplasm.3 Information is limited on distribution of geminiviruses in Nigeria.4 Information on the transmission efficiency of a less predominant leaf hopper, C .dabrowski, in MSV transmission is required to assess its role in MSV epidemiology.5 Effect of ACMV and MSV immunization on physiological status of the experimental animal, Oryctolagus cuniculus (Domestic rabbit), used routinely for antibody production. These include the effects of the immunogens administered and the quantification of the viruses used in immunizations. These will ensure minimal adverse effects on health and also enable optimisation of immunisation protocols. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  8. 8. Aims:• To produce antibodies for the detection and differentiation of geminiviruses• Determine the virus-vector interactions of MSV and leaf hoppersObjectives: Produce polyclonal and monoclonal antibodies against cassava mosaic begomoviruses and MSV for detection and differentiation of EACMV and MSV. Conduct surveys for geminivirus distribution in Nigeria and the food crops they infect Compare the differences in acquisition, transmission abilities of MSV between C. triangular and C. dabrowski Determine the physiological status of rabbits used in routine immunisations with ACMV and MSV International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  9. 9. 3. Production polyclonal antibodies (PABS)1. PURIFICATION OF ACMV: Method adapted from Thottappilly, 1986) Density gradient step removed. Method 1; From test plants, Nicotiana benthamiana Method 2; Directly from Manihot esculenta2. MSV PURIFICATION: Method by (Bock et al.,1974)3. IMMUNISATIONS: Mus musculus (Mouse); Oryctolagus cunniculus (Rabbit) Immunized at 2 week intervals for 12 weeks4 Bleeding5 Storage of serum International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  10. 10. Fig: 1HAT IN MEDIA,Only HGPRT +ve cells survive;- salvage pathway TAS/ACP-ELISA LIMITING DILUTION International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  11. 11. Table 1; Experimental animals used in immunisations Conc. Of virus in Animal immunized (mean value: n=2) (mean value: n=2) purified prep. Healthy sap Host plant *(A405) Virus Mice 4 /ACMV 0.861 0.065 N. benthamianaGroup A Mice 3/ MSV 1.300 0.28 Zea mays Group A Rabbit 1/ ACMV 0.861 0.065 N. benthamiana Rabbit 1/ACMV 0.887 0.321 N.benthamiana Mice 3 /EACMV N. benthamianaGroup B Mice 2 /MSV 1.83 0.27 Zea mays Group B Rabbit 1/ EACMV 0.504 0.226 N. benthamiana Mice 3EACMVGroup C /ACMV 3.498 0.143 M.esculenta Group C Rabbits 2EACMV /ACMV *(A405) values at 1 hr sustrate incubation International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  12. 12. Results Table 2 ACMV/EACMV AND MSV RABBIT AND MOUSE POLYCLONAL ANTISERA PRODUCED RECIPROCAL ANTISERUM RECIPROCAL ANTISERUM TITRE IN TAS ELISA TITRE IN ACP ELISAANIMAL 1hr 0/N 1hr 0/N incubation incubation 1nc 1ncACMV/EACMV(1) ACMV/R/A1 1,000 –(2) ACMV/R/B1 8,000 (1.95) 32,000 (1.85)(3) EACMV/R/B1 NDT* 16,000(4) EA/ACMV/R/C1 256,000 (4.07) 256,000 (5.01) 64,000 (2.02) 512,000 (2.51) (Unadsorbed) (Unadsorbed) 16,000 (2.53) 32,000 (2.38) (Adsorbed) (Adsorbed)(5) EA/ACMV/R/C2 1,600 (1.90) 128,000 (2.3) 512,000 (1.93) 1,024,000 (2.27) (Unadsorbed) (Unadsorbed) 1,000 (1.96) NDT (Adsorbed) (Adsrobed)(6) ACMV/M/A1-A4 1,000 (3.2) –(7) EACMV/M/B4 8,000 (2.03) –(8) EA/ACMV/M/C3 256,000 (2.84) –(9) EA/ACMV/M/C4 64,000 (1.99) –MSV(1) MSV/R/A1 – – 64,000 (2.1) – (Unadsorbed) 256,000 (4.3) 256,000 (2.5) (Adsorbed) (Adsorbed)(2) MSV/M/A1 256,000 (4.73) –(3) MSV/M/A2 256,000 (3.82) –(4) MSV/M/B1 NDT 250 (1.84)(5) MSV/M/B2 NDT 4,000 (2.44) * Not detectable titre – Not tested ( ) D/H values International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  13. 