Viroids and prions

JOSHUAALBERT&SONUS.S
2014-2015
VIROIDSANDPRIONS
“Project submitted in partial fulfillment
of the requirements for the central
board of senior secondary school class
XI in biology"
INDIAN SCHOOL DARSAIT,
MUSCAT

Acknowledgement
At the outset, we would like to express our sincere gratitude to our school, for providing us
with such an opportunity toshowcaseour skills, our teachers for their guidance; and to each
and every one who contributed to making this project a reality. Above all, we would like to
thank God theAlmighty for giving us the strength and endurance to undertaken this project
and execute it to the best of our abilities.
___________

CONTENTS
Sl. No TOPIC Page No.
1 Abstract 5
2 Introduction 7
3 Viroid the Plant Invaders 8
4 Spindle Tuber Disease of Potato 10
5 Prion 14
6 Bovine Spongiform Encephalopathy 15
7 Bibliography 18
___________

LIST OF FIGURES
Sl. No Description Page No.
1 Dmitri Iwanowsky 5
2 Tobacco mosaic disease 5
3 Examples of Viroids 8
4 Timeline of Discovery of Viroids 9
5 Potato Spindle Tuber Disease 10
___________
LIST OF TABLES
Sl. No Description Page No.
1 Differences Between Bacteria & Viruses 7
2 Comparison of Cows’ Brain 16
___________
ABBREVIATIONS USED
Sl. No Abbreviation Abbreviated Word Page No.
1 PLMVd Pelamoviroids 8
2 ASBVd Avsunviroids 8
3 CBVd Coleviroids 8
4 PSTVd Potato Spindle Tuber Viroid 9
5 BSE Bovine Spongiform Encephalopathy
___________

ABSTRACT
Viruses are too small to be seen with a light microscope and cannot be cultured outside their
hosts. Therefore, although viraldiseases arenot new, theviruses themselves could notbestudied
until the twentieth century. In 1886, the Dutch chemist Adolf Mayer showed that tobacco
mosaic disease (TMD) was transmissiblefroma diseased plant toa healthy plant. In 1892, in an
attempt to isolate the cause of TMD, the Russian bacteriologist Dmitri Iwanowsky filtered the
sap of diseased plants through a porcelain filter that was designed to retain bacteria. He
expected to find the microbe trapped in the filter; instead, he found that the infectious agent
had passed through the minute pores of the filter. When he infected healthy plants with the
filtered fluid, they contracted TMD. The first human
diseaseassociated with a filterableagent was yellowfever.
Advances in the molecular biological techniques in the
1980s and 1990s led to the recognition of several new
viruses, including human immunodeficiency virus (HIV)
and SARS associated corona virus. Israeli acute paralysis
virus becamea concern in 2006, when it killed up to90%
of the pollinating bees in some U.S. hives. This new virus
was first seen in bees in Israel in 2002 and seems to have
been in theUnited States sincethen.
Viroids are nucleic acid species of
low molecular weight and unique
structure that cause several
important diseases of cultivated
plants. Viroids are the smallest
known agents of infectious disease.
Unlikeviral nucleic acids,
Figure 1: Dmitri Iwanowsky
Figure 2 : Tobacco mosaic disease

Viroids arenot encapsulated. Despitetheir smallsize, Viroids replicateautonomously in cells of
susceptibleplant species. Known Viroids aresingle-stranded, covalently closed, circular, as well
as linear, RNA molecules with extensive regions of intramolecularcomplementarily; they exist
in their native state as highly base-paired rods. The biological properties of Viroids are
determined by their primary structures; Viroids thus constitute genetic systems of minimal
complexity. Sofar, Viroids havebeen identified only as pathogens of higherplants, but it is likely
that certain animal(including human) diseases arecaused by similar agents.
Prions are unconventional infectious agents responsible for transmissible spongiform
encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-
resistant prion protein (PrPres) that replicates in the body by inducing the misfolding of the
cellular prion protein (PrPC). Although compelling evidence supports this hypothesis,
generation of infectious prion particles in vitro has not been convincingly demonstrated. Here
we show that PrPC --> PrPres conversion can be mimicked in vitro by cyclic amplification of
protein misfolding, resultingin indefiniteamplificationof PrPres. Thein vitro-generated forms
of PrPres share similar biochemical and structural properties with PrPres derived from sick
brains. Inoculationof wild-typehamsters with in vitro-produced PrPres led toa scrapiedisease
identical to the illness produced by brain infectious material. These findings demonstrate
that prions can be generated in vitro and provide strong evidence in support of the protein-
only hypothesis of prion transmission.
___________

