Animal Viruses

Properties of Obligate Intracellular Parasites

Property Rickettsiae Chlamydiae Viruses

Generation of metabolic energy

+ - -

Independent protein synthesis

+ + -

Rigid cell envelope + + Variable

Antibiotic susceptibility

+ + -

Mode of replication Fission Fission Host cell synthesis

Nucleic acids in mature particle

DNA/RNA DNA/RNA DNA or RNA, not usually both

Definitions of Terms for Viruses and Viral Infections

Virion – mature infectious virus particleCapsid – protein shell that encloses and protects the viral nucleic acidCapsomer – polymers of polypeptide chains which are the morphological units of icosahedral capsidsCore – internal part of a virus partciel, which consists of the nucleic acid and closely associated proteinsNucleocapsid – structure composed of the capsid containing the nucleic acid or core

Definitions continued

Envelope – viral membrane, consisting of a lipid bilayer, proteins and glycoproteinsPeplomers (spike proteins)– viral proteins or glycoproteins that project from the envolope

Definitions continued+ssRNA – single stranded RNA of the same polarity as messenger RNA-ssRNA – single stranded RNA complementary to messenger RNAvRNA – RNA of an intact ssRNA virus particlecRNA – RNA that is complementary to the RNA of an intact ssRNA virus particlecDNA – complementary DNA made from a viral RNA by recombinant procedures

Definitions continued

DNA dependent DNA polymerase (DNA polymerase) – an enzyme that uses DNA as a template for producing DNADNA dependent RNA polymerase (RNA polymerase) – an enzyme that uses DNA as a template for producing RNARNA dependent RNA polymerase (reverse transcriptase) – an enzyme that uses RNA as a template for producing DNA

Definitions continued

Transfection – in infection of mammalian or bacterial cells by vare viral nucleic acid Transformation – stable hereitable change in the genetic makeup and phenotype of a cell resulting from the infection of that cell by a virusPermissive cells – cells that support the complete virus life cycle, with production of infectious virus particles

Definitions continued:Nonpermissive cells – cell which permit only part of the virus life cycle, usually transformed by viruses, especially DNA virusesProductive infection – infection that results in th eproduction of infectious virusNonproductive infection – infection that has no infectious virus, cells may be transformedDefective virus – virus that is not capable of going through its entire replicative cycle unless the cell is infected with a complete virus particle as well.Cytopathic effect – observable damage to a cell resulting from virus infection

Structure of Animal Viruses

Size – not seen under the microscopeNucleic Acid – varies

12 codons -numerous codonsSegmented genomesEither DNA or RNA, not both

CapsideCapsomeres protect nucleic acid

Baltimore’s Classification

Class I – ds DNAClass II – ss DNAClass III – ds RNAClass IV – ss+ RNAClass V – ss- RNAClass VI – RNA tumor viruses (retroviruses)

Baltimore’s Classification SystemClass I – ds DNAClass II – ss DNAClass III – ds RNAClass IV – ss+ RNAClass V – ss- RNAClass VI – RNA tumor viruses (retroviruses)

CapsideCapsomeres protect nucleic acid

• Complex – found in pox viruses

• Isometric/Icosahedral– 20 facets with 12 vertices– Each hexomer has six

neighbors– Number of capsomeres per

capsid is used to classify icosahedral animal viruses

• Helical – Naked helical viruses are all

resistent– No naked human helical

viruses

Viral envelopesSurrounded by nucleocapsidFormed by modified host cellular membraneContain host derived phosphlipid bilayerContains virus derived proteins and glycoproteins

• Some are enzymes• Some provide attachment to

cellsEnvelopes are more fragile than naked viruses and are often inactivated by lipid solvents

Viral envelopesCharacteristics

Surrounded by nucleocapsidFormed by modified host cellular membraneContain host derived phospholipid bilayerContains virus derived proteins and glycoproteins

• Some are enzymes• Some provide attachment to cells

Envelopes are more fragile than naked viruses and are often inactivated by lipid solvents

• Ether sensitive – have membranes• Ether resistent – have no membranes

Viral ProteinsMatrix proteins – M proteins

Found associated with the inner layer of the envelope, seem to make the envelope more rigid and help with organization of the virus particle

Fusion proteins – F proteinsFound on the envelope surface in some virus groups, cause viruses and virus-infected cells to fuse with uninfected cells

Nonstructural viral proteins Enzymes found in the core of the virions of some virus types

Physiochemical Classification6 DNA and 13 RNA virus families, with one unclassified virus, classification is based on:

Chemical nature of the nucleic acidSymmetry of the nucleocapsidPresence or absence of the envelopeNumber of capsomeres for isometric virionsDiameter of the nucleocapsids for helical viruses

Icosahedral viruses may be either DNA or RNA, enveloped or notHelical viruses are all RNA, enveloped

Replication cycle, Productive Cycle

AttachmentPenetrationUncoatingTranscription/translation of early mRNAReplication of viral nucleic acidTranscription/translation of late mRNAAssembly of virionsRelease

Replication cycle; Productive Infection

Attachment to specific receptor sites on the host cell membrane

Presence of specific receptor sites on the cell is the most important determinant of host specificitySpecie, tissue and physiological state of the cells determine the type of receptors presentViruses tend to be host specific

Replication cycle; Productive Infection

Penetration by one of several methods:Nonenveloped

• Naked viruses may have rearrangement of the capsid protein after binding to cells, virus slips through by direct penetration of the membrane

• Most are engulfed by receptor mediated endocytosis, with partial breakup of the capsid in the vacoule, followed by migration into the cyctplasm

Enveloped • Engulfement of virions by receptor mediated

endocytosis via coated pits to coated vesicles which then fuse with lysosomes to form phagosomes

• Fusion of the viral envelope and cell membrane, leaves the nucleocapsid inside the cell

Replication cycle; Productive Infection

Uncoating the viral nucleic acidAttachment seems to lead to a conformation change in the capsidSometimes• cellular enzymes uncoat the viral nucleic

acid• Synthesis of virus products may take

place without completely uncoating the viral nucleic acid

Replication cycle; Productive Infection

Synthesis (Eclipse)Use host cell enzymes in replcationSynthesis of virus encoded macromolecules proceeds synthesis of:• early proteins• Viral genome proteins• Late proteins

Requires viral RNA and host machinery

Replication cycle; Productive Infection

AssemblyRelease

Disintegration of infected cell (burst), especially for naked nucleocapsidsSlow release, with aquision of the envolope as the nucleocapsid buds through a virus-modified cellular membraneReverse phagocytosis

Mixed Virally Infected CellsMixed Viruses

Interference with replication of second virusComplementation as some viruses are incomplete and can replicate only in the presence of another virus

Multiple Same VirusesRecombination due to crossing overReassortment in viruses with segmented genomesReactivation can lead to rescue of a marker on an inactivated particle by superinfection of a live viris particle or virus particle inactivated by a different regionInterference at a large multpilicity of infection, may have defective particles produced

Cultivation of Viruses

Cell systemsIntact animals – very expensiveEmbryonated egg Tissue culture

• Primary cell lines edrived directly from animals• Diploid cell strains from embryonic tissues, such

as from a chick embryo can grow for 40-50 generations

• Permament (cancerous) cell lines that may divide indefinately, such as Hela cells

Consequences of a Viral InfectionAs seen in tissue culture

Cell death and lysis CPEProliferation of host cell masses of cells piled on each otherFusion of membranes of adjacent cells leading to a multinucleate giant cell, thereby forming a syncytiaTransformation into malignant cancer cells

• Viral nucleic acid integrated into host DNA• Viral nucleid acid arranged in circular duplex (similar

to a nucleosome)Silent infection (latency) with no morphologogical change in the cellSteady-state persistent infection – infected cells produce and release virus

Measurement of Animal Viruses

Infectious unitsEnumeration of total number of particlesObservation of viruses & products as antigensDNA probesPCR

Measurement of Animal VirusesInfectious units

Plaque formation in tissue culturePock formation on chorioallantoic membrane in chick embryoFocus formation if virus causes proliferation of cellsSerial dilution end point method

• Cytopathic effects in tissue culture• Characteristic symptoms in experimental animals or eggs

Enumeration of total number of virus particlesElectron microscopyhemagglutionation

Measurement of Animal Viruses

Observation of viruses and virus products as antigens

Complement fixationDirect flourescent antibodyGel immunodiffusion or immunoelectrophoresisRadioimmunoassayELISA

Measurement of Animal Viruses

DNA ProbesDot hybridization with autoradiography or ELISA readout

• HPV 16,18 most common with cervical cancer

PCR gene amplification using small primer pairs of ssDNA

Nucleic Acid Amplification Testing (NAT) for calculation of viral load, such as HIV in bloodOther amplification methodologies, with HIV

Antibody Titer:Response to Animal Viruses

Types of testsNeutralization of infectivityComplement fixationHemagglutination-inhibitionLatex agglutinationIndirect ELISAIndirect flourescent antibodyRadioimmunoassayWestern blot

Uses of Tests

DiagnosisNeed four-fold rise in titerHigh concentrations on IgM

SurveysDetermine need for immunizationLookback

Control of Virus Diseases

Prevention of transmissionPublic health surveillance of the environmentEducationIsolation of cases of the diseasePassive immunization of contractsActive immunization to creat an immune population

• Vaccines may be either:– Live attentuated– Inactivated– Subunit vaccines

Live Attenuated vs Inactivated Vaccines

Property Attenuated Inactivated

Duration of Immunity

Many years Usually less

Antibody response IgG, IgA IgG only

Cell mediated response

Good Poor

Interference Occasionally No

Reversion to Virulence

Rarely No

Treatment of Viral Disease

SymptomaticImmune serum DrugsInterferons

Type 1 – α and β interferonsType 2 – gamma interferons

Treatment of Viral Disease

InterferonsType 1 – α and β interferons

• Important cytokines for antiviral response• Synthesis induced by an infected cell with either a active or

inactive virus, ds RNA or other compounds• Species – specific, not virus specific• Binds to recipient cell, activates a protein transcription

factor• Have both a + and – effect on cells of the immune system,

such as flu-like symptoms– α interferons used to treat hairy cell leukemia, Kaposi’s

sarcoma, Hepatitis B, genital warts– β interferons used to treat multiple sclerosis

Treatment of Viral Disease

InterferonsType 2 - gamma interferons• Important cytokines for antiviral response

with activated T cells– gamma interferons used to treat chronic

granulomatous disease

Patterns of Pathogenesis

Localized infectionsDisseminated infectionsInapparent infectionsPersistent infections

Patterns of Pathogenesis of Viral Infections

Localized infections – viral replication near site of entryDisseminated Infections

local multiplication at siteextension through lymphaticsmultiplication at second sitesecondary viremiaInfectuon at target organ

Patterns of Pathogenesis of Viral Infections

Inapparent InfectionsVery common, result from infection by attenuated virusConsidered to be very important since:

• Represent an unrecognized source of dissemination of a virus

• Confer immunity to the host

Persistent infectionsLatent infections - HerpesChronic infections – Hepatitis B

Immunity to Virus Infectiuons

InterferonsDefective interference particleViral neutralizationIg bindingAntibody complement mediated cylolysisAntibody dependent cell mediated cytotoxicityLysis by natural killer cellsCell mediated immunity

Immunity to Virus Infections

Interferons inhibit viral multiplication, temporary localized recurrencesDefective interference particles allow for temporary localized protectionViral neutralization by antibody prevents viral infection from entering susceptable cellsIg binding to virus can enhance defense host mechanisms by promoting phagocytosis

Immunity to Virus Infections

Antibody complement mediated cylolysisAntibody dependent cell mediated cytotoxicityLysis by natural killer cellsCell mediated immunity

Important for recovery from host viral infection, especially when host cells are not killed

Unusual immune reactions to viral diseases

Immunological toleranceExamples of exposure in utero include:

• Lymphocytic choriomeningitis (LCM)• Rubella

Diseases from virally-induced immunological response

Cell mediated response – hep BEnhancing antibodies – flavovirusInactivated virus vaccine – respiratory syncytial virus

Dynamics of Parasitism

TransmissibiltyMobility of the host, coupled with loss of virulenceResistence to the effects of the parasite, so that the host lives long enough for viral transmissionInfects a variety of animals, although specific to speciesIf infect new species, tend to be more virulent

New virus diseases, zoonoses

Environmental changes increase human contact with vectorGenetic changes in virus

Point mutationsIntramolecular recombinationGenetic reassortment

How do we eliminate a virus from a population?

Limited antigenic typesLifelong immunityLimited subclinical casesNo carrier stateNo animal reservoirGood vaccine