13. (1 hr incubation) 1.4 8.00 7.26 1.2 6.88 7.00 H D 6.00 1 5.78 Titre points Titre points 5.00 4.81 0.8 A405nm 4.00 0.6 3.46 2.96 3.00 0.4 2.41 1.99 2.00 1.74 1.38 0.2 1.00 0 0.00 1/500 1/1000 1/2000 1/4000 1/80001/160001/32000 1/64000 /128000 1 1/256000 Antiserum DilutionsFIG 2: EA/ACMV/M/C4 ANTISERUM TITRE BY INDIRECT ELISA (TAS)ANTISERUM PRODUCED FROM MICE BY INTRAPERITONEAL IMMUNISATIONS International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  14. 14. (O/N incubation) 4 8.00 3.5 7.00 6.68 Titre points (A405nm) H 3 6.09 D 6.00 5.57 Titre points 2.5 5.00 A 405nm 4.92 2 3.93 4.00 3.44 1.5 2.92 3.00 2.72 1 2.00 1.81 1.53 0.5 1.00 0 0.00 1/500 1/1000 1/2000 1/4000 1/8000 1/16000 1/32000 1/640001/128000 512000 1/ Antiserum DilutionsFIG 3: EA/ACMV/M/C4 ANTISERUM TITRE BY INDIRECT ELISA (TAS)ANTISERUM PRODUCED FROM MICE BY INTRAPERITONEAL IMMUNISATIONS International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  15. 15. All the polyclonal antisera raised against ACMV, EACMV and MSV are useful for virus detection by ACP-ELISA “Compared to TAS-ELISA, ACP-ELISA for geminivirus detection is convenient and cost-effective” International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  16. 16. BA Plate 1; Growing hybridoma cells. (a) Lower left corner: dividing cells (b) Top right: cells forming a colony International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  17. 17. Table 31st screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mousehybridomas WELLS WITH WELLS +VE % OF TOTAL WELLS GROWING BY ELISA TO SECRETING SPECIFIC HYBRIDS ACMV ANTIBODIES AGAINST ACMVPlate 1 24 /96 1/24 1.04%Plate 2 27/96 3/27 3.13%Plate 3 49/96 4/49 4.17%Plate 4 22/96 2/22 2.1%Plate 5 35/96 4/35 4.2%Plate 6 42/96 6/42 6.25% International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  18. 18. TABLE 4; 2nd screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mousehybridomas CELL ACMV R. ACMV R. ACMV Ms ACM Ms Poly/ DSMZ DSMZ poly/ LINES Poly/ACMV Poly/EACM Poly/ACMV EACMV ANTG Poly/ACMV EACMV ANTG ANTG ANTG ANT G ANTG 2nd 3rd 2nd 3rd 2nd screen 2nd 3rd 2nd 3rd 2nd 3rd screen. screen. screen. screen. screen. screen. screen. screen. screen. screen. 1H3 - - - - - - - - - - - 2C5 + - - + - - - - - - 2F6 - - - - - - - - - - - 2F61 - - - - - - - - - - - 2F62 - - - - - - - - + - - 2EE5 - - - - - - - - + - - 3A10 + - + - - + - - - - - 3A101 - + - - - - - - - - - 3B12 ++ - ++ - - - - - - + - 3F1 - - - + + - - - - + - 3F11 - - - - - - - - - - - 3B10 - - - - - - - - - - - 4F6 +++ - ++ - + - - + - + - 4F61 - - - - - - - - + - - 4A12 - - - - - - - - + - - 4A121 - - - - - - - - - - - 5B2 - - - - - ++ - - - - - 5G11 - - - - - - - - + - - 6B3 + - ++ - - ++ - - - ++ - 6B31 - + - - - - - - + - - 6B32 - + - - - - - - - - 6B33 - + - + - - - - + - - 6B34 - + - - - + - - ++ - - 6B35 - - - + + - - - + - -Key:ACMV R. POLY =ACMV rabbit polyclonal antibody; ACMV MS POLY =ACMV mouse polyclonal antibody; DMSZ IGg =DMSZ rabbit immunoglobulin; ACMVANTG =ACMV Antigen; EACMV ANTG =EACMV Antigen International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  19. 19. Table 5CLONING: EACMV/ cell lines in TAS-ELISA (O/N incubation)Cell lines 1st Cloning 2nd cloning6B3 ++ ++3F1 +++ +++ 1.5 – 1.9: + weak positive 2.0 – 3.0: ++ positive 3.0<: +++ strong positive International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  20. 