1. Human Retroviruses Dr. Medhat K. Shier Virology Consultant

2. Retroviruses RNA viruses single stranded, positive sense, enveloped, icosahedral. Distinguished from all other RNA viruses by presence of an unusual enzyme, reverse transcriptase. Retro = reversal RNA is serving as a template for DNA synthesis. Two genera of human interest LentivirusLentus = slow Human immunodeficiency viruses 1 & 2 (HIV-1 & -2) Human T-cell lymphotropic virus-bovine leukemia virus group (HTLV-BLV) Human T-cell leukemia viruses 1 & 2 (HTLV-1 & -2)

3. Structure The viral envelope formed from host cell membrane; contains 72 spiked knobs. These consist of a transmembrane protein TM (gp 41), which is linked to surface protein SU (gp 120) that binds to a cell receptor during infection. The virion has cone-shaped, icosahedral core, containing the major capsid protein CA (p24). Between capsid and envelope is an outer matrix protein, MA (p17). Two identical copies of positive sense ssRNA genome (retroviruses are diploid). Enzymes: reverse transcriptase, integrase and protease.

4. HIV particles

7. HIV Genome Three major genes Gag gene codes for CA, MA and NC proteins Gag gene codes for CA, MA and NC proteins Pol gene codes for reverse transcriptase, protease, integrase and ribonuclease. Pol gene codes for reverse transcriptase, protease, integrase and ribonuclease. Env gene codes for TM and SU Env gene codes for TM and SU

8. HIV Replication The first phase of viral entery, reverse transcription and integration into host genome is accomplished by viral proteins. The second phase of synthesis and processing of viral genomes, mRNAs and structural proteins, uses host cell machinery. Attachment to specific cell surface receptor: gp120 binds CD4 molecule on the helper T cells, monocytes and dendritic cells Attachment to specific cell surface receptor: gp120 binds CD4 molecule on the helper T cells, monocytes and dendritic cells Viral entery. Viral entery. Reverse transcription of viral RNA into DNA. The resulting double stranded DNA is called provirus. Reverse transcription of viral RNA into DNA. The resulting double stranded DNA is called provirus. Integration of provirus into host cell DNA. The viral integrase cleaves the chromosomal DNA and covalently inserts the provirus. The insertion site is random. Integration of provirus into host cell DNA. The viral integrase cleaves the chromosomal DNA and covalently inserts the provirus. The insertion site is random. Transcription and translation of viral DNA sequences. The provirus is transcribed into a full length mRNA by the cell RNA polymerase II. Transcription and translation of viral DNA sequences. The provirus is transcribed into a full length mRNA by the cell RNA polymerase II. Assembly and maturation of progeny virus. Assembly and maturation of progeny virus.

10. HIV Acquired immunodeficiency syndrome (AIDS) was first reported in US in 1981. By 1984, AIDS was recognized as an infectious disease caused by a virus.

11. Transmission of HIV Sexual contact: HIV is present in semen and vaginal secretions; either homoxesual or heterosexual contact Sexual contact: HIV is present in semen and vaginal secretions; either homoxesual or heterosexual contact Transfusions: whole blood, plasma, clotting factors or cellular fractions of blood. Transfusions: whole blood, plasma, clotting factors or cellular fractions of blood. Contaminated needles: accidentally or sharing needles by drug users. Contaminated needles: accidentally or sharing needles by drug users. Perinatal: Transplacental, during delivery or via breast milk. Perinatal: Transplacental, during delivery or via breast milk.

12. Pathogenesis and clinical significance (1) Initial infection: Initial infection: genital tract macrophages genital tract macrophages HIV disseminates via blood HIV disseminates via blood Dendritic cells in lymphoid tissue Dendritic cells in lymphoid tissue CD4+ lymphocytes CD4+ lymphocytes Acute phase viremia: several weeks after the initial infection, 1/3 – 2/3 of individuals experience an acute disease syndrome similar to infectious mononucleosis. Circulating antibody appears in 1 – 10 weeks after the initial infection (seroconversion). Acute phase viremia: several weeks after the initial infection, 1/3 – 2/3 of individuals experience an acute disease syndrome similar to infectious mononucleosis. Circulating antibody appears in 1 – 10 weeks after the initial infection (seroconversion). Latent period: lasts from months to many years (average 10 years). During this period, 90% of HIV proviruses are transcriptionally silent. Although there is continous loss of CD4+ cells in which HIV is replicating, active replacement through stem cell multiplication is occurring. The infection remains clinically asymptomatic as long as the immune system is functional. Latent period: lasts from months to many years (average 10 years). During this period, 90% of HIV proviruses are transcriptionally silent. Although there is continous loss of CD4+ cells in which HIV is replicating, active replacement through stem cell multiplication is occurring. The infection remains clinically asymptomatic as long as the immune system is functional.

