Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15

IntroductionPoliomyelitis – one of the earliest evidences

- Egyptian priest- From hieroglyph

Rabies also led to the discovery of the virus when it could not be explained by the presence of bacteria.

1940s Electron Microscopy- start of study (chemical nature of virus)- infectivity mainly studied in animal models

General Structure of VirusVirus – diverse in size and structure

- Poliovirus – smallest virus (28 nm)- Mimivirus – largest virus (750 nm)- Capsid – aka coat

protein core w/c holds the viral nucleic acid possibly surrounded by lipidic envelope

- Viral Classification- based on morphology, structure, physical and chemical properties

Viral Nucleic AcidViral Genome- composed of either DNA or RNA

- Maybe: double or single stranded linear (Poliovirus) or circular (Hepa B virus) segmented (Influenza- 8 segments) or one molecule(Poliovirus)

*Virus that contains Plus-sense ss RNA can have their genome translated directly by the host ribosome

Retroviridae- ex. HIV- requires a specific virus-encoded enzyme (reverse transcriptase) to convert it’s ss RNA to ss

DNA, to be able to replicate within the host cell.- This enzyme is a primary target of antiviral drugs

Mulitplicity Reactivation- in poliovirus- Random damage to viral capsid and nucleic acid following treatment with Hypochlorite (a biocide

intensively used for surface disinfection) resulted in complementary reconstruction of an infectious particle by hybridization of gene pool of inactive virus.

Viral Capsid - protect the viral nucleic acid from detrimental physical and chemical conditions.

- for attachment to host cell Capsomere – subunit that makes up a viral capsid

- Gives the shape of the capsid- Provide the virus with resistance to chemical and physical agents- 3 different architectural styles:

»Icosahedral - have capsomeres in the form of Pentons & Hexons- Adenovirus- 240 hexons & 12 pentons- Poiliovirus- 20 hexons & 12 pentons

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15 »Helical symmetries- influenza and mumps virus

- Subunits symmetrically packed in helical array, like coils of wound rope under electron microscopy

- Core is hallow, viral nucleic acid is embedded into the ridges on the inside of each subunit

»Complex -mammalian virus; several percutaneous structure envelopes the viral genome core

Bacteriophages- aka bacteria virus- Shows a complex structure consisting of:

» Capsid head» Tail» Tail fibres

Viral Envelope- a lipidic envelope that surrounds the viral capsid

- originated from the host cell- added during replication or after excision of viral progeny from the host cell- may come from Cell Nuclear Membrane (Herpes simplex virus) or the Cytoplasmic Membrane

(influenza virus)- Enveloped Viruses are the most susceptible to chemical and physical conditions and do not survive

well on their own, although they can persist longer in inorganic soil (blood, exudates, feces)

Viral Receptors- glycoproteins protruding from the viral capsid or embedded in the envelope

- important for viral infectivity- recognizes the host cell receptor site conveying the viral specificity

Virus-host cell interaction

*Viruses are True intracellular parasites that grow within living cells and use their energy and synthetic machinery to produce viral components

Virus progeny/Virions- new virus from the replication of one virus within the host cell- can be released and infect adjacent cells-

Major viral groups based on host specificity:1. Viruses of bacteria & blue-green algae2. Plant viruses3. Animal(insects included) viruses

Rabies & Infulenza – can cause diseases in both animals and humans- these are exceptions because virus rarely cross species barriers due to their extreme specificity

Virus-Host cell interaction:1. Multiplication of virus and destruction of the host cell upon release of viral progeny2. Multiplication of the virus and release of the virions w/o the immediate destruction of the host cell3. Survival of the virus in a latent stage w/o noticeable changes in a dramatically altered or transformed

state4. Incorporation of the viral nucleic acid to the host cell genome w/o noticeable changes in the infected

cell

Asymptomatic Viral Infections- virus replicates w/in the host but doesn’t produce symptoms of a disease

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15

HIV- enveloped virus with a cone-shaped nucleoplasmid

- 2 copies of positive-sense ss RNA & enzyme reverse transcriptase- the viral genome encodes for the ff. genes:

»env, envelope proteins»gag, capsid proteins»pol, enzymes involves in viral multiplication»tat, enzymes regulating host metabolism

- 70 glycoprotein spikes (gp120) projects from the envelope & interacts specifically with the CD4 protein receptor on the T-lymphocyte

- the provirus may lie dormant or can produce viral mRNA (polycistronic) & proteins to resume the multiplication cycle producing virions

HAART Highly Activated Antiretroviral Therapy- antiviral txt, combining a protease inhibitor & 2 reverse transcriptase inhibitor

- reduce the number of HIV particles (1010 viral particles produced continuously per day) & slow the

progression of the disease by restoring & maintaining the number of T4 helper lymphocytes.

