Rubella & Flavivirus

Rubella Contents Infectious Agent Properties Epidemiology Clinical Features Immune Response Complications Prevention/Control

Introduction n n n n n n n n n

Causes a mild exanthematous disease Low grade fever, enlarged LN, rash German measles Childhood disease Endemic worldwide Rubivirus Family Togaviridae 1 serotype Humans only natural host & reservoir

Infectious agent n

EM n n n

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Virus n

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Heat labile, inact by lipid solvents

Biological Characteristics n n n n

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Spherical, core with lipid envelope 2 glycoproteins – E1 & E2 Core – T3 icosahedral symmetry – 32 capsomeres

Restricted host range Replicate in many mammalian cell types in vitro Grows slowly High G+ C content – 70%

Effect on host cell n n n n

High MOI – rounding, detachment after -24-72 hrs Low MOI visible CPE Dominant response – cell survival rather than apoptosis EM- ER distention, Golgi membrane distention, vesicles with virus, chromosome brakages

Infectious agent n

Strains n n

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1 serotype, different strains Phenotypic variation – reduced virulence, milder symptoms, < complications All strains cross react serologically Major neutralization epitopes – conserved

Srtucture & organization of genome n n

ssRNA, positive stranded, 10kb 70% G+C content

Infectious agent n

Structure & organization of genome (contd) n n

2 long polycistronic ORF 5’ ORF – 2 NS proteins – p150 & p90 n

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conserved

3’ ORF – 24S subgenomic mRNA Between the 2 – 123 n’tides NS ORF Clustered at 5’end – long ORF – 200kd n Cleavage into 2 n

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p150 – encodes a cysteine protease, MT, X P90 – encodes a replicase, helicase motifs

Infectious agent n

Srtucture & organization of genome (contd) n

Structural gene ORF n n n n

Clustered within 3’ ORF Translated only from subgenomic 24 S RNA Order of genes : NH2 – C – E1 – E2 – COOH C n n

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E1 n n n n

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32 kd capsid protein Release from polyprotein requires cellular signal peptidases 58kd env glycoprotein Heterodimer with E2 Forms spike on surface of virion Class 1 membrane protein

E2 n n n

Second part of E2 Heavily glycosylated Buried beneath E1

Infectious agent n

Life Cycle n n n n n n n

Eclipse phase of 10-12 hrs Max virus released after 36-48 hrs Poor uptake Cellular receptor not identified Entry – receptor mediated endocytosis Fusion with membrane glycoproteins Core change in conformation at low pH – adherence to endosomal membrane

Infectious agent n

Life Cycle n n

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genomic RNA – mRNA template for translation of NS P90 – input RNA – used as template for –ve str complementary RNA Localization in vesicles on ER Progeny n n n

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Sequestered into replicase complex – templates Undergoes translation – NS precurssor Encapsidation process for assembly

Subgenomic RNA n n n n

Template for struc proteins Occurs in hypertonic salt soln High affinity for ribosomes Requires cellular signal peptidases

Infectious agent n

Life Cycle n

Post-translational processing 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 n

Infectious agent n

Persistence n n

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Infection with low MOI – viral persistence Type 1 IFN & IFN-       n

   

Infectious agent n

Antigens n n n n

No reliable animal model Particulate CF antigens HAI Ag – CF, C, E1 B cell epitopes E2 - polyclonal Ab- antigenic n E1 – dominant surface Ag n

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T cell epitopes n

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17 epitopes – E1, E2, C

Antigenic mimicry n

Induce autoimmune disorders

Epidemiology

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Endemic worldwide Age n n n

Natural acquisition – 5-9 yrs of age Uncommon in preschool children Problem in dev countries n n n n

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Mortality – 0.1% Origin & Spread n n

Humans only known natural host Congenitally infected infants – source & maintenance n

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Low economic status No national policy 10-25% of women of child bearing age Incidence of congenital rubella syndrome – 1-2/1000 live births

Shed high amounts of virus – for many months

Molecular aspects n n n

E1 gene – 2 genotypes – differs by 8-10 & at n’tide level 1-3 aa substitutions High degree of conservation

Clinical Features

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Early childhood/adult life – mild infection/mild exanthem Most cases subclinical/unrecognized Clinically apparent – combination of symptoms n

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Maculopapular rash, enlarged LN, fever, conjunctivitis, arthralgia, sore throat

Incubation – 14-18 days Infection during pregnancy Transmission n

Aerosols

Clinical Features

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Pathogenesis n

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Mucosa of URT, NP lymphoid tissue – site of virus replication Spread via lymphatics Transient viremia Seeding of LN – enlargement 7-9 days later – virus in serum Onset of virus in nasopharynx & stool – source of virus Rash on day 14-21 after exposure Virus not found in serum – NPS for 1 week Other sites where virus found – conjunctiva, urine, synovium, lung, csf Ab appear

Immune Response n n

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Ab seen at onset of rash – few weeks IgM – can be detected 1 year after acute infection IgG – by about 2 weeks – directed to E1 Males have a more robust Ab response Females have a stronger response to E2 Immune complexes – detected 3 months after – more in those with complications Transient IS