INTRODUCTION
One hundred years ago, researchers could not imaginesubmicroscopic particles, and thus
they described theinfectious agent as contagiumvivumfluidum—a contagious fluid. By the
1930s, scientists had begunusing theword virus, theLatin word for poison, to describethese
filterableagents. Thenatureof viruses, however, remained elusive until1935, when Wendell
Stanley, an American chemist, isolated tobaccomosaic virus, making it possible for the first
time to carry out chemicaland structuralstudies on a purified virus. At about the same time,
theinvention of the electron microscopemade it possible to see viruses.
Life can be defined as a complex set of processes resulting fromtheactions of proteins
specified by nucleic acids. Thenucleic acids of living cells are in action all thetime. Because
viruses are inert outsideliving host cells, in this sense they are not considered to be living
organisms. However, once viruses enter a host cell, the viral nucleic acids becomeactive, and
viral multiplication results. In this sense, viruses are alive when they multiply in the host cells
they infect. From a clinicalpoint of view, viruses can be considered alive becausethey cause
infection and disease, just as pathogenicbacteria, fungiand protozoa do. Depending on one’s
viewpoint, a virus may be regarded as an exceptionally complex aggregation of nonliving
chemicals, or as an exceptionally simple living microorganism.
Table 1 : Differences Between Bacteria &
VirusesViruses were originally
distinguished from other infectious
agents because they are especially
small (filterable) and because they
are obligatory intracellular
parasites—that is, they absolutely
require living host cells in order to
multiply. However, certain small
bacteria, such as some rickettsias,
share both of these properties.
Viruses and bacteria are compared
in Table 1.

VIROIDS - the Plant Invaders
A Viroid is a virus (VIR) like (OID) particle. Viroids are“sub-viruses” composed exclusively of
a single circularstrandof nucleic acid (RNA) that codes for a singleprotein or small, circular
RNA molecules without a protein coat.
 No coding capacity - do not programtheir
own polymerase.
 Use host-encoded polymerasefor
replication.
 Mechanically transmitted;often seed
transmitted.
 More than 40 viroid species and many
variants havebeen characterized.
 “Classical” Viroids have been found only in
plants.
 Viroids differ from viruses in that viruses, at
their most basic level, consist of genetic
material (DNA or RNA) contained within a
protective protein shell.
 Viroids differ from prions, another typeof
sub viral infectious agent, in that prions are
made only of protein lacking nucleic acid.
DID YOU KNOW?
VIROIDS are
 Very small, covalently
closed, circularRNA
molecules capableof
autonomous replication and
induction of disease.
 Rangein size from
approximately 20 nm.
 Are infectious particles.
 Causecertain plant and
human diseases.
 It consists only of Nucleic
Acid (RNA).
The smallest viroid identified so far is
a 220-nucleobase scRNA (small
cytoplasmic RNA) associated with the
rice yellow mottle sobemovirus
(RYMV) (Collins et al. 1998).
In comparison, the genomes of the
smallest known viruses capable of
causing an infection by themselves
are around two kilobases in size.
Figure 3 : Examples of Viroids
Genus Coleviroids:
CbVd 1 (coleusblumei 1)
Genus Avsunviroids:
ASBVd (avocado sunblotch)
Genus Pelamoviroids:
PLMVd (peach latent mosaic)

1974:
Confirmation
that Viroids
are non-
coding.
1973:
Electron
micrograph
shows Viroid’s
hairpin
structure
Discovery
Early 1960s:
Raymer and
O’Brien
develop a
bioassay for
the agent
causing potato
spindle tuber
disease. 1965:
Raymer
teams up
with Diener;
they show
that agent is
not a typical
Viroid.
1971:
Diener
demonstrate
s that the
agent is a
free non-
coding RNA,
coins the
term Viroid.
1968:
Characterization
of
chrysanthemum
stunt and citrus
exocortis as non-
typical viruses.
1978: PSTVd
is sequenced.
1976: EM
shows that
viroids
form
closed
circular
RNAs
Theodore O. Diener
Figure 4 : Timeline of Discovery of Viroids