Tumour Virus- virus infected cells change dramatically, acquiring the characteristic of tumour cells exhibiting

uncontrolled growth.- Acquisition of viral genes by the host must be followed by other events as environmental or dietary

exposures to chemical carcinogen for cancer to occur

Multiplication of human viruses

Objective of replication cycle: to ensure the multiplication of the virus with the formation of the identical viral progenyMultiplication time of Human Virus is 4- more than 40 hours; Bacterial Virus takes only 20 minutes.

Six distinct phases of the replication cycle: 1. Attachement to the host cell

-Virus-cell Recognition Event- similar to any protein-protein interaction in that it occurs through a stereospecific network of hydrogen bonds and lipophilic association

2. Penetration of the viral particle- Direct Injection- only the naked DNA will enter the host cell

HIV core penetrates cell

cytoplasm

Membrane fusion

Uncoating, release of 2

RNA & reverse transcriptase

into cytoplasm

Reverse transcriptase

copies RNA itno ss DNA

ss DNA duplicated to form ds DNA (copy of the original viral

RNA genome)

DNA moves into host cell nucleus

DNA integrated as PROVIRUS

into a host cell chromosome

Initial contact mainly dependent on Brownian motion

Reversible phase during w/c electrostatic repulsion is reduced

Irreversible changes in virus-receptor-host-receptor configuration that initiated viral penetration through the cell membrane

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15 - Fusion- viral envelope will bind with host cell membrane, liberating the viral capsid w/in the

cell cytoplasm- Endocytosis- the whole virus will be engulfed by the host cell, forming a Cystolic Vacuole.

3. Uncoating of the viral particle- Viral nucleic acid needs to be released from the capsid- Penetration by Endocytosis: acidification of cystolic vacuole induces a conformational change in

the capsid & the release of viral nucelocapsid into the cytoplasm- Some like Reovirus only needs a partial uncoatingfor the expression of the viral genome

4. Replication of viral nucleic acids & translation of the genome- the host cell synthesis is taken over by the virus- the viral genome is replicated

- some viruses can encode for genes for the products of w/c regulate the synthetic process according to the needs of the virus

- host cell mRNA encodes directly for functional proteins, whereas viral mRNA is polycistronic, w/c means several distinct proteins are encoded w/in a single piece of mRNA. Thus, virus needs a virus-specific protease to cut the correct place

5. Maturation/Assembly of virions- Replicated viral genome and some viral proteins become packaged w/in the capsid- Most non-enveloped viruses accumulate in the cytoplasm or nucleus and are only released

during cell lyses.6. Release of the virions into the surrounding environment

- Mature virions are released from the host cell- Some viruses produce a lytic enzyme or protease to lyse the host enabling the release of

infectious particles- Enveloped viruses are released by budding process - Ultimately cell will die following damage to its metabolism and house keeping function during

viral replication

Cultivation of human viruses

» The study & ID of viruses depends on our ability to propagate them» Cultivation of many viruses enabling a better understanding of their replication properties, more rapid

diagnosis and the easier production of vaccine.Cell Culture

» Sources of cell samples: Humans, primates and rodents» Cell culture according to history

a. Primary- diploid cell line- Derived directly from an intact tissue (human embryo, kidney or monkey kidney)

b. Secondary- diploid cell line- Derived from primary culture, usually those arising from embryonic tissue- Limited number of subcultures can be performed w/ these cells generally up to a mmaximum of about 50 before the cells degenerate

c. Continuous Cell Line- usually derived from malignant tissue- has the capacity to multiply in vitro indefinitely

» In principle, cell culture for propagating viruses replies on the growth of cells in a Semiconfluent Monolayer attached to a surface

transcription of viral genes into viral mRNA translation of the viral genome into proteins replication of the viral genome

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15

»To subculture, the cells are separated from the monolayer w/ the use of Trypsin to form a suspension

of single cells. Growth temp of 37∘C»Bovine Albumin- serum to supplement a culture media»The established cell monolayer will support viral replication from w/c viruses can be harvested»Cytopathic Effect- characteristic morphological change in the infected cells, & usually indicates cell

death.- Cell shrinkage, ballooning, detachment of cells from the surface of the flask

