Viroids:

pathogen at the frontier of

life
Ganesan Vadamalai
Dept. of Plant Protection
Faculty of Agriculture
Universiti Putra Malaysia

Outline:
Plant pathology and pathogens
What is a viroid?
Importance of viroids
Economic
Life science

Summary

Plant pathology
A science that studies plant diseases and
their causes.

Plant Pathogens
Organism that cause disease in plants.

Fungi, Bacteria, Mollicute (Phytoplasma &

Spiroplasma), Nematodes, Virus and Viroid.

What are viroids?

Subviral plant pathogen
First described in 1970

~38 viroids

low molecular weight, single stranded,

circular RNAs
Transmitted mechanically
Some are seed and pollen transmissible
Replicate in all cells of host plant
Pathogenic

Discovery
Early 1960s:

1968:

1973: Electron

1976: EM

Raymer and
OBrien develop a
bioassay for the
agent causing
potato spindle
tuber disease

Characterization of
chrysanthemum
stunt and citrus
exocortis as nontypical viruses

micrograph shows
viroids hairpin
structure

shows that
viroids form
closed circular
RNAs

1965: Raymer

1971: Diener

1974:

1978: PSTVd is

teams up with
Diener; they show
that agent is not a
typical virion

demonstrates that
the agent is a free
non-coding RNA,
coins the term
viroid

Confirmation that
viroids are noncoding

sequenced

Randles and Hatta

(1977)

Classification of Viroids
Classification is based on the absence or
presence of a conserved central region.
1. Avsunviroids (ASBVd-type species)

Avsunviroideae
Avsunviroid
Pelamoviroid

2. Pospiviroids (PSTVd-type species)

Pospiviroideae:
5 subgroups: Pospiviroid, Apscaviroid, Cocadviroid,
Coleviroid, Hostuviroid

3. Unassigned viroids

Avsunviroideae

Nomenclature

Viroid =
virus- like

Adapted from Flores R et al (1998) Arch Virol 143, 623-629.

Type species
Genus Pospiviroids:

Genus Coleviroids:

PSTVd (potato spindle tuber)

CbVd 1 (coleus blumei 1)

Genus Hostuviroids:

Genus Avsunviroids:

HSVd (hop stunt)

ASBVd (avocado sunblotch)

Genus Cocadviroids:

Genus Pelamoviroids:

CCCVd (coconut cadang-cadang)

PLMVd (peach latent mosaic)

Genus Apscaviroids:
ASSVd (apple scar skin)

Pospiviroidae: Nuclear, CCR, no

hammerhead self-cleavage

Avsunviroidae: Chloroplastic, no
CCR, hammerhead self-cleavage

Symptomology
slowly developing lethal disease to the
symptomless infection.

Chlorosis
Mottling
Mosaic

Detection
Biological tests
Polyacrylamide gel electrophoresis (PAGE)

2D-PAGE,
bi-directional PAGE

Polymerase chain reaction (PCR)

RT-PCR
Real Time PCR

Nucleic acid hybridisation tests

Northern blot

Ribonuclease Protection Assay (RPA)

Sequencing

conventional
NGS

Viroid vs Virus
Difference

smaller than viruses, contain no genes

Virus has nucleic acid (DNA or RNA) protected by a
coat protein
Viroid lacks a protein coat (exist as free or naked
RNA)

Similarity

Both disrupts protein synthesis mechanism in host

Rolling circle mechanism

Symmetric variant
RZ

(Family Avsunviroidae)
RZ

2
5 OH

2
5 OH

P
3

HF

HF
2
5 OH

P
3

Asymmetric variant (Family Pospiviroidae)

Why viroids are special?

Smallest known pathogen

246-400 bases
Comparison with other organism
Human : 3.2 billion base pairs
(Human Genome Project Website, 2008)

Smallest virus : 4000 base

No known functional genes

0.0001mm

VIRUSES & VIROIDS

Importance of viroids
Causes economically important diseases

Coconut cadang-cadang disease in the

Philippines
Meaning: dying-dying
caused by Coconut cadang-cadang viroid
(CCCVd)
Losses > 40 million palms

Cadang-cadang disease
Slow lethal disease of coconut palms
reported from 1914 in the Philippines
Rate of spread ~ 0.5km pa.
New outbreaks still occur
Mode of field spread not known
Managed by replanting

