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Page1 JOSHUA ALBERT & SONU S.S 2014-2015 VIROIDS AND PRIONS Project submitted in partial fulfillment of the requirements for the central board of senior secondary school class XI in biology" INDIAN SCHOOL DARSAIT, MUSCAT

Viroids and prions

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JOSH

UA A

LBER

T & S

ONU

S.S

20

14

-201

5

VIR

OID

S A

ND

PR

ION

S

“Project submitted in partial fulfillment of the requirements for the central

board of senior secondary school class

XI in biology"

INDIAN SCHOOL DARSAIT,

MUSCAT

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Acknowledgement At the outset, we would like to express our sincere gratitude to our school, for providing us with such an opportunity to showcase our 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 the Almighty for giving us the strength and endurance to undertaken this project

and execute it to the best of our abilities.

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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

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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

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LIST OF TABLES

Sl. No Description Page No.

1 Differences Between Bacteria & Viruses 7

2 Comparison of Cows’ Brain 16

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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

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ABSTRACT

Viruses are too small to be seen with a light microscope and cannot be cultured outside their hosts. Therefore, although viral diseases are not new, the viruses themselves could not be studied until the twentieth century. In 1886, the Dutch chemist Adolf Mayer showed that tobacco mosaic disease (TMD) was transmissible from a diseased plant to a 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 disease associated with a filterable agent was yellow fever. 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 became a concern in 2006, when it killed up to 90% 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 the United States since then.

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.

Unlike viral nucleic acids,

Figure 1: Dmitri Iwanowsky

Figure 2 : Tobacco mosaic disease

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Viroids are not encapsulated. Despite their small size, Viroids replicate autonomously in cells of susceptible plant species. Known Viroids are single-stranded, covalently closed, circular, as well as linear, RNA molecules with extensive regions of intramolecular complementarily; 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. So far, Viroids have been identified only as pathogens of higher plants, but it is likely that certain animal (including human) diseases are caused 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, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease 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.

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INTRODUCTION

One hundred years ago, researchers could not imagine submicroscopic particles, and thus they described the infectious agent as contagium vivum fluidum—a contagious fluid. By the 1930s, scientists had begun using the word virus, the Latin word for poison, to describe these filterable agents. The nature of viruses, however, remained elusive until 1935, when Wendell Stanley, an American chemist, isolated tobacco mosaic virus, making it possible for the first time to carry out chemical and structural studies on a purified virus. At about the same time, the invention of the electron microscope made it possible to see viruses. Life can be defined as a complex set of processes resulting from the actions of proteins specified by nucleic acids. The nucleic acids of living cells are in action all the time. Because viruses are inert outside living host cells, in this sense they are not considered to be living organisms. However, once viruses enter a host cell, the viral nucleic acids become active, and viral multiplication results. In this sense, viruses are alive when they multiply in the host cells they infect. From a clinical point of view, viruses can be considered alive because they cause infection and disease, just as pathogenic bacteria, fungi and 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 & Viruses Viruses 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.

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VIROIDS - the Plant Invaders A Viroid is a virus (VIR) like (OID) particle. Viroids are “sub-viruses” composed exclusively of a single circular strand of nucleic acid (RNA) that codes for a single protein or small, circular RNA molecules without a protein coat.

No coding capacity - do not program their own polymerase.

Use host-encoded polymerase for replication.

Mechanically transmitted; often seed transmitted.

More than 40 viroid species and many variants have been 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 type of sub viral infectious agent, in that prions are made only of protein lacking nucleic acid.

DID YOU KNOW?

VIROIDS are

Very small, covalently closed, circular RNA molecules capable of autonomous replication and induction of disease.

Range in size from approximately 20 nm.

Are infectious particles. Cause certain 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 (coleus blumei 1)

Genus Avsunviroids:

ASBVd (avocado sunblotch)

Genus Pelamoviroids:

PLMVd (peach latent mosaic)

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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

demonstrates 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

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Diseases Caused by Viroids - Potato Spindle Tuber Disease

Of the many diseases caused by Viroids, the spindle tuber disease of potatoes was the first to be recognized and studied by plant pathologists. Nearly 50 years were to elapse between initial description of this disease in the early 1920's and the identification of its causal agent, a small, highly-structured, covalently closed circular RNA molecule known as Potato spindle tuber viroid (PSTVd). PSTVd remains an important pathogen of potato and tomato, and a recent increase in the number of reported latent infections of ornamental species is creating new challenges for current disease management strategies. PSTVd is also a favorite object of study for viroid molecular biologists, thanks in large part to its ability to replicate to high titers in tomato where certain strains rapidly induce the appearance of a characteristic disease syndrome that includes stunting and epinasty.

