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.