Complications n

Joint symptoms n

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Thrombocytopenia n

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1:1500, self limiting

Encephalopathy n

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Arthralgia, arthritis, - 60%

Most serious complication, 1:6000, 1-6 days after rash

Congenital Rubella Syndrome

Congenital Rubella Syndrome n

Early gestation infection of placenta n n

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Necrotic syncytiotrophoblast cytotrophoblast

Damage to vascular endothelium Later stages Clinical consequences – depend on the stage of pregnancy n

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First trimester – resorption of embryo, spontaneous abortion, abnormal fetal dev, Later months – premature delivery, still births If survive to full tyerm - 67-85 % born with birth defects

Congenital Rubella Syndrome n

Most common clinical manifestations n n n n n n n n n

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Hearing loss Congenital heart disease Pulmonary/valvular stenosis Psychomotor retardation Cataract, retinopathy Neonatal thrombocytopenia Hepatosplenomegaly Intra-uterine growth retardation Less common – bony radiolucencies, hepatitis, hemolytic anemia

Laboratory findings n n

Increase in csf proteins, abnormal ECG Isolate virus from csf – for as long as 20 months

Congenital Rubella Syndrome n

Most common clinical manifestations n

Teratogenesis n n

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Multifactorial Direct effects of viral replication during critical stage of ontogeny of specific organs Chronic infection – slower growth rate, decreased survival Non-cytopathic effect – upsets balance of cellular growth & differentiation – affect organogenesis Direct cytolytic effects – cataracts, myocardium, muscle, ear, CNS etc IC deposition IgM detected – 20 weeks CMI – 27 weeks Hence prior to 20 week of gestation – fetus highly vulnerable Progressive Rubella Panencephalitis – late onset – second decade of life

Diagnosis n

Differential n n n

Other common viral/non-viral pathogens – give rise to rash Measles, scarlet fever, roseola, erythema infectiosum, enteroV/AdenoV If no lab data – need 2 of the following to confirm n

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Cataract, glaucoma, CHD, hearing loss etc

Laboratory n n n n n n n

IgM – up to 5-6 weeks after symptoms dev Cross-reacts with parvo B19 & EBV, RF Confirmation requires – 4-fold rise Low avidity IgG HAI or Neutralization Virus isolation – NPS etc Molecular tests

Prevention/Control n n n n n

Most cases – subclinical, mild –no specific treatment Complications – treat symptomatically Pooled human Ig that contains neutralizing Ab Anti-virals – none so far Vaccines n n n n n n n

Virus isolate – serial passages in cell culture Attenuating virus, less virulent HPV 77/DE5 – predom in N America Cendehill Takahashi To336 HPV 77/DK12

Prevention & Control n

Cendehill & Takahashi n

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best tolerated in adult female

HPV 77/DE5 n n

Predom in N America till 1979 Replaced with RA 27/3 n

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Adverse reactions n n n n

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95% recipients prod Ab, mild symptoms, >12 years protection

Less than natural rubella Fever, enlarged LN, arthritis, arthralgia, etc Virus persisits in PBMCs for up to 6 years IC persisits – seen in complications

Policy for vaccination n n n n

Young children – herd immunity Protect dev fetus Contraindicative in pregnancy 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 n

34 mosquito-borne, 17 tick-borne, 22 zoonotic 40 spp cause human disease n

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First virus to be typed – Yellow Fever n

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First filterable agent to cause human disease

Family Flaviviridae Cause a variety of human deisease – fever – encephalitis – haemorrhagic fevers Global concern – DF, DHF/DSS, JE, YF, WN, TBE Others – Kyasanur Forest Disease, MVE, SLE, Emergence n

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22 mosquito-borne, 13 tick-borne, 5 zoonotic

Decrease in mosquito control, societal factors, increased and faster transportation, dense urbanization

Vaccination n

Only for YF

Family Characteristics n

Flaviviridae – 3 genera n n n n

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Flavivirus Pestivirus Hepacivirus Unassigned group – GB agents

Parsimony analysis – tree of NS3 helicase regions Positive strand virus Similar in n n

RNA dep RNA polymerases Unique mode of replication

Replication Cycle n

Virus n n n n n n n

Spherical, 40-60nm in diameter Lipid envelope – protects it from cellular proteases 6% RNA, 66% protein, 9% CBH, 17% lipid Readily inact – temp, solvents Infectivity – stable at pH 8.4 – 8.8 Sensitive to bile, proteolytic enzymes, lipolytic enzymes 3 viral proteins n n n

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E – envelope – major surface protein M – membrane - maturation C - Capsid

Antigenic composition/classification n n n

Common antigenic sites – group specific, serocomplex specific, type specific 14 Clades Evolutionary tree

Replication Cycle

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Genomic structure n n n n n

+ve ssRNA, 11kb 5’ cap – lacks poly A tail Acts as an mRNA for translation of long ORF 1 large poly protein Non-coding regions n

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5’NCR – poorly conserved, common secondary structures, site of initiation of plus strand sysnthesis 3’NCR – sequence divergence & heterogeneity, consensus sequences

