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

Production of Monoclonal and Polyclonal
Antibodies 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

Outline

1. Introduction
2. Justification & Work plan
3. Production of antibodies
4. Immunization effects on animals
5. Vector transmission studies
6. Surveys for geminiviruses in Nigeria
7. Conclusions


1. Introduction
Geminiviruses

Family: 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, BGMV
Mastrevirus: MSV
Curtovirus: BCTV
Topocuvirus: TPCTV


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).


Vector of CMD
Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae)

• Most economically important
virus vector (Geddes, 1990)

• It is a pest on 350 plant species,
including cassava, around the
world


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)


2. Justification & Work Plan
1 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.

Aims:
• To produce antibodies for the detection and differentiation of
geminiviruses
• Determine the virus-vector interactions of MSV and leaf hoppers

Objectives:
 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


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 esculenta

2. MSV PURIFICATION: Method by (Bock et al.,1974)
3. IMMUNISATIONS: Mus musculus (Mouse); Oryctolagus
cunniculus (Rabbit)
Immunized at 2 week intervals for 12 weeks
4 Bleeding
5 Storage of serum


Fig: 1

HAT IN MEDIA,Only HGPRT +ve cells survive;- salvage pathway

TAS/ACP-ELISA
LIMITING DILUTION


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. benthamiana

Group 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. benthamiana
Group B
Mice 2 /MSV 1.83 0.27 Zea mays
Group B

Rabbit 1/ EACMV 0.504 0.226 N. benthamiana

Mice 3EACMV
Group C /ACMV 3.498 0.143 M.esculenta
Group C

Rabbits 2EACMV
/ACMV

*(A405) values at 1 hr sustrate incubation

Results
Table 2
ACMV/EACMV AND MSV RABBIT AND MOUSE POLYCLONAL
ANTISERA PRODUCED

RECIPROCAL ANTISERUM RECIPROCAL ANTISERUM
TITRE IN TAS ELISA TITRE IN ACP ELISA
ANIMAL 1hr 0/N 1hr 0/N
incubation incubation 1nc 1nc
ACMV/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

(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 Dilutions

FIG 2: EA/ACMV/M/C4 ANTISERUM TITRE BY INDIRECT ELISA (TAS)
ANTISERUM PRODUCED FROM MICE BY INTRAPERITONEAL IMMUNISATIONS

(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 Dilutions

FIG 3: EA/ACMV/M/C4 ANTISERUM TITRE BY INDIRECT ELISA (TAS)
ANTISERUM PRODUCED FROM MICE BY INTRAPERITONEAL IMMUNISATIONS


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”


B

A

Plate 1; Growing hybridoma cells.
(a) Lower left corner: dividing cells
(b) Top right: cells forming a colony

Table 3
1st screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mouse
hybridomas
WELLS WITH WELLS +VE % OF TOTAL WELLS
GROWING BY ELISA TO SECRETING SPECIFIC
HYBRIDS ACMV ANTIBODIES AGAINST
ACMV
Plate 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%


TABLE 4; 2nd screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mouse
hybridomas
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; ACMV
ANTG =ACMV Antigen; EACMV ANTG =EACMV Antigen International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

Table 5
CLONING: EACMV/ cell lines in TAS-ELISA (O/N incubation)

Cell lines 1st Cloning 2nd cloning
6B3 ++ ++
3F1 +++ +++

1.5 – 1.9: + weak positive
2.0 – 3.0: ++ positive
3.0<: +++ strong positive


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
MSV
Plate 1 10/96 2/10 2.1%
Plate 2 8/96 1/8 1.04%
Plate 3 6/96 2/6 2.1
Plate 4 7/96 5/7 5.2%
Plate 5 5/96 1/5 1.04%
Plate 6 8/96 3/8 3.13%


Table 7; CLONING: MSV cell lines in ACP-ELISA

Cell Ist 2ND
lines CLONING CLONING

Healthy Diseased Diseased/Healthy Healthy Diseased Diseased/Healthy
sap sap sap sap
3F4 0.209 0.617 2.9 0.126 1.239 9.8

3E11b 0.282 2.1 7.4 0.123 0.404 3.3

1H2 0.049 0.455 9.2 0.103 0.406 3.9

5F2 0.039 0.315 8.0 0.152 0.586 3.9


Plate 2

Fusions with Swiss Albino strain of mice yielded no Hybridomas


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”


4. Effects of Immunisation on Rabbits


Fig 4; Bi-monthly Temperature Differences In Immunized
Animals Before And After Immunisations
of two rabbits)

1.4 Group 1: Control; Distilled water alone
Group 2: Adjuvant alone
1.2 Group 3: Antigen + Adjuvant
Temp 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

TABLE 8 : PERCENTAGE CHANGE OF PHYSICAL AND HAEMATOLOGICAL PARAMETERS
BETWEEN 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 B

MEANS IN THE SAME COLUMN WITH DIFFERENT LETTERS ARE SIGNIFICANTLY DIFFERENT (p<0.05).


5. TRANSMISSION EFFICIENCY OF MSV in
C. dabrowski and C. triangular


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
AAPs

IAP (Inoculation Access Period) * = 24 hrs; AAP (Acquisition Access Period)**; No of replicates = 3

Significantly higher transmission efficiency in C.triangular at all AAPs (p<0.05)

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)

C. triangular is an efficient vector of MSV
C. dabrowiski is relatively poor vector of MSV


6. SURVEY FOR GEMINIVIRUSES IN NIGERIA


Fig 7: MAP OF NIGERIA SHOWING STUDY SITES FOR GEMINIVIRUSES

Surveyed during Oct-Nov 2002
Cassava, maize, tomato, pepper, okra, cowpea and jatropha

Methods for virus detection

1. Serological tests (ELISA):
ACMV poly; MSV poly; SCRI Mabs; DMSZ MAbs

2. 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


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
GEMINIVIRUSES

PRIMER SEQUENCES FROM PITA ET AL ( 2001a).


Table 9: Occurrence of Geminiviruses in Nigeria

COWPEA TOMATO PEPPER OKRA JATROPHA
STATE TSC ACMV Pab TSC ACMV Pab TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR
OYO 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 - - - -
NASSARA
WA 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


Table 9 (Continued) : Occurrence of Geminiviruses in Nigeria

COWPEA TOMATO PEPPER OKRA JATROPHA
STATE TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR TSC ACMV Pab PCR
JIGAWA 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


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 M

1KB 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

Total positive samples detected by PCR using
universal 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).


% 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
Zones

Fig 8: SEROLOGICAL INDEXING FOR ACMV AND EACMV IN LEAF SAMPLES
DSMZ MAb 2- REACTS WITH ACMV & EACMV DSMZ MAb 4- REACTS WITH ACMV; DOES NOT REACT WITH EACMV
IN 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

% 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


This survey demonstrated usefulness of
antibodies produced in this study:
MAbs 6B3 and 3F1 MAbs were very specific and detected only 2
EACMV 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


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

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.


ACKNOWLEDGEMENTS
IITA, Ibadan

Scottish Crop Research Institute (SCRI), Scotland, UK

Deushe Sammlung von Microrganismen und Zellkulturen
(DSMZ), Germany


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

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