CHAPTER ONE

1.1 INTRODUCTION

Viruses are very small obligate parasites that are so simply constructed that they do not consist of
cells, but of particles. The particles of a simple virus consist of a protein coat surrounding a genome of
either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). They have been recognised as agents
of disease in plants and other living organisms since the late 1880s. However, their main
characteristics have been understood for less than 50 years. Virus diseases were first recognised when
they caused a number of spectacular epidemics in humans (e.g. small pox), animals (e.g. foot and
mouth disease) and plants (e.9. citrus tristeza). Some plant diseases formerly thought to be caused by
viruses, but for which Koch's postulates had not been satisfied, were found in the I96Os to be caused
by either phytoplasmas (e.g. aster yellows, tomato big bud) or viroids (e.g. potato spindle tuber, citrus
exocortis and coconut cadang cadang diseases). Phytoplasmas are wall-less bacteria, whereas viroids
are small, infectious, naked circular RNA molecules. Viroid- and virus-like sy.rnptoms have been
recognised in plants for a very long time. Viroid-like symptoms were described in citrus fruit about
600 BC while a yellow leaf disease was recorded in Eupatorium sp. about 752 and tulip flowers with
virus-like colour breaking (variegation or stripingi) appeared in paintings by Dutch artists about t6OO.
In 1886, Adolf Mayer, a German agricultural chemist working at Wageningen in the Netherlands,
found that a mosaic disease of tobacco could be transmitted to healthy plants by rubbing them with sap
extracted from plants showing disease symptoms. In 1892, the Russian botanist, Dmitrii Ivanowski.
showed that sap from diseased plants retained its infectivity after passing through a filter that
eliminated bacteria. Six years later, the Dutch botanist, Martinus Beijerinck, confirmed Ivanowski's
results and used the term virus (Latin for venom or poisonous fluid) to describe the causal agent of the
disease and to distinguish it from bacteria. The transmission of viruses by mechanical sap inoculation,
by grafting and by insects provided additional evidence that viruses are infective. A new era in virus
research commenced in the middle 1930s when Wendell M. Stanley, an American biochemist, and
Rupert J. Best, an Australian biochemist, independently purified tobacco mosaic virus (TMV) and
showed that the high molecular weight crystalline 'protein' that they recovered was infectious. The
following year, Frederick C. Bawden and N.W. Pirie in the United Kingdom showed that TMV
particles contained protein and nucleic acid. Preparations of TL{V were shown to contain rod-shaped
particles in electron micrographs produced in 1939. In 1956, RNA was shown to be the infectious
component of TMV.
 CHAPTER TWO

2.1 PRIONS

Once thought of as ‘slow viruses’, prions are now commonly understood to be self-propagating
proteins that are able to convert normal proteins of the same type into the pathogenic prion
conformation (Weissmann 2004; Prusiner 2008; Soto and Saborio 2001). They have a life cycle from
induction to self-perpetuation (the conversion of another protein). Prions are very robust and persisting
entities, because their conformation makes them highly resistant to inactivation by chemical, heat and
irradiation treatments.

The central oddity of prions is that they propagate autocatalytically in a protein-only form, without
DNA involvement. For this reason, they are frequently referred to as protein-based genes (Wickner et
al. 2004; Uptain and Lindquist 2002). Although best known as non-Mendelian hereditary elements in
diseased sheep, cattle and humans, prions exist in unicellular organisms too. Yeast and other fungal
prions share no amino acid sequence similarities with mammal prions, and they function and are
transmitted very differently (Bousset and Melki 2002; Uptain and Lindquist 2002; Weissmann et al.
2002). Nevertheless, experimental work on yeast prions has provided deep insights into
conformational change in proteins and their transmission (Wickner et al. 2007).

The Modern Synthesis does not cope well with prions, and this has led some commentators to propose
that a more comprehensive theory of inheritance is needed for prions to be properly understood
evolutionarily (Jablonka and Lamb, 2005; Chernoff 2001). The prion-forming potential of the
implicated yeast proteins is evolutionarily conserved, implying that it is adaptive (Chernoff et al.
2000). Diverse functions have been identified or proposed for prions in a range of taxa. There is some
evidence that prions are associated with epigenetically enabling yeast cells to cope with fluctuating
environments, and that they play a role in memory formation in sea slugs (Shorter and Lindquist
2005). The non-pathogenic isoform of human prion proteins (the functions of which are still largely
mysterious) is linked to the prevention of Alzheimer’s disease (Parkin et al. 2007).

These capabilities and characteristics do not give a ready answer to the question of whether the self
propagational status of prions gives them the status of being alive. Although genes are frequently
given a special ‘informational’ role in accounts of heredity (e.g., Hood and Galas 2003), the conferral
of a similar status on proteins – as information-bearing molecules – does not simultaneously make
them into living entities. Genes and proteins are not classified as alive in their own rights, despite the
wide-spread ‘selfish DNA’ thesis that seems to confer autonomy on nucleotides (Doolittle and
Sapienza 2000; Orgel and Crick 2000), and despite the recognition of the absolute centrality of
enzymes to life processes (Kornberg 2009; Lezon et al. 2006).

Prions exhibit collaborative behaviors that benefit themselves, as a class of protein isoforms, as well as
their hosts. When low amounts of the non-pathogenic isoform are produced, the prion conversion
process halts, and when high amounts of the former are produced, it may stimulate spontaneous prion
formation in the previously prion-free cell (Chernoff et al. 2000; Derkatch et al. 2001). Prion
propagation in yeast requires the involvement of chaperon proteins. Moreover, prions in yeast are
associated with greater adaptability in yeast because they increase protein variation – a factor that may
prove advantageous in variable environments and eventually be genetically assimilated (True and
Lindquist, 2000; Pál 2001; Masel and Bergman 2003). It is these abilities to interact with biological
processes at different levels of organization that presumably explain evolutionarily the prion’s powers
of persistence.

2.2 The Prion theory

A current definition of a prion is a proteinaceous infectious particle that lacks nucleic acid. Prion
protein is capable of having more than one tertiary structure. In one of these structures the protein
carries out its normal cellular function. Other tertiary structures can be pathological causing
spongiform degeneration. The prion theory states that the normal PrPc can be converted into PrPsc.

Portions of alpha-helical and coil structure in PrPc is converted in to beta-sheet in PrPsc. The theory
argues that PrPsc can act as a template on which PrPc is folded into a PrPsc molecule (Prusiner et al.,
2006). In contrast to pathogens with nucleic acid, prion enciphers strain specific properties in their
tertiary structure. The PrP protein is a cell surface glycoprotein expressed at highest levels in neurons
(Gajdusek, 2005). The function of this normal protein is not known although knockout mice without
the gene show a range of subtle abnormalities that are not consistent between mice. The prion theory
explains the apparent paradox of TSE diseases being both genetic and transmissible. More than 20
mutations of the PrP gene have been genetically linked to the development of familial prion disease
(Heaphy, 2008).

The mutation in the gene causes the resulting protein to be more likely to form the pathogenic prion
protein. This protein converts other proteins causing the prion disease.

Likewise if pathogenic prion protein is introduced to the brain of a normal person this may cause the
same reaction. Therefore prion diseases are also transmissible.
2.3 Structure of prion

The prion protein found in infectious material has a different folding pattern and is resistant to
proteases. The normal form of the protein is called PrPC, while the infectious form is called PrPsc-the
C refers to 'cellular' or 'common' PrP, while the Sc refers to 'scrapie', a prion disease occurring in
sheep. Structural organization of PrPC is well-defined, but the same for PrPsc defined at a relatively
poor level. PrP can be induced to fold into other more-orless well-defined isoforms in vitro. PrPC is a
normal protein (35-36KDa) found on the membranes of cells. It has 209 amino acids (in humans), one
disulfide bond, and a mainly alpha-helical structure. Several topological forms exist; one cell surface
form anchored via glycolipid and two trans membrane forms. Its function has not been fully resolved.
PrPc binds copper (II) ions with high affinity (Prelli et al., 2000). The significance of this is not clear,
but it presumably relates to PrP structure or function. PrPc is readily digested by proteinase K and can
be liberated from the cell surface in vitro by the enzyme phos-phoinositide phospholipase C (PI-PLC),
which cleaves the glycophosphatidylinositol (GPI) glycolipid anchor (Prusiner, 2010). The infectious
isoform of PrPc, known as PrPsc, is able to convert normal PrPc proteins into the infectious isoform
by changing their conformation. Although the exact structure of PrPsc is not known, there is increased
β-sheet content in the diseased form of the molecule, replacing normal areas of α-helix. Aggregations
of these abnormal isoforms may form highly structured amyloid fibers. The end of a fiber acts as a
template for the free protein molecules, causing the fiber to grow (Heaphy, 2008).