Diseases Caused by Viroids - Potato Spindle Tuber Disease
Of themany diseases caused
by Viroids, thespindle tuber
disease of potatoes was the
first to be recognized and
studied by plant pathologists.
Nearly 50 years were to
elapse between initialdescription of this disease in theearly 1920's and theidentificationof
its causalagent, a small, highly-structured, covalentlyclosed circularRNA moleculeknown
as Potato spindletuber viroid (PSTVd). PSTVd remains an important pathogen of potatoand
tomato, and a recent increasein thenumber of reported latent infections of ornamental
species is creating newchallenges for currentdisease management strategies. PSTVdis also a
favoriteobject of study for viroid molecular biologists, thanks in largepart to its ability to
replicateto high titers in tomato where certain strains rapidly inducetheappearanceof a
characteristic diseasesyndromethat includes stuntingand epinasty.
Symptoms and Signs
The naturalhost rangeof PSTVd includes many solanaceous species. Theviroid may cause
disease in Solanumtuberosum (potato), S. lycopersicum (syn. Lycopersiconesculentum,
tomato), and Capsicumannuum (pepper) wheresymptoms may
vary considerably depending on plant species, variety, viroid
strain and environmentalconditions. Infections in other hosts
are symptomless; e.g., Brugmansia spp., Datura sp., Lycianthes
rantonneti (syn. S. rantonneti),Persea
americana (avocado), Physalis peruviana (Capegooseberry), S.
jasminoides, S. muricatum(pepino), and Streptosolen jamesonii.
DISEASE Potatospindle tuber
PATHOGEN Potatospindle tuber viroid
HOSTS potato, tomato, ornamentals (Solanaceae)
Figure 5:- Potato Spindle
Tuber Disease

In potato, growth of infected plants may be severely reduced or even cease entirely; however,
reduction in growth may also be hardly visible. The vines of infected plants may be smaller,
more upright, and producesmaller leaves than their healthy counterparts. Infected tubers
may be small, elongated (from which thedisease derives its name), misshapen, and cracked.
Their eyes may be more pronounced thannormaland may be borneon knob-like
protuberances thatmay even develop intosmall tubers.
The first symptoms of PSTVd infection in tomato(Figure2) are growth reduction and chlorosis
in thetop of the plant. Subsequently, this growth reduction may develop into stunting, and the
chlorosis may become more severe, turningintoreddeningand/or purpling. In this stage,
leaves may becomebrittle. Generally, this stunting is permanent; occasionally,however, plants
may either die or partially recover. As stuntingbegins, flower and fruit initiation stop.
Disease Transmission
PSTVd can be transmitted in four different ways:
Vegetative propagation- Propagationby tubers, cuttings, and
micro-plants provides a very efficient means of viroid
transmission. Onceestablished, PSTVd infection is persistent;
therefore, plants frominfected lots act as a permanent sourceof inoculumfor other lots
and crops. Vegetativepropagation has been themajor pathway for PSTVd transmission
in potatoand ornamentals suchas Brugmansia spp. and S. jasminoides. The absenceof
symptoms increases therisk that infected plants will be used for
propagation
 Mechanical transmission- Under favorableconditions, PSTVd
is readily transmittedby normalcultivation activities.This is most
clearly seen with potatoes and tomatoes, whereviroid spread is
mainly along therow
 Infected seed and pollen- PSTVdis assumed to havespread
among potato germplasmcollections allover theworld via
infected trueseed. Oncepresent in a germbank, theviroid can be
transmitted toother (wild) potatoplants either mechanically or

