topic 8; Viruses pt1 Introduction to Viruses (pt2)

What is a virion?

A fully assembled infective virus that can infect host cells and replicate.

What is the fundamental component of a virion?

A nucleoprotein core.

What is the function of the capsid in viruses?

It is a shell of proteins that encloses the genome of vertebrate viruses.

What does the term 'nucleocapsid' refer to?

The packaged form of the genome within the capsid.

Name the two types of capsid symmetry.

Icosahedral and helical.

What is an envelope in the context of viruses?

A lipid bilayer covering the nucleocapsid, acquired when it buds through a cellular membrane.

What are the functions of envelope glycoproteins?

They bind to host cell receptors, mediate membrane fusion, participate in uncoating, and can destroy host cell receptors.

Why are epitopes on envelope glycoproteins important?

They are crucial for inducing protective immune responses in infected animals.

What is a capsomere?

Structural features such as protrusions seen on the surface of virus particles in electron micrographs.

Adenoviridae family?

● Virion size: seventy to ninety nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Type of genome: linear, double stranded DNA

Adenoviridae family

No, it is absent.

Asfarviridae family

● Virion size: one hundred seventy five to two hundred fifteen

nanometers

● Capsid symmetry: icosahedral

● Envelope: present

● Type of genome: linear, double stranded DNA

What is the virion size for the Circoviridae family?

● Virion size: seventeen to twenty two nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Type of genome: circular molecule of positive sense or ambisense,

single stranded

Herpesviridae family

● Virion size: one hundred twenty to two hundred nanometers

● Capsid symmetry: icosahedral

● Envelope: present

● Type of genome: single molecule of linear, double stranded DNA

poxviridae

● Virion size: three hundred by two hundred nanometers (large, brick shaped, complex)

● Capsid symmetry: complex

● Envelope: present

● Type of genome: single molecule of linear, double stranded DNA

Parvoviridae

● Virion size: eighteen to twenty six nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Type of genome: single molecule of linear, positive sense or negative sense, single stranded DNA

Papillomaviridae

● Virion size: fifty five nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Type of genome: single molecule of circular, double stranded DNA

Arteriviridae family

● Virion size: forty to sixty nanometers

● Capsid symmetry: icosahedral

● Envelope: present

● Genome: linear, single molecule of positive sense, single stranded

RNA

Bunyaviridae

Virion size: eighty to one hundred twenty nanometers

● Capsid symmetry: helical

● Envelope: present

● Genome: three segments of linear, negative sense or ambisense, single stranded RNA

Astroviridae

● Virion size: twenty eight to thirty nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Genome: linear, single molecule of positive sense, single stranded RNA

Birnaviridae

● Virion size: sixty nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Genome: two segments of linear, double stranded RNA

Bornaviridae

● Virion size: ninety nanometers

● Capsid symmetry: icosahedral

● Envelope: present

●Genome: linear, single molecule of negative sense, single stranded

RNA

Bunyaviridae

● Virion size: eighty to one hundred twenty nanometers

● Capsid symmetry: helical

● Envelope: present

● Genome: three segments of linear, negative sense or ambisense, single stranded RNA

Caliciviridae

● Virion size: twenty seven to forty nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Genome: linear, single molecule of positive sense, single stranded RNA

Coronaviridae

● Virion size: one hundred twenty to one hundred sixty nanometers

● Capsid symmetry: helical

● Envelope: present

● Genome: linear, single molecule of positive sense, single stranded RNA

Flaviviridae

● Virion size: forty to sixty nanometers

● Capsid symmetry: icosahedral

● Envelope: present

● Genome: linear, single molecule of positive sense, single stranded RNA

Orthomyxoviridae

● Virion size: eighty to one hundred twenty nanometers

● Capsid symmetry: helical

● Envelope: present

● Genome: six to eight segments of linear, negative sense, single stranded RNA

Paramyxoviridae

● Virion size: one hundred fifty to three hundred nanometers

● Capsid symmetry: helical

● Envelope: present

● Genome: linear, single molecule of negative sense, single stranded RNA

Picornaviridae

● Virion size: thirty nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Genome: linear, single molecule of positive sense, single stranded RNA

Reoviridae

● Virion size: sixty to eighty nanometers

● Capsid symmetry: icosahedral

● Envelope: absent

● Genome: ten to twelve segments of linear, double stranded RNA

Retroviridae

● Virion size: eighty to one hundred nanometers

● Capsid symmetry: icosahedral

● Envelope: present

● Genome: diploid (two copies), linear, positive sense, single stranded RNA

Rhabdoviridae

● Virion size: one hundred eighty by seventy five nanometers

● Capsid symmetry: helical

● Envelope: present

● Genome: linear, single molecule of negative sense, single stranded RNA

Togaviridae

● Virion size: seventy nanometers

● Capsid symmetry: icosahedral

● Envelope: present

● Genome: linear, single molecule of positive sense, single stranded RNA

What is the Baltimore Classification of viruses based on?

