Introduction to Virology

Pathogenesis of Viral Diseases

DR. MELANIE JANE A. TENDENCIA August 3, 2011

Microorganisms bacteria, protozoans & worms, fungi

- either single cells or composed of many cells

Cells capable of independent replication

- can synthesize their own energy & proteins - can be seen in the light microscope Viruses are not cells - not capable of independent replication - cannot synthesize own energy & proteins - too small to be seen in the light microscope

VIRAL COMPONENTS
Capsid protein shell, or coat, that encloses the nucleic acid genome Capsomeres - morphologic units seen in the electron microscope on the surface of icosahedral virus particles - represent clusters of polypeptides Defective virus a virus particle that is functionally deficient in some aspects of replication Envelope lipid-containing membrane that surrounds some virus particles - acquired during viral maturation by a budding process through a cellular membrane Peplomers virus-encoded glycoproteins or projections exposed on the surface of the envelope Nucleocapsid the protein-nucleic acid complex representing the packaged form of the viral genome Structural units The basic protein building block of the coat - also called protomer Subunit a single folded viral polypeptide chain Virion the complete virus particle; serve to transfer the viral nucleic acid from one cell to another

Characteristics of Viruses
1) Viruses - particles composed of an internal core containing

either DNA or RNA (but not both) covered by a

protein coat. Some viruses have an outer lipoprotein membrane, called an envelope, external to the coat.

- do not have a nucleus, cytoplasm, mitochondria, or

ribosomes

Cells both prokaryotic & eukaryotic have both DNA & RNA = eukaryotic cells have nucleus, cytoplasm, mitochondria & ribosomes = prokaryotes not divided into nucleus & cytoplasm - (-) mitochondria - (+) ribosomes can synthesize own proteins

2) Viruses must reproduce (replicate) w/in cells, because they cannot generate energy or synthesize proteins obligate intracellular parasites
- vs. chlamydiae & rickettsiae cannot synthesize own energy to replicate independently

3) Viruses replicate in a manner different from that of cells - they do not undergo binary fission or mitosis - One virus can replicate to produce hundreds of progeny viruses, whereas one cell divides to produce only two daughter cells

Comparison of Viruses & Cells

Property

Viruses

Cells

Type of nucleic acid Proteins

DNA or RNA but not both Few

DNA and RNA Many Cell membrane in all Present Present in eukaryotes Many

Lipoprotein membrane Envelope present in some Ribosomes Mitochondria Enzymes Absent Absent None or few

Multiplication by binary
fission or mitosis No Yes

Classification of Viruses Basis of classification 1.Virion morphology size, shape, type of symmetry, presence or absence of peplomers, & presence or absence of membranes 2.Virus genome properties type of nucleic acid (DNA or RNA), size of genome in kilobases or kilobase pairs, strandedness (single or double), whether linear or circular, sense (positive, negative or ambisense), segments (number, size), nucleotide sequence 3.Physicochemical properties of the virion molecular mass, buoyant density, pH stability, thermal stability & susceptibility to physical & chemical agents, esp. ether & detergents 4. Virus protein properties number, size & functional activities of structural & non-structural proteins, amino acid sequence, modifications, & special functional activities.

5. Genome organization & replication, including gene order, number & position of open reading frames, strategy of replication & cellular sites (virion assembly & release) 6. Antigenic properties 7. Biologic properties natural host range, mode of transmission, vectors relationships, pathogenicity, tissue tropisms & pathology Families - -viridae (virion morphology, genome structure, strategies of replication) , Herpesviridae, Paramyxoviridae Genera - - virus (physicochemical & serologic differences), Herpesvirus, Paramyxovirus

DNA- Containing Viruses

Parvoviruses

Polyomaviruses
Papillomaviruses

Adenoviruses
Hepadnaviruses

Herpesviruses
Poxviruses

Parvoviruses very small, 18-26 nm - cubic symmetry, 32 capsomeres, no envelope - Human parvovirus B 19 replicates in immature erythroid cells & causes several adverse consequences aplastic crisis, fifth disease, & fetal death Polyyoma viruses small, 45 nnm, nonenveloped, cubic symmetry, 72 caps - JC virus progressive multifocal leukoencephalopathy - BK virus nephropathy in transplant patients Papillomaviruses 55 nm - wart viruses - certain genotypes cause genital cancers Adenoviruses medium sized, 70-90 nm, nonenveloped, cubic, 252 caps - acute respiratory diseases, conjunctivitis, gastroenteritis

Hepadnaviruses small, 40-48 nm, enveloped - acute & chronic hepatitis; persistent infections associated w/ a high risk of developing cancer Herpesviruses 150-200 nm, cubic , 162 capsomeres, lipid-cont envelope - herpes simplex types 1 and 2 (oral and genital lesions) - varicella-zoster virus (chicken pox & shingles) - cytomegalovirus, Epstein-Barr virus (inf mononucleosis), human herpesviruses 6 &7 (T lymphotropic) & human herpesvirus 8 (Kaposi sarcoma) Poxviruses large brick-shaped or ovoid, 220-450 nm (L) x 140260 nm (W) x 140-260 nm thick - all poxviruses tend to produce skin lesions - smallpox, vaccinia, molluscum contagiosum

RNA-Containing Viruses Picornaviruses Arenavirus Astroviruses Coronaviruses Caliciviruses Retroviruses Hepeviruses Orthomyxoviruses Reoviruses Bunyaviruses Arboviruses Bornaviruses Togaviruses Filoviruses Paramyxoviruses Rhabdoviruses Flaviviruses

Viroids

Prions

Picornaviruses small, 28-30 nm, cubic - enteroviruses (polioviruses, coxsackieviruses, echoviruses - rhinoviruses common colds - hepatovirus hepatitis A Reoviruses medium sized, 60-80 nm - rotaviruses gastoenteritis Arboviruses have a complex cycle involving arthropods as vectors transmit the viruses to vertebrate hosts by their bite - dengue, yellow fever, encephalitis viruses - not a virus family, ecologic grouping Coronaviruses - 120-160 nm particles, enveloped - SARS Retroviruses spherical, enveloped, 80-110 nm in diameter - the viron contains a reverse transcriptase enzyme that produces a DNA copy of the RNA genome HIV AIDS

Structure
Size & Shape

= 20 to 300 nm in diameter = sphere, rod, bullet, bricks

= complex structures of precise geometric symmetry

= shape of virus particles - determined by the arrangement of the repeating subunits that form the protein

coat, capsid

HIV Influenza

Phage O29 Smallpox

Phage P22

Filamentous Phage Coronavirus

T4 Phage

Adenovirus

Viral Nucleic Acids = viral nucleic acid (genome) located internally - can either be single- or double-stranded DNA or single or double-stranded RNA - the NA can either be linear or circular

DNA always a single molecule

RNA can exist either as a single molecule or in several pieces Almost all viruses contain only a single copy of their genome

haploid
Exception: Retrovirus family members have two copies of their RNA genome diploid

Capsid & Symmetry

Capsid a protein coat which surrounds the nucleic acid - made up of subunits called capsomers - each capsomer consist of one or several proteins E/M spherical particle, w/ a central hole - the arrangement of capsomers gives the virus structure its geometric symmetry 2 forms of symmetry in viral capsids: 1. icosahedral (cubic) capsomers are arranged in 20 triangles that form a symmetric figure (icosahedron) w/ the approx outline of a sphere

2. helical capsomers are arranged in a hollow coil that appears rod-shaped

The helix can be either rigid or flexible.

Both the icosahedral & the helical forms can exist either as a naked nucleocapsid or with an outer envelope layer

Viral Proteins
Functions: 1. The outer capsid proteins protect the genetic material & mediate the attachment of the virus to specific receptors on the host cell surface. This interaction of the viral proteins w/the cell receptor is the major determinant of species & organ specificity. 2. Outer viral proteins are also important antigens that induce neutralizing antibody & activate cytotoxic T cells to kill virus-infected cells. = also the target of antibodies (antibodies bind to these viral proteins & prevent (neutralize) the virus from entering the cell & replicating. (after both natural infection & immunization)

3.

internal viral proteins = some are structural (capsid proteins of enveloped viruses)
= Enzymes (polymerases that synthesize the viral mRNA) = Superantigens produced by some viruses, similar in their action to the superantigens of bacteria - herpesvirus family Epstein-Barr virus & CMV - retrovirus mouse mammary tumor virus - activation of CD4+ T cells is required for replication of these viruses to occur

Envelope = a lipoprotein membrane composed of lipid derived from

the host cell membrane & protein that is virus-specific

= glycoproteins - found in the spike-like projections on the surface, which attach to host cell receptors during the entry of the virus into the cell = matrix proteins mediates the interaction between the capsid proteins & the envelope

In general, the presence of an envelope confers instability on the virus

Enveloped virus are more sensitive to heat , detergents, & & lipid solvents such as alcohol & ether than are nonenveloped (nucleocapsid) viruses, w/c are composed only of NA & capsid proteins

The surface proteins of the virus, the capsid proteins or the envelope glycoproteins the principal antigens against w/c the host mounts its immune response to viruses. They are also the determinants of type specificity
(serotype)

