Rubella

Contents
Infectious Agent
Properties
Epidemiology
Clinical Features
Immune Response
Complications
Prevention/Control
 Introduction
n Causes a mild exanthematous disease
n Low grade fever, enlarged LN, rash
n German measles
n Childhood disease
n Endemic worldwide
n Rubivirus
n Family Togaviridae
n 1 serotype
n Humans only natural host & reservoir
 Infectious agent
n EM
n Spherical, core with lipid envelope
n 2 glycoproteins – E1 & E2
n Core – T3 icosahedral symmetry – 32 capsomeres
n Virus
n Heat labile, inact by lipid solvents
n Biological Characteristics
n Restricted host range
n Replicate in many mammalian cell types in vitro
n Grows slowly
n High G+ C content – 70%
n Effect on host cell
n High MOI – rounding, detachment after -24-72 hrs
n Low MOI visible CPE
n Dominant response – cell survival rather than apoptosis
n EM- ER distention, Golgi membrane distention, vesicles with virus,
chromosome brakages
 Infectious agent
n Strains
n 1 serotype, different strains
n Phenotypic variation – reduced virulence, milder
symptoms, < complications
n All strains cross react serologically
n Major neutralization epitopes – conserved
n Srtucture & organization of genome
n ssRNA, positive stranded, 10kb
n 70% G+C content
 Infectious agent
n Structure & organization of genome (contd)
n 2 long polycistronic ORF
n 5’ ORF – 2 NS proteins – p150 & p90
n conserved
n 3’ ORF – 24S subgenomic mRNA
n Between the 2 – 123 n’tides
n NS ORF
n Clustered at 5’end – long ORF – 200kd
n Cleavage into 2
n p150 – encodes a cysteine protease, MT, X
n P90 – encodes a replicase, helicase motifs
 Infectious agent
n Srtucture & organization of genome (contd)
n Structural gene ORF
n Clustered within 3’ ORF
n Translated only from subgenomic 24 S RNA
n Order of genes : NH2 – C – E1 – E2 – COOH
n C
n 32 kd capsid protein
n Release from polyprotein requires cellular signal peptidases
n E1
n 58kd env glycoprotein
n Heterodimer with E2
n Forms spike on surface of virion
n Class 1 membrane protein
n E2
n Second part of E2
n Heavily glycosylated
n Buried beneath E1
 Infectious agent
n Life Cycle
n Eclipse phase of 10-12 hrs
n Max virus released after 36-48 hrs
n Poor uptake
n Cellular receptor not identified
n Entry – receptor mediated endocytosis
n Fusion with membrane glycoproteins
n Core change in conformation at low pH –
adherence to endosomal membrane
 Infectious agent
n Life Cycle
n genomic RNA – mRNA template for translation of NS
n P90 – input RNA – used as template for –ve str
complementary RNA
n Localization in vesicles on ER
n Progeny
n Sequestered into replicase complex – templates
n Undergoes translation – NS precurssor
n Encapsidation process for assembly
n Subgenomic RNA
n Template for struc proteins
n Occurs in hypertonic salt soln
n High affinity for ribosomes
n Requires cellular signal peptidases
 Infectious agent
n Life Cycle
n Post-translational processing
n E1 & E2 translocation into ER lumen occurs
simultaneously
n Transported into Golgi stack

n Glycosylation

n Transported to plasma membrane

n Assembly on intracellular area of membrane

n Budding from all membranes

 Infectious agent
n Persistence
n Infection with low MOI – viral persistence
n Type 1 IFN & IFN-


n 

n  
n 
 

 Infectious agent
n Antigens
n No reliable animal model
n Particulate CF antigens
n HAI Ag – CF, C, E1
n B cell epitopes
n E2 - polyclonal Ab- antigenic
n E1 – dominant surface Ag