Nature of the genome and pathways of messenger RNA synthesis and Originally six classes, expanded to seven groups

Group I

● Double stranded DNA Messenger RNA production

● Messenger RNA produced by transcription in much the same way as cellular DNA

Group II

● Single stranded DNA Replication pathway

● Single stranded DNA genome is converted into a double stranded DNA intermediate

● Transcription to messenger RNA occurs from the double stranded DNA intermediate

Group III

● Double stranded RNA Replication and messenger RNA production

● Strands of double stranded RNA separate

● One strand is used as template for generation of messenger RNA

● Uses an RNA dependent RNA

polymerase encoded by the virus

Group IV

● Single stranded RNA of positive polarity

Positive polarity

● Genomic RNA can serve directly as messenger RNA

Replication

● Intermediates of double stranded RNA, called replicative intermediates, are formed during copying of the genomic RNA

● From these intermediates, multiple full length RNA strands of negative polarity are produced

● These negative strands can serve as templates for production of RNA with positive polarity

● Positive polarity products include

● Full length genomic RNA

● Shorter viral messenger RNAs

Group V

● Single stranded RNA of negative polarity

● Sequence is complementary to messenger RNA

Replication and messenger RNA production

● Double stranded RNA

intermediates are used to make copies of the genome and to

produce messenger RNA

● Negative stranded genome can be converted directly to messenger RNA

● Additionally, full length positive RNA strands are made to serve as templates for production of the

negative stranded genome

Group VI

● Diploid (two copies) single stranded RNA

Replication pathway

● Genome must be converted to double stranded DNA

● Uses the enzyme reverse transcriptase

● Double stranded DNA is transported to the nucleus of the host cell

● Integrated into the host genome

● Messenger RNA is produced by transcription of the integrated viral DNA

Group VII

● Partial double stranded DNA Replication pathway

● Virus makes single stranded RNA intermediates that act as messenger RNA

● These single stranded RNA

intermediates are also converted back into double stranded DNA genomes by reverse transcriptase

● This is necessary for genome replication

Replication of Viruses General Principles

● Viruses can multiply only in host cells

● They utilize host cell organelles, enzymes, and other

macromolecules for replication

● Effects of viral replication range from changes in cellular metabolism to cytolysis

● In infected cell cultures, duration of the replication cycle may range from six to forty hours

What is the primary requirement for viral multiplication?

Viruses can only multiply in host cells.

What are the major stages of virus replication?

The stages include attachment, penetration, uncoating, replication, assembly, and release.

How many groups are in the Baltimore Classification of Viruses?

Seven groups.

What characterizes Group I viruses?

Double stranded DNA; messenger RNA produced by transcription from DNA.

What is the replication pathway for Group II viruses?

Single stranded DNA is converted into a double stranded DNA intermediate.

How do Group III viruses produce messenger RNA?

They use one strand of double stranded RNA as a template for messenger RNA generation.

What is unique about Group IV viruses?

They have single stranded RNA of positive polarity that can serve directly as messenger RNA.

What defines Group V viruses?

Single stranded RNA of negative polarity; their sequence is complementary to messenger RNA.

What is the replication pathway for Group VI viruses?

Diploid single stranded RNA must be converted to double stranded DNA using reverse transcriptase.

What is the key feature of Group VII viruses?

They have partial double stranded DNA and make single stranded RNA intermediates that act as messenger RNA.

What occurs during the attachment stage of virus replication? (Step 1)

The virus interacts randomly with host cell receptors.

