Production of Monoclonal and Polyclonal Antibodies for Detection of Geminiviruses and the Virus...

International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

Adeola AlaPh.D Student

Virology Unit, IITA-Ibadan, Nigeria

&

Animal Physiology Unit, Department of Zoology

University of Ibadan, Nigeria

Production of Monoclonal and Polyclonal

Antibodies for Detection of Geminiviruses and

the Virus –Vector Relationships


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).


• Most economically important

virus vector (Geddes, 1990)

• It is a pest on 350 plant species,

including cassava, around the

world

Vector of CMDBemisia tabaci (Gennadius) (Homoptera: Aleyrodidae)


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


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

3. Production polyclonal antibodies (PABS)


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

TAS/ACP-ELISALIMITING DILUTION

Fig: 1


Anim

al im

mun

ized

Viru

s

Conc

. Of

vir

us

in

purif

ied p

rep.

*(A40

5)

(mea

n va

lue:

n=2

)

Healt

hy sa

p

(mea

n va

lue:

n=2

)

Host

plan

t

Mice

Mice

4 /ACMV

3/ MSV

Grou

p A

Rabbit 1/ ACMV

0.861

1.300

0.861

0.065

0.28

0.065

N. benthamiana

Zea mays

N. benthamiana

Rabbit 1/ACMV

Mice

Mice

3 /EACMV

2 /MSV

Grou

p B

Rabbit 1/ EACMV

0.887

1.83

0.504

0.321

0.27

0.226

N.benthamiana

N. benthamiana

Zea mays

N. benthamiana

Mice 3EACMV

/ACMV

Grou

p C

Rabbits 2EACMV

/ACMV

3.498

0.143

M.esculenta

*(A405) values at 1 hr sustrate incubation

Table 1; Experimental animals used in immunisations

Group A

Group B

Group C


ACMV/EACMV AND MSV RABBIT AND MOUSE POLYCLONAL

ANTISERA PRODUCED

RECIPROCAL ANTISERUM

TITRE IN TAS ELISA

RECIPROCAL ANTISERUM

TITRE IN ACP ELISA

ANIMAL 1hr

incubation

0/N

incubation

1hr

1nc

0/N

1nc

ACMV/EACMV

(1) ACMV/R/A1

(2) ACMV/R/B1

(3) EACMV/R/B1

(4) EA/ACMV/R/C1

(5) EA/ACMV/R/C2

(6) ACMV/M/A1-A4

(7) EACMV/M/B4

(8) EA/ACMV/M/C3

(9) EA/ACMV/M/C4

MSV

(1) MSV/R/A1

(2) MSV/M/A1

(3) MSV/M/A2

(4) MSV/M/B1

(5) MSV/M/B2

1,000

8,000 (1.95)

NDT*

256,000 (4.07)

1,600 (1.90)

1,000 (3.2)

8,000 (2.03)

256,000 (2.84)

64,000 (1.99)

–

–

32,000 (1.85)

16,000

256,000 (5.01)

128,000 (2.3)

–

–

–

–

–

64,000 (2.02)

(Unadsorbed)

16,000 (2.53)

(Adsorbed)

512,000 (1.93)

(Unadsorbed)

1,000 (1.96)

(Adsorbed)

64,000 (2.1)

(Unadsorbed)

256,000 (4.3)

(Adsorbed)

256,000 (4.73)

256,000 (3.82)

NDT

NDT

512,000 (2.51)

(Unadsorbed)

32,000 (2.38)

(Adsorbed)

1,024,000 (2.27)

(Unadsorbed)

NDT

(Adsrobed)

–

256,000 (2.5)

(Adsorbed)

–

–

250 (1.84)

4,000 (2.44)

* Not detectable titre – Not tested

( ) D/H values

ResultsTable 2


(1 hr incubation)

7.266.88

5.78

4.81

3.46

2.96

2.411.99

1.741.38

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1/500 1/1000 1/2000 1/4000 1/80001/160001/320001/640001/1280001/256000Antiserum Dilutions

A4

05

nm

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Tit

re p

oin

ts

HDTitre points

FIG 2: EA/ACMV/M/C4 ANTISERUM TITRE BY INDIRECT ELISA (TAS)

ANTISERUM PRODUCED FROM MICE BY INTRAPERITONEAL IMMUNISATIONS


(O/N incubation)

4.92

6.09

6.68

5.57

3.93

3.44

2.922.72

1.811.53

0

0.5

1

1.5

2

2.5

3

3.5

4

1/500 1/1000 1/2000 1/4000 1/8000 1/160001/32000 1/640001/1280001/512000Antiserum Dilutions

A 4

05

nm

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Tit

re p

oin

ts (

A4

05

nm

)

HDTitre points

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”


Plate 1; Growing hybridoma cells.(a) Lower left corner: dividing cells

(b) Top right: cells forming a colony

B

A


1st screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mouse

hybridomas

WELLS WITH

GROWING

HYBRIDS

WELLS +VE

BY ELISA TO

ACMV

% OF TOTAL WELLS

SECRETING SPECIFIC

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 3


CELL LINES

ACMV R. Poly/ACMV

ANTG

ACMV R. Poly/EACM

ANTG

ACMV Ms Poly/ACMV

ANT G

ACM Ms Poly/ EACMV ANTG

DSMZ Poly/ACMV

ANTG

DSMZ poly/ EACMV ANTG

2nd

screen.

3rd

screen.

2nd

screen.