by pollen exchange. Seed is also a potentialsourceof infection for other crops such as
tomatoand pepper that arepropagated by seed
 Aphid transmission- This routeof transmission requires thesourceplant tobe infected
by both Potatoleafroll virus (PLRV) and PSTVd, thereby limiting thenumber of potential
infection sources. PSTVd is assumed to be encapsidated by the viralcoat protein; such
encapsidation protects theviroid from digestion by micrococcalnucleasein vitro,
suggestingthata similar protectiveeffect may occur in vivo.
Disease Management
Disease management can bedivided intotwo parts: prevention of infection and viroid
eradication.
Prevention of infection includes allmeasures to prevent the introduction of PSTVd intoa
specific crop. It is very important to start a new cultivation with viroid-freeplantingmaterial
(tubers, seeds or plants). PSTVd is considered a quarantine'organism' in many countries, and
therefore, governmentalmeasures toprevent introduction of PSTVd with plants from other
countries will often be applied. Certification schemes includingtestingmay be required to
provide further guarantees thattheplanting materialis free from PSTVd.
In addition tothe use of healthy planting material, it is also important toprevent viroid
introduction via human activities. BecausePSTVd is mechanically transmissible, it can be
introduced intopotentialhost plants via the hands, clothes, or equipment used by people
working in or visiting thegreenhouse. Theuseof disposablegloves and specific clothing and
equipment that stays insidea greenhousecompartmentmay prevent PSTVd introductioninto
greenhouse-growncrops. Furthermore, increasingthenumber of plants species grown in a
greenhousecompartment increases therisk of introduction of PSTVd. Growers should either
growa singlecrop or they should separatedifferent crops and lots, preferably in different
compartments. BecausePSTVd can betransmitted by aphids, plantingPLRV-freeseed potatoes
and controlling aphid populations alsocontributes tothemanagement of PSTVd in potato
crops.

Viroid eradication is based on destruction of PSTVd-infected plantsand thorough cleaningof
equipment and greenhouses whereinfected plants havebeen grown. All infected plants
together with thosefrom an adequate buffer zoneshould be destroyed. In caseof field-grown
potatoes, crop rotations involving non-PSTVd host species help eliminateinfected volunteer
plants. In case of symptomless infections such as thosecommonly observed in ornamentals, all
plants in the lot should be destroyed. Any rock wool or plastic used to cover the soil should
also be removed from thegreenhouseand destroyed. Ideally, all materialslated for disposal
should be transported in a closed container toan incinerator; alternatively, such materialcan
be taken to a refuse dump and covered with a layer of soil.
When PSTVd is identified in a greenhouse-growncrop, allparts of thegreenhouseshould be
thoroughly cleaned, preferably usinga steam cleaner and a scrub brushfor parts that are
difficult toclean (Figure7). A regular acid treatmentcan beused for watering tubes and
drippers. After cleaning thegreenhouseand associated equipment,application of a
disinfectantcompletes theeradication procedure. When cultivationof crops susceptibleto
PSTVd infection resumes, extra monitoringfor PSTVd symptoms and/or testing areadvisable.
___________

Prions
A prion is an infectious agent, specifically a protein in a misfolded form. The word prion,
coined in 1982 by Stanley B. Prusiner, is derived from thewords protein and infection. The
protein itself, whether in its misfolded or its correctly folded form, can be referred to as the
prion protein (PrP). A protein as an infectious agent stands in contrasttoall other known
infectious agents, likeviruses, bacteria, fungi, or parasites—allof which must contain nucleic
acids (either DNA, RNA, or both). Prions are responsiblefor mammalian transmissible
spongiformencephalopathies, including bovinespongiformencephalopathy(BSE,alsoknown
as "mad cow disease") and scrapiein sheep. In humans, prions cause Creutzfeldt-Jakob
Disease (CJD), variant Creutzfeldt-JakobDisease (vCJD), Gerstmann–Sträussler–Scheinker
syndrome, FatalFamilial Insomnia and kuru. All known prion diseases in mammals affect the
structureof the brain or other neural tissueand all arecurrently untreatableand universally
fatal. In 2013, a study revealed that 1 in 2,000 peoplein theUnited Kingdommight harbour
theinfectious prion protein that causes vCJD.

