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Medical Virology Introduction to Basics

Dr.T.V.Rao MD

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History Virology • Smallpox was endemic in China by 1000BC. In response, the practice of variolation was developed. Recognizing that survivors of smallpox outbreaks were protected from subsequent infection, variolation involved inhalation of the dried crusts from smallpox lesions like snuff, or in later modifications, inoculation of the pus from a lesion into a scratch on the forearm of a child. Dr.T.V.Rao MD

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Virus infections are Universal …….

Introduction to Virology • A virus is an obligate intracellular parasite containing genetic material surrounded by protein

• Virus particles can only be observed by an electron microscope 4

Introduction to Virology • Recognizing the shape, size, and structure of different viruses is critical to the study of disease – Viruses have an inner core of nucleic acid surrounded by protein coat known as an envelope – Most viruses range in sizes from 20 – 250 nanometers 5

Viral Properties • Viruses are inert (nucleoprotein ) filterable Agents • Viruses are obligate intracellular parasites • Viruses cannot make energy or proteins independent of a host cell • Viral genome are RNA or DNA but not both. • Viruses have a naked capsid or envelope with attached proteins • Viruses do not have the genetic capability to multiply by division. • Viruses are non-living entities 6

Viruses are Ultramicroscopic

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The size of viruses

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VIRAL STRUCTURE – SOME TERMINOLOGY

• virus particle = virion • protein which coats the genome = capsid • capsid usually symmetrical • capsid + genome = nucleocapsid • may have an envelope 9

Virion • The complete infectious unit of virus particle • Structurally mature, extracellular virus particles. 10

Virion envelope

Capsid

Viral core

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Virion Structure Lipid Envelope

Nucleic Acid

Protein Capsid Virion Associated Polymerase

Spike Projections

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Distinguishing characteristics of viruses

• Obligate intracellular parasites • Extreme genetic simplicity • Contain DNA or RNA • Replication involves disassembly and reassembly • Replicate by "one-step growth” 13

How are viruses named? • Based on: - the disease they cause poliovirus, rabies virus

- the type of disease murine leukemia virus

- geographic locations Sendai virus, Coxsackie virus

- their discovers Epstein-Barr virus

- how they were originally thought to be contracted dengue virus (“evil spirit”), influenza virus (the “influence” of bad

air)

- combinations of the above Rous Sarcoma virus

Virus particle = virion

White, DO and Fenner, FJ. Medical Virology, 4th Ed. 1994

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5 BASIC TYPES OF VIRAL STRUCTURE icosahedral nucleocapsid

nucleocapsid

lipid bilayer

ICOSAHEDRAL

ENVELOPED ICOSAHEDRAL

helical nucleocapsid

COMPLEX nucleocapsid

lipid bilayer glycoprotein spikes = peplomers

HELICAL

ENVELOPED HELICAL 16

Dr.T.V.Rao MD

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Icosahedral • Adeno-associated Virus (AAV) • Herpes Simplex Virus 1 (HHV1) Adenovirus Herpes Simplex Virus 2 (HHV2) Human Immunodeficiency Virus (HIV) B19 Human T-lymphotrophic Virus (HTLV) Coxsackievirus - A Norwalk Virus Coxsackievirus - B Papilloma Virus (HPV) Cytomegalovirus (CMV) Polio virus Eastern Equine Encephalitis Rhinovirus Rubella Virus Virus (EEEV) Saint Louis Encephalitis Virus Echovirus Varicella-Zoster Virus (HHV3) Epstein-Barr Virus (EBV) Western Equine Encephalitis Virus Hepatitis A Virus (HAV) (WEEV) Hepatitis B Virus (HBV) Yellow Fever Virus Hepatitis C Virus (HCV) Hepatitis Delta Virus (HDV) Hepatitis E Virus (HEV) 19

Viral Structure • Varies in size, shape and symmetry • VIP for classification • 3 types of capsid symmetry: – Cubic

(icosahedral)

• Has 20 faces, each an equilateral triangle. Eg. adenovirus

– Helical • Protein binds around DNA/RNA in a helical fashion eg. Coronavirus

– Complex • Is neither cubic nor helical eg. poxvirus

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The Baltimore classification system Based on genetic contents and replication strategies of viruses. According to the Baltimore classification, viruses are divided into the following seven classes: 1. dsDNA viruses