n T cell epitopes
n 17 epitopes – E1, E2, C
n Antigenic mimicry
n Induce autoimmune disorders
 Epidemiology
n Endemic worldwide
n Age
n Natural acquisition – 5-9 yrs of age
n Uncommon in preschool children
n Problem in dev countries
n Low economic status
n No national policy
n 10-25% of women of child bearing age
n Incidence of congenital rubella syndrome – 1-2/1000 live births
n Mortality – 0.1%
n Origin & Spread
n Humans only known natural host
n Congenitally infected infants – source & maintenance
n Shed high amounts of virus – for many months
n Molecular aspects
n E1 gene – 2 genotypes – differs by 8-10 & at n’tide level
n 1-3 aa substitutions
n High degree of conservation
 Clinical Features
n Early childhood/adult life – mild
infection/mild exanthem
n Most cases subclinical/unrecognized
n Clinically apparent – combination of
symptoms
n Maculopapular rash, enlarged LN, fever,
conjunctivitis, arthralgia, sore throat
n Incubation – 14-18 days
n Infection during pregnancy
n Transmission
n Aerosols
 Clinical Features
n Pathogenesis
n Mucosa of URT, NP lymphoid tissue – site of virus
replication
n Spread via lymphatics
n Transient viremia
n Seeding of LN – enlargement
n 7-9 days later – virus in serum
n Onset of virus in nasopharynx & stool – source of virus
n Rash on day 14-21 after exposure
n Virus not found in serum – NPS for 1 week
n Other sites where virus found – conjunctiva, urine,
synovium, lung, csf
n Ab appear
 Immune Response
n Ab seen at onset of rash – few weeks
n IgM – can be detected 1 year after acute
infection
n IgG – by about 2 weeks – directed to E1
n Males have a more robust Ab response
n Females have a stronger response to E2
n Immune complexes – detected 3 months after
– more in those with complications
n Transient IS
 Complications
n Joint symptoms
n Arthralgia, arthritis, - 60%
n Thrombocytopenia
n 1:1500, self limiting
n Encephalopathy
n Most serious complication, 1:6000, 1-6 days after
rash
n Congenital Rubella Syndrome
 Congenital Rubella Syndrome
n Early gestation infection of placenta
n Necrotic syncytiotrophoblast
n cytotrophoblast
n Damage to vascular endothelium
n Later stages
n Clinical consequences – depend on the stage of
pregnancy
n First trimester – resorption of embryo, spontaneous
abortion, abnormal fetal dev,
n Later months – premature delivery, still births
n If survive to full tyerm - 67-85 % born with birth defects
 Congenital Rubella Syndrome
n Most common clinical manifestations
n Hearing loss
n Congenital heart disease
n Pulmonary/valvular stenosis
n Psychomotor retardation
n Cataract, retinopathy
n Neonatal thrombocytopenia
n Hepatosplenomegaly
n Intra-uterine growth retardation
n Less common – bony radiolucencies, hepatitis, hemolytic
anemia
n Laboratory findings
n Increase in csf proteins, abnormal ECG
n Isolate virus from csf – for as long as 20 months
 Congenital Rubella Syndrome
n Most common clinical manifestations
n Teratogenesis
n Multifactorial
n Direct effects of viral replication during critical stage of ontogeny
of specific organs
n Chronic infection – slower growth rate, decreased survival
n Non-cytopathic effect – upsets balance of cellular growth &
differentiation – affect organogenesis
