Retroviridaeiacld.ir/DL/modavan/viruses/92/hivdrparsania.pdfRetroviridae • Retroviruses...
Transcript of Retroviridaeiacld.ir/DL/modavan/viruses/92/hivdrparsania.pdfRetroviridae • Retroviruses...
RetroviridaeRetroviridae
• Retroviruses (family Retroviridae) areRetroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses that replicate through a DNA intermediatethat replicate through a DNA intermediate using reverse transcriptase.
• This large and diverse family includes• This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorderswith a variety of immune system disorders, and cause degenerative and neurological syndromessyndromes.
Retrovirus Virions Thin Section EM of Some Retrovirusesf
Type A
Type B (eccentric)MMTV
T C ( t l)Type C (central)ALV,RSV
Type D (bar)
Lentivirus (cone)HIV
Retrovirus structure
• Retrovirus virions are 80-120 nm in diameter have spherical morphology a• Retrovirus virions are 80-120 nm in diameter, have spherical morphology, a phospholipid envelope with knobs
• Contain around 2000 molecules of nucleocapsid (NC) protein that bind to the two copies of (+) strand RNA genomecopies of (+) strand RNA genome
•Retroviral ribonucleoproteins are encased within a protein shell built from the capsid protein to form an internal core, which can have different shapes and has a conical shape in HIVa conical shape in HIV
Retrovirus Structure and Function
Figure 10.23a
Retrovirus ClassificationFamily: RetroviridaeFamily: Retroviridae
Genus Features Examples1. Alpharetrovirus Simple,
OncoAvian leucosis virus, RSV
2. Betaretrovirus Simple, Mouse Mammary Tumor Onco Virus
3. Gammaretrovirus Simple, Onco
Murine leukemia virus (Moloney, Harvey)( y y)
4. Deltaretrovirus Complex, Onco
Bovine Leukemia, Human T Cell Leukemia (HTLV)
5 Epsilonretrovirus Complex Walleye Dermal Sarcoma5. Epsilonretrovirus Complex, Onco
Walleye Dermal Sarcoma
6. Lentivirus Complex HIV, Visna, EIAV
7. Spumavirus Complex Simian Foamy Virus
• RNA virus, 120nm in diameter
Family : Retroviridae
• Envelope gp160; gp120 & gp41
Subfamily : Lentivirus
gp41
• Icosahedral symmetry
N l id• Nucleocapsid
Outer matrix protein (p17)
M j id t i ( 24)Major capsid protein (p24)
Nuclear protein (p7)
Di l id RNA i h l• Diploid RNA with several copies of reverse transcriptasetranscriptase
Retrovirus replication cycle
1. Attachment of the virion to a specific cell surface receptor
2. Penetration of the virion core into the cell
3. Reverse transcription within the core structure to copy the genome RNA into DNA
i f h h l4. Transit of the DNA to the nucleus
5. Integration of the viral DNA into random sites in cellular DNA to form the provirus
6. Synthesis of viral RNA by cellular RNA polymerase II using the integrated provirus as a template
7. Processing of the transcripts to genome and mRNAs
8. Synthesis of virion proteins
9 A bl d b ddi f i i9. Assembly and budding of virions
10. Proteolytic processing of capsid proteins
Retroviruslife cycle:f y
Electron micrograph of HIV buddingElectron micrograph of HIV budding
HIV Structure
surface transmembrane
matrix protein
capsid protein
nucleocapsid protein
RTIntegraseprotease
Retroviral Proteins• gag, pol, and env
Gag protein proteolytically processed into– Gag protein proteolytically processed into• MA (matrix)• CA (capsid)• NC (nucleocapsid)NC (nucleocapsid)
– Pol protein encodes enzymes• PR (protease) • RT (Reverse Transcriptase which has both DNA polymerase and RNase( p p y
H activities)• IN (Integrase)
– Env protein encodesSU f l i• SU surface glycoprotein
• TM transmembrane protein• “Accessory” genes (in Complex Retroviruses) - regulate and
coordinate virus expression; function in immune escapecoordinate virus expression; function in immune escape• Oncogene products (v-Onc, in Acutely Transforming