20. Table 6; MSV/M/A1 AND MSV/M/A2 BALB/C (1st Screening) WELLS WITH WELLS +VE % OF TOTAL WELLS GROWING BY ELISA TO SECRETING SPECIFIC HYBRIDS MSV ANTIBODIES AGAINT MSVPlate 1 10/96 2/10 2.1%Plate 2 8/96 1/8 1.04%Plate 3 6/96 2/6 2.1Plate 4 7/96 5/7 5.2%Plate 5 5/96 1/5 1.04%Plate 6 8/96 3/8 3.13% International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  21. 21. Table 7; CLONING: MSV cell lines in ACP-ELISACell Ist 2NDlines CLONING CLONING Healthy Diseased Diseased/Healthy Healthy Diseased Diseased/Healthy sap sap sap sap3F4 0.209 0.617 2.9 0.126 1.239 9.83E11b 0.282 2.1 7.4 0.123 0.404 3.31H2 0.049 0.455 9.2 0.103 0.406 3.95F2 0.039 0.315 8.0 0.152 0.586 3.9 International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  22. 22. Plate 2Fusions with Swiss Albino strain of mice yielded no Hybridomas International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  23. 23. Plate 3 MAbs 6B3 and 3F1 differentiated EACMV from ACMV and they are useful for specific detection of EACMV.“This is the first monoclonal antibody specific to EACMV” International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  24. 24. 4. Effects of Immunisation on Rabbits International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  25. 25. Fig 4; Bi-monthly Temperature Differences In Immunized Animals Before And After Immunisationsof two rabbits) 1.4 Group 1: Control; Distilled water alone Group 2: Adjuvant alone 1.2 Group 3: Antigen + AdjuvantTemp change (mean 1 0.8 Group 1 Group 2 Group 3 0.6 0.4 0.2 0 0 2 4 6 8 10 12 Period/weeks International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  26. 26. TABLE 8 : PERCENTAGE CHANGE OF PHYSICAL AND HAEMATOLOGICAL PARAMETERSBETWEEN GROUPS OF RABBITS IN 12 WEEKS GIVEN DIFFERENT TREATMENTS PARAMETER GROUP 1 GROUP 2 GROUP 3 WEIGHT 5.248 BA 3.411 B 9.022 A TEMPERATURE 0.0929 B 0.7143 A 0.9143 A DIFFERENCE PCV -0.766 B 3.095 B 23.497 A PLATELETS 39.97 A 93.38 A 41.54 A HB 0.912 B 7.757 B 41.619 A RBC 1.188B 9.790 B 56.563 A MCV -1.149A -5.691BA -8.192B MCHC 4.645 A 4.301 A 9.788 A TOTAL WBC 3.99 B 27.21 BA 60.49 A NEUTROPHILS 76.81 BA 108.11A 8.90 B LYMPHOCYTES -3.97 B 19.67 B 114.23 A EOSIN -57.8 B -63.1 B 453.50 A MONOCYTES -33.33 B 27.78 A -20.83 BMEANS IN THE SAME COLUMN WITH DIFFERENT LETTERS ARE SIGNIFICANTLY DIFFERENT (p<0.05). International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  27. 27. 5. TRANSMISSION EFFICIENCY OF MSV in C. dabrowski and C. triangular International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  28. 28. Fig 5: Percent transmission of MSV by C. triangular and C. dabrowski given different virus acquisition access periods 80 25 70 20 20 20 20 20 60 Total insects used % transmission 50 15 C.triangular 40 C.dabrowski 12 12 Total insects used 10 30 20 5 10 0 0 30 secs 15 mins 1 hr 24 hr 48 hr 96 hr AAPsIAP (Inoculation Access Period) * = 24 hrs; AAP (Acquisition Access Period)**; No of replicates = 3Significantly higher transmission efficiency in C.triangular at all AAPs (p<0.05) International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  29. 29. Fig 6: Percent transmission of MSV by C. triangular and C. dabrowski given different virus inoculation access periods 60 25 50 20 20 20 20 20 20 20 40 Total insects used % transmission 15 C. triangular 30 C. dabrowski Total insects used 10 20 5 10 0 0 30 secs 15 mins 1 hr 24 hr 48 hr 96 hr Inoculation Access Periods (IAPs)Significantly higher transmission efficiency in C.triangular at all IAPs (p<0.05) International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  30. 30. C. triangular is an efficient vector of MSVC. dabrowiski is relatively poor vector of MSV International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  31. 