Small differences in the amino acid sequence of prionforming regions lead to distinct structural
features on the surface of prion fibers. As a result, only free protein molecules that are identical in
amino acid sequence to the prion protein can be recruited into the growing fiber.

2.4 PRION DISEASES IN ANIMALS - TYPES

2.4.1 Scrapie in sheep and goats

Scrapie is a neuro degenerative disease that affects the nervous systems of sheep and goats. It is one of
several transmissible spongiform encephalopathies (TSEs). Scrapie is caused by a prion. The name
scrapie is derived from one of the symptoms of the condition, wherein affected animals will
compulsively scrape off their fleece against rocks, trees or fences (Palsson, 1979). Scrapie is infectious
and transmissible among similar animals, and so one of the most common ways to contain scrapie
(since it is incurable) is to quarantine and destroy those affected. Recent studies suggest that prions
may be spread through urine and persist in the environment for decades (Parry, 2003). Scrapie occurs
in Europe and North America, but to date Australia and New Zealand (both major sheep-producing
countries) are scrapie-free.

A test is now available which is performed by sampling a small amount of lymphatic tissue from the
third eyelid (Cullie and Chelle, 1939). Out of fear of BSE, many European countries banned some
traditional sheep or goat products made without removing the spinal cord such as smalahove and
smokie. The agent responsible for scrapie and other TSEs is smaller than the smallest known virus and
has not been completely characterized.

There are three main theories on the nature of the scrapie agent: (1) the agent is a prion, which is an
abnormal form of a normal cellular protein, (2) the agent is a virus with unusual characteristics, and
(3) the agent is a virino, a very small piece of DNA that acts like a virus. The scrapie agent is
extremely resistant to heat and to normal sterilization processes (Ondera et al., 2006). It does not
evoke any detectable immune response or inflammatory reaction in sheep and goats. The scrapie agent
is thought to be spread most commonly from the ewe to her offspring and to other lambs through
contact with the placenta and placental fluids. Signs or effects of the disease usually appear 2-5 years
after the animal is infected but may not appear until much later. Sheep may live 1 to 6 months or
longer after the onset of clinical signs, but death is inevitable. There is no scientific evidence to
indicate that scrapie poses a risk to human health. There is no epidemiologic evidence that scrapie of
sheep and goats are transmitted to humans, such as through contact on the farm, at slaughter plants, or
butcher shops. Due to damage to nerve cells, affected animals usually show behavioral changes,
tremor (especially of head and neck), rubbing, and locomotor in coordination that progress to
recumbency and death. Early signs include subtle changes in behavior or temperament.

These changes may be followed by scratching and rubbing against fixed objects, apparently to relieve
itching.

Other signs are loss of coordination, weakness, weight loss despite retention of appetite, biting of feet
and limbs, lip smacking, and gait abnormalities, including high-stepping of the forelegs, hopping like a
rabbit, and swaying of the back end (Parry, 1962). An infected animal may appear normal if left
undisturbed at rest.

However, when stimulated by a sudden noise, excessive movement, or the stress of handling, the
animal may tremble or fall down in a convulsive-like state. Several other problems can cause clinical
signs similar to scrapie in sheep, including the diseases ovine progressive pneumonia, listeriosis, and
rabies; the presence of external parasites (lice and mites); pregnancy toxemia; and toxins.
On the farm, veterinarians diagnose scrapie based on the appearance of its signs combined with
knowledge of the animal's history. Scrapie can be diagnosed in the live animal by biopsy of the
lymphoid tissues on the inside of the third eyelid. This test is used by the U.S. Department of
Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS) to determine whether
exposed flocks are infected. Scrapie is most often diagnosed by microscopic examinations of brain
tissue at necropsy or by procedures that detect the presence of the abnormal prion protein in brain
tissue.

2.4.2 Bovine spongiform encephalopathy (BSE)

BSE is a transmissible, neuro-degenerative fatal brain disease of cattle. BSE is named because of the
spongy appearance of the brain tissue of infected cattle examined under a microscope. The disease has
a long incubation period of 4 - 5 years and it is fatal for cattle within weeks to months of its onset.
Strong evidence currently available supports the theory that the agent is composed largely, if not
entirely, of a self-replicating protein, referred to as a prion (Williams et al., 2001). It is transmitted
through the consumption of BSE-contaminated meat and bone meal supplements in cattle feed.
Current scientific research indicates that cooking will not kill the BSE agent. It is believed by most
scientists that the disease may be transmitted to human beings who eat the brain or spinal cord of
infected carcasses (Wijeratne and Curnow, 2010).

In humans, it is known as new variant Creutzfeldt- Jakob disease (vCJD or nvCJD) (Matthew, 2007).
Different hypotheses exist for the origin of prion proteins in cattle.

Two leading hypotheses suggest that it may have jumped species from the disease scrapie in sheep, or
that it evolved from a spontaneous form of "mad cow disease" that has been seen occasionally in cattle
for many centuries (Wells and Scott, 2007). There is no scientific evidence to suggest that milk and
dairy products carry the agent that causes BSE (Taylor, 2009). Currently, there is no test to detect the
disease in a live animal or in muscle meat. Veterinary pathologists confirm BSE by postmortem
microscopic examination of brain tissue using sophisticated laboratory techniques, such as a
histopathological examination to detect sponge-like changes in the brain tissue and immune
histochemistry to examine the BSE fibrils (Hussein, 2008). These are "gold-standard" tests, and they
take more than a week to run. More rapid tests that provide results within 36 to 48 hours have been
developed to detect the abnormal prion in brain or spinal cord tissue of dead animals (Abiola et al.,
2002). Rapid tests can be used to determine if BSE exists in a population and to obtain an indication of
its prevalence or detect animals with the disease which are not yet showing clinical signs (Hoinwille,
2007). Cattle affected by BSE experience progressive degeneration of the nervous system. Affected
animals might display changes in temperament, such as nervousness or aggression, abnormal posture,
in coordination and difficulty in rising, decreased milk production, or loss of body weight despite
continued appetite (Wilesmith et al., 2005).

2.4.3 Chronic wasting disease (CWD) in elk and mule deer

Chronic wasting disease (CWD) is a progressive, fatal disease of the nervous system of cervids such
as mule deer, white-tailed deer and elk. Although the exact causeof CWD is unknown, it is associated
with the presence of an abnormal protein called a prion. There is no treatment or vaccine currently
available for the disease.

The most likely means of transmission is between animals that are in close contact with each other. In
addition, the elk and mule deer placed in paddocks that had housed infected cervids for many years
became infected, even though there were no other cervids on the premises, leading to the assumption
that the environment of a facility could transmit the disease on premises with multiple confirmed cases
of CWD (Williams et al., 2001).