Retroviruses) - produce transformed phenotype
Regulatory proteins: Tat
• HIV LTR functions as a promoter in a variety of cell types in vitro
• It includes an enhancer sequence that binds a number of cell type specific q yp ptranscription activators
Stimulation of transcription by HIV‐1 Tat protein:by HIV 1 Tat protein:
• Before Tat is made proviral transcripts are terminated within 60 bp of the initiation site
d f h ll• Production of the Tat protein allows transcription complexes to synthesize full length RNA
• Binding of Tat to TAR together with the cyclin T b it f T k l d t ti l ti fsubunit of Tak leads to stimulation of
phosphorylation of the largest subunit of RNA polymerase II
A lt th t i ti l l b• As a result, the transcriptional complexes become competent to carry out transcription
Regulatory proteins: Rev
• Rev Protein is an RNA binding protein that recognizes a specific sequence within the structural element in env called the Rev‐responsive element (RRE)
HIV accessory proteins
Nef protein:
• Translated from multiply spliced• Translated from multiply spliced early transcripts
• myristylated at its N‐terminus and myristylated at its N terminus andanchored to the inner surface of the plasma membrane
• Nef deleted HIV and SIV are much less pathogenic in vivo
• Nef downregulates expression of CD4 by enhancing endocytosis
C ti t CD4 T l h t b• Can activate CD4+ T lymphocytes by modulating signaling pathways
Vif Protein:HIV accessory proteins
• Viral infectivity factor
• Accumulates in the cytoplasm and at the plasma membrane of infected cells
• Mutant viruses lacking the vif gene were less infectious d d f ti iand defective in some way
• vif‐defective virions enter cells, initiate reverse transcription but do not produce full‐length doubletranscription, but do not produce full‐length double stranded DNA
• vif inhibits antiviral action of a cytidine deaminase, which vif inhibits antiviral action of a cytidine deaminase, which is synthesized in nonpermissive cells
•This enzyme deaminates deoxycytidine to deoxyuridiney y y yand leads to endonucleolytic digestion or G to A transitions
9200 l tid (HIV 1)
HIV Genome• 9200 nucleotides (HIV‐1):
• env ‐ gp160 (gp120:outer membrane part, gp41: transmembrane part)
• gag core proteins – p55, p17 and p24
• pol – p66 (protease), p31,p51 (integrase/endonuclease)
EARLY Accessory Genes LATEEARLY Accessory Genes LATEtat ‐ trans activator of transcription vif ‐ viral infectivity factor
vpr‐ viral protein Rl t f i l t i i i l t i Urev‐ regulator of viral protein expression vpu‐ viral protein U
nef – negative regulatory factor vpx – HIV ‐ 2 TAT and REV are essential for HIV replicationand REV are essential for HIV replication
Subtyping of isolatesSubtyping of isolates
• RFLP AnalysisRFLP Analysis• Nucleotide sequence analysisS b ifi i b• Subtype specific genetic probes
Genetic variation in HIVGenetic variation in HIV
Mutations occur 65 times more than that observed in Influenza virusf
HIV‐1 – Three groups HIV‐ 2 –eightM (M j )M (Major) subtypes A‐HO (Outlier)( )N (New)
Subtypes of HIVSubtypes of HIV
Subtypes of HIV‐1 – MSubtypes of HIV 1 M
• 10 Subtypes/Clades/Genotypes10 Subtypes/Clades/GenotypesDesignated A‐K Differ
in Geographical distribution and majorin Geographical distribution and major mode of TransmissionAfrica ‐ A C D US and WesternAfrica A,C , D US and WesternThailand ‐ E, B Europe ‐ BI di CIndia ‐ C
TransmissionTransmission
• Sexual Activity – both homosexual and yheterosexual. – Women are more easily infected through intercourse than are menthan are men.
• Injecting drug use• Tainted blood transfusions – extremely rare sinceTainted blood transfusions extremely rare since 1986.
• Transmission across the placental barrier, during the p gbirth process, or through mother’s milk.
