Retroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses. members that are oncogenic, are associated

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2 Retroviridae Retroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase. This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorders, and cause degenerative and neurological syndromes.

3 Retrovirus Virions Thin Section EM of Some Retroviruses Type A Type B (eccentric) MMTV Type C (central) ALV,RSV Type D (bar) Lentivirus (cone) HIV

4 Retrovirus structure Retrovirus virions are 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 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 HIV

5 Retrovirus Structure and Function Figure 10.23a

6 Retrovirus Classification Family: Retroviridae Genus Features Examples 1. Alpharetrovirus Simple, Avian leucosis virus, RSV Onco 2. Betaretrovirus Simple, Mouse Mammary Tumor Onco Virus 3. Gammaretrovirus Simple, Onco 4. Deltaretrovirus Complex, Onco Murine leukemia virus (Moloney, Harvey) Bovine Leukemia, Human T Cell Leukemia (HTLV) 5. Epsilonretrovirus Complex, Walleye Dermal Sarcoma Onco 6. Lentivirus Complex HIV, Visna, EIAV 7. Spumavirus Complex Simian Foamy Virus

7 RNA virus, 120nm in diameter Envelope gp160; gp120 & gp41 Family : Retroviridae Subfamily : Lentivirus Icosahedral symmetry Nucleocapsid Outer matrix protein (p17) Mj Major capsid protein ti (p24) Nuclear protein (p7) Diploid idrna with ihseveral copies of reverse transcriptase

8 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 4. 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. Assembly and budding of virions ii 10. Proteolytic processing of capsid proteins

9 Retrovirus life cycle:

11 Electron micrograph of HIV budding

12 HIV Structure surface transmembrane matrix protein capsid protein nucleocapsid protein RT Integrase protease

13 gag, pol, and env Retroviral Proteins Gag protein proteolytically processed into MA (matrix) CA (capsid) NC (nucleocapsid) Pol protein encodes enzymes PR (protease) RT (Reverse Transcriptase which has both DNA polymerase and RNase H activities) IN (Integrase) Env protein encodes SU surface glycoprotein TM transmembrane protein Accessory genes (in Complex Retroviruses) - regulate and coordinate virus expression; function in immune escape Oncogene products (v-onc, in Acutely Transforming Retroviruses) - produce transformed phenotype

14 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 p transcription activators

15 Stimulation of transcription by HIV 1 Tat protein: Before Tat is made proviral transcripts are terminated within 60 bp of the initiation site Production of the Tat protein allows transcription complexes to synthesize full length RNA Binding of Tat to TAR together with the cyclin T subunit of Tak Tkleads 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

16 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)

17 HIV accessory proteins Nef protein: Translated from multiply spliced early transcripts myristylated at itsn N terminus terminusand anchored 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

18 Vif Protein: Viral infectivity factor HIV accessory proteins Accumulates in the cytoplasm and at the plasma membrane of infected cells Mutant viruses lacking the vif gene were less infectious and df defective in some way vif defective virions enter cells, initiate reverse transcription, butdonotproducefull lengthdouble double stranded DNA vif inhibitsantiviral action of a cytidinedeaminase, deaminase, which is synthesized in nonpermissive cells This enzyme deaminates deoxycytidine to deoxyuridine y yy y and leads to endonucleolytic digestion or G to A transitions

19 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 LATE tat trans activator of transcription vif viral infectivity factor vpr viral protein R rev regulator lt of viral protein ti expression vpu viral protein ti U nef negative regulatory factor vpx HIV 2 TAT and REV are essential for HIV replication

20 Subtyping of isolates RFLP Analysis Nucleotide sequence analysis Subtype specific genetic probes

21 Genetic variation in HIV Mutations occur 65 times more than that observed in Influenza virus HIV 1 Three groups HIV 2 eight M (Major) subtypes A H O (Outlier) N (New)

22 Subtypes of HIV

23 Subtypes of HIV 1 M 10 Subtypes/Clades/Genotypes Designated A K Differ in Geographical distribution and major mode of Transmission Africa A,C, D US and Western Thailand E, B India C Europe B

25 Transmission Sexual Activity both homosexual and heterosexual. Women are more easily infected through intercourse than are men. Injecting drug use Tainted bloodtransfusions extremely rare since Transmission across the placental barrier, during the birth process, or through mother s milk.

26 HIV Transmission HIV enters the bloodstream through: Open Cuts Breaks in the skin Mucous membranes Direct injection

27 HIV Transmission Common fluids that are a means of transmission: i Blood Semen Vaginal Secretions Breast Milk

28 HIV in Body Fluids Blood 18,000 Semen 11,000 Vaginal Fluid Amniotic 7,000 Fluid 4,000 Saliva 1 Average number of HIV particles in 1 ml of these body fluids

29 ENTRY OF HIV INSIDE T CELL

31 HIV Life History CCR5 the co receptor HIV chemokine CD4 CCR5 CD4 CCR5 CD4 Mutant CCR5 macrophage

32 Virion interaction with CD4 receptor and CXCR4 co receptor

34 HIV and AIDS The cellular and immunological picture - The course of the disease

35 July 2008 e

36 Serology

37 Laboratory diagnosis of HIV infection Direct demonstration of infective agent - Virus isolation- virus culture -Antigen detection- P24 detection - viral nucleic acid detection- PCR Indirect demonstration of infective agent - Anti -HIV antibody detection

38 Types of HIV Diagnostic Tests HIV Antibodies HIV 1 RNA HIV p24 Antigen Most Common Test for Established Infection Used for Acute HIV and Indeterminate WB Rarely Used. Future use: 4 th Generation EIA

39 Specimens to be collected for Blood / Serum / Plasma Saliva / Urine Antibody detection Note! Specimen requiring storage before shipping to lab: -No 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.