Replication Cycle n

Cycle of replication n

Binding & uptake n n n n n

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Receptor mediated endocytosis Via E with one or more host cell surface molecules E reacts with sulfated glycosaminoglycans For infection with mammalian cells – need heparan sulfate Also by ADE

Fusion – of envelope with cell membrane Clathrin-coated pits on cell surface – uptake Prelysosomal vesicles Membrane is acis catalyzed Capsid released into cytoplasm Translation & Processing of viral proteins Assembly & Release

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Translation and Proteolytic processing n n

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1 large polyprotein Processed by cellular proteases, 1 virally encoded serine protease 10 products C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4BNS5 C protein 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

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Translation and Proteolytic processing n n n n n n n n n n

prM M E NS1 NS2A NS2B NS3 NS4A NS4B NS5

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Translation and Proteolytic processing n

prM n n n

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M n

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Short ectodomain – spans membrane in 2 areas

E n n n n n

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Generated in ER by host signalase peptidase Cleaved by trans-Golgi enzyme – furin – M Pr is secreted into extracell medium

Transmebrane domain in C terminus – anchor Signal seq for NS1 transloc 12 highly conserved Cys residues Glycosylated 3 struc domains – receptor binding, fusogenic, contact site of homodimer Protects genome from cellular proteases

NS1 n n n

Cell-assoc, cell surface, extracellular Released from C terminus by signal peptidase Elicits strong HIR

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Translation and Proteolytic processing n

NS2A n n

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NS2B n

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Hydrophobic, unknown function Cleaved from NS2B by cytoplasmic serine protease Membrane assoc, required cofactor for NS3 function as serine protease

NS3 n n n

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Has several enzymatic activities Involved in proteolytic processing, RNA replication Active protease – 40aa of NS2A + 167-181 residue of NS3 N terminal Mediates cleavage of NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4B/NS5 Cleaves after pairs of basic residues and before aa with small side chains C terminus – RNA replication – RNA helicases – unwind RNA

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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

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Assembly & Release n n n n

In ER lumen – mature virus Membrane bound vesicles Follow the secretory pathway Exocytosis/budding

Molecular Basis of Virulence n

Strain differences in neurovirulence/neuroinvasiveness n

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Attenuated by serial passage

Mutations n

YF (67), Den2 (53), JE(45) n

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Between parental strain and vaccine strain

Location of mutation – domain II, III, I Impt for determining extent of reduction of virulence n E – Domain III n

Virus Cell Interactions n n n n

No CPE – arthropod or vertebrate cell culture CPE – mammalian cells Proliferation/hypertrophy of rough ER Ultramicroscopic level n n n n n

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Microscopically n n

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Mitochrondrial damage Fragmentation of reticular memb Distended vacuoles Inclusion bodies Increase in lysosomal bodies Cell rounding, shrinkage, pyknosis of nuclei Dislodgement from growth surface, cell fusion, syncytia

Others n n

Inhibit fusion with ammonium chloride Virus concentrates in perinuclear areas

Pathology n

3 patterns of encephalitis damage n n n

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Virus factors n

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Age, sex, genetic suscep, pre-existing infection, immunity to heterologous agent, physiol factors etc Skin Langerhans – primary targets

Extraneural sites n

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High dose, intracerebral, intrnasal

Host factors n

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Fatal encephalitis Subclinical encephalitis Inapparent infection

Connective tissue, skeletal muscle, myocardium, smooth muscle, lymphoreticular tissue, endocrine, exocrine

Damage n n n

Neuronel, glial damage Central chromatolysis, eosinophilia, cell shrinkage Inflammation, perivascular infiltration, nodule formation, oedema

Pathology n

Residual effects n n

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Persistent/congenital n

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Neurological Psychiatric disturbances Neural tissue

Immune Response n

Protection, recovery, pathogenesis of encephalitis

Arthropod infection n

Need for blood n n

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Biological transmission n n n n n

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For energy Egg development Dep on ingestion of infected blood Dep on infection of epithelial cells lining midgut Dep on escape of virus from midgut into hemocoel Dep on infection of cells of salivary gland Dep on secretion of virus insaliva during feeding

Process of viral transference n n n n

Piercing mouthparts – cannulate small vessel – microhematoma Enzyme apyrase released by mosq – prevent platelet aggregation & coagulation Virus released intravascularly & intrvascularly Spread via lymphatic channels

Vertical transmission Veneral transmission

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Steps required

Disease syndromes n

Encephalitis n n

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Fever, Arthralgia, rash n n

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SLE, JE, MVE, TBE, Rocio Virus, Louping Ill Virus, Modoc virus, Powassan virus etc DF, WN, Banzi, Kokobera, Edgehill virus, Kunjin virus etc

Hemorrhagic fever n

YF, DHF/DSS, KFDV, Omsk HFV

Disease syndromes n

Encephalitis n

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Fever, Arthralgia, rash n

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JE- virus,epid, diagnosis, transmission, control DF- virus,epid, diagnosis, transmission, control

Hemorrhagic fever n

YF- virus,epid, diagnosis, transmission, control