By: Dr.Malak El-Hazmi

( Foundation Block , Microbiology : 2012)

OBJECTIVESOBJECTIVES  

General characteristics of viruses.General characteristics of viruses. StructureStructure & symmetry of viruses.& symmetry of viruses. Classification of viruses.Classification of viruses. Steps of virus replication .Steps of virus replication . laboratory diagnosis of viral infections.laboratory diagnosis of viral infections.

Properties of MicroorganismsProperties of Microorganismscharacteristiccharacteristic ParasitesParasites Fungi Fungi BacteriaBacteria VirusesViruses

CellCell YesYes YesYes YesYes NoNo

Type of Type of

nucleusnucleus

EukaryoticEukaryotic EukaryoticEukaryotic ProkaryoticProkaryotic ----------

Nucleic acidNucleic acid BothBoth

DNADNA

& RNA & RNA

Both Both DNADNA

& RNA& RNA

Both Both DNADNA

& RNA& RNA

DNA DNA or or RNARNA

RibosomeRibosomess PresentPresent PresentPresent PresentPresent AbsentAbsent

MitochondriaMitochondria PresentPresent PresentPresent AbsentAbsent AbsentAbsent

ReplicationReplication MitosisMitosis Budding orBudding or

mitosismitosis

Binary Binary fissionfission

specialspecial

Characteristics of virusesCharacteristics of viruses Acellular organismsAcellular organisms Tiny particles Tiny particles

• Internal coreInternal core• Protein coatProtein coat• Some Vs have lipoprotein mb Some Vs have lipoprotein mb

Obligate intracellular organismsObligate intracellular organisms Replicate in a manner diff from cells Replicate in a manner diff from cells

( 1V many Vs )( 1V many Vs )

Size ; 20-300 nmSize ; 20-300 nm

Viral StructureViral Structure

1-Viral genome 1-Viral genome

2-Capsid2-Capsid

3-Envelope3-Envelope

Viral StructureViral Structure1-Viral genome1-Viral genome

RNA RNA (Ribonucleic acid)(Ribonucleic acid) All RNA Vs have ss All RNA Vs have ss

except Reovirusesexcept Reoviruses single / multiplesingle / multiple (+) polarity (+) polarity (-) polarity(-) polarity

DNADNA (Deoxyribonucleic acid)(Deoxyribonucleic acid) All DNA Vs have ds All DNA Vs have ds

except Parvovirusesexcept Parvoviruses Single moleculeSingle molecule

or

All Vs are haploid ,except retroviruses are diploid

Viral structureViral structure2-Capsid 2-Capsid a protein coat a protein coat Subunits (capsomeres)Subunits (capsomeres) Genome (NA) + capsid Genome (NA) + capsid

= nucleocapsid= nucleocapsid Function;Function;

Protects NAProtects NA Facilitates its entry into cellFacilitates its entry into cell

SymmetrySymmetrybased on arrangement of capsomeresbased on arrangement of capsomeres

Cubic symmetryCubic symmetry

( Icosahederal ) ( Icosahederal )

Helical symmetryHelical symmetry

Complex symmetryComplex symmetry

SymmetrySymmetrybased on arrangement of capsomeresbased on arrangement of capsomeres

1-1-Cubic symmetryCubic symmetry

( Icosahedral )( Icosahedral )

Adenovirus HerpesvirusAdenovirus Herpesvirus

SymmetrySymmetrybased on arrangement of capsomeresbased on arrangement of capsomeres

2- Helical symmetry2- Helical symmetry

3- Complex symmetry3- Complex symmetry

poxvirusespoxviruses

ElongatedElongated (filoviruses)(filoviruses)

PleomorphicPleomorphic ( influenza v.)( influenza v.)

Viral structureViral structure3-Envelope 3-Envelope Lipoprotein mbLipoprotein mb

(host lipid ,virus specific protein )(host lipid ,virus specific protein )

Budding Budding Envelope is derived from cell mbEnvelope is derived from cell mb

except herpesviruses from nuclear mbexcept herpesviruses from nuclear mb Enveloped Vs are more sensitive toEnveloped Vs are more sensitive to

heat ,dry & ether than nonenveloped Vsheat ,dry & ether than nonenveloped Vs Glycoprotein attaches to host cell receptorGlycoprotein attaches to host cell receptor

Viral proteinsViral proteins The outer viral psThe outer viral ps Mediate attachment to specific RsMediate attachment to specific Rs Induce neutralizing AbsInduce neutralizing Abs Target of AbsTarget of Abs

The internal viral psThe internal viral ps Structural ps ( capsid ps of enveloped Vs )Structural ps ( capsid ps of enveloped Vs ) Nonstructural ps ( enzymes)Nonstructural ps ( enzymes)