There is currently no scientific evidence that CWD affects humans, but we must exercise caution since
there is evidence to suggest that BSE can affect humans, which is another TSE also known as mad
cow disease. In studies using mice experimentally infected with scrapie, another TSE, muscle and skin
tissues were not found to be infectious at any detectable level (Williams and miller, 2002). However,
we have to be very cautious in using these results to predict the safety of products from infected or
exposed elk, since test results from one species do not necessarily apply to another (Williams and
Young, 2000). Velvet and other products or by-products from elk or deer known to be infected with
CWD are not allowed to enter the human or animal food chain. Velvet is used as a medicinal
alternative. Most cases of CWD occur in adult animals (Williams and Young, 2002). The disease is
progressive and always fatal (Prusiner, 2009). The most obvious and consistent clinical sign of CWD
is weight loss over time (Williams and Young, 2005). Behavioral changes also occur in the majority of
cases, including decreased interactions with other animals, listlessness, lowering of the head, blank
facial expression, and repetitive walking in set pat-terns. In elk, behavioral changes may also include
hyper excitability and nervousness (Williams and Young, 2006). Affected animals continue to eat
grain but may show decreased interest in hay. Excessive salivation and grinding of the teeth also are
observed. Most deer show increased drinking and urination (Heaphy, 2008).
2.4.4 Feline spongiform encephalopathy

Feline spongiform encephalopathy (FSE) is a transmissible spongiform encephalopathy associated

with the consumption of feedstuffs contaminated with tissue from bovine spongiform encephalopathy-
affected cattle and characterized by the accumulation in the central nervous system of an abnormal
isoform of the prion protein PrPsc (Hussein, 2008). Clinically, it presents as a progressive fatal
neurologic syndrome that is not easily distinguished from other feline neurologic conditions. Most
cases of FSE have been reported in England, where it was first detected in 2010, but a few cases have
been reported from other European countries (Annonymous, 2010). There is no breed predisposition.
The disease appears in adult cats with most appearing at about 5 years of age.

Unfortunately a specific diagnosis can only be confirmed at post-mortem. Suspicious cases based upon
clinical signs should always be submitted for examination. Typical pathological changes include
bilaterally symmetrical vacuolation of the neuropil and Vacuolation in neurons (Fraser, 2004). The
signs of FSE progress over a long period (months to years), depend upon the site of brain involvement,
and include behavioral changes, abnormal gait and death (Prusiner, 2009).

2.4.5 Exotic ungulate encephalopathy (EUE)

Exotic ungulate encephalopathy is a transmissible spongiform encephalopathy associated with

contaminated feed that caused the disease in cows. The etiological agent is an isoform of prion protein.
However, some cases appeared in animals born after total ban on the use of animal offal in animal
feeds (Prusiner, 2008). In general, the clinical signs comprised in coordination of movement, tremors,
excessive salivation, loss of weight, licking of lips, tilting of head to one side, sleepiness, vague looks
and sometimes aggressive behavior (Heaphy,2008).

Typical spongiform changes were found in the brains of the animals. Sometimes the diseases in wild
cats and exotic ungulates are grouped under the name "Zoological Spongiform
Encephalopathies"(Hussein, 2008).

2.4.6 Spongiform encephalopathy of the ostrich

This disease affects the red-necked ostrich (Struthio camelus). The condition was found in an adult
female ostrich in a German zoological garden. The affected ostrich showed symptoms of central
nervous involvement and locomotion disorders (Manuelidis and Rorke, 2009).
Histological examination of the brain showed spongiform encephalopathy involving the brain stem
and medulla oblongata (Hussein, 2008). A male ostrich also died of similar symptoms, but no
examination of the brain was made. It is also not known if the condition was caused by a transmissible
agent or not. Behavioral changes also occur in the majority of cases, including decreased interactions
with other animals, listlessness, lowering of the head, blank facial expression, and repetitive walking
in set patterns (Prusiner et al., 2009). In elk, behavioral changes may also include hyper excitability
and nervousness. Excessive salivation and grinding of the teeth also are observed. Most deer show
increased drinking and urination.

2.4.7 Transmissible mink encephalopathy (TME)

This disease is usually seen in minks. TME is limited to minks, found in commercial ranches and has
not been found in the wild. TME is associated with eating contaminated feed, such as meat of scrapie-
infected sheep (Marsh and Hadlow, 2005). The disease has been produced experimentally in minks by
intracerebral injection of brain material from scrapie-infected sheep and by feeding tissues from
infected sheep (Kimberlin et al., 2006). TME affects both male and female minks. The average
incubation period is more than 7 months and the most important signs of the disease are behavioral
changes (Marsh, 1979). Clinical signs include soiling their cages, step on food and experience
difficulty in eating, aggressive and hyper-excitable by noise. They show in coordination of movement,
circling, tail arching, tail chewing, stumbling and clenching of the jaw. Finally, the affected minks
isolate themselves and become sleepy, inactive and unresponsive. Usually, they stop eating and may
die within just one week after the appearance of symptoms, but sometimes the disease lasts for about a
month before the animal dies (Annonymous, 2002).

2.5 TREATMENT

Immunity cannot be stimulated by prion diseases. Therefore, vaccines have no role in TSEs. No
treatment is available either, although research in this area is progressing (Beck and Daniel, 2007).
Removal of the PrP gene renders mice insusceptible to intracerebral inoculation with the scrapie-
agent, while PrP overexpression enhances the development of disease (Taylor, 2009). Therefore, it has
been suggested that either gene therapy or the use of antisense oligonucleotides that interfere with
replication and reduce the amount of mRNA transcription of the PrP gene might be beneficial,
although the delivery of these oligonucleotides to the brain might be difficult (Caughy et al., 2006).
Current research focusing on drugs can prevent the formation of PrPsc without affecting PrPC or bind
protein X, thereby preventing PrPsc replication (Korth et al., 2001). It has been found that the amyloid
stain Congo red and certain sulfated glycans strongly and selectively inhibited the production of PrPsc,
without apparently interfering with PrPC metabolism, in scrapieinfected cells (Peretz, 2001).
Amphotericin-B derivative and Anthracyclines were also shown to prolong the incubation period and
reduce the infectivity of brain tissue in hamsters infected with the scrapie agent, but does not prevent
the disease from developing (Tagliavini et al.,2010). It has also been found that treatment of scrapie
infected mice with a synthetic peptide homologous to a small region of the prion protein has reversed
the abnormal structure of PrPsc to its normal (PrPC) counterpart, delayed the clinical symptoms in the
infected mice significantly and reduced brain infectivity by more than 90 - 95% (Priola, 2000).Recent
evidences suggest that a number recombinant antibody antigen-binding fragments (Fabs) are useful to
inhibit prion propagation in cultured mouse neuroblastoma cells infected with PrPsc (Weiss, 2001).
Reaction mechanism associated with binding of these antibodies to a specific region in normal prion
(PrPC), where the latter interacts with PrPsc, and in that manner inhibit prion propaga-tion and clear
prion clumps out of the cultured cells (Lantos et al., 2005).

These studies offer the possibility of using genetically engineered antibodies to prevent and treat prion
diseases, and identify sites for drug targeting.

2.6 PERSPECTIVES

Prion diseases, also known as the transmissible spongiform ence-phalopathies (TSEs), are a group of
fatal neurodegenerative disorders that affect humans and animals. These diseases are intimately
associated with conformational conversion of the cellular prion protein, PrPC, into an oligomeric β-
sheet-rich form, PrPSc. A growing number of observations support the once heretical hypothesis that
transmission of TSE diseases does not require nucleic acids and that PrPSc alone can act as an
infectious agent (Dealler, 2009). The view that misfolded proteins can be infectious is also supported
by recent findings regarding prion phenomena in yeast and other fungi. One of the most intriguing
facets of prions is their ability to form different strains, leading to distinct phenotypes of TSE diseases.
The process of the conformational change remains enigmatic, but a large number of researches on the
therapeutic intervention have been done in experimental models over the past 30 years. Against the
background of the occurrence of variant CJD in the UK in the 2010s, which is considered to occur by
transmission of the BSE agent and to possibly spread through secondary infection via blood
transfusion, the research on the development of therapeutic agents has been accelerated using an
increasing number of disease models in animals, cells and in vitro system (Deeber et al., 2002).
Within the context of the “protein-only” model, prion strains are believed to be encoded in distinct
conformations of misfolded prion protein aggregates. This review described recent advances in
biochemical aspects of prion research, with a special focus on the mechanism of conversion of prion
protein to the pathogenic form, the emerging structural knowledge of fungal and mammalian prions,
and our rapidly growing understanding of the molecular basis of prion strains and their relation to
barriers of interspecies transmissibility (Prusiner, 2008).