The Chick Embryo – 9-11 days old fertile chicken eggs

- Used as a convenient cell system to grow human pathogenic viruses

Control of VirusAntiviral Chemotherapy- most are pro drugs that need to be activated within the cell (by Kinase & Other cellular enzymes)

- Role: slow or halt disease progression. Not cure.- target Viral enzymes: Protease; Polymerases; Reverse Transcriptase- Protease- particularly important for uncoating process preventing the release of viral

nucleocapsids- Neuraminidase Inhibitors- prevents the release of mature virions

HIVAntiretrovirals- antiviral drugs

- Aim to reduce HIV plasma levels for as much and as long as possible- Side effects:

Immune Recinstitution syndrome Lipodystrophy syndrome- includes:

fat redistribution insulin resistance; hyperglycaemia; dyslipidaemia

Liver damage Osteonecrosis GI Disturbance Anorexia Pancreatitis Dyspnoea Cough

Headache Insomnia Dizziness Fatigue Blood disorder Myalgia Arthralgia Rash Urticaria fever

- Protease Inhibitors- metabolized by Cytochrome P-450 & therefore have significant potential for drug Interaction

- Non-nucleoside Reverse Transcriptase Inhibitor- interact with a number of drugs metabolized in the liver

Side effects: Rash Psychiatric disturbance CNS disturbance

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15 Fatal hepatitis

Herpesvirus InfectionHerpesviridae- a family of viruses that include:

Herpes simplex virus Chickenpox (Varicella) Shingles (Herpes zoster) Cytomegalovirus

Mild herpes simples- txt with topical antiviral drug

Herpetic gingivostomatitis- txt with change of diet and analgesics

Systemic antivirals- given within 24 hrs onset of rashes, for adults infected w/ chickenpox- Given within 27hrs onset of rashes, for adults infected w/ shingles

Side effects of Herpes Antivirals:» Nausea» Vomiting» Stomach pain» Headache

» Fatigue» Rash» Increase in serum» Increase in urine uric acid

InfluenzaOseltamivir- Antiviral that was recommended for txt of influenza by National Institute & Clinical Excellence (NICE)

- extensively used for prevention and control of the swine flu outbreak in UK 2009- LIMITATIONS: Drug needs to be taken w/in a few hours of the onset of symptoms

Severe side effects for children and adolescents

Respiratory Syncytial Virus- responsible for severe bronchiolitis notably in infants

- Txt: Palivizumab (monoclonal antibody) & Ribavirin (antiviral drug), both associated with side effects

Vaccination- most successful measure against viral infections

- Contains antigens that elicit a specific and active immunity against an infecting agent- Can induce the innate and adaptive (cellular, humoral) parts of the immune system

Small pox vaccine- by Jenner- Inoculation of cowpox well before the “germ theory” of diseases were postulated

Most common methods of viral vaccine preparation:1. Use of Live attenuated viruses- will cause a strong immune response w/o causing the disease

- used in prep. for MMR vaccine and Poliomyelitis vaccine2. Use of inactivated viruses- Hepatitis A virus & Influenza vaccine3. Use of viral components

Hepatitis B virus vaccine- recombinant vaccine where the viral DNA encoding for a virus surface antigen (HBsAg) is

expressed in yeast (Saccharomyces cerevisiae) or mammalian cells (Chinese hamster ovary cells)

Human Papillomavirus (HPV)- contains virus-like particles & recombinant capsid protein expressed in yeast or using a

baculovirus as an expression system.

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15

Adjuvants in vaccines:»Aluminum Salts- antigens are absorbed to the aluminum salts»Monophosphoryl lipid A- increase or modulate host immunes response to the antigen

Human normal immunoglobulin (HNIG)- prepared from a pool of donated human plasma and contains immunoglobulin G (igG) and antibodies against viruses like:

» Hepa A» Measles» Mumps» Rubella» Varicella

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15

Intramuscular normal immunoglobulin- used to protect against hepa A in immunocompromised patients- can also be used for pregnant ladies against rubella virus

Disease-specific immunoglobulins- prepared from a pool of plasma obtained from a human donor who have high-levels of the specific antibody requiredDisease-specific Hepatitis B immunoglobulins- treating infants born from mothers infected with the virusDisease-specific Varicella Zoster immunoglobulins- treatment for individuals who are at a high risk such as:

a. neonates whose mothers developed chickenpoxb. for those exposed to the virus while requiring intensive care or proloned special carec. for immunocompromised individuals