● Initial virus cell interaction is a random event

● Depends on the number of virus particles present

● Depends on the availability of

appropriate receptor molecules on host cells

Determinants of specificity

● Virus cell interaction determines host range of the viral species

● Also determines tissue tropism of the viral species

Ligands and receptors

● Some viruses have more than one type of ligand molecule

● May bind to several cell surface receptors

Detachment

● In some viruses, individual virions can detach and adsorb to another cell

● In orthomyxoviruses and paramyxoviruses, detachment is mediated by viralneuraminidase

● Neuraminidase is a receptor destroying enzyme

Stage 2: Entry into the Cell

General characteristics

● Virus uptake or penetration is an energy dependent process

What is the main mechanism of entry for viruses into host cells?

● Receptor mediated endocytosis

● Involves coated pits and vesicles

● Clathrin is involved in forming coated pits and vesicles

● Fusion of viral envelope with the plasma membrane

● Used by enveloped viruses

What is the process of uncoating in viral replication? (Stage 3)

● Process whereby the viral genome is released in a form suitable for transcription

– Enveloped viruses

● In enveloped viruses in which the nucleocapsid is discharged directly into the cytoplasm

● Transcription can usually proceed without complete uncoating

Viruses that replicate in the nucleus

● In some viruses which replicate in the cell nucleus, uncoating may be completed at the nuclear pores

What is required for the synthesis of viral proteins? (Stage 4)

– Central role

● Central event in replication of viruses

– Requirement

● Requires production of viral messenger RNA

– Messenger RNA synthesis in DNA viruses

● For DNA viruses that replicate in

the nucleus, host cell transcriptases can be used to synthesize viral messenger RNA

– Viral enzymes

● Some viruses utilize their own enzymes to generate messenger RNA

Protein Synthesis and Processing

– Site of synthesis

● Membrane proteins and glycoproteins

● Synthesized on membrane bound ribosomes

● Soluble proteins such as enzymes

● Synthesized on ribosomes free in the cytoplasm

– Post-translational modification

● Most viral proteins undergo post translational modification

● Modifications include

● Proteolytic cleavage

● Phosphorylation

● Glycosylation Glycosylation

● Sugar side chains are added to viral proteins in a programmed manner

● Occurs as proteins are transferred from the rough endoplasmic reticulum to the Golgi apparatus

● Prepares proteins for final assembly and release from the cell

Where are membrane proteins and glycoproteins synthesized?

On membrane-bound ribosomes.

What is the role of post-translational modification in viral proteins?

It prepares proteins for final assembly and release from the cell.

How do non-enveloped viruses typically release from host cells?

They are released following cellular disintegration.

Assembly and Release of Virions (stage 5)

General Differences

● Enveloped and non enveloped

viruses assemble and are released in distinct ways

– Non enveloped animal viruses

● Usually have icosahedral structure

● These viruses are usually released following cellular disintegration

Sites of Assembly for Some Non Enveloped Viruses

Picornaviruses and Reoviruses

● Assembly occurs in the cytoplasm of the cell

Parvoviruses, Adenoviruses, Papovaviruses

● Assembled in the nucleus

Assembly and Release in Enveloped Viruses

– Acquisition of envelope

● Final step in virion assembly

involves acquisition of an envelope by budding from cell membranes

– Membrane modification prior to budding

● Cell membranes are modified by insertion of virus specified

transmembrane glycoproteins

● These glycoproteins aggregate in patches in the plasma membrane

Cytopathic effects

● Many enveloped viruses are non cytopathic and may be associated with persistent infections

● However, togaviruses, paramyxoviruses, and

rhabdoviruses are cytolytic

Enveloped Viruses Budding at Internal Membranes

Flaviviruses, Coronaviruses, Arteriviruses, Bunyaviruses

uses, Coronaviruses, Arteriviruses, Bunyaviruses

● Acquire their envelopes inside cells

● Bud through membranes of the rough endoplasmic reticulum or the Golgi apparatus

● Virions are transported in vesicles to the cell surface

● Vesicle fuses with the plasma membrane

● Virion is released by exocytosis

What is the final step in the assembly of enveloped viruses?

Acquisition of an envelope by budding from cell membranes.

How do herpesviruses replicate and release?

They replicate in the nucleus and bud through the nuclear membrane.