2. ssDNA viruses 3. dsRNA viruses 4. (+) sense ssRNA viruses (codes 5. (-) sense ssRNA viruses 6. RNA reverse transcribing viruses 7. DNA reverse transcribing viruses

directly for protein)

where "ds" represents "double strand" and "ss" denotes "single strand". 21

Virus Classification I - the Baltimore classification

• All viruses must produce mRNA, or (+) sense RNA • A complementary strand of nucleic acid is (–) sense • The Baltimore classification has + RNA as its central point • Its principles are fundamental to an understanding of virus classification and genome replication, but it is rarely used as a classification system in its own right

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From Principles of Virology Flint et al ASM Press 23

Virus classification II the Classical system • This is a based on three principles -

– 1) that we are classifying the virus itself, not the host – 2) the nucleic acid genome – 3) the shared physical properties of the infectious agent (e.g capsid symmetry, dimensions, lipid envelope) 24

Virus classification III the genomic system • More recently a precise ordering of viruses within and between families is possible based on DNA/RNA sequence • By the year 2000 there were over 4000 viruses of plants, animals and bacteria - in 71 families, 9 subfamilies and 164 genera

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Viral Structure - Overview Nucleic acid Nucleocapsid Capsid

Envelope protein Membrane protein Spike protein

Viral envelope**

Fig 1. Schematic overview of the structure of animal viruses ** does not exist in all viruses

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Icosahedral capsids

a) Crystallographic structure of a simple icosahedral virus.

b) The axes of symmetry

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Cubic or icosahedral symmetry

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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Adenovirus

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Adenovirus

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Helical symmetry

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Helical • California Encephalitis Virus Coronavirus Hantavirus Influenza Virus (Flu Virus) Measles Virus ( Rubeola) Mumps Virus Para influenza Virus Rabies Virus Respiratory Syncytial Virus(RSV) 36

• Helical symmetry

How to assemble 37

Helical symmetry In 1955, Fraenkel, Conrat, and Williams demonstrated that tobacco mosaic virus (TMV) spontaneously formed when mixtures of purified coat protein and its genomic RNA were incubated together.

TMV, a filamentous virus

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Enveloped helical virus

Enveloped icosahedral virus

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Properties of naked viruses • • • • • • • •

Stable in hostile environment Not damaged by drying, acid, detergent, and heat Released by lysis of host cells Can sustain in dry environment Can infect the GI tract and survive the acid and bile Can spread easily via hands, dust, fomites, etc Can stay dry and still retain infectivity Neutralizing mucosal and systemic antibodies are needed to control the establishment of infection

Naked viruses( Non Enveloped ) • Adeno-associated Virus (AAV) Adenovirus B19 Coxsackievirus - A Coxsackievirus - B Echovirus Hepatitis A Virus (HAV) Hepatitis E Virus (HEV) Norwalk Virus

COMPLEX SYMMETRY surface view

White, DO and Fenner, FJ. Medical Virology, 4th Ed. 1994

cross section

POXVIRUS FAMILY

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ENVELOPE • OBTAINED BY BUDDING THROUGH A CELLULAR MEMBRANE (except poxviruses) • POSSIBILITY OF EXITING CELL WITHOUT KILLING IT • CONTAINS AT LEAST ONE VIRALLY CODED PROTEIN – ATTACHMENT PROTEIN

• LOSS OF ENVELOPE RESULTS IN LOSS OF INFECTIVITY 43

CLASSIFICATION NUCLEIC ACID

• RNA or DNA

• • • •

segmented or non-segmented linear or circular single-stranded or double-stranded if single-stranded RNA – is genome mRNA (+) sense or complementary to mRNA (-) sense 44

Genome • The genome of a virus can be either DNA or RNA • DNA-double stranded (ds): linear or circular Single stranded (ss) : linear or circular • RNA- ss:segmented or non-segmented ss:polarity+(sense) or polarity –(nonsense) ds: linear (only reovirus family) 45