Diseases Caused by Prions – Bovine Spongiform Encephalopathy
The word BSE is short but it stands for a disease with a long name, bovine spongiform
encephalopathy. "Bovine" means that thedisease affects cows, "spongiform" refers to theway
thebrain from a sick cow looks spongy under a microscope, and "encephalopathy" indicates
that it is a disease of thebrain. BSE is commonly called “mad cow disease.” BSE is a progressive
neurologic diseaseof cows. Progressivemeans that it gets worse over time. Neurologic means
that it damages a cow’s centralnervous system .
Most scientists think thatBSE is caused by a protein called a prion. For reasons that arenot
completely understood, thenormalprion protein changes intoan abnormalprion protein that
is harmful. Thebody of a sick cow does not even knowtheabnormalprion is there. Without
knowing it is there, thecow’s body cannot fight off thedisease.
Symptoms of BSE
A common symptom of BSE is incoordination. Asick cow has troublewalking and getting
up. A sick cow may also act very nervous or violent, which is why BSE is often called “mad
cow disease.”
It usually takes four to six years from the time a cow is infected with theabnormalprion to
when it first shows symptoms of BSE. This is called the incubation period. Duringthe
incubationperiod, thereis no way to tell that a cow has BSE by looking at it. Oncea cow
starts toshow symptoms, it gets sicker and sicker untilit dies, usually within two weeks to six
months. Thereis no treatment for BSE and no vaccinetoprevent it.
Currently, thereis no reliable way to test for BSE in a live cow. After a cow has died, scientists
can tell if it had BSE by looking at its brain under a microscopeand seeing thespongy
appearance. Scientists can alsotell if a cow had BSE by using test kits that can detect the
abnormalprion in thebrain.

Brain from a healthy cow, as seen under a
microscopeusing specialstains.
Brain from a cow sick with BSE, as seen
under a microscopeusing special
stains. This brain is sponge-like, and the
largewhite spaces are like the "holes" of a
sponge.
Disease Transmission
The parts of a cow that arenot eaten by people are cooked, dried, ground intoa powder, and
used for many purposes, includingas ingredients in animalfeed. A cow gets BSE by eating
feed contaminated with parts that camefromanother cowthat was sick with BSE. The
contaminatedfeed contains theabnormalprion, and a cow becomes infected with the
abnormalprion when it eats the feed. If a cow gets BSE, it most likely ate thecontaminated
feed during its first year of life. Remember, if a cow becomes infected with theabnormal
prion when it is one year old, it usually will not show signs of BSE untilit is five years old or
older.
Table 2:- Comparison of cow brain
brain tissues

Prevention and control
 The measures in the strategy for dealing with BSE are early detection and warning
systems and prevention and rapid responsemeasures and mechanisms in place.
 Targeted surveillanceof occurrences of clinicalneurologicaldisease;
 Awareness programs to enhancesurveillance;
 Screeningtests at routineslaughter;
 Transparency in reportingfindingsof BSE;
 Safeguardson importation of live ruminantspecies and their products, in
accordancewith theOIE TerrestrialCode;
 Removal of specified risk material(SRM) (brain, spinalcolumn) duringslaughter
and processing of carcasses;
 Prohibit theinclusion of SRM in animalfeeds, thus removing potentially
contaminatedmaterialfromthe food chain;
 Humanedestructionof all suspected and susceptibleanimals exposed to
contaminatedfeed (cohorts);
 Appropriatedisposal of carcasses and all animalby-products;
 Livestock identificationtoenable effectivesurveillanceand tracing of suspected
livestock.
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BIBILOGRAPHY
i. http://en.wikipedia.org/wiki/Viroid
ii. http://www.ncbi.nlm.nih.gov/pubmed/7121568
iii. http://ghr.nlm.nih.gov/condition/prion-disease
iv. http://en.wikipedia.org/wiki/Prion
v. http://en.wikipedia.org/wiki/Potato_spindle_tuber_viroid
vi. http://www.ncbi.nlm.nih.gov/pubmed/15851027
vii. http://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathy
viii. http://www.who.int/zoonoses/diseases/bse/en/
ix. http://www.apsnet.org/edcenter/intropp/lessons/viruses/pages/potatospindletuber.aspx
x. http://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathy
xi. http://www.fda.gov/AnimalVeterinary/ResourcesforYou/AnimalHealthLiteracy/ucm136
222.htm
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Viroids and prions

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