Viricidal effects of chemicals & physical agents on viruses» Viruses are generally transmitted via surfaces & therefore the use of viricidal disinfectants on hard

surfaces & viricidal antiseptics on skin is important » Susceptibility to viricidal agents:

a. Small non-enveloped viruses are more resistant than large enveloped virusesb. Lipid-rich envelope being damaged easily by chemical & physical agents, large enveloped

virus» Biocidal activity depends upon a number of factors, such as:

a. Concentrationb. Contact timec. Presence of soilingd. Formulation

» Target site for biocide: a. Envelopeb. Glycoproteinc. Capsidd. Viral nucleic acid

» Highly reactive biocide that damages the capsid: a. Aldehyde – ex. Gulataldehydb. Oxidizing agents- ex. Peracetic acid, Hydrogen peroxide

- H 2O2 & Chlorine dioxide have been shown to penetrate within the capsid and

damage viral nucleic acid» Lesser reactive biocide that can only damage the capsid to a certain extent:

a. Quaternary ammonium compounds (QACs)b. Biguanides- ex. Chlorhexidine

» Viruses with a HELICAL CAPSID STRUCTURE are more susceptible since the destruction/alteration of capsid is more likely to cause damage to the viral nucleic acid w/c is closely associated to this type of structures

» More viruses are susceptible to exposure to temperatures above 60℃for 30 minutes

- used for inactivation of viral contaminants (HIV) in blood products» Viruses survive well at low temperatures and they can be routinely stored at -40℃ to -70℃

Control of Viruses in Pharmaceutical Products

» Factors that affect contamination of pharmaceutical products:

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15 a. origin of product componentb. history of donorc. amount of material usedd. manufacturing processe. capacity to remove/destroy and contaminate

» A risk assessment/stringent control are applied to samples containing a component from human or animal origin

» For preparation of viruses, the inactivation process must ensure that it does not affect antigenicity while killing the virus and other potential contaminants such as mycoplasmas

» Prep of Influenza Vaccine, the inactivation process must cause minimum alteration of the HAEMAGGLUTININ & NEURAMINIDASE antigens

Viruses and Gene Therapy

» certain viruses are used as vectors for the delivery of genes to targeted cells» Viruses used for Gene Transfer Medicinal Products:

a. (AAV) Adenovirusesb. poxvirusesc. retrovirusesd. lentivirusese. adeno-associated virusesf. herpesviruses

» Viral vectors- for human use are freeze-dried/liquid prep of recombinant viruses, genetically modified to transfer

genetic material to human somatic cells in vivo or ex vivo» Stringent control:

a. Complete sequence of the genome of the viral vectorb. Verification of the genomic integrity of the vectorc. Determination of the concentration of the infectious vector d. Residual host cell protein and DNAe. Residual reagent including antibiotics

» Retroviridae-derived vector- genetic modifications aims to ensure that the recombinant retroviruses are rendered replication-incompetent.

» Adeno-associated virus vectors- deficient adenovirus in which certain genes necessary for viral replication have been replaced

Viruses as Antimicrobial

» Bacteriophages- viruses that infect only bacteria- 20-200 nm - all bacterial species can be infected by a phage

» Phages- tadpole-shaped consisting of a head that contains the viral genome & a tail which recognizes the host receptor, attach and subsequently serve as a nucleic acid injection device

» Lytic Cycle- phage replication resulting to the lysis of the host cell» Lysogenic Cycle- phage replication resulting in the viral nucleic acid being integrated into the host

genome- Prophage- the viral nucleic acid that has integrated the hos genome- Lysogenic- the host cell that contains the viral nucleic acid

» Virulent phage- aka Lytic Phage- Infection of these result in the replication of the phage within the susceptible bacteria and

the release of infectious phage progeny from the host cell following lysis

Chapter 5: VIRUS 18 | Lacap, Dixie Mae N. Lacap 02.∞.15

- Each lysis will for plaques (holes) in the sample» Transduction- when prophage takes the adjacent bacterial gene that becomes incorporated in the virion

and transmits it to a new susceptible host cell

- Responsible for the gene transfer between bacteria

Epidemiological Uses and Diagnosis

» Phage typing- method that differentiates distinct strains of the same bacterial species on the basis of their susceptibility to phages

Prions- can be recovered from the brains of infected individuals as rod-like structures which are oligomers of a 30kDa glycoprotein

Devoid of nucleic acid Extremely resistant to heating and ultraviolet irradiation Fail to produce an immune response in the host Autocatalytic replication

end

praise the lord

hallelujah