31. 6. SURVEY FOR GEMINIVIRUSES IN NIGERIA International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  32. 32. Fig 7: MAP OF NIGERIA SHOWING STUDY SITES FOR GEMINIVIRUSESSurveyed during Oct-Nov 2002Cassava, maize, tomato, pepper, okra, cowpea and jatropha International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  33. 33. Methods for virus detection1. Serological tests (ELISA): ACMV poly; MSV poly; SCRI Mabs; DMSZ MAbs2. PCR:• DNA Extraction Extraction of DNA was by the method of Dellaporta et al., (1983).• PCR REACTION MIXTURE AND THERMAL CYCLES – To amplify the DNA extracted• AGAROSE GEL ELECTROPHORESIS OF THE AMPLIFIED DNA International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  34. 34. Primers used for PCR amplification of geminiviruses TARGET VIRUS PRIMER SEQUENCE (5’-3’) IN DNA ACMV ACMV -AL1/F GCG GAA TCC CTA ACA TTA TC AC1 ACMV -ARO/R GCT CGT ATG TAT CCT CTA AGG CC TG AV2 EACMV UV -AL3/F TAC ACA TGC CTC RAA TCC TG AC3 UV -AL1/R2 CTC CGC CAC AAA CTT ACG TT AC1 WHITEFLY PRIMER A /F TAA TAT TAC CKG WKG VCC CR TRANSMITTED PRIMER B /R TGG ACY TTR CAW GGB CCT TCA CA CR GEMINIVIRUSESPRIMER SEQUENCES FROM PITA ET AL ( 2001a). International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  35. 35. Table 9: Occurrence of Geminiviruses in Nigeria COWPEA TOMATO PEPPER OKRA JATROPHASTATE TSC ACMV Pab TSC ACMV Pab TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCROYO 9 1/9 - 6 0/6 - 8 0/8 - 12 1/12 - - - -KWARA 4 0/4 - 2 0/2 - 5 0/5 - 2 1/2 - - - -KOGI 7 0/7 - 6 0/6 - 5 1/5 - 4 0/4 - - - -NASSARAWA 18 1/18 - 4 0/4 - 0/3 - 3 0/3 - 2 0/2 -BENUE 8 0/8 - 4 0/4 - 2 0/2 - 2 1/2 - - - -ENUGU 2 0/2 - 2 0/2 - 2 0/2 - 3 0/3 - 1 0/1 -EBONYI - - - - - - 2 0/2 - - - - - -ONDO 4 1/4 - 5 2/5 -- 7 1/7 1/7 2 1/2 - 5 1/5 -OGUN - - - 2 0/2 - - - - - - - - - -NIGER 5 0/5 - 3 0/3 - - - - 3 2/3 - - - -KADUNA 5 0/5 - 2 0/2 - 4 1/4 1/4 8 0/8 - 6 0/6 -KANO 16 0/16 - 5 0/5 - 5 0/5 - 2 0/2 - - - - PAb= POLYCLONAL ANTIBODY TSC= TOTAL SAMPLES COLLECTED International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  36. 36. Table 9 (Continued) : Occurrence of Geminiviruses in Nigeria COWPEA TOMATO PEPPER OKRA JATROPHASTATE TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCRJIGAWA 5 0/5 - 2 0/2 - - 2 0/2 - - - -BAUCHI 3 0/3 - - - - 3 0/3 - 3 0/3 - - - -YOBE 3 0/3 - 3 0/3 - - - - - - - - - -GOMBE 8 0/8 - - - - 4 0/4 - 3 0/3 - - - -ADAMAWA 2 0/2 - - - - - - - 3 0/3 - - - -TARABA 14 0/14 - 2 -- - - - - 3 0/3 - - - -PLATEAU - - - - - - - - - 3 0/3 - - - -EDO - - - - - - 2 0/2 - 4 0/4 - - - -DELTA - - - 2 -- - - - 2 0/2 - - - -IMO 5 0/5 - - - - - - - - - - -ABIA - - - 2 - 2 0/2 - 4 0/4 - 2 0/2 -AKWA IBOM - - - - - - 2 0/2 - 2 0/2 - - - -CROSS RIVERS - - - - - - 4 0/4 - 3 0/3 - - - -RIVERS - - - - - - 2 0/2 - 4 1/4 - 2 0/2 -OSUN - - - - - - - 2 0/2 - - -TOTAL 118 3/118 52 3/52 -- 62 3/62 --- 79 5/79 -- 18 1/18 -- PAb= POLYCLONAL ANTIBODY TSC= TOTAL SAMPLES COLLECTED International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  37. 37. Plate 4: PCR Indexing for Other Geminiviruses in Survey Samples Using Universal Primer Pair (Broad Spectrum Detection) Uv-ali/f1/r1 M 8 10 24 32 M1KB marker used 500bp 500bp M 42 47 48 M•46 samples from all zones were randomly picked for analyses.•universal primers for geminiviruses (Primer A/F; Primer B/R).•5 samples were positive, three cassava samples (M115-lane 24; M300-lane 32, and S154-lane 42) one pepper sample (N31- lane 10) and one tobacco sample (N22-lane 8).•Lane 47; Negative control / healthy casava Lane 48; Positive control/ ACMV diseased cassava International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  38. 