DNA

double-stranded

line ar

circular

singl sing e le

RNA

single-stranded

line ar

circular

multip singl sing le e le

multip le

doublestranded

single-stranded

linear

linear (circular)*

singl e

multipl e

(+)sense

sing le

multip le

(-)sense

sing le

multip le 46

Viral genome strategies • • • • • • • •

dsDNA (herpes, papova, adeno, pox) •ssDNA (parvo) •dsRNA (reo, rota) •ssRNA (+) (picorna, toga, flavi, corona) •ssRNA (-) (rhabdo, paramyxo, orthomyxo, bunya, filo) •ssRNA (+/-) (arena, bunya) •ssRNA (+RTase) (retro, lenti) 47

DNA VIRUSES DOUBLE STRANDED

ENVELOPED

HERPESVIRIDAE HEPADNAVIRIDAE

NON-ENVELOPED

CIRCULAR

PAPILLOMAVIRIDAE POLYOMAVIRIDAE (formerly grouped together as the PAPOVAVIRIDAE)

SINGLE STRANDED NON-ENVELOPED

COMPLEX ENVELOPED

PARVOVIRIDAE

POXVIRIDAE

LINEAR

ADENOVIRIDAE

All families shown are icosahedral except for poxviruses

Modified from Volk et al., Essentials of Medical Microbiology, 4th Ed. 1991 48

DNA viruses

From Principles of Virology Flint et al ASM Press

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RNA VIRUSES SINGLE STRANDED positive sense

ENVELOPED

ICOSAHEDRAL

FLAVIVIRIDAE TOGAVIRIDAE RETROVIRIDAE

HELICAL

CORONAVIRIDAE

SINGLE STRANDED negative sense

DOUBLE STRANDED

NONENVELOPED

ENVELOPED

NONENVELOPED

ICOSAHEDRAL

HELICAL

ICOSAHEDRAL

PICORNAVIRIDAE CALICIVIRIDAE ASTROVIRIDAE

ORTHOMYXOVIRIDAE PARAMYXOVIRIDAE RHABDOVIRIDAE FILOVIRIDAE BUNYAVIRIDAE ARENAVIRIDAE

REOVIRIDAE

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RNA viruses

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BASIC STEPS IN VIRAL LIFE CYCLE • • • •

ADSORPTION PENETRATION UNCOATING AND ECLIPSE SYNTHESIS OF VIRAL NUCLEIC ACID AND PROTEIN • ASSEMBLY (maturation) • RELEASE 52

RECEPTOR

VIRUS

ICAM-1

polio

CD4

HIV

acetylcholine

rabies

EGF

vaccinia

CR2/CD21

EpsteinBarr herpes

HVEM

Sialic acid

Influenza, reo, corona

Virus Replication 1 Virus attachment 2 3

1

5 4 5 6

4

2 3

7 8

7 6

and entry Uncoating of virion Migration of genome nucleic acid to nucleus Transcription Genome replication Translation of virus mRNAs Virion assembly Release of new virus particles

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ADSORPTION

• TEMPERATURE INDEPENDENT • REQUIRES VIRAL ATTACHMENT PROTEIN • CELLULAR RECEPTORS 55

PENETRATION - ENVELOPED VIRUSES •FUSION WITH PLASMA MEMBRANE •ENTRY VIA ENDOSOMES

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PENETRATION

herpesviruses, paramyxoviruses, HIV 57

PENETRATION - ENVELOPED VIRUSES •FUSION WITH PLASMA MEMBRANE •ENTRY VIA ENDOSOMES, FUSION WITH ACIDIC ENDOSOME MEMBRANE

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VIRUS UPTAKE VIA ENDOSOMES

• CALLED –VIROPEXIS / ENDOCYTOSIS / PINOCYTOSIS

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PENETRATION NON-ENVELOPED VIRUSES entry directly across plasma membrane:

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Replicative cycle • As obligate intracellular parasites, Virus must enter and replicate in living cells in order to “reproduce” themselves. This “growth cycle” involves specific attachment of virus, penetration and uncoating, nucleic acid transcription, protein synthesis, maturation and assembly of the virions and their subsequent release from the cell by budding or lysis 64