Symptoms and Signs The natural host range of PSTVd includes many solanaceous species. The viroid may cause disease in Solanum tuberosum (potato), S. lycopersicum (syn. Lycopersicon esculentum, tomato), and Capsicum annuum (pepper) where symptoms may vary considerably depending on plant species, variety, viroid strain and environmental conditions. Infections in other hosts are symptomless; e.g., Brugmansia spp., Datura sp., Lycianthes rantonneti (syn. S. rantonneti),Persea americana (avocado), Physalis peruviana (Cape gooseberry), S. jasminoides, S. muricatum(pepino), and Streptosolen jamesonii.

DISEASE Potato spindle tuber

PATHOGEN Potato spindle tuber viroid

HOSTS potato, tomato, ornamentals (Solanaceae)

Figure 5:- Potato Spindle Tuber Disease

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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 produce smaller leaves than their healthy counterparts. Infected tubers may be small, elongated (from which the disease derives its name), misshapen, and cracked. Their eyes may be more pronounced than normal and may be borne on knob-like protuberances that may even develop into small tubers.

The first symptoms of PSTVd infection in tomato (Figure 2) are growth reduction and chlorosis in the top of the plant. Subsequently, this growth reduction may develop into stunting, and the chlorosis may become more severe, turning into reddening and/or purpling. In this stage, leaves may become brittle. Generally, this stunting is permanent; occasionally, however, plants may either die or partially recover. As stunting begins, flower and fruit initiation stop.

Disease Transmission PSTVd can be transmitted in four different ways:

Vegetative propagation- Propagation by tubers, cuttings, and micro-plants provides a very efficient means of viroid transmission. Once established, PSTVd infection is persistent;

therefore, plants from infected lots act as a permanent source of inoculum for other lots and crops. Vegetative propagation has been the major pathway for PSTVd transmission in potato and ornamentals such as Brugmansia spp. and S. jasminoides. The absence of

symptoms increases the risk that infected plants will be used for propagation Mechanical transmission- Under favorable conditions, PSTVd is readily transmitted by normal cultivation activities. This is most clearly seen with potatoes and tomatoes, where viroid spread is mainly along the row Infected seed and pollen- PSTVd is assumed to have spread among potato germplasm collections all over the world via infected true seed. Once present in a germ bank, the viroid can be transmitted to other (wild) potato plants either mechanically or

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by pollen exchange. Seed is also a potential source of infection for other crops such as tomato and pepper that are propagated by seed

Aphid transmission- This route of transmission requires the source plant to be infected by both Potato leafroll virus (PLRV) and PSTVd, thereby limiting the number of potential infection sources. PSTVd is assumed to be encapsidated by the viral coat protein; such encapsidation protects the viroid from digestion by micrococcal nuclease in vitro, suggesting that a similar protective effect may occur in vivo.

Disease Management Disease management can be divided into two parts: prevention of infection and viroid eradication.

Prevention of infection includes all measures to prevent the introduction of PSTVd into a specific crop. It is very important to start a new cultivation with viroid-free planting material (tubers, seeds or plants). PSTVd is considered a quarantine 'organism' in many countries, and therefore, governmental measures to prevent introduction of PSTVd with plants from other countries will often be applied. Certification schemes including testing may be required to provide further guarantees that the planting material is free from PSTVd.

In addition to the use of healthy planting material, it is also important to prevent viroid introduction via human activities. Because PSTVd is mechanically transmissible, it can be introduced into potential host plants via the hands, clothes, or equipment used by people working in or visiting the greenhouse. The use of disposable gloves and specific clothing and equipment that stays inside a greenhouse compartment may prevent PSTVd introduction into greenhouse-grown crops. Furthermore, increasing the number of plants species grown in a greenhouse compartment increases the risk of introduction of PSTVd. Growers should either grow a single crop or they should separate different crops and lots, preferably in different compartments. Because PSTVd can be transmitted by aphids, planting PLRV-free seed potatoes and controlling aphid populations also contributes to the management of PSTVd in potato crops.

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Viroid eradication is based on destruction of PSTVd-infected plants and thorough cleaning of equipment and greenhouses where infected plants have been grown. All infected plants together with those from an adequate buffer zone should be destroyed. In case of field-grown potatoes, crop rotations involving non-PSTVd host species help eliminate infected volunteer plants. In case of symptomless infections such as those commonly 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 the greenhouse and destroyed. Ideally, all material slated for disposal should be transported in a closed container to an incinerator; alternatively, such material can be taken to a refuse dump and covered with a layer of soil.

When PSTVd is identified in a greenhouse-grown crop, all parts of the greenhouse should be thoroughly cleaned, preferably using a steam cleaner and a scrub brush for parts that are difficult to clean (Figure 7). A regular acid treatment can be used for watering tubes and drippers. After cleaning the greenhouse and associated equipment, application of a disinfectant completes the eradication procedure. When cultivation of crops susceptible to PSTVd infection resumes, extra monitoring for PSTVd symptoms and/or testing are advisable.

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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 the words 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 contrast to all other known infectious agents, like viruses, bacteria, fungi, or parasites—all of which must contain nucleic acids (either DNA, RNA, or both). Prions are responsible for mammalian transmissible spongiform encephalopathies, including bovine spongiform encephalopathy(BSE, also known as "mad cow disease") and scrapie in sheep. In humans, prions cause Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann–Sträussler–Scheinker syndrome, Fatal Familial Insomnia and kuru. All known prion diseases in mammals affect the structure of the brain or other neural tissue and all are currently untreatable and universally fatal. In 2013, a study revealed that 1 in 2,000 people in the United Kingdom might harbour the infectious prion protein that causes vCJD.

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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 the disease affects cows, "spongiform" refers to the way the brain from a sick cow looks spongy under a microscope, and "encephalopathy" indicates that it is a disease of the brain. BSE is commonly called “mad cow disease.” BSE is a progressive neurologic disease of cows. Progressive means that it gets worse over time. Neurologic means that it damages a cow’s central nervous system .

Most scientists think that BSE is caused by a protein called a prion. For reasons that are not completely understood, the normal prion protein changes into an abnormal prion protein that is harmful. The body of a sick cow does not even know the abnormal prion is there. Without knowing it is there, the cow’s body cannot fight off the disease.

Symptoms of BSE A common symptom of BSE is incoordination. A sick cow has trouble walking 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 the abnormal prion to when it first shows symptoms of BSE. This is called the incubation period. During the incubation period, there is no way to tell that a cow has BSE by looking at it. Once a cow starts to show symptoms, it gets sicker and sicker until it dies, usually within two weeks to six months. There is no treatment for BSE and no vaccine to prevent it.

Currently, there is 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 microscope and seeing the spongy appearance. Scientists can also tell if a cow had BSE by using test kits that can detect the abnormal prion in the brain.

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Brain from a healthy cow, as seen under a microscope using special stains.

Brain from a cow sick with BSE, as seen under a microscope using special stains. This brain is sponge-like, and the large white spaces are like the "holes" of a sponge.

Disease Transmission The parts of a cow that are not eaten by people are cooked, dried, ground into a powder, and used for many purposes, including as ingredients in animal feed. A cow gets BSE by eating feed contaminated with parts that came from another cow that was sick with BSE. The contaminated feed contains the abnormal prion, and a cow becomes infected with the abnormal prion when it eats the feed. If a cow gets BSE, it most likely ate the contaminated feed during its first year of life. Remember, if a cow becomes infected with the abnormal prion when it is one year old, it usually will not show signs of BSE until it is five years old or older.

Table 2:- Comparison of cow brain

brain tissues

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Prevention and control

The measures in the strategy for dealing with BSE are early detection and warning systems and prevention and rapid response measures and mechanisms in place.

Targeted surveillance of occurrences of clinical neurological disease; Awareness programs to enhance surveillance; Screening tests at routine slaughter; Transparency in reporting findings of BSE; Safeguards on importation of live ruminant species and their products, in

accordance with the OIE Terrestrial Code; Removal of specified risk material (SRM) (brain, spinal column) during slaughter

and processing of carcasses; Prohibit the inclusion of SRM in animal feeds, thus removing potentially

contaminated material from the food chain; Humane destruction of all suspected and susceptible animals exposed to

contaminated feed (cohorts); Appropriate disposal of carcasses and all animal by-products; Livestock identification to enable effective surveillance and 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/ucm136222.htm

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