All ssRNA Vs (-) polarity have transcriptaseAll ssRNA Vs (-) polarity have transcriptase

( RNA dependent RNA polymerase) inside virions( RNA dependent RNA polymerase) inside virions RetroVs & HBV contain reverse transcriptase RetroVs & HBV contain reverse transcriptase

Classification of virusesClassification of viruses

Type of NAType of NA The no. of strand The no. of strand The polarityThe polarity

of viral genomeof viral genome The presence or The presence or

absence of envelope absence of envelope Type of symmetryType of symmetry

Medically Important VirusesMedically Important Viruses

ComplexComplexPoxviridaePoxviridae

Icosahedral Icosahedral HerpesviridaeHerpesviridae

HepadnaviridaeHepadnaviridae

IcosahedralIcosahedralAdenoviridaeAdenoviridae

PapillomaviridaePapillomaviridae

PolyomaviridaePolyomaviridae

EnvelopedEnveloped

RNADNA

NonenvelopedNonenveloped

Single-strandedSingle-stranded double-strandeddouble-stranded

IcosahedralIcosahedralParvoviridaeParvoviridae

NonenvelopedNonenveloped

HelicalHelicalOrthomyxoviridaeOrthomyxoviridae

ParamyxoviridaeParamyxoviridae

RhabdoviridaeRhabdoviridae

FiloviridaeFiloviridae

BunyaviridaeBunyaviridae

ArenaviridaeArenaviridae

HelicalHelicalCoronaviridaeCoronaviridae

Icosahedral Icosahedral Togaviridae Togaviridae

Flaviviridae Flaviviridae

RetroviridaeRetroviridae

IcosahedralIcosahedral

ReoviridaeReoviridae

EnvelopedEnveloped

RNADNA

NonenvelopedNonenveloped

Medically Important VirusesMedically Important Viruses

double-strandeddouble-strandedSingle-strandedSingle-stranded

NonenvelopedNonenveloped

IcosahedralIcosahedralPicornaviridae Picornaviridae

Hepeviridae Hepeviridae

Caliciviridae Caliciviridae

Astroviridae Astroviridae

Pos- strandPos- strandNeg - strandNeg - strand

EnvelopedEnveloped

Replication Replication Adsorption Adsorption

(Attachment)(Attachment) PenetrationPenetration UncoatingUncoating Synthesis of viral Synthesis of viral

componentscomponents• mRNAmRNA• Viral proteinsViral proteins• NANA

AssemblyAssembly ReleaseRelease

Viral growth cycleViral growth cycle

Attachment site ; Attachment site ;

- glycoprotein - glycoprotein

- folding in the capsid proteins- folding in the capsid proteins ..

AdsorptionAdsorption

PenetrationPenetration2-Endocytosis2-Endocytosis

Viral Viral envelopeenvelope

fuses with endosome mbfuses with endosome mb NonenvelopedNonenveloped V. lysis ,pore V. lysis ,pore

1-Fusion 1-Fusion

((envelopedenveloped Vs ) Vs )

Replication Replication Adsorption (Attachment)Adsorption (Attachment) PenetrationPenetration UncoatingUncoating

Release of viral genome - cytoplasmRelease of viral genome - cytoplasm

- nucleus- nucleus

Synthesis of viral componentsSynthesis of viral components

mRNAmRNA Viral genome transcription mRNAViral genome transcription mRNA

+ssRNA acts directly+ssRNA acts directly

Viral proteinsViral proteins mRNA translation viral proteinsmRNA translation viral proteins

cell ribosome - enzymescell ribosome - enzymes

- structural ps - structural ps

replication of viral genomereplication of viral genome

Replication Replication Adsorption (Attachement)Adsorption (Attachement) PenetrationPenetration UncoatingUncoating Synthesis of viral componentsSynthesis of viral components

• mRNAmRNA• Viral proteinsViral proteins• NANA

Assembly Assembly

NANA + + V. proteinsV. proteins = = VirionsVirions ReleaseRelease

Release Release 1-Budding1-Budding

(enveloped Vs)(enveloped Vs)

-cell mb*-cell mb*

-nuclear mb-nuclear mb

(herpesVs)(herpesVs)

2- 2- Cell lysisCell lysis

or or rupturerupture

(nonenveloped)(nonenveloped)

laboratory diagnosis of viral laboratory diagnosis of viral infectionsinfections

Microscopic examination.Microscopic examination.Cell culture.Cell culture.Serological tests .Serological tests .Detection of viral Ag.Detection of viral Ag.Molecular method .Molecular method .

Microscopic examinationMicroscopic examination Light microscopyLight microscopy;; Histological appearanceHistological appearance

Ex. Inclusion bodiesEx. Inclusion bodies

Owl’s eye (CMV) (CMV)

Electron microscopy; Electron microscopy; Morphology& size of virionsMorphology& size of virions Ex. Dx of viral GEx. Dx of viral GE ,rotav. , adenov. E ,rotav. , adenov.

Dx of skin lesion caused by herpesv, poxv. Dx of skin lesion caused by herpesv, poxv. It is replaced by Ag detection & molecular testsIt is replaced by Ag detection & molecular tests

Electron micrographs Electron micrographs

PoxvirusPoxvirus

RotavirusRotavirus

HerpesvirusHerpesvirus

AdenovirusAdenovirus

Virus cultivationVirus cultivationLaboratory animalLaboratory animalEmbryonated eggEmbryonated eggCell cultureCell culture

Cell cultureCell culture No of sub passagesNo of sub passages

Primary C/CPrimary C/C 1 or 21 or 2

Diploid C/CDiploid C/C

[semi continuous][semi continuous]

20 to 5020 to 50

Continuous cell Continuous cell lineline

Indefinite Indefinite

Variation in Sensitivity of cell cultures to infection by viruses Variation in Sensitivity of cell cultures to infection by viruses commonly isolated in clinical virology laboratoriescommonly isolated in clinical virology laboratories

VirusCell culturea

PMK HDF HEp-2

RNA virus

DNA virus

Enterovirus Rhinovirus Influenza virus RSV

Adenovirus HSV VZV CMV

+++ ++ +/-+ +++ ++++ + -++ + +++

+ ++ +++

+ ++ +++ +++ -- +++ -

PMK, primary MK. Degree of sensitivity: +++, highly sensitive;++, moderately sensitive; +, low sensitivity; +/-, variable; -, not sensitive

Detection of viral growthDetection of viral growth

Cytopathic effectsCytopathic effects

IFIFOtherOther

Uninfected cc Cell rounding Syncytium

Problems with cell culture Problems with cell culture ;;

Long incubation Long incubation Sensitivity is variableSensitivity is variable Susceptible to bacterial contaminationSusceptible to bacterial contamination Some Vs do not grow in c/c ex. HCV Some Vs do not grow in c/c ex. HCV

Rapid culture techniqueRapid culture technique

Shell Vial AssayShell Vial Assay Detect viral antigensDetect viral antigens 1-3 days1-3 days

Serological test;Serological test;Antigen detection;Antigen detection;

sample sample virus virus testtest Nasopharyngeal aspirate Influenza V. IFNasopharyngeal aspirate Influenza V. IF Skin scrapings HSV IFSkin scrapings HSV IF Faeces Rotavirus ELISAFaeces Rotavirus ELISA Blood HBV(Blood HBV(HBsAgHBsAg) ELISA) ELISA

Serological test;Serological test;

Ex of techniquesEx of techniques Complement fixation test (CFT) Complement fixation test (CFT) Immunofluorescence (IF)Immunofluorescence (IF) Enzyme- linked immunosorbent assay (ELISA)Enzyme- linked immunosorbent assay (ELISA)

Antibody detection;Antibody detection;

Immunofluorescence ; IFImmunofluorescence ; IF

A- Direct A- Direct

Ag detection;Ag detection; Sample (Ag)Sample (Ag)

B- IndirectB- Indirect

Ab detection;Ab detection; Sample (Ab)Sample (Ab)

ELISAELISAAb detection Ag detection Ab detection Ag detection

Indirect ELISA for Ab detection ; Indirect ELISA for Ab detection ;

coloured wells indicate reactivitycoloured wells indicate reactivity

Molecular test;Molecular test;

Polymerase chain reaction (PCR)Polymerase chain reaction (PCR) Amplification tech.Amplification tech. Viral genomeViral genome

Uses;Uses; DxDx Monitoring response to RxMonitoring response to Rx

Reference book and the Reference book and the relevant page numbersrelevant page numbers

Review of Medical Microbiology and Review of Medical Microbiology and

Immunology Immunology

By: Warren Levinson .By: Warren Levinson .

1010thth Edition, 2008. Edition, 2008.

Pages;192-195,199-207, 216-220,233-235Pages;192-195,199-207, 216-220,233-235..

Lippincott’s Illustrated Reviews: Microbiology Lippincott’s Illustrated Reviews: Microbiology

By: Richard A.Harvey ,By: Richard A.Harvey ,

Pamela C Champe & Pamela C Champe &

Bruce D. FisherBruce D. Fisher

22ndnd Edition, 2007 . Edition, 2007 .

Pages;233-242Pages;233-242