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UNCOATING • NEED TO MAKE GENOME AVAILABLE • ONCE UNCOATING OCCURS, ENTER ECLIPSE PHASE • ECLIPSE PHASE LASTS UNTIL FIRST NEW VIRUS PARTICLE FORMED

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SYNTHESIS OF VIRAL NUCLEIC ACID AND PROTEIN • MANY STRATEGIES • NUCLEIC ACID MAY BE MADE IN NUCLEUS OR CYTOPLASM • PROTEIN SYNTHESIS IS ALWAYS IN THE CYTOPLASM 68

ASSEMBLY AND MATURATION • NUCLEUS • CYTOPLASM • AT MEMBRANE

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RELEASE • LYSIS • BUDDING THROUGH PLASMA MEMBRANE • NOT EVERY RELEASED VIRION IS INFECTIOUS

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Transmission of Viruses • Respiratory transmission – Influenza A virus • Faecal-oral transmission – Enterovirus • Blood-borne transmission – Hepatitis B virus • Sexual Transmission – HIV • Animal or insect vectors – Rabies virus 71

Viruses enter the body of the host in a variety of ways, for example...

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The commonest forms of transmission are via...

INHALED DROPLETS in sneezing of coughing for example the COMMON COLD or INFLUENZA VIRUSES. 73

or by

...

drinking water or eating raw food, for example, HEPATITIS A and POLIOVIRUS. 74

The commonest forms of transmission are also via...

sexual intercourse for example HIV and HEPATITIS B and... 75

also...

vertical transmission from mother to baby for example HIV, HEPATITIS B and RUBELLA... 76

also...

bites of vector arthropods such as mosquitoes for example YELLOW FEVER, RIFT VALLEY FEVER and DENGUE. 77

Most viral infections...

do not lead to such serious complications and the host... 78

get well after a period of sickness to be immune for the rest of their lives. Examples are MEASLES INFECTION, RUBELLA or German measles, MUMPS and many others...

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A bacteriophage • A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA ('ss-' or 'ds-' prefix denotes singlestrand or double-strand) along with either circular or linear arrangement. 80

Structure of Bacteriophage

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Classification of Bacteriophages • The dsDNA tailed phages, or Caudovirales, account for 95% of all the phages reported in the scientific literature, and possibly make up the majority of phages on the planet. However, other phages occur abundantly in the biosphere, with different virions, genomes and lifestyles. Phages are classified by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid. 82

Sub-viral agents • Satellites – – – – –

Contain nucleic acid Depend on co-infection with a helper virus May be encapsidated (satellite virus) Mostly in plants, can be human e.g. hepatitis delta virus If nucleic acid only = virusoid

• Viroids – Unencapsidated, small circular ssRNA molecules that replicate autonomously – Only in plants, e.g. potato spindle tuber viroid – Depend on host cell polII for replication, no protein or mRNA

• Prions – No nucleic acid – Infectious protein e.g. BSE

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Viroids & Prions • Viroids

– ss RNA genome and the smallest known pathogens. – Affects plants

• Prions – – – – –

Infectious particles that are entirely protein. No nucleic acid Highly heat resistant Animal disease that affects nervous tissue Affects nervous tissue and results in

• Bovine spongiform encepahltits (BSE) “mad cow disease”, • scrapie in sheep • kuru & Creutzfeld-Jakob Disease (CJD) in humans

Dr.T.V.Rao MD

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Viroids • Viroids are small (200-400nt), circular RNA molecules with a rodlike secondary structure which possess no capsid or envelope which are associated with certain plant diseases. Their replication strategy like that of viruses - they are obligate intracellular parasites.

Dependovirus /Virusoids • Viroids are small (200-400nt), circular RNA molecules with a rod-like secondary structure which possess no capsid or envelope which are associated with certain plant diseases. Their replication strategy like that of viruses - they are obligate intracellular parasites.

(Prions) • Prions are rather ill-defined infectious agents believed to consist of a single type of protein molecule with no nucleic acid component. Confusion arises from the fact that the prion protein & the gene which encodes it are also found in normal 'uninfected' cells. These agents are associated with diseases such as Creutzfeldt-Jakob disease in humans, scrapie in sheep & bovine spongiform encephalopathy (BSE) in cattle.

THANK YOU

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