13. Pathogenesis and clinical significance (2) Clinical complications during the latent period: there are multiple non-specific conditions such as persistent generalized lymadenopathy, diarrhea, chronic fevers, night sweats and weight loss. The more common opportunistic infections such as herpes zoster and candidiasis may occur repeatedly during this period. The CD4+ cell count remains normal or gradually declines but is greater than 200 / ul. Progression from asymptomatic infection to AIDS is not sudden. Clinical complications during the latent period: there are multiple non-specific conditions such as persistent generalized lymadenopathy, diarrhea, chronic fevers, night sweats and weight loss. The more common opportunistic infections such as herpes zoster and candidiasis may occur repeatedly during this period. The CD4+ cell count remains normal or gradually declines but is greater than 200 / ul. Progression from asymptomatic infection to AIDS is not sudden. AIDS: Coinfection with HHV-6 can transactivate transcription from the silent HIV provirus, increasing HIV replication. Any stimulation of an immune response causing activation of resting T cells also activates HIV replication. Appearance of HIV mutants with altered antigenic specificity which are not recognized by the existing humoral antibody or cytotoxic T lymphocytes; also contributes to progression with CD4+ count falling below 200 / ul and appearance of serious diseases and opportunistic infections. AIDS: Coinfection with HHV-6 can transactivate transcription from the silent HIV provirus, increasing HIV replication. Any stimulation of an immune response causing activation of resting T cells also activates HIV replication. Appearance of HIV mutants with altered antigenic specificity which are not recognized by the existing humoral antibody or cytotoxic T lymphocytes; also contributes to progression with CD4+ count falling below 200 / ul and appearance of serious diseases and opportunistic infections.

14. Opportunistic Infections (1) Bacteria: Mycobacterium avium complex Mycobacterium avium complex Disseminated miliary disease Disseminated miliary disease Chronic bronchitis, pneumonia Chronic bronchitis, pneumonia Chronic osteomyelitis, renal infection Chronic osteomyelitis, renal infection Mycobacterium tuberculosis Mycobacterium tuberculosis Chronic pneumonitis, osteomyelitis Chronic pneumonitis, osteomyelitis Meningitis, miliary disease Meningitis, miliary disease Streptococcus pneumoniae Streptococcus pneumoniae Salmonella spp. Salmonella spp. Haemophilus influenza (pneumonia) Haemophilus influenza (pneumonia) Campylobacter spp. (diarrhea) Campylobacter spp. (diarrhea) Shigella spp. (diarrhea) Shigella spp. (diarrhea)

15. Opportunistic Infections (2) Fungi: Candida spp. Oral, vaginal or systemic candidiasis Candida spp. Oral, vaginal or systemic candidiasis Histoplasma capsulatum (disseminated disease) Histoplasma capsulatum (disseminated disease) Cryptococcus neoformans (meningitis) Cryptococcus neoformans (meningitis) Pneumocystis carinii Unicellular eukaryote Unicellular eukaryote Taxonomic status is uncertain Taxonomic status is uncertain Most common opportunistic pathogen in AIDS patients Most common opportunistic pathogen in AIDS patients Fatal pneumonia Fatal pneumonia

16. Opportunistic Infections (3) Parasites: Toxoplasma gondii (focal encephalitits) Toxoplasma gondii (focal encephalitits)Viruses: HHV-8 (Kaposi’s sarcoma-associated herpesvirus) HHV-8 (Kaposi’s sarcoma-associated herpesvirus) HSV (oral, genital ulcers) HSV (oral, genital ulcers) JCV (progressive multifocal leukoencephalopathy, affect the white matter of the brain) JCV (progressive multifocal leukoencephalopathy, affect the white matter of the brain) CMV (Chorioretinitis, encephalitis, enterocolitis, gastritis) CMV (Chorioretinitis, encephalitis, enterocolitis, gastritis)

17. Malignancies associated with AIDS Kaposi’s sarcomaHHV-8 Kaposi’s sarcomaHHV-8 LymphomasEBV LymphomasEBV

21. Laboratory Diagnosis Antigen / antibody detection ELISA, serum ELISA, serum HIV-1 & -2 antibodies, HIV-1 CA (p24) antigen HIV-1 & -2 antibodies, HIV-1 CA (p24) