Viral Virulence

Relative nature of virulenceLa Crosse Virus(-)ve strand RNA virus (Bunyaviridae)Same family as Hantavirus, but is arthropod-borneneurotropicAttenuated Clone B.5Reduced capacity to produce viremia and cross the blood brain barrier Virulence revealed by intracerebral innoculation, but only in suckling miceNo difference in virulence with wt strain when innoculated subcutaneously in adult miceChoice of host and route can dramatically influencesusceptibilty and resistanceEqual HighVirulenceEqual LowVirulenceAttentuationobserved

Factor influencing VirulenceViral FactorsViral StrainRoute of InfectionDose of VirusHost FactorsSpeciesAgeGenetic Susceptibility

Measures/Quantitation of VirulenceSymptoms (e.g.)Paralysis (poliovirus)Jaundice (hepatitis)Rash (measles)Case/infection ratio Death/Survival# of IU/PFU per LD50 (50% fatality in cohort) Pathogenic lesionsRabbit Myxoma Virus1951

Biological control of wild rabbits: co-evolution of viral virulence and host resistance1859, 12 European rabbits were introduced into an Australia farm; by 1928 more than a billion rabbits (>500/sq.mile) were ruining agriculture1950, rabbit myxoma virus (>99% mortality rate) was introduced; by 1953, >95% of rabbit population was eliminated, by 1955, rabbit population began to increase. Reasons: Virulence of rabbit myxoma virus decreased; surviving rabbits developed increased resistance; changes in vector activity (mosquitoes) decreased efficiency of transmission

Measures/Quantitation of VirulenceSymptoms (e.g.)Paralysis (poliovirus)Jaundice (hepatitis)Rash (measles)Case/infection ratio Death/Survival# of IU/PFU per LD50 (50% fatality in cohort) Pathogenic lesionsRabbit Myxoma Virus1951

Experimental Manipulation of Viral VirulencePassage in AnimalsAdaptation to survival in host; may be tissue specific Passage in Cell CultureAttentuation due to lack of host immune responseVIRULENCE

Experimental Manipulation of Viral VirulencePassage in AnimalsAdaptation to survival in host; may be tissue specific Yellow FeverAdaptation to neurovirulence by intracerebral passagingSHIV (chimeric Simian-Human Immunodeficiency Virus)Repeated passaging results in severely pathogenic virus (SHIV 89.6 to 89.6P) that causes CD4 depletion and death within 6 monthsPassage in Cell CultureAttentuation due to lack of host immune responseVaccinia (small pox vaccine strain) MVA (Modified Vaccinia Ankara)250 passages in Chick Embryonic Fibroblast results in ability to infect but not replicate in mammalian cellsResults in loss of immune evasion genes; more immunogenic vaccine vector (?)HIVT-cell line adapted virus More neutralization sensitive than primary strains grown in fresh PBMCs

Selection of Attentuated Viral VariantsTemperature Sensitive VariantsAntibody-resistant virusSome neutralization resistant viruses can have increased attentuation in vivoNeutralization resistance can also lead to increased virulenceMutagenized viruses and selection

Study of Attentuated VirusesVariant viruses (wt. vs attentuated) should be genetically pureVariant viruses should differ by as little as possibleVariant viruses should differ only under non-permissive conditions; i.e. there should be culture or innoculation conditions where replication is comparable

Comparative pathogenesis(Virulent vs attentuated viruses)Portal of entryUpper vs lower respiratory tract for influenza; ability to replicate in lower respiratory tract (higher temp.) results in increased pathogenictiyViremiaMost viremic can be most pathogenic (not always) (poliovirus strains)Ability to produce peripheral viremia may affect end-organ pathology (La Crosse vs Tahyna virus)Neural SpreadTarget OrganTropism relative pathogenicity for different tissuesEvasion of host immune responses

Comparative pathogenesis(Virulent vs attentuated viruses)Portal of entryUpper vs lower respiratory tract for influenza; ability to replicate in lower respiratory tract (higher temp.) results in increased pathogenictiyViremiaMost viremic can be most pathogenic (not always) (poliovirus strains)Ability to produce peripheral viremia may affect end-organ pathology (La Crosse vs Tahyna virus)Neural SpreadTarget OrganTropism relative pathogenicity for different tissuesEvasion of host immune responsesLog10 PFU per mlLog10 PFU per mg brainTahyna virus actually replicates better than La Crossevirus in brain, but inability to produce fatal encephalitis after subcutaneous injection is due to lack of replicationin periphery

Comparative pathogenesis(Virulent vs attentuated viruses)Portal of entryUpper vs lower respiratory tract for influenza; ability to replicate in lower respiratory tract (higher temp.) results in increased pathogenictiyViremiaMost viremic can be most pathogenic (not always)Neural SpreadIM injection of wt vs avirulent strain of rabies virus (e.g. MAR variant RV 194-2) results in equal speed of spread to CNS, but once there, spreads more slowly to contiguous neuronsTarget OrganTropism relative pathogenicity for different tissuesEvasion of host immune responsesavirulentvirulent

Comparative pathogenesis(Virulent vs attentuated viruses)Portal of entryUpper vs lower respiratory tract for influenza; ability to replicate in lower respiratory tract (higher temp.) results in increased pathogenictiyViremiaMost viremic can be most pathogenic (not always)Neural SpreadIM injection of wt vs avirulent strain of rabies virus (e.g. MAR variant RV 194-2) results in equal speed of spread to CNS, but once there, spreads more slowly to contiguous neuronsTarget OrganBunyavirus neurotropismNeurotropism = neuroinvasivenessPoliovirus enterotropism vs neurotropismTropism relative pathogenicity for different tissuesEvasion of host immune responsesBunyavirusPoliovirusLog10 PFU per mg brainLD50 based on IC injection

Comparative pathogenesis(Virulent vs attentuated viruses)Portal of entryUpper vs lower respiratory tract for influenza; ability to replicate in lower respiratory tract (higher temp.) results in increased pathogenictiyViremiaMost viremic can be most pathogenic (not always)Neural SpreadIM injection of wt vs avirulent strain of rabies virus (e.g. MAR variant RV 194-2) results in equal speed of spread to CNS, but once there, spreads more slowly to contiguous neuronsTarget OrganBunyavirus neurotropismPoliovirus enterotrpism vs neurotropismTropism relative pathogenicity for different tissuesEvasion of host immune responses(Early)(Late)(~50%)Clinical AIDSSexualTransmissionHIV

Comparative pathogenesis(Virulent vs attenuated viruses)Portal of entryUpper vs lower respiratory tract for influenza; ability to replicate in lower respiratory tract (higher temp.) results in increased pathogenictiyViremiaMost viremic can be most pathogenic (not always)Neural SpreadIM injection of wt vs avirulent strain of rabies virus (e.g. MAR variant RV 194-2) results in equal speed of spread to CNS, but once there, spreads more slowly to contiguous neuronsTarget OrganBunyavirus neurotropismPoliovirus enterotrpism vs neurotropismTropism relative pathogenicity for different tissuesEvasion of host immune responsesLCMV (Clone 13 vs Armstrong strain)Clone 13 replicates better/faster in macrophages; rapid destruction of macs leads to atttenuation of antigen presentation, suppression of immune response and thus results in viral escapeArmstrong strain leads to immunizing infection and viral clearanceSIV/HIV (wt vs Dnef)Also, Sidney Blood Bank cohort example

Viremia(Log10 RNA copies/ml)Alveolar macsVirus titer

Genetic Determinants of VirulenceMutant vs wt ClonesAttenuation or virulence can be due to changes in viral proteins or UTR of viral genomesGenerally, increased number of mutations is correlated with increased attenuation and reduced chance of reversionConsideration for recombinant life-virus vaccine devlopmentSIV Dnef

Reversion to virulence does not necessarily require back mutation; compensatory mutations in same protein (or even different proteins) is possibleAttenuating mutations are generally host range alterations (replication is affected only in some tissues, or cells)

Virulence Genes of Cellular OriginVirokinesMimic the action of cytokines; increases host cell proliferation and virus productionViroceptorsCytokine decoysAb or Complement scavenger

Virulence Genes of Cellular OriginPox VirusesVCP (Vaccinia Complement Control Protein)Abrogates complement mediated atttack on viral infected cellsHomolog of C4-BP that inactivates C4b, a critical player in the complement cascadeTNF ViroceptorsTNF is proinflammatory cytokine that activates immune networksSoluble TNF-receptor homology encoded by poxviruses can TNF secreted by host cell and dampen subsequent immune response (IL-4): Super-poxTH2 cytokine which suppresses Th1 (cell-mediated) immunityMousepox engineered to express IL-4 becomes extremely virulent (Super-pox)

HerpesvirusesgE/gI glycoprotein can act as Fc receptors; prevent effector functions of antiviral antibodies produced by the host

AUSTRALIA:Engineered Mouse Virus Spurs Bioweapon FearsElizabeth Finkel

MELBOURNE, AUSTRALIA--The surprising virulence of a virus genetically altered to reduce rodent infestations in Australia has raised alarm over whether such research could be hijacked to produce biological weapons. In an unusual twist, those sounding the alarm are not environmental activists but the scientists themselves. Despite their warning, it's not clear whether the unexpected result, which turned a vector into a potent killer, could be duplicated in viruses that affect humans. But scientists say it should serve as a warning to the community to be more aware of the potentially harmful consequences of their work.Science 2001 Jan 26;291(5504):585

Mousepox-sensitive(BALB/c)Mousepox-resistant(C57BL/6))Early activation of virus-specifc CTLsProduction of high levels of type 1 cytokines IL-2, IL-12, IFN-g and TNF-aIL-4 is a Type 2 cytokine enhances humoral immunityCan Vaccinia (the attenuated version of smallpox that is used in smallpox vaccine) engineered toexpress IL-4 become a super smallpox--overcome people who has already been vaccinated??

Bioterrorism agent??