Herpesviruses (Unique Pattern)

– Replication site

● Replicate in the nucleus – Budding pattern

● Bud through the inner lamella of the nuclear membrane

● Accumulate in the space between inner and outer lamellae

● Also accumulate in cisternae of the endoplasmic reticulum and in cytoplasmic vesicles

– Release

● Released by exocytosis or by cytolys

Poxviruses (Very Complex Assembly)

– Time course

● Assembly and release is a complex process taking several hours

– Enveloping

● Following assembly, virus particles move out of the assembly area

● Become enveloped in a double membrane derived from the trans Golgi network

– Release

● At the periphery of the cell, fusion with the plasma membrane occurs

● This results in loss of the outer layer of the double membrane

● Leads to release of extracellular enveloped viru

What are the general considerations for laboratory diagnosis of viral infections?

Diagnosis can be based on clinical signs, postmortem findings, and histopathological changes.

What is the purpose of collecting oropharyngeal or nasopharyngeal aspirates?

They are suitable for diagnosing respiratory viral diseases.

What is the purpose of collecting feces

● Virus particles shed in feces

● Used for enteric viral diseases

Detection of Virus, Viral Antigens, or Nucleic Aci

Method 1: Isolation of live virus

Method 2: electron microscopy

Method 3: Immunofluorescence and Immunohistochemistr

Method 4: Immunodiffusion

Method 5: Hemagglutination and Hemadsorption Hemagglutination

Method 6: Polymerase Chain Reaction (PCR

Method 7: Diagnostic Serology Serology

Method 1: Isolation of Live Virus

– Systems used

● Cell culture

● Fertile eggs

● Experimental animals Characteristics

● Sensitive procedure when cultural conditions are optimal for a particular virus

● A number of blind passages may

be required before a virus becomes adapted to a particular cell line

Method 2: Electron Microscopy

– Uses

● Can be used to recognize mixed viral infections

● Can detect viruses which cannot be grown in vitro

– Limitations

● Large numbers of viral particles must be present in clinical samples

Method 3: Immunofluorescence and Immunohistochemistry

– Principle

● Antiviral antibodies labelled with fluorochromes can be used

– Uses

● Detect viral antigens in clinical specimens

● Demonstrate virus or viral antigen in specimens

Method 4: Immunodiffusion

● Carried out in agar

– Procedure

● Place a fluid sample containing the virus under test in a well in the agar

● Place antiserum in an opposite well in the agar

● Fluids diffuse out of the wells

● A line of precipitate forms if the sample under test contains viral antige

Method 5: Hemagglutination and Hemadsorption Hemagglutination

● Certain viruses can interact with erythrocytes of many animal species, causingagglutination of red blood cells

–Viral families with hemagglutinating ability:

● Orthomyxoviridae

● Paramyxoviridae

● Adenoviridae

● Parvoviridae

●Togaviridae Hemadsorption

● Used to describe binding of erythrocytes to cells infected with hemagglutinating viruses

Method 6: Polymerase Chain Reaction (PCR)

– Type of method

● In vitro method

– Principle

● Based on amplification of a particular nucleic acid sequence

– Components

● Thermostable DNA polymerase

● Primers specific for the target sequence

What is the purpose of the polymerase chain reaction (PCR) in viral diagnostics?

To amplify a particular nucleic acid sequence.

Method 7: Diagnostic Serology Serology

● Detection of antigen–antibody reactions in serum

● Used to check past or recent infection by identifying specific

antibodies

● Antibodies take time to form, so early infection may show antigen

without detectable antibodies

(window period)

● For influenza, serology is not used for acute diagnosis

● Often requires paired sera to show a rise in antibody levels

Examples of serologic tests:

● Enzyme linked immunosorbent assay

● Serum neutralization test

● Hemagglutination inhibition test

● Complement fixation tests

● Western blott

What does serology detect in the context of viral infections?

Antigen-antibody reactions in serum to check for past or recent infections.

What is the window period in serology?

The time when antigen may be present without detectable antibodies.

What are some examples of serologic tests?

Enzyme linked immunosorbent assay, serum neutralization test, hemagglutination inhibition test.

What is hemagglutination?

The agglutination of red blood cells caused by certain viruses.

What is hemadsorption?

The binding of erythrocytes to cells infected with hemagglutinating viruses.

What is the role of reverse transcriptase in Group VI viruses?

It converts single stranded RNA to double stranded DNA.

What happens to virus particles during the assembly of enveloped viruses?

They acquire their envelope by budding from the cell membrane.

What is the significance of cytopathic effects in viral infections?

They can indicate whether a virus is cytolytic or associated with persistent infections.

What are replicative intermediates in viral replication?

Double stranded RNA formed during the copying of genomic RNA.