Early

Mid

Late

Site of a Cadang-Cadang epidemic in the Philippines

SYMPTOMS

Leaf:
-Spots
fine to large yellow
non-necrotic spots

-Brooming
not common
lamina severely reduced

CC

Nuts:
initially become
small and rounded,
production then stops

Threat of viroid in Malaysian

Agriculture?
Potential threat to Malaysian oil palm
industry

CCCVd reported in commercial oil palm

plantation in Malaysia (Vadamalai et al, 2006)
Causes orange spotting disease

Orange Spotting

Effects of OS

4 year old OS affected oil palm

Healthy oil palm

Oil palm CCCVd variants

Present in both OS palms and asymptomatic palms
Sequencing showed a number of variants were present
- 4 variants of 297nt, 293nt, 270nt and 246nt
- > 90% sequence similarity with CCCVd from coconut
246nt from an OS palm; 297nt, 293nt, 270nt from an
asymptomatic palm
All oil palm CCCVd variants have C31U base substitution
(Vadamalai et al 2006; Arch. Virol. 151, 1447-1456)
(Wu et al 2013; Arch. Virol. 158,1407-1410)

CCCVd 246 - coconut palm

CCCVd 296 - coconut palm

CCCVd 297 - oil palm

(Vadamalai et al 2006; Arch. Virol. 151, 1447-1456)

a) OP

Energy: -134.4 kcal/mole

293
CCCVd
293 - oil palm
-A

UG
AU

CU

AU

GU

+G

10
20
30
40
50
60
70
80
90
100
110
120
130
140
CU
|AAAU
--CCCAAAA
- U
A
U
GGGAAACAA U
A
A
UAC
U A
U- UU
AGCU
UC GC
UU
G
GGGG
CUACA
GGGCC
ACUA C GCAGG GAG GCCGC UGAG
AUCC CCGGG
CUCAAGCG UC GGGA GGG GCG
CUGGG CG UCG GCU CG GGAGG
GGGUCGA GU GCG GGAG A
CCCC
GAUGU
CCUGG
UGAU G UGUCC UUC CGGCG ACUC
UAGG GGCCC
GGGUUCGC AG UCCU CCC CGC
GGCCC GC AGC CGA GC CCUCC
CCCAGCU CG UGC CCUC G
U---UUC
AAAAAAA
U U
A
U
C
ACA
U
AUCAGA
C- C
C-- UU
U UU
GCCU- AUU
A
290
280
270
260
250
240
230
220
210
200
190
180
170
160
150

UC

-GU

-A

GU

GC

AC

CG

GA

270
- oil
palm
b)CCCVd
OP270 Energy:
-119.0
kcal/mole
CU
CU
U.

10
20
30
40
50
60
70
80
90
100
110
120
|AAAU
--CCAAAA- U
A
U
GG-GAAACAA U
A
A
UAC
U A
.-UGC
UU
G
GGGG
CUACA
GGGCACC
ACUA C GCAGG GAG GCCGC UGAG
AUCC CCGGG
CUCAAGCG UC GGGA GGG GCG
CUGGG CG UCG
GCG GGAG A
CCCC
GAUGU
CCUGUGG
UGAU G UGUCC UUC CGGCG ACUC
UAGG GGCCC
GGGUUCGC AG UCCU CCC CGC
GGCCC GC AGC
UGC CCUC G
---UUC
AAAAAAA
U U
A
U
U
AACA
U
AUCAGA
C- C
C-- - / \--UU
A
260
250
240
230
220
210
200
190
180
170
U
A
130
U
CG
160
GC
CU
AU
U
U
GC
140
CG
UA
U
C
C
G
C
C
C
C
150
C
C
CG

Mutations in all OP variants

Mutations in specific variants

Mutations in single clones

UC
AC

(Vadamalai et al 2006; Arch. Virol. 151, 1447-1456)

10

Structural variation associated with sequence variation

a) CCCVd

246

Coconut 246

b) CCCVd

296

Coconut 296

c) OP 297

Oil palm 297

d) OP 293

Oil palm 293

e) OP 270

Oil palm 270

(Vadamalai et al 2006; Arch. Virol. 151, 1447-1456)

Sequence of a 246nt CCCVd variant

from an Orange Spotting Oil Palm
J02049
TL

TR

(Wu et al 2013; Arch. Virol. 158,1407-1410)

11

Viroids and Life Science

May be the living fossils of the RNA world

small size, imposed by error-prone replication

their circular structure
which assures complete replication without genomic tags
their lack of protein-coding ability, consistent with a ribosomefree habitat
replication mediated in some by ribozymesthe fingerprint of
the RNA world.

(Flores et al, 2005)

Fungi

Viroids: opening the matryoshka of plant

pathology
Bacteria & Mollicute

Virus
Viroid
??

Summary
Smallest pathogen

Difficult to detect
No control measures

Potential treat to Malaysian oil palm

industry

12

The importance of the infinitely small is

infinitely large (Pasteur)

Size does not matter!

THANK YOU

13