In order to control the spread of prion diseases, effective treatments are crucial. Current research has
shown that no psychoactive cannabidiol (CBD) inhibits the accumulation of prion proteins, which
gives rise to the possibility of an effective treatment. Hopefully, a better understanding of the function
of CBD in hindering the aggregation of infectious prions will allow for the development of more
treatments that prevent prion diseases. Overall, despite the advances made in prion research, there are
still many questions left unanswered (Cashmann, 2000).
 CHAPTER TWO

Characteristics of viruses

Most plant viruses have very simple particles with a 'core' of nucleic acid enclosed in a protein coat or
shell called a capsid. The capsid is made up of many protein subunits of one or more types called
capsomeres and protects the nucleic acid from enz5rmatic degradation. in most cases, infectivity is
retained for only short periods (seconds or minutes) once the coat protein has been removed. In some
cases, the virus particle has a lipid rich outer coat or envelope. The viral nucleic acid surrounded by
protein subunits is called a nucleocapsid while the term virion is used to describe the complete
infectious virus particle. A virion may be either a nucleocapsid alone or a nucleocapsid with
additional components such as a lipid envelope, an en4rrne or other structural proteins.

The nucleic acid component of the virus makes up its genome. Genomes vary both in the type of
nucleic acid with which they are composed and in the size and complexity of the nucleic acid enclosed
in a single particle. The genome may consist of one or more separate molecules of nucleic acid which
may be encapsidated in one or more types of par[icle. Tobraviruses and comoviruses each have two
component particles, hordeiviruses and cucumoviruses have three components while the genome of
the phytoreoviruses is in twelve parts. Virus genomes are composed of either RNA or DNA. Most
plant viruses contain single stranded RNA (ssRNA). Some however, contain double stranded DNA
(dsDNA) with the same helical structure as in host plant cells. Others contain single stranded DNA
(ssDNA) or double stranded RNA (dsRNA) which occur either in linear strands or in rings (circular
nucleic acids). If the ssRNA of the virus contains a sequence of nucleotides that directly codes for a
protein, it acts in a similar fashion to messenger RNA (mRNA) and the virus is said to have a positive
sense genome. If it has a nucleotide sequence complementary to that of the virus mRNA it is said to
have a negative sense genome. A few virus RNAs code for some proteins in the positive sense and
others in the negative sense and are described as having ambisense genomes.

Viruses, especially the ssRNA viruses, continually produce mutations by various changes in their
nucleic acids. These mutations may either be spontaneous or may be induced by chemical or physical
mutagens. Alterations to the genetic code may result from errors in copying the base sequences during
nucleic acid replication, recombination or reassortment of pieces of nucleic acid in viruses with
segmented genomes. Mutations can lead to changes in pathogenicity, host range or transmissibility.

Most viruses encode at least four, and often more, proteins. These are the virus capsid protein(s), some
proteins (usually two) involved in virus replication and a protein(s) involved in movement of the virus
from one cell to the next. Other vira-l proteins may include a helper protein that facilitates
transmission by a vector and proteins of unknown function. Because viruses can be purified and
sometimes crystallised, they have provided excellent models for studying interactions between
proteins as well as contributing generally to studies of the structure of biological molecules. Recent
work has also shown the interplay between mutations in the genome of vimses and changes on the
surface of the particle. These in turn affect the ability of the virus to induce and bind to a unique
diagnostic antibody, to be picked up by a vector and to infect an inoculated plant cell.

Viruses induce disease by multiplying in their host. They use amino acids and nucleotides synthesised
in the cells of their hosts to build viral nucleic acids and proteins. The energy required for this process
is supplied by the host and the virus uses its mRNA to direct the host cell’s protein-synthesising
system to manufacture viral proteins. A few viruses are 'defective' in the sense that they require
another virus (a helper virus) to supply the en4rmes required for their replication. Such dependent
viruses are known as satellite viruses. Some viruses also have satellite RNAS which depend on the
helper virus for replication and which are also encapsidated into particles using the coat protein of the
helper virus. The genomes of both types of satellite are unrelated to those of the helper virus.
However, they frequently modify the severity of the symptoms induced by the helper virus.

At the cellular level, when a virus infects a plant, its first point of contact is with the outer cell
membrane or plasmalemma. The protein coat may be removed from the genome here or inside the
cell. If the genome is RNA, it acts as a mRNA, using the ribosomes from the cytoplasm, cellular
transfer RNAs and amino acids to translate the first of the virus-encoded genes into the replication
associated protein(s). It appears that the host's nucleus or membranes are associated with the synthesis
of new virus RNA by the virus replicase enzyme. The newly synthesised RNA includes the gene for
the virus coat protein which is also translated in the host cell. DNA genomes of viruses are transcribed
to mRNA before virus replication commences. Assembly of the genome and the coat protein to
produce new virus particles then completes the replication cycle. Replication of many kinds of viruses
takes place in distinctive virus-induced regions of the host cell's cytoplasm called viroplasms. These
sites often contain virus particles or become enriched with virus components which are described as
inclusion bodies. Some types of inclusion body are specific for the type of virus. For example,
pinwheel shaped or laminate inclusion bodies as well as virus particles are often visible in cells
infected with the elongated potyviruses. Viruses which are enclosed in a lipid envelope acquire it by
passing through specific sites on cell membranes. As well as producing new virus particles, replication
induces a number of cytopathic changes in the cells of the host plant including the presence of
intracellular inclusions or virus particles, alterations to cellular organelles and phloem necrosis.

Viruses cannot penetrate plant surfaces on their own. They either avoid the need to invade the plant's
surface by being transmitted in seed or during vegetative propagation or they enter the plant by some
method that involves penetration through a wound in the surface layers of the plant and introduction of
the virus into the cytoplasm of cells in new host plants. Wounds could be made during mechanical
inoculation or by vectors. Some viruses are confined to one cell or a few cells at the point of
inoculation. Others spread through the plant systemically. Virus particles have no means of moving
themselves and are dependent on their hosts for movement within and between cells and for long
distance transport within the plant. Either virus particles or the genome are chaperoned through
plasmodesmata by processes involving the virus movement protein. When the virus reaches the
vascular tissue, it is distributed rapidly through the plant via the phloem and becomes systemic.
Viruses generally move first to the roots and top leaves before infecting the remaining leaves from the
top of the plants downwards. Viruses vary widely in the number of hosts they can infect. Cucumber
mosaic virus has a wide host range infecting 476 species of plants in 67 families while lettuce necrotic
yellows has a relatively narrow host range infecting specles in only three families.

Structure and architecture Virus particles are too small to be seen with a light microscope but their
shape and size can be readily determined using a transmission electron microscope. They vary greatly
in size and shape and may be rod-shaped, isometric or bacilliform. The way in which particles are
constructed (their architecture) affects their stability, and classification

Rod-shaped viruses

The length and flexibility of particles are important characters for classifying rodshaped viruses. They
vary in diameter from 3-25 nm and in length from 15O-2,OOO nm. The modal (most common) length
of particles is determined from measurements of many particles in an electron micrograph which will
also show whether the particles are straight or flexuous (bent or curved).