HIV TransmissionHIV Transmission• HIV enters the bloodstream through:
– Open Cuts
– Breaks in the skin
– Mucous membranes
– Direct injectionDirect injection
HIV TransmissionHIV Transmission• Common fluids that are a means of t i itransmission:
Blood– Blood
Semen– Semen
– Vaginal SecretionsVaginal Secretions
– Breast MilkBreast Milk
HIV in Body FluidsHIV in Body Fluids
Blood Semen11,000 Vaginal
Fluid7 000
Blood18,000
Amniotic Fl id7,000 Fluid4,000 Saliva
1
Average number of HIV particles in 1 ml of these body fluids
ENTRY OF HIV INSIDET CELLT CELL
HIV ‐ Life Historyh
HIV ‐ Life HistoryhCCR5 ‐ the co receptorCCR5 ‐ the co receptor
HIVHIV
chemokineMutant
CD4
CD4CD4
CCR5CCR5
CCR5
macrophage
Virion interaction with CD4 receptor and CXCR4 co‐receptorreceptor
HIV and AIDSThe cellular and immunological picture - The course of the disease
July 2008 e
SerologySerology
Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection
• Direct demonstration of infective agent Direct demonstration of infective agent- Virus isolation- virus culture- Antigen detection- P24 detectiong- viral nucleic acid detection- PCR
• Indirect demonstration of infective agent- Anti -HIV antibody detection
Types of HIV Diagnostic TestsTypes of HIV Diagnostic Tests
HIV Antibodies HIV‐1 RNA HIV p24 Antigen
Most Common Test for Established Infection
Most Common Test for Established Infection
Rarely Used. Future use: 4th
Generation EIARarely Used. Future use: 4th
Generation EIAUsed for Acute HIV and
Indeterminate WBUsed for Acute HIV and
Indeterminate WBEstablished InfectionEstablished Infection Generation EIAGeneration EIAIndeterminate WBIndeterminate WB
Specimens to be collected forA ib d d iAntibody detection
• Blood / Serum / Plasma Blood / Serum / Plasma• Saliva / Urine
Note!Note!Specimen requiring storage before shipping to lab:-No longer than 7 days at 4◦CNo longer than 7 days at 4 C- No longer than 3 days at RT- For longer period serum or plasma must be separated from
clot or cells and stored at -20 ◦C- For PCR testing samples need to be processed within 48 h 0f
collection.collection.
Initial and Supplemental HIV Tests
• Initial Test
pp
• Initial Test- Enzyme Immunoassay (EIA)
Ch il i t I (CIA)- Chemiluminescent Immunoassay (CIA)
• Supplemental Tests• Supplemental Tests- Western blot
I di I fl A (IFA)- Indirect Immunofluorescence Assay (IFA)- Qualitative HIV-1 RNA
Generation of EIA Tests
First Second Third *Fourth
U d i lU d i l D t t I M d I GD t t I M d I GU bi t HIVU bi t HIV D t t HIV tib diD t t HIV tib di
*Not US FDA‐approved as of 10/1/09
Uses crude viral lysate
Uses crude viral lysate
Detects IgM and IgG in “Sandwich” EIA
Detects IgM and IgG in “Sandwich” EIA
Uses recombinant HIV antigens or peptides
Uses recombinant HIV antigens or peptides
Detects HIV antibodies and p24 antigen
Detects HIV antibodies and p24 antigen
Rapid testsRapid testsAdvantages:1. Quick 2. Easy to perform3 N hi ti t d i t t i d3. No sophisticated instruments are required4. Can be done on single sample
Disadvantages:1. Costly1. Costly2. Tedious if large no. samples have to be tested at one time
FACTORS KNOWN TO CAUSE FALSE POSITIVE HIV ANTIBODY TEST RESULTS
Passive immunization: receipt of gamma globulin or immune globulin (as prophylaxis against infection which contains g ( p p y gantibodies)Leprosy Tuberculosis Mycobacterium aviumSystemic lupus erythematosusRenal (kidney) failureRenal (kidney) failure Hemodialysis/renal failure Alpha interferon therapy in hemodialysis patients FluFlu Flu vaccination Herpes simplex I & Herpes simplex II Upper respiratory tract infection (cold or flu)Upper respiratory tract infection (cold or flu)Recent viral infection or exposure to viral vaccinesMalaria
FACTORS KNOWN TO CAUSE FALSE
Pregnancy in multiparous women
POSITIVE HIV ANTIBODY TEST RESULTS Pregnancy in multiparous women Hepatitis B vaccinationTetanus vaccination Organ transplantationOrgan transplantationRenal transplantationAnti‐lymphocyte antibodiesAnti‐collagen antibodies Serum‐positive for rheumatoid factor, antinuclear antibody (both found in rheumatoid arthritis and other autoantibodies) ( )Autoimmune diseases lupus erythematosus, scleroderma, connective tissue diseaseAcute viral infections DNA viral infectionsAcute viral infections, DNA viral infections Malignant neoplasms