n Direct cytolytic effects – cataracts, myocardium, muscle, ear, CNS
etc
n IC deposition
n IgM detected – 20 weeks
n CMI – 27 weeks
n Hence prior to 20 week of gestation – fetus highly vulnerable
n Progressive Rubella Panencephalitis – late onset – second decade
of life
 Diagnosis
n Differential
n Other common viral/non-viral pathogens – give rise to rash
n Measles, scarlet fever, roseola, erythema infectiosum,
enteroV/AdenoV
n If no lab data – need 2 of the following to confirm
n Cataract, glaucoma, CHD, hearing loss etc
n Laboratory
n IgM – up to 5-6 weeks after symptoms dev
n Cross-reacts with parvo B19 & EBV, RF
n Confirmation requires – 4-fold rise
n Low avidity IgG
n HAI or Neutralization
n Virus isolation – NPS etc
n Molecular tests
 Prevention/Control
n Most cases – subclinical, mild –no specific treatment
n Complications – treat symptomatically
n Pooled human Ig that contains neutralizing Ab
n Anti-virals – none so far
n Vaccines
n Virus isolate – serial passages in cell culture
n Attenuating virus, less virulent
n HPV 77/DE5 – predom in N America
n Cendehill
n Takahashi
n To336
n HPV 77/DK12
 Prevention & Control
n Cendehill & Takahashi
n best tolerated in adult female
n HPV 77/DE5
n Predom in N America till 1979
n Replaced with RA 27/3
n 95% recipients prod Ab, mild symptoms, >12 years protection
n Adverse reactions
n Less than natural rubella
n Fever, enlarged LN, arthritis, arthralgia, etc
n Virus persisits in PBMCs for up to 6 years
n IC persisits – seen in complications
n Policy for vaccination
n Young children – herd immunity
n Protect dev fetus
n Contraindicative in pregnancy
n Reinfection – rare, but reported
 Flaviviruses
Contents
Introduction
Family characteristics
Replication cycle
Molecular basis of virulence
Virus-cell interactions
Pathogenesis
Pathology
Arthropod infection
Syndromes
 Introduction
n 73-80 viruses
n 34 mosquito-borne, 17 tick-borne, 22 zoonotic
n 40 spp cause human disease
n 22 mosquito-borne, 13 tick-borne, 5 zoonotic
n First virus to be typed – Yellow Fever
n First filterable agent to cause human disease
n Family Flaviviridae
n Cause a variety of human deisease – fever – encephalitis –
haemorrhagic fevers
n Global concern – DF, DHF/DSS, JE, YF, WN, TBE
n Others – Kyasanur Forest Disease, MVE, SLE,
n Emergence
n Decrease in mosquito control, societal factors, increased and faster
transportation, dense urbanization
n Vaccination
n Only for YF
 Family Characteristics
n Flaviviridae – 3 genera
n Flavivirus
n Pestivirus
n Hepacivirus
n Unassigned group – GB agents
n Parsimony analysis – tree of
NS3 helicase regions
n Positive strand virus
n Similar in
n RNA dep RNA polymerases
n Unique mode of replication
 Replication Cycle
n Virus
n Spherical, 40-60nm in diameter
n Lipid envelope – protects it from cellular proteases
n 6% RNA, 66% protein, 9% CBH, 17% lipid
n Readily inact – temp, solvents
n Infectivity – stable at pH 8.4 – 8.8
n Sensitive to bile, proteolytic enzymes, lipolytic enzymes
n 3 viral proteins
n E – envelope – major surface protein
n M – membrane - maturation
n C - Capsid
n Antigenic composition/classification
n Common antigenic sites – group specific, serocomplex specific, type
specific
n 14 Clades
n Evolutionary tree
 Replication Cycle