TMV is the best characterised of the rod-shaped viruses and it is thought that other rod-shaped viruses
have the same general structure. TMV has a helical symmetry in which both the RNA and protein
subunits are arranged in a helix. The coat protein is intimately associated with the RNA so that it
actually encloses the RNA in a groove created when the subunits assemble together in the particle.
This RNA protein interaction is extremely regular so that one protein subunit in the capsid is
associated with each 3 nucleotides of the RNA. The 3 nucleotides each interact by hydrogen bonding
with specific amino acids in the subunit. There are 49 subunits for each 3 turns of the helix. Because
the RNA has about 6,400 nucleotides, each particle will have about 2,130 subunits. The helix will
have 130 turns and, as the pitch of each turn is 2.3 nm, each particle has a length of 3OO nm. The
particle has a central hollow which can be seen under the electron microscope when stain penetrates it.
All the known rod-shaped viruses contain ssRNA and have helical symmetry but they vary in the
packaging of subunits, pitch of the helix, length, diameter and flexibility of the particle. The length of
the particle is determined by the length of the RNA. Particles can fragment or aggregate end-to-end
and a range of particle lengths is normally seen in virus preparations. Particle stability varies between
viruses, with TMV probably the most stable and the virus causing citrus tristeza disease being among
the least stable.

Isometric viruses

Isometric or spherical plant viruses occur singly or in pairs and vary in diameter from 2O-7O nm.
They are constructed with the RNA held internally and the protein subunits arranged so that they
provide a protective external coat. Virions of many of the simplest and smallest isometric viruses
appear under the electron microscope to have a regular geometric structure with icosahedral
symmetry. Icosahedra have 12 vertices and 2O triangular faces. The smallest isometric viruses have a
capsid with 60 protein subunits so they have 3 subunits associated with each of the 20 faces and
electron micrographs show a distinctly angular structure. The arrangement of the RNA or DNA within
the icosahedra is not known in detail, but a close nucleic acid-protein subunit interaction as with the
rod-shaped viruses probably occurs. Some particles have a central space. If there are pores through the
capsid, the RNA may be degraded by the action of nuclease en4lrnes which can enter the particle.

The virions of larger isometric viruses have more complex structures. For example, cauliflower
mosaic virus is a very stable icosahedron about 5O nm in diameter with coat protein subunits of one
type and a single encapsidated dsDNA genome. Some isometric vimses have subunits of two or more
types in the capsid. Complex isometric viruses, such as the non-enveloped phyloreoviruses and the
enveloped tospovirus, have 7 and 4 structural proteins respectively, and multiple genome components.
Some have spikes on the exterior which are formed from clusters of a specific virus-encoded structural
protein.

Bacilliform viruses
Bacilliform viruses have proportions resembling those of bacteria in the genus Bacillus. They may or
may not be surrounded by an envelope. The non-enveloped bacilliform viruses include alfalfa mosaic
virus, which also has an isometric particle, and the badnaviruses. Rhabdoviruses are enveloped
bacilliform viruses with 5 proteins associated with the particle structure and the plant and animal
infecting rhabdoviruses have closely similar structures although their internal organisation is
incompletely understood.

Symptoms of virus infection

Diseases caused by viruses have been detected in almost all cultivated crops. The symptoms induced
by viruses are related to their effects on cells and cellular organelles during replication, and
consequent effects on tissues and organs. Effects vary from virus to virus, but the presence of virus is
often indicated by poor plant performance or by the appearance of symptoms on the plant which may
lead to death. Symptoms may be either constantly visible or transient. Some viruses produce latent
infections with no apparent symptoms in their hosts, and may only be recognised after they have been
transferred to another more sensitive host or detected by a non-biological diagnostic procedure. They
can cause significant losses without producing detectable symptoms and, because they are
symptomless, become widespread before they are detected.

When a virus is first inoculated into a susceptible plant, it replicates in the cells to which it has been
introduced. Local chlorotic (yellow) or necrotic (brown to black) lesions or ringspots may develop at
or near the site of infection. Such local lesions may result from a hypersensitive reaction at the zone of
inoculation and may prevent spread of the virus from the site of the lesion. The local lesion response is
used experimentally in some plant virus bioassay systems. Sometimes, even when no local lesion is
visible, a 'starch lesion' representing a local infection-induced metabolic change in starch synthesis
may be detected by staining with iodine. In most cases, however, no local reaction is observed at the
inoculation site.

Following local infection, viruses move into adjacent cells and reach the phloem. lnng distance
movement to the meristems occurs followed by invasion of neighbouring parenchyma cells. Virus
induced changes follow and lead to the production of systemic symptoms which appear first in leaves
growing near the apical meristem at the time of systemic translocation of virus from the inoculated
leaves. A range of other symptoms may develop, depending on the host species and the virus and may
be used to distinguish virus infection.
and the virus and may be used to distinguish virus infection One of the more obvious effects of viruses
is their influence on the structure and pigmentation of chloroplasts. This occurs even though the
replication of the virus occurs principally in the cytoplasm of the cell. The types of effects observed
include the production of vesicles, reduction of the number of grana, reduction in the number of
chloroplast ribosomes, reduction in ribulose bisphosphate carboxylase which is involved in the
conversion of carbon dioxide to organic carbon, reduction of starch accumulation and transport,
modification of the photosynthetic pathway, aggregation or fragmentation of chloroplasts and changes
in the chlorophyll and carotene content. The results of these changes to the chloroplasts become
obvious first in the upper leaves of the plant. A typical sequence of symptoms in affected leaves is
vein clearing, vein yellowing, vein banding and the development of a persistent mosaic (patchy
variation in colour).

Changes in other organelles are not as obvious, although mitochondria may develop vesicles and
degenerate and the nucleolus may disintegrate. As the virus spreads systemically through the plant
these changes occur in the infected cells so that there are major changes in the whole plant. Overall,
the main metabolic changes detected in infected plants are an increase in respiration, a decline in
photosynthetic carbon fixation and a resultant decline in net assimilation rate. Growth substances vary
in amount and their levels may be correlated with changes in growth rate.

Changes at the cellular level may lead to changes in tissues. In general, these changes may be
classified as either necrosis (tissue death), hypoplasia (reduced growth as shown by smaller leaf areas,
thinner laminae or stunting) or hyperplasia (distortion or production of outgrowths such as galls or
enations). On leaves, symptoms may include yellowing, reddening, chlorotic striations, mottling,
ringspots, interveinal chlorosis, rolli.ng or distortion of the lamina, hypoplasia and the production of
galls or enations. Floral symptoms include colour 'break' in the corolla and reduction in flower size. In
trunks or stems, stem-pitting, stemgrooving, bark-peeling, inhibition of lignification or gall formation
may occur. Fruit dappling (mottling), discolouration, size reduction or distortion may reduce fruit
quality.

Some viruses are confined to specific tissues, but may induce general poor performance or gall-
formation, yellowing or leafrolling in other parts of the plant. For example, the luteoviruses (so named
because some viruses in the group induce leaf-yellowing symptoms) replicate specifically in the
parenchyma cells and companion cells associated with the phloem. These viruses induce changes in
the phloem such as necrosis of sieve tubes, thickening of the cell wails and accumulation of
carbohvdrate.
The productivity of virus-infected plants may be reduced by virus-induced hormonal changes, reduced
availability of photoslmthates, direct effects on the chloroplasts, reduced translocation of starch,
reduced stomatal opening, less effective leaf posture, reduced leaf initiation, reduced fixation of
nitrogen in legumes, reduced nutrient uptake, epinasty (downward bending of the petiole) and leaf
rolling, increased senescence and premature abscission of leaves, or tumour, gall or enation
production. In addition to lost productivity, there are adverse effects on the quality of produce, the
competitive ability of the plant in a community such as a pasture or crop and the longevity of the plant.

Some viruses are lethal. One example of a lethal virus disease is 'quick decline' of citrus trees
propagated on sour orange rootstocks, in which cytopathic effects of the citrus tristeza virus on the
rootstock cause rapid death of the scion. Another is coconut foliar decay virus infection of certain
coconut varieties in Vanuatu which is associated with damage to the phloem, frond necrosis and death
of palms.