Confirmatory Tests
Western Blot, Line immunoassay
• WB use antigens from whole virus lysates electrophoretically transferred to a membrane
• LIA use recombinant or synthetic HIV antigens mechanically applied on to support membranemechanically applied on to support membrane
• Presence or absence of bands is scored
• Highly specific, Labor intensive, expensive -WHO criteria-presence of at least 2 envelope bands (gp120 gp160 gp41)presence of at least 2 envelope bands (gp120, gp160, gp41)
HIV-1 Western Blot AntigensHIV 1 Western Blot Antigens
p = protein
gp = glycoprotein
Number = molecular weightNumber = molecular weight
Components Used in HIV-1 Western BlotComponents Used in HIV 1 Western Blot
HIV Western blot StripHIV Western blot Strip
Color Reagent
Human HIV AntibodiesY YY YYYYY Y
Antihuman IgG AntibodiesEnzyme Detector
Human HIV Antibodies(from patient serum)
Y YY YYY
HIV Antigens( W bl )(on Western blot)
HIV-1 and HIV-2 Gene Products &W BlWestern Blot
Interpretive Criteria for HIV-1 Western Blot
Positive ControlPositive Control PositivePositiveNegativeNegative IndeterminateIndeterminateAt least two of the Following bands: p24, gp41, gp120/160
No bands:One or more bands presentNot meeting positive criteria
Direct Methods of HIV diagnosis:24 i d ip24 antigen detection
• EIA for detection of p24 antigen in serum, plasma, CSF or p g pcell culture
• Can detect infection in window period in late stage of• Can detect infection in window period, in late stage of disease ,and in newborns
• To monitor response to anti-retroviral therapy
• To monitor disease progression• To monitor disease progression
• Negative test does not rule out HIV infection
Detection of HIV nucleic acid( A A)(RNA or DNA)
• Polymerase chain reaction (PCR)y ( )
– To detect and quantify the viral nucleic acid in infected lymphocytes in blood in serum and in culture supernatantlymphocytes in blood, in serum and in culture supernatant
• Application of PCR–HIV detection in newborn
– Window period– Resolution of indeterminate ELISA/WB– Resolution of indeterminate ELISA/WB– Characterization of isolates– Measurement of virus load.
Virus isolationVirus isolation
• HIV can be cultured from blood (PBMC), semen, vaginal/cervical specimen, tissue, CSF and plasma
• Direct stimulation of patient’s lymphocytes or co-cultivation of ti t’ l h t ith h lth l h tpatient’s lymphocytes with healthy lymphocytes
• 98% positivity
• Virus can be isolated in window period
• Procedure is expensive, labor intensive and time consuming
• Procedure is used only in research settings
Virus isolationVirus isolation
CO CULTURE METHOD•CO-CULTURE METHOD
– PBMCs from heterologous HIV un-infected donors are stimulated with PHA and after 48–72 hrs the stimulated cells are cultured along with the PBMCs from the patientg p
HIV Diagnosis during window periodHIV Diagnosis during window period
• Need for laboratory diagnosis in window period Need for laboratory diagnosis in window period- Following untested blood transfusion- Risky heterosexual/homosexual exposurey p- Needle stick injury
• By demonstrating virus and virus components- PCR- p24 antigen assay (40%)- Viral culture
Window periodWindow period
• Early ELISA & WB- 2.1 months (3wks- 3 mths)y ( )
• Sandwich ELISA (III gen ELISA)- 6wks
• IV generation ELISA- 16-18 days
• NAT- 12-14 days
Disinfection & InactivationHIV is completely inactivated ( 105 units of infectivity) by treatment for 10 minutes at room temperature with any of the p yfollowing:
10% household bleach50% ethanol50% ethanol35% isopropanol0.5% paraformaldehyde0.3% hydrogen peroxide
The virus is also inactivated by extremes of pH (pH 1.0, pH y p (p , p13.0).However, when HIV is present in clotted or unclotted blood in a needle or syringe exposure to undiluted bleach for at leasta needle or syringe, exposure to undiluted bleach for at least 30 seconds is necessary for inactivation.
HIV is readily inactivated in liquids or 10% b h i 56 °C f 10 i bserum by heating at 56 °C for 10 minutes, but
dried proteinaceous material affords marked t ti L hili d bl d d t ldprotection. Lyophilized blood products would
need to be heated at 68 °C for 72 hours to i ti ti f t i ti iensure inactivation of contaminating virus.