n Genomic structure
n +ve ssRNA, 11kb
n 5’ cap – lacks poly A tail
n Acts as an mRNA for translation of long ORF
n 1 large poly protein
n Non-coding regions
n 5’NCR – poorly conserved, common secondary structures, site of
initiation of plus strand sysnthesis
n 3’NCR – sequence divergence & heterogeneity, consensus sequences
 Replication Cycle
n Cycle of replication
n Binding & uptake
n Receptor mediated endocytosis
n Via E with one or more host cell surface molecules
n E reacts with sulfated glycosaminoglycans
n For infection with mammalian cells – need heparan sulfate
n Also by ADE
n Fusion – of envelope with cell membrane
n Clathrin-coated pits on cell surface – uptake
n Prelysosomal vesicles
n Membrane is acis catalyzed
n Capsid released into cytoplasm
n Translation & Processing of viral proteins
n Assembly & Release
n Translation and Proteolytic processing
n 1 large polyprotein
n Processed by cellular proteases, 1 virally encoded
serine protease
n 10 products
n C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-
NS5
n C protein
n 11 kd
n Forms the ribonucleoprotein complex

n Highly basic – N & C termini – binds to genomic RNA

n Central region – hydrphobic – binds to cell memb

n Anchor C – signal seq – for transloc of prM into ER

n Translation and
Proteolytic processing
n prM
n M
n E
n NS1
n NS2A
n NS2B
n NS3
n NS4A
n NS4B
n NS5
n Translation and Proteolytic processing
n prM
n Generated in ER by host signalase peptidase
n Cleaved by trans-Golgi enzyme – furin – M
n Pr is secreted into extracell medium
n M
n Short ectodomain – spans membrane in 2 areas
n E
n Transmebrane domain in C terminus – anchor
n Signal seq for NS1 transloc
n 12 highly conserved Cys residues
n Glycosylated
n 3 struc domains – receptor binding, fusogenic, contact site of
homodimer
n Protects genome from cellular proteases
n NS1
n Cell-assoc, cell surface, extracellular
n Released from C terminus by signal peptidase
n Elicits strong HIR
n Translation and Proteolytic processing
n NS2A
n Hydrophobic, unknown function
n Cleaved from NS2B by cytoplasmic serine protease
n NS2B
n Membrane assoc, required cofactor for NS3 function as serine
protease
n NS3
n Has several enzymatic activities
n Involved in proteolytic processing, RNA replication
n Active protease – 40aa of NS2A + 167-181 residue of NS3 N
terminal
n Mediates cleavage of NS2A/NS2B, NS2B/NS3, NS3/NS4A,
NS4B/NS5
n Cleaves after pairs of basic residues and before aa with small
side chains
n C terminus – RNA replication – RNA helicases – unwind RNA
n Translation and Proteolytic processing
n NS4A & NS4B

n Functions in RNA replication

n Anchors replicase components ?

n Also found dispersed in cytoplasm and

nucleus
n C termini is transmembrane domain

n NS5

n Largest

n Most conserved

n Probably also involved in 5’ capping

n Assembly & Release
n In ER lumen – mature virus
n Membrane bound vesicles
n Follow the secretory pathway
n Exocytosis/budding
 Molecular Basis of Virulence
n Strain differences in
neurovirulence/neuroinvasiveness
n Attenuated by serial passage
n Mutations
n YF (67), Den2 (53), JE(45)
n Between parental strain and vaccine strain
n Location of mutation – domain II, III, I
n Impt for determining extent of reduction of virulence
n E – Domain III
 Virus Cell Interactions
n No CPE – arthropod or vertebrate cell culture
n CPE – mammalian cells
n Proliferation/hypertrophy of rough ER
n Ultramicroscopic level
n Mitochrondrial damage
n Fragmentation of reticular memb
n Distended vacuoles
n Inclusion bodies
n Increase in lysosomal bodies
n Microscopically
n Cell rounding, shrinkage, pyknosis of nuclei
n Dislodgement from growth surface, cell fusion, syncytia
n Others
n Inhibit fusion with ammonium chloride
n Virus concentrates in perinuclear areas
 Pathology
n 3 patterns of encephalitis damage
n Fatal encephalitis
n Subclinical encephalitis
n Inapparent infection
n Virus factors
n High dose, intracerebral, intrnasal
n Host factors
n Age, sex, genetic suscep, pre-existing infection, immunity to
heterologous agent, physiol factors etc
n Skin Langerhans – primary targets
n Extraneural sites
n Connective tissue, skeletal muscle, myocardium, smooth muscle,
lymphoreticular tissue, endocrine, exocrine
n Damage
n Neuronel, glial damage
n Central chromatolysis, eosinophilia, cell shrinkage
n Inflammation, perivascular infiltration, nodule formation, oedema
 Pathology
n Residual effects
n Neurological
n Psychiatric disturbances
n Persistent/congenital
n Neural tissue
n Immune Response
n Protection, recovery, pathogenesis of encephalitis
 Arthropod infection
n Need for blood
n For energy
n Egg development
n Biological transmission
n Dep on ingestion of infected blood
n Dep on infection of epithelial cells lining midgut
n Dep on escape of virus from midgut into hemocoel
n Dep on infection of cells of salivary gland
n Dep on secretion of virus insaliva during feeding
n Process of viral transference
n Piercing mouthparts – cannulate small vessel – microhematoma
n Enzyme apyrase released by mosq – prevent platelet aggregation
& coagulation
n Virus released intravascularly & intrvascularly
n Spread via lymphatic channels
Vertical transmission
Veneral transmission
n Steps required
 Disease syndromes
n Encephalitis
n SLE, JE, MVE, TBE,
n Rocio Virus, Louping Ill Virus, Modoc virus,
Powassan virus etc
n Fever, Arthralgia, rash
n DF, WN,
n Banzi, Kokobera, Edgehill virus, Kunjin virus etc
n Hemorrhagic fever
n YF, DHF/DSS, KFDV, Omsk HFV
 Disease syndromes
n Encephalitis
n JE- virus,epid, diagnosis, transmission, control
n Fever, Arthralgia, rash
n DF- virus,epid, diagnosis, transmission, control
n Hemorrhagic fever
n YF- virus,epid, diagnosis, transmission, control