Mixed virus infections are common in vegetatively propagated plants, and historically the 'running out'
of potato varieties and the decline in productivity of strawberry crops, are good examples of the effect
of cumulative infections with a number of viruses on plant performance. Mixed infections may result
in only an additive effect of the combined viruses, but there are many examples of synergism, where
mixed infections produce a more severe disease than expected from the additive effects. Co-infection
of tobacco with potato virus Y caused up to a 10-fold increase in the amount of potato virus X and this
was correlated with the development of very severe symptoms. However, interference with virus
multiplication typically occurs between strains of the same virus and this 'cross protection' response
forms the basis of one means of reducing the severity of losses due to virus infection.

Factors affecting symptom expression

The environment affects the susceptibility of plants to infection and the expression of s1'rnptoms in
plants. For example, shading plants before inoculation can significantly increase their susceptibility to
mechanical inoculation. The time of day when inoculation is carried out can also affect the number of
successful inoculations obtained. Sometimes high light intensity and long days may favour virus
replication, but high light intensity may also lead to the induction of milder symptoms than at low light
intensity. Drought reduces replication and symptom severity, but this effect has been little studied.
Nutritional conditions favourable for plant growth generally also favour virus replication. Temperature
has a major effect on virus replication. Up to about 32'C the speed of virus replication generally
increases with increasing temperature and the time between inoculation and producilon of first
symptoms decreases correspondingly. Temperatures above about 33'C inhibit replication. As plants
age they become less susceptible to virus infection. The developmental stage is also important as
susceptibility of experimental hosts has been found to be low or nil after flowering has commenced.

Transmission of viruses

The virus life-cycle includes a phase within the protoplast of the host, during which replication,
maturation of virions and translocation takes place, and a phase outside the host when the intact virion
moves to a new host. The transmission of viruses to new hosts is an essential part of the cycle of
disease, and usually involves a vector, or carrier, of the virion to a new host. Unlike fungi and bacteria,
viruses are not transmitted by wind and water. The need for a vector complicates the ecology of plant
viruses and the study of the movement of plant viruses between plants involves a knowledge of the
web of interactions which exist between the plant, the virus, the vector and the environment.

Direct transmission in living higher plant material includes the transmission of viruses in seeds,
pollen and vegetative propagation material. When viruses are transferred from one generation of plants
to another only by vegetative or sexual propagation, the virus is retained within a cultivar or species.
This mode of spread as a circle indicating the limited ability of such viruses to move outside the
species. Nevertheless, it is known that pollen containing infectious virus particles can be transferred to
other species and inoculated mechanically to leaves sometimes via insect feeding damage. A number
of seed-borne viruses are also transmissible by specific nematode species and insects. Direct
transmission is used experimentally by virologists to transfer viruses from one plant to another. It can
be achieved in several ways. Grafting (including budding) is the most effective way to demonstrate
that a transmissible agent is the cause of a disease, and many viruses and virus-like endoparasites can
be transmitted in this way even if they are not mechanically transmissible using a sap extract.
Approach grafting in which two independent self-sustaining plants are grafted together has been used
to transfer viruses between different species. Under nursery and field conditions, graft propagation and
rootgrafting are both known to spread viruses of dicotyledonous plants. Graft propagation is not
possible with monocot5rledonous plants, so experimental demonstration of transmissibility depends
on other methods. The parasitic plant, dodder (Cuscuta spp.) will transfer a number of viruses when
haustoria are allowed to develop on adjacent plants.

Mechanical transmission is a common method for transferring viruses between plants

experimentally. It normally involves rubbing the leaf epidermis with infective sap, virus particles or
virus nucleic acid and a fine abrasive powder such as carborundum to create wounds through which
the virus may enter the leaf. Not all viruses are mechanically transmissible, particularly those that are
limited to the phloem or other specific tissues. Despite the ease of mechanical transmission under
experimental conditions, transfer of virus from one host to another without the intervention of a vector
is not common in nature. It is only known to be important for very stable viruses such as tobacco
mosaic virus and potato virus X. These viruses can contaminate structures, tools and soil debris, and
wounding of a host plant allowing contact of the tissue with a source of virus can lead to infection.

Vectors can transmit viruses across plant species barriers, and their activity and type of association
with the virus determines the range, rate, timing and pattern of spread of the viruses they transmit.
This mode of transmission is described as non-persistent, and is typical of transmission of many
viruses by aphid vectors. The virus is associated with ducts within the vector's stylet and most viruses
transmitted in this way have a number of different vector species. Vectors remain infective for up to
about 4 hours. A variant of this type of transmission is the semi-persistent transmission found in some
leafhopper-virus and aphid-virus systems in which infectivity is retained for up to about 1OO hours.
Alternatively, the vector may have a much longer feeding time followed by a period during which it is
unable to infect plants on which it feeds. This is described as persistent transmission because the
vectors typically retain the ability to transmit virus for the rest of their lives.

Identification of viruses

Disease symptoms caused by viruses are frequently similar to the symptoms associated with nutrient
deficiencies or excesses, or to chemical toxicities associated with agricultural chemicals such as
herbicides. Plant pathologists have two ways to implicate a virus as the probable cause of a disorder.
The first way is to map the distribution of the affected plants. Viruses which are vectorborne
frequently show patterns either with distinct foci, or random distributions, depending on how far the
vector has travelled or how much plant to plant spread has occurred within the crop. Rarely are all
plants affected in a site. In contrast, abiotic nutrient or chemically induced diseases generally show a
pattern associated with soil type or the application of chemicals where all plants show symptoms. It is
only when the incidence of a virus disease approaches 1OO0/o that it is difficult to determine whether
a virus is the cause of a disease. For example, severe cucumber mosaic virus epidemics in narrow
leafed lupin crops cause almost all plants to be stunted and infertile as might be expected after a
herbicide application. The second method is to demonstrate transmissibility of .the s1'rnptom either by
graft or mechanical sap inoculation to plants of the same and related species under experimental
conditions. This is the first step in applying Koch's postulates for establishing the cause of disease.

Virologists rely on good insect-free greenhouse and associated facilities for diagnosis by infectivity
assays, host range studies, vector studies and the propagation of plants so that virus concentrations
reach sufficiently high levels for either purification or component analysis. Herbaceous indicator
plants which express different symptoms when inoculated with different viruses assist virus
identification. In the laboratory, routine diagnostic methods based on serology and electron
microscopy are now being supplemented by component analysis in which nucleic acids unique to an
infected plant may be isolated and sequenced for use as a probe and for placing the virus in a
taxonomic group.

Classification and nomenclature

The International Committee on Taxonomy of Viruses (ICTV) controls virus classification and
nomenclature under the auspices of the Virologg Division of the International Union of
Microbiological Societies. The ICTV recognises about 3,600 virus species in plants, animals and
bacteria and it has been estimated that about 9OO of these are plant viruses. A database on plant
viruses MIDE: Virus Identification Data Exchange) is now available on the Internet at the Australian
National University address (http:/ /biologr.anu.edu.aulGroups/MES-/vide). The ICTV aims to set up a
universal virus database.

The properties used to classify viruses are the morphology of the virion, its chemical and physical
properties including the characteristics of its nucleic acids, proteins, lipids and carbohydrates, its
overall pattern for transcribing and translating information on the genome into viral proteins, its
antigenic properties and its biological characteristics such as host range and methods of transmission.

A number of earlier systems used to classify viruses are now obsolete. Viruses are currently classified
in a hierarchical system in which each taxonomic grouping possesses a majority of a set of characters.
The levels (and their suffixes) adopted in the hierarchy are

Order (-virales)

Family (-viridae)

Subfamily (-virinae)

Genus (-virus)
Species (synonymous with the term 'virus')

Species can be distinguished by genome properties, structure, physicochemical and serological

properties. Biological properties are usually not sufficiently stable for them to be used for defining a
species. The names given to species of plant viruses are vernacular and are usually based on the host
first reported with the disease and the type of symptom observed e.g. cucumber mosaic, lettuce
necrotic yellows and tomato spotted wilt viruses. The acronyms (CMV, LNYV and TSWV,
respectively) of these viruses have been standardised and each is unique within the plant viruses.
Stable variants of a species with distinct biological properties such as type of vector, symptoms
induced in plants or host range are referred to as strains of the virus and are distinguished by a letter or
number after the species name (e.g. cucumber mosaic virus strain Q is a distinct strain originating
from Queensland).