38. Total positive samples detected by PCR usinguniversal primers for geminiviruses (Primer A/F;Primer B/R)93 samples analysed•Three tomato samples (S8; S28; and S43)•Two pepper samples (S6, N31).•one tobacco sample (N22). International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  39. 39. % incidence of ACMV/EACMV using MAbs 80 120 113 70 100 60 Total samples analysed 82 80 50 DSMZ 2 MAb% incidence DSMZ 4 MAb SCRI 17 MAb 40 60 SCRI 20 MAb SCRI 33 MAb SCRI 60 MAb 30 Total plants analysed 40 20 20 10 11 5 0 0 0 Arid/semi Arid Northern Guinea Southern Guinea Derived savannah Humid forest savannah savannah ZonesFig 8: SEROLOGICAL INDEXING FOR ACMV AND EACMV IN LEAF SAMPLESDSMZ MAb 2- REACTS WITH ACMV & EACMV DSMZ MAb 4- REACTS WITH ACMV; DOES NOT REACT WITH EACMVIN SINGLE INFECTIONS SCRI MAb 17- REACTS WITH ACMV & EACMV SCRI MAb 20– REACTS WITH ACMV, EACMV,& ICMV SCRI MAb 33- REACTS WITH ACMV ALONE SCRI MAb 60- REACTS WITH ICMV ALONE International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  40. 40. % incidence of MSV using MSV polyclonal antibody 120 100 94 90 100 80 70 80 Total samples analysed 60% incidence % positive samples 60 50 S Total samples analysed 40 40 30 20 20 17 18 9 10 4 5 0 0 Arid/semi Arid Northern Guinea Southern Guinea Derived Mid Altitude Humid forest savannah savannah savannah Zones FIG 9: SEROLOGICAL INDEXING FOR MSV IN LEAF SAMPLES International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  41. 41. This survey demonstrated usefulness of antibodies produced in this study:MAbs 6B3 and 3F1 MAbs were very specific and detected only 2EACMV positive samples in 40 randomly selected samples from the humid Forest and Derived Savannah Zones Further it showed occurrence of several geminiviruses infecting several economically important crops, knowledge on which are scanty in Nigeria International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  42. 42. 7. CONCLUSIONS• Eliminating the density gradient step in purifications of antigens is not detrimental in the production of monoclonal antibodies.• Each animal is unique in it’s immune response. Different antibody titres obtained with the same type and quantity of immunising antigen• A maximum of 6 immunisations of experimental animals with ACMV/EACMV and MSV is adequate for high titre polyclonal antibody production. All antibodies produced detected immunising antigen.• Protocol used for fusions is efficient (all plates produced hybridomas) and is recommended. EACMV specific MAb was produced.• The Swiss albino strain of mice is unsuitable for MAb production using X63 myeloma cell lines. Homologous fusion partners yield the highest numbers of stable hybridomas (Maden, 1985)• PAbs and MAbs produced efficiently detected geminiviruses in indirect ELISA International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  43. 43. CONCLUSIONS C. dabrowski is inefficient as a vector of MSV and is unlikely to contribute to disease epidemics. The antigens and adjuvant administered in study do not affect experimental animals in a clinically important or preclusive manner. Field surveys demonstrated usefulness of diagnostic reagents and also showed occurrence of diverse geminiviruses infecting several economically important crop species. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  44. 44. ACKNOWLEDGEMENTS IITA, Ibadan Scottish Crop Research Institute (SCRI), Scotland, UKDeushe Sammlung von Microrganismen und Zellkulturen (DSMZ), Germany International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

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