Ex. Poliovirus types 1, 2, 3 distinguished by the antigenicity of their capsid proteins

Atypical viruslike agents 1. Defective NA & proteins but cannot replicate without a helper virus, w/c provides the missing function 2. Pseudovirions contain host cell DNA instead of viral DNA within the capsid; can infect cells but they dont replicate 3. Viroids consists solely of a single molecule of circular RNA without a protein coat or envelope 4. Prions infectious particles that are composed solely of proteins; (-) NA = implicated as the cause of certain slow diseases called transmissible spongiform encephalopathies - Creutzfeldt-Jacob disease humans - BSE mad cow disease - scrapie - sheep

Replication
Viral Growth Curve

= shows the amount of virus produced at different times after infection

= presented in a typical growth curve the amount of virus produced is plotted on a logarithmic scale as a function of time after infection. = the time required for the growth cycle varies it is minutes for some bacterial viruses and hours for some human viruses

First event the disappearance of the virus

(solid line dropping to the x axis)

- although the virus particle is no longer present , the viral NA continues to function & begins to accumulate within the cell (dotted line) Eclipse period the time during which no virus is found inside the cell - ends with the appearance of virus (solid line)

Latent period the time from the onset of infection to the appearance of
the virus extracellularly - toward the end of this period, there is alteration of cell morphology & marked derangement of cellular function Infection begins with one virus particle & ends with several hundred virus particles having been produced

Stages of the viral growth cycle

Attachment & penetration by parental virion

Uncoating of the viral genome - viral DNA in the nucleus

Early transcription synthesis of early mRNA Early translation - synthesis of early proteins

Viral genome replication

Late viral mRNA synthesis late viral protein synthesis Progeny virion assembly Virion release from cell

Viral Life Cycle

Overview - A virus needs to replicate & create progeny

- A virus cannot live on its own

- Active only when replicating within a host using a hosts resources & food - Inside a host a virus sole purpose is to make as many copies of itself & infect other host cells

- A viral life cycle is dependent on a host cell - A virus will remain dormant until it is able to infect the next host, activate & replicate
- Viruses use the most efficient method to locate a

host, create progeny, & spread to other hosts

Viral infection occurs when a virus enters a host: * through a physical breach (cut in the skin) * direct inoculation (mosquito bite) * direct infection of the surface itself (inhalation of the virus onto trachea) It is only after a virus enters a host that it can gain access to possible susceptible cells

Viral entry Virus must enter cells of the host organism in order for it to reproduce & establish infection use the cells materials Proteins found on the surface of the virus interact with proteins of the cell

- Attachment or adsorption occurs between the viral particle & the host cell membrane A hole forms in the cell membrane then the virus particle or its genetic contents are released into the host cell in the host cell viral reproduction may commence

Viral replication A virus must take control of the hosts replication mechanisms A distinction between susceptibility & permissibility of a host cell is made Permissibility determines the outcome of the infection After control is established & the environment is set for the virus to begin making copies of itself, replication occurs quickly

Viral Shedding -After a virus has made many copies of itself exhaust the cell of its resources -Cell is no longer useful to the virus must find new host

Shedding the process by which virus progeny are

released to find new hosts

- final stage in the viral life cycle

Viral latency
- Virus hide within another cell - evade the host cell defense or immune system

- it is not the best interest of the virus to continually

replicate -hiding latency = virus will not produce any progeny = will remain inactive until external stimuli (light or

stress) prompts it into activation

Viral Entry

- the earliest stage of the infection in the viral life cycle

- virus comes into contact with the host cell &

introduces viral material into the cell

Major steps:
1. Attachment or adsorption 2. Membrane fusion or hemifusion state 3. Entry pore formation 4. Viral Penetration

Attachment or adsorption receptors on the viral envelope become connected to complementary receptors on the cell membrane - this attachment causes the two membranes to remain in mutual proximity, favoring further interactions between surface points - this is also the first requisite that must be satisfied before a cell can become infected makes the cell susceptible

- enveloped viruses exhibiting this: HIV, Herpes simplex virus, influenza virus
- non-enveloped viruses: bacteriophages or phages virus that infect bacteria - they have long tails on which to attach to receptors on the bacterial surface

Membrane fusion or hemifusion state the cell membrane is punctured & made to further connect with the unfolding viral envelope Entry pore formation an opening is established for the stabilization of an opening for which viral particles can enter Viral penetration viral capsid or genome is injected into the host cells cytoplasm

Entry via Membrane Fusion - viral receptors attach to the receptors on the surface of the cell & secondary receptors may be present to initiate the puncture of the cell membrane or fusion with the host cell followed by the unfolding of the viral envelope

- the virus envelope blends with the cell membrane releasing its contents
- can be done only with viruses that contain an envelope - HIV, Herpes simplex, influenza virus

Entry via Endocytosis

- the virus tricks the cell into thinking that the virus knocking at the door is nothing more than nutrition or harmless goods - a cell takes in resources from the environment attach goods into surface receptors

engulf them into the cell inside a vacuole

- inside the cell the virus breaks out of the vacuole to gain access to the cytoplasm

- Examples: poliovirus, Hepatitis C virus, Foot-andmouth disease virus

Entry via Genetic Injection

- virus simply attaches to the surface of the cell via receptors on the cell, & inject only its gene into the cell, leaving the rest of the virus on the surface - restricted to viruses in which only the gene is required for infection of a cell (most all positive-sense, single-stranded RNA viruses - Example: phages

- Once a virus is in a cell will activate formation of proteins to gain full control of the host cell - Control mechanisms include: - suppression of intrinsic cell defenses

- suppression of cell signalling

- suppression of host cellular transcription & translation cytotoxic effects lead to the death & decline of a cell infected by a virus After the introduction of the viral particle unpacking of the viral proteins & the viral genome via some form of nucleic acid occurs as preparation for viral replication

Viral Replication

- Formation of biological viruses during the infection process in the target host cells -Purpose: to allow production & survival of its own kind = by generating abundant copies of its genome & packaging these copies into viruses to be able to continue infecting new hosts - Replication between viruses is greatly varied & depends on the type of genes involved

Once a virus has entered a target cell it must replicate its genome & proteins Replication strategies used by single stranded RNA-containing viruses depend on whether the genome can be used as messenger RNA (mRNA) Translation-competent genomes (alphaviruses, flavivruses, & picornaviruses) are termed plus sense or (+) sense are translated by cellular ribosomes immediately after the entry of the genome into the cytoplasm Genome replication of (+) sense RNA-containing viruses requires the synthesis of a minus sense or (-) sense , RNA intermediat w/c acts as template for the production of (+) sense genomic RNA

Retroviruses are RNA-containing viruses that replicate using a DNA intermediate

= viral genomic RNA is (+) sense & single-stranded -however does not serve as m-RNA = The retrovirus RNA genome is the template for the synthesis of a double- stranded DNA copy, termed as the provirus

- synthesis of the provirus is mediated by a

virus- encoded RNA-dependent DNA polymerase or reverse transcriptase so named because of the reversal of genetic information from RNA to DNA - provirus is translocated to the nucleus & is integrated to the host chromosomal DNA

Class 1: Double stranded DNA viruses

- must enter the host nucleus before it its able to replicate - requires host cell polymerases to replicate its genome highly dependent on the cell cycle - Proper infection & production of progeny requires that the cell be in replication as that is when the cells polymerases are active - virus may induce the cell to forcefully undergo cell division chronically this may lead to transformation of the cell cancer - Example: Adenoviridae - Poxvirus family class I virus which does not replicate within the nucleus. (e.g. smallpox virus)

Class 2: Single stranded DNA viruses

- replicate within the nucleus & form a double stranded DNA intermediate during replication - Circoviridae, Parvoviridae
Class 3: Double stranded RNA viruses - replicates in the cytoplasm as w/ most RNA viruses

- do not use the host replication polymerases to as much degree as DNA viruses
- Reoviridae, Birnaviridae - replication is monocistronic & includes individual, segmented genomes each of the genes code for only one protein

Class 4 & 5: Single stranded RNA viruses - two types = replication is primarily in the cytoplasm = replication is not as dependent on the cell cycle as other DNA viruses Class 4: Single stranded RNA viruses positive (+) sense

- can be directly accessed by host polymerases to immediately form proteins two groups:
> viruses where the genome RNA forms the mRNA & is translated into a polyprotein product that is subsequently cleaved to form the mature proteins. > viruses with complex transcription, for which subgenomic mRNAs, ribosomal frameshifting & proteolytic processing of polyproteins may be used - Examples: Coronaviridae, Flaviviridae, Picornaviridae

Class 5: Single stranded RNA viruses Negative (-) sense

- cannot be directly accessed by host polymerases to immediately form proteins. Instead they must be transcripted by viral polymerases into a readable form, which is the positive sense reciprocal two groups:
> viruses containing non segmented genomes for which the first step in replication is transcription from the (-) stranded genome by the viral RNA-dependent RNA polymerase to yield monocistronic mRNAs that code for the various viral proteins. A (+) sense genome copy is then produced that serves as template for the production of the (-) strand genome. Replication is within the cytoplasm > viruses with segmented genomes for which replication occurs in the nucleus & for which the viral RNA- dependent RNA polymerase produces monocistronic m RNAs from each genome segment.

- Examples: Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Filoviridae & Rhabdoviridae

Class 6: Positive (+) sense single stranded RNA viruses that replicate through a DNA intermediate - use reverse transcriptase to convert the positive sense RNA into DNA - use DNA to create templates of proteins (instead of using RNA), which is spliced into the host genome using integrase replication can then commence with the help of the host cells polymerases = Example: HIV

Class 7: Double-stranded DNA viruses that replicate through a single stranded RNA intermediate
- viruses have a double-stranded, gapped genome that is filled in to form a covalently closed circle (ccc DNA) that serves a a template for production of viral mRNAs & a subgenomic RNA. The pregenomic RNA serves as template for the viral reverse transcriptase & for production of DNA genome = Example: Hepatitis B virus

Viral Shedding

- refers to the successful production of virus progeny & that the progeny is leaving the cell to infect other host cells - shedding from a single cell, from one part of the body to another part, and shedding from bodies into the environment where the viruses may infect other bodies

= via budding

= via apoptosis
= via reverse endocytosis

Via budding - budding through the cell envelope - use the cells membrane for the virus itself most effective for viruses that need an envelope - Prior to budding, the virus may put its own receptor on to the surface of the cell in preparation for the virus to bud through forming an envelope with the viral receptors already on it - This process will slowly use up the cell membrane & eventually lead to the demise of the cell - this is also how antiviral responses are able to detect virus

infected cells

= Examples: HSV, SARS, smallpox

Via apoptosis

- cell suicide release of progeny into the extracellular space

- usually controlled results in the cells genome being chopped up, before apoptotic bodies of dead cell material clump off the cell to be absorbed by macrophages. - way for a virus to get into macrophages either to infect them or simply travel to other tissues in the body - primarily used by non-enveloped viruses but enveloped viruses may also use this = HIV enveloped virus that exhibits this process for the infection of macrophages

Via reverse endocytosis

- viral progeny are synthesized within the cell & the host cells transport system is used to enclose vacuoles of virus progeny for release into the extracellular space - used primarily by non-enveloped viruses, although enveloped viruses may do this too

= Example: use of recycling viral particles in the enveloped Varicella-Zoster virus

Viral latency

- the ability of a pathogenic virus to lie dormant within a cell, denoted as the lysogenic part of the viral life cycle - a phase in certain viruses life cycles in which after initial infection, virus production ceases, however the the viral genome is nor fully eradicated

- the virus can reactivate & begin producing large amounts of viral progeny without the host being infected by new outside virus stays within the host indefinitely
- Mechanisms: episomal, proviral

Episomal latency

- refers to the use of genetic episomes during latency

- viral genes are floating in the cytoplasm or nucleus as distinct objects - more vulnerable to marauding ribozymes or host foreign gene degradation than provirus latency - Herpesviridae Herpes simplex virus undergoes episomal latency in neuron cells & leaves genetic material floating in the cytoplasm - Advantages: > the virus does not need to enter the nucleus & hence may avoid ND10 domains from activating interferon via that pathway > far easier to maintain & reactivate than proviral latency - Disadvantages: more exposure to cellular defenses leading to possible degadation of viral gene via cellular enzymes

Proviral latency

- begins when the virus genome integrates into the host genome becomes a provirus

- requires that the viral gene get into the nucleus & insert itself into the host genome
- Example: Retroviruses HIV the nucleus inserts its gene between Long Terminal Repeats using integrase & remains within the host own gene - Advantages: automatic host cell division results in replication of the viruses gene - Disadvantages: include the need to enter the nucleus & increased difficulty in maintaining the latency

Antiviral Agents 1.Nucleoside Analogs inhibit NA replication by inhibition of polymerases for NA replication - acyclovir, lamivudine, zidovudine 2.Nucleotide attached phosphate group - cidofovir 3. Nonnucleoside Reverse transcriptase inhibitor - binds directly to reverse transcriptase, disrupt catalysis - nevirapine 4. Protease inhibitors - saquinavir 5. Fusion inhibitor - blocks the virus & cellular membrane fusion step 6. Others Amantadine & rimantadine - foscarnet INTERFERON host-coded proteins that are members of the large cytokine family which inhibit viral replication

Viral Vaccines

Killed-Virus Vaccines Attenuated Live-virus vaccines

Table 30-9