Viruses resembling each other in genome properlies and virion morpholory are grouped into genera
and given genus names. Genus names are often, but not always, derived from the name of the type
genus. For example, the genus Ctrcumouittts includes ail the viruses closely related to cucumber
mosaic virus including the serologically and biologically distinct tomato asperrny virus which has
essentially the same genome organisation and virion morphology. The family level of classification is
used to group similar genera. Viruses in the genera Cucumovirus, Bromovirus,Ilarvirus and
Alfamovirus all have positive sense ssRNA that occurs in three segments and are placed in the family
Bromoviridae. The hierarchical structure above the level of family or genus is not yet well developed.
Only one order, the Mononegavirales, which includes the family Rhabdoviridae, is recognised.

Viruses may be referred to by their full generic name e.g. potato Y potyvirus, tobacco necrosis
necrovirus, cucumber mosaic cucumovirus, but the name of the genus is generally omitted e.g. potato
Y virus, tobacco necrosis virus, cucumber mosaic virus, respectively.
 CHAPTER THREE

Viroids as agents of disease

A group of diseases resembling those caused by viruses are now known to be caused by agents called
viroids. The first to be recognised was potato spindle tuber viroid which was found because the
infective particle associated with it sedimented much more slowly than expected for a virus and was
therefore much smaller than a virus particle. All viroids are small, circular, single-stranded, naked,
infectious RNAs, which do not act as mRNAs, but which nevertheless induce diseases ranging from
stunting to distortion and death. They have been found only in plants. They range in size from 246 to
about 380 nucleotides. This partial double-strandedness is thought to make the molecule more resistant
to degradation than if the molecule existed in uiuo as a single-stranded RNA molecule. Viroids are
isolated from plants as either circles or broken circles (linear molecules) both of which are infectious.
They are most readily recognised in plants by separating the nucleic acid components using
polyacrylamide gel electrophoresis and observing the viroids as specific bands in the region between
the transfer RNA and the smallest of the ribosomal RNAs of the host plant. Sensitive stains such as
silver are routinely used for these gels, but a number of variations of these methods are available.

About 30 viroid diseases are known and the associated viroids have been sequenced for most of these.
Despite their differences in sequences, certain domains have been recognised as common to all of
them. The central conserved domain has been used as the basis for grouping viroids, but their
taxonomy is still under study.

The mode of replication of viroids seems to involve host coded polymerase enzyrnes. Replication by a
rolling circle mechanism has been proposed and this is based on the detection in infected tissue of
linear multimers of the complementary strand of the viroid RNA. The pathogenic changes leading to
symptoms are not understood. It is likely that viroids act as regulatory molecules and exert their
pathogenic effects by altering cell nucleic acid metabolism. Pathogenicity can vary dramatically as a
result of two or three nucleotide changes (mutations) suggesting that disease induction involves
specific recognition of the viroid sequence by some host molecule. A simplistic distinction may be
made between viruses and viroids. Viruses can be defined as parasites of cellular translation (a step in
protein synthesis) systems whereas viroids are probably parasites of cellular transcription (synthesis of
RNA from a DNA template). No viroids have been found outside the angiosperrns and they have no
similarities to the prions which are associated with animal diseases such as scrapie and Creutzfeld-
Jakob disease.
Examples of viroid diseases include potato spindle tuber, citrus exocortis, avocado sunblotch,
grapevine yellow speckle, tomato apical stunt, coconut cadang-cadang and tinangaja, apple scar skin,
hop stunt and chrysanthemum stunt. The coconut diseases are the only lethal viroid diseases known.
Viroids can be latent in some hosts and only recognised when transferred to a sensitive indicator plant.
Hop latent viroid has a worldwide distribution but is symptomless in hops. It is likely that many
viroids are present in nature, but their detection will probably rely on the use of molecular diagnosis
rather than biological methods. These methods have already detected viroid complexes in citrus and
grapevine, for example, and sequence analysis has shown that recombination between viroids
occurring in the same host is the probable explanation for the origin of some new viroids. Citrus
exocortis viroid has been proposed as a possible dwarfing agent for citrus, as it has no effect on the
fruit of sweet orange scion varieties, but it reduces growth of some rootstock varieties.

Cytopathic changes associated with viroid infection include alterations to the cell wall, plasmalemma,
chloroplasts and cellular proteins. The nucleus and nucleolus accumulate viroid in some systems,
whereas the chloroplast accumulates it in others. Viroids are translocated in plants but the method is
thought to differ from that of viruses because no viroid coded movement protein is likely to occur in
the plant. Most viroids are spread via vegetative propagation but they are also readily transmitted by
mechanical inoculation and citrus exocortis is reported to spread during hedging operations in citrus
groves. Some are pollen and seed-borne. Insect transmission is generally not thought to be important.
Coconut cadang-cadang viroid can be transmitted by a high pressure injection procedure to young
coconut seedlings, but this method does not work for mature palms. The natural mode of spread of this
important disease is not known. No transmission has been demonstrated by the use of contaminated
tools.

Diagnostic procedures for viroids are based on detecting viroid-like molecules in plants. The
properties of size and circularity can be identified most readily by gel electrophoretic procedures.
Viroids change from structured partially basepaired rods to open unstructured circles as a result of
denaturation of the internal hydrogen bonds. Procedures such as 'return' and '2-dimensional' gel
electrophoresis use this property to detect and isolate viroids. The unique nucleotide sequences of
viroids can be exploited for diagnosis by hybridisation and polymerase chain reaction (PCR) based
assays.

Control of virus and viroid diseases

Annual losses due to viruses and viroids are thought to be of the order of US$60 billion per year
worldwide. They are regarded as being second only to the fungi in importance as plant pathogens. The
types of losses vary. For example, in perennial crops, each diseased plant can take years to replace.
Where infection is chronic, as in vegetatively propagated crops, mild non-apparent infections may
reach a high incidence and cause constant losses of about lO-2Oo/o a year. In annual crops, losses
vary from year to year, depending on how severely disease develops. The most appropriate means of
controlling viruses and viroids depend on the type of loss, but the principles are the same for both
groups of pathogens. Accurate diagnosis is the key to the control of viruses and viroids because
control strategies depend on knowing the biology and epidemiology of the pathogen. Symptoms are
unreliable for diagnosis and specific diagnostic tests are needed. Control measures can be broadly
defined as direct or indirect. Direct control involves removal of infected material, 'cure' of infection or
host resistance to the disease while in indirect control measures, an attempt is made to interfere with
the disease cycle.

Direct control measures

Disease will not develop if the crop is immune to virus infection or is free from virus when planted
and there is no source of infection in the field or near enough to allow virus to spread to the crop.

Resistant cultivars provide one of the cheapest and most effective solutions to the problem of virus
diseases. Resistant cultivars suppress or retard the multiplication of a virus or the development of
pathogenic symptoms so plants show little or no sign of disease and virus replication within the plant
is reduced or negligible. Some plants prevent the virus from infecting. For example, some lines of
tobacco are not infected by cucumber mosaic, tobacco mosaic and tobacco necrosis viruses. The
underlying mechanism is not fully understood but is thought to be related to the thickness of the leaf
cuticle and the nature of the epidermal hairs. Some host cultivars are readily infected by virus, but the
virus cannot replicate as readily as in other cultivars. In other cultivars, the systemic spread of the
virus may be limited. Maize breeders have used this property to breed cultivars resistant to maize
dwarf mosaic virus. The net effect is that the plant is resistant to disease. Attempts have been made to
develop cultivars resistant to the major virus diseases.