40 Initial and Supplemental HIV Tests Initial Test - Enzyme Immunoassay (EIA) - Chemiluminescent i Immunoassay (CIA) Supplemental Tests - Western blot - Indirect Immunofluorescence Assay (IFA) - Qualitative HIV-1 RNA

41 Generation of EIA Tests First Second Third *Fourth *Not US FDA approved as of 10/1/09 Uses crude viral Uses recombinant HIV Detects t IgM and IgG Detects t HIV antibodies lysate antigens or peptides in Sandwich EIA and p24 antigen

42 Advantages: Rapid tests 1. Quick 2. Easy to perform 3. No sophisticated t instruments t are required 4. Can be done on single sample Disadvantages: 1. Costly 2. Tedious if large no. samples have to be tested at one time

43 FACTORS KNOWN TO CAUSE FALSE POSITIVE HIV ANTIBODY TEST RESULTS Passive immunization: receipt of gamma globulin or immune globulin (as prophylaxis p against infection which contains antibodies) Leprosy Tuberculosis Mycobacterium avium Systemic lupus erythematosus Renal (kidney) failure Hemodialysis/renal failure Alpha interferon therapy in hemodialysis patients Flu Flu vaccination Herpes simplex I & Herpes simplex II Upperrespiratory respiratory tract infection (cold or flu) Recent viral infection or exposure to viral vaccines Malaria

44 FACTORS KNOWN TO CAUSE FALSE POSITIVE HIV ANTIBODY TEST RESULTS Pregnancy in multiparous women Hepatitis B vaccination Tetanus vaccination Organ transplantation Renal transplantation Anti lymphocyte antibodies Anti collagen antibodies Serum positive for rheumatoid factor, antinuclear antibody (both found in rheumatoid arthritis and other autoantibodies) Autoimmune diseases lupus erythematosus, scleroderma, connective tissue disease Acute viral infections, DNA viral infections Malignant neoplasms

45 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 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)

46 HIV-1 Western Blot Antigens p = protein gp = glycoprotein Number = molecular weight

47 Components Used in HIV-1 Western Blot HIV Western blot Strip Color Reagent Y Y Y Enzyme Detector Antihuman IgG Antibodies Y Y Y Y YY Human HIV Antibodies (from patient serum) HIV Antigens (on Western blot)

48 HIV-1 and HIV-2 Gene Products & Western Blot

49 Interpretive Criteria for HIV-1 Western Blot Positive Control Negative Indeterminate Positive No bands: One or more bands present Not meeting positive criteria At least two of the Following bands: p24, gp41, gp120/160

50 Direct Methods of HIV diagnosis: p24 antigen detection EIA for detection of p24 antigen in serum, plasma, CSF or cell culture Candetect infection in window period, in late stage of disease,and in newborns To monitor response to anti-retroviral therapy Tomonitor disease progression Negative test does not rule out HIV infection

51 Detection of HIV nucleic acid (RNA or DNA) Polymerase chain reaction (PCR) To detect and quantify the viral nucleic acid in infected lymphocytes in blood, in serum and in culture supernatant Application of PCR HIV detection in newborn Window period Resolution of indeterminate ELISA/WB Characterization of isolates Measurement of virus load.

52 Virus 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 patient s t 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

53 Virus isolation CO-CULTURE CULTURE METHOD PBMCs from heterologous HIV un-infected donors are stimulated with PHA and after hrs the stimulated cells are cultured along with the PBMCs from the patient

54 HIV Diagnosis during window period Need for laboratory diagnosis in window period - Following untested blood transfusion -Risky heterosexual/homosexual exposure - Needle stick injury By demonstrating virus and virus components -PCR - p24 antigen assay (40%) - Viral culture

55 Window period Early ELISA & WB- 2.1 months (3wks- 3 mths) Sandwich ELISA (III gen ELISA)- 6wks IV generation ELISA days NAT days

56 Disinfection & Inactivation HIV is completely inactivated ( 10 5 units of infectivity) by treatment for 10 minutes at room temperature with any of the following: 10% household bleach 50% ethanol 35% isopropanol 0.5% paraformaldehyde 0.3% hydrogen peroxide The virus is also inactivated by extremes of ph (ph 1.0,,pH 13.0). However, when HIV is present in clotted or unclotted blood in a needle or syringe, exposure to undiluted bleach for at least 30 seconds is necessary for inactivation.

57 HIV is readily inactivated in liquids or 10% serum by heating at 56 C for 10 minutes, but dried proteinaceous material affords marked protection. ti Lyophilized blood products would need to be heated at 68 C for 72 hours to ensure inactivation of contaminating ti virus.

Immunologic Methods in Diagnosis of HIV Infection. Tehran Medical Sciences Branch, Islamic Azad

Immunologic Methods in Diagnosis of HIV Infection M Parsania, Ph.D. Tehran Medical Sciences Branch, Islamic Azad University Retroviridae Retroviruses (family Retroviridae) id ) are enveloped, single stranded