3rd

screen.

2nd screen 2nd

screen.

3rd

screen.

2nd

screen.

3rd

screen.

2nd

screen.

3rd

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 - - - + + - - - + - -

TABLE 4; 2nd screening of EA/ACMV/M/C1 and EA/ACMV/M/C2 Balb/C mouse

hybridomas

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


Cell lines 1st Cloning 2nd cloning

6B3 ++ ++

3F1 +++ +++

1.5 – 1.9: + weak positive

2.0 – 3.0: ++ positive

3.0<: +++ strong positive

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

Table 5


WELLS WITH

GROWING

HYBRIDS

WELLS +VE

BY ELISA TO

MSV

% OF TOTAL WELLS

SECRETING SPECIFIC

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 6; MSV/M/A1 AND MSV/M/A2 BALB/C (1st Screening)


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

Cell

lines

Ist CLONING

2ND

CLONING

Healthy sap

Diseased sap

Diseased/Healthy Healthy sap

Diseased sap

Diseased/Healthy

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


Fusions with Swiss Albino strain of mice yielded no Hybridomas

Plate 2


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”

Plate 3


4. Effects of Immunisation on Rabbits


0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 Period/weeks

Te

mp

ch

an

ge

Group 1 Group 2 Group 3

(mea

n o

f tw

o r

abb

its)

Fig 4; Bi-monthly Temperature Differences In Immunized

Animals Before And After Immunisations

Group 1: Control; Distilled water alone

Group 2: Adjuvant alone

Group 3: Antigen + Adjuvant


PARAMETER GROUP 1 GROUP 2 GROUP 3

WEIGHT 5.248 BA 3.411 B 9.022 A

TEMPERATURE DIFFERENCE

0.0929 B 0.7143 A 0.9143 A

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

TABLE 8 : PERCENTAGE CHANGE OF PHYSICAL AND HAEMATOLOGICAL PARAMETERS

BETWEEN GROUPS OF RABBITS IN 12 WEEKS GIVEN DIFFERENT TREATMENTS

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


12

20 20 20 20

12

0

10

20

30

40

50

60

70

80

30 secs 15 mins 1 hr 24 hr 48 hr 96 hr

AAPs

% t

ran

sm

iss

ion

0

5

10

15

20

25

To

tal in

se

cts

us

ed

C.triangular

C.dabrowski

Total insects used

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 5: Percent transmission of MSV by C. triangular and C.

dabrowski given different virus acquisition access periods


20 20 20 20 20 20

0

10

20

30

40

50

60

30 secs 15 mins 1 hr 24 hr 48 hr 96 hr

Inoculation Access Periods (IAPs)

% t

ran

sm

issio

n

0

5

10

15

20

25

To

tal in

sects

used

C. triangular

C. dabrowski

Total insects used

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

Fig 6: Percent transmission of MSV by C. triangular and

C. dabrowski given different virus inoculation access periods


C. triangular is an efficient vector of MSV

C. dabrowiski is relatively poor vector of MSV


6. SURVEY FOR GEMINIVIRUSES IN NIGERIA


Surveyed during Oct-Nov 2002

Cassava, maize, tomato, pepper, okra, cowpea and jatropha

Fig 7: MAP OF NIGERIA SHOWING STUDY SITES FOR GEMINIVIRUSES


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


PRIMER SEQUENCES FROM PITA ET AL ( 2001a).

Primers used for PCR amplification of geminiviruses

VIRUS PRIMER SEQUENCE (5’-3’)TARGET 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 T G 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 TRANSMITTED GEMINIVIRUSES

/F

PRIMER B /R

TAA TAT TAC CKG WKG VCC

TGG ACY TTR CAW GGB CCT TCA CA

CR

CRPRIMER A


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 - - - -

Table 9: Occurrence of Geminiviruses in Nigeria

PAb= POLYCLONAL ANTIBODY

TSC= TOTAL SAMPLES COLLECTED


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 --

COWPEA TOMATO PEPPER OKRA JATROPHA

Table 9 (Continued) : Occurrence of Geminiviruses in Nigeria

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

•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

M 8 10 24 32 M

M 42 M

500bp

500bp

47 48

1KB marker used


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

11

0

5

113

82

0

10

20

30

40

50

60

70

80

Arid/semi Arid Northern Guinea

savannah

Southern Guinea

savannah

Derived savannah Humid forest

Zones

% in

cid

en

ce

0

20

40

60

80

100

120

To

tal s

am

ple

s a

na

lyse

d

DSMZ 2 MAb

DSMZ 4 MAb

SCRI 17 MAb

SCRI 20 MAb

SCRI 33 MAb

SCRI 60 MAb

Total plants analysed

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

9

4

17

94

5

18

0

20

40

60

80

100

120

Arid/semi Arid Northern Guinea

savannah

Southern Guinea

savannah

Derived

savannah

Mid Altitude Humid forest

Zones

% i

nci

de

nc

e

0

10

20

30

40

50

60

70

80

90

100

To

tal

sam

ple

s an

alys

ed

% positive samples

Total samples analysedS

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


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.

CONCLUSIONS


ACKNOWLEDGEMENTS

IITA, Ibadan

Scottish Crop Research Institute (SCRI), Scotland, UK

Deushe Sammlung von Microrganismen und Zellkulturen

(DSMZ), Germany

Date post:	11-May-2015
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Production of Monoclonal and Polyclonal Antibodies for Detection of Geminiviruses and the Virus...

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