More recently, virus-derived genes such as the coat or movement protein genes have been transformed
into plants by genetic engineering procedures to inhibit virus replication in infected plants, giving rise
to what is termed transgenic resistance.
Resistance should not be confused with tolerance. Some cultivars can be infected by viruses, but show
negligible or mild symptoms. Virus tolerance is heritable and has been used to breed citrus cultivars
that show few symptoms even though infected with tristeza virus. Similarly, barley cultivars have been
developed that are tolerant of infection with barley yellow dwarf virus.

Crops can be kept free from virus by preventing the entry of material containing virus. This is
achieved by imposing quarantine restrictions which may prevent the entry of infected material into a
country, state, region or even an individual farm. Planting material known to be infected by virus can
be freed of infection in a number of ways. Seeds free from infection can be obtained if a seed crop can
be grown in an area where the disease does not occur. Lettuce mosaic virus has been controlled
through the use of virus-free seed. If the virus is carried on the outside of the seed as tomato mosaic
virus is. the seed can be treated with hydrochloric acid, trisodium phosphate or sodium hypochlorite to
inactivate the virus. Heat therapy can be used to remove viruses from infected tissues. Heat therapy
often reduces the amount of virus in infected tissues, but rarely totally removes the virus infection.
New growth produced while plants are grown at 35- 39'C for several weeks is sometimes virus-free
and can be removed for separate propagation. This technique works because replication of most, but
not all, viruses is inhibited above 33'C. Potato viruses A and Y and potato spindle tuber viroid can be
eliminated from infected potatoes by storage at 5-15'C followed by meristem-tip culture, probably due
to reduced replication.

Plants can also be freed from virus infection by interfering with virus replication biochemically in a
process often referred to as chemotherapy. Some antibiotics inhibit either translation or transcription in
virus-infected cells. Analogues which replace nucleotides with spurious nucleotides can inhibit virus
replication. One of these, virazole, which blocks purine ring synthesis has been used in association
with meristem culture to obtain virus-free explants from virus-infected plants. Generally, the
phytotoxicity of inhibitory chemicals precludes their use except in specialised applications.

Once plant material is free from viruses, it can be protected from reinfection by maintenance in tissue
culture or by culturing in insect-free glass or gauze houses. Living hosts of the virus such as weeds or
ornamental or crop plants should be removed and plant remains that harbour the virus also should be
destroyed to ensure that there is no source of inoculum to reinfect virus-free plants.

Indirect control measures

Indirect control measures are directed at breaking the disease cycle so that infection does not continue.
It may be possible to avoid a disease by modifying cropping procedures. In tropical and subtropical
climates, sequential planting of crops to ensure continuous production also means that there is always
a source of inoculum for a growing crop. Overlapping onion and shallot crops often cause outbreaks of
onion yellow dwarf virus in New Zealand. The disease cycle can be broken by introducing a crop-free
period. This may be difficult to do where crops such as taro (Colocasia escuLenta) are grown all year
round. Alternatively, disease can be avoided by growing the crop in areas where the disease does not
occur. Similarly, it may be possible to grow a crop in an area free from vectors that may carry the
disease from plant to plant. These practices are often used in the production of disease-free planting
material.

Airborne virus vectors may be avoided by changing the sowing or transplanting time of the crop. Late
planting will avoid early vector flights and vice versa. However, the effects of changing planting date
on yield must be taken into consideration. For example, virus infection may be reduced by early
planting, but yields may become uneconomic. Changing plant density may also alter virus infection,
with less infection occurring at higher densities. However, competition between plants may lower
yields.

Vectors must also be controlled. This may involve the use of chemicals to reduce the vector
population below the level at which damage occurs or the use of a variety of non-chemical barriers to
limit spread by airborne vectors. In general, insecticides are most effective against viruses that are
carried in a persistent manner in their vector. Vectors that transmit in a non-persistent manner will die
eventually after feeding on a plant sprayed with a systemic insecticide, but may transmit the virus in
the mean time. In fact, the spray may agitate the insect so that it moves around and probes more plants
than normal leading to a higher transmission rate than expected. The costs and environmental effects
of spraying must also be considered. Vectors of non-persistent viruses can be prevented from
transmitting by spraying plants with paraffin or mineral oils. The oil does not directly inactivate the
virus, but may interfere with transmission as the vector's stylet probes the leaf. However, the oils may
be phytotoxic and it may be difficult to keep a continuous coverage on plants.

Soil may be treated by steaming, fumigation or solarisation to kill vectors and keep the production
system virus-free.

Barriers may be effective in preventing the spread of aphid-borne viruses. The barriers may be other
crops or they may be physical barriers. For example, tall growing maize crops protect lower-growing
crops such as cucurbits. Sticky yellow polythene strips distributed around crops or on the windward
side of crops reduce the incidence of potato virus Y and cucumber mosaic virus in capsicums because
the aphids are attracted to the strips and get caught on the sticky surface. Coarse white nets suspended
over crops will reduce infection by reducing the chances of flying vectors detecting host plants.

Reflective mulches laid on the ground around crop plants are effective in controlling aphid-borne
viruses. The mulches are thought to act as a repellent by reflecting UV light which discourages the
aphids from landing. Mulches tend to be expensive and their effectiveness decreases as plants grow
and cover the mulch with their leaves. Straw mulches successfully control the whitefly transmitted
tomato yellow leaf curl virus in tomato crops in Israel. It is believed the yellow colour of the straw
attracted the whiteflies which were killed by heat reflected from the mulch. Straw eventually loses its
effectiveness because its colour fades, but yellow plastic mulches may be as effective and longer-
lasting.

Plants can be protected from infection by a severe strain of a virus by preinoculation with a mild strain
of the same virus. The interference between strains of the same virus is termed 'cross protection' and
when the phenomenon is used to control a virus disease, it is termed 'mild strain protection'. Satellite
RNAs can modify the symptoms induced by their helper virus by making the disease either more
severe or less severe. When disease amelioration is induced by a satellite RNA, it is possible to
introduce it by inoculation with a mild strain of the virus containing the satellite or to transform the
plant with the gene of the satellite so that it will be activated when the plant is infected with the
pathogenic helper virus. This phenomenon has been demonstrated for cucumber mosaic and tobacco
ring spot viruses and the effectiveness of this type of protection has been demonstrated with cucumber
mosaic virus infection of tomatoes in Italy and the United States.
 CHAPTER FOUR

CONCLUSION

Prions are indeed responsible for transmissible and inherited disorders of protein conformation. They
also cause sporadic disease, in which neither transmission between individuals nor inheritance is
evident. Moreover, there are hints that the prions causing the diseases explored thus far may not be the
only ones. Prions made of rather different proteins may contribute to other neurodegenerative diseases
that are quite prevalent in humans.

Ongoing research may also help determine whether prions consisting of other proteins play a part in
more common neurodegenerative conditions, including Alzheimer's disease, Parkinson's disease and
amyotrophic lateral sclerosis. There are some marked similarities in all these disorders. As is true of
the known prion diseases, the more widespread ills mostly occur sporadically but sometimes "run" in
families. All are also usually diseases of middle to later life and are marked by similar pathology:
neurons degenerate, protein deposits can accumulate as plaques, and glial cells (which support and
nourish nerve cells) grow larger in reaction to damage to neurons. In spite of the amount and quality of
the work that has been carried out to prove that an abnormal prion protein is the sole cause of prion
diseases and the acceptance of this theory by the majority of the scientific community, the prion
hypothesis still faces some crucial problems.

Many scientists argue that a definitive proof that prions could cause disease by themselves is still
lacking and that an associated factor such as a virion cannot be ruled out. There are also several other
newly emerging diseases that represent a risk to human or animal health that should also be given
similar attention. While this is highly desirable in order to protect man and animals from these
emerging diseases, older maladies like tuberculosis, malaria, bilharziasis, sleeping sickness and river
blindness, which are devastating millions of people and their animals in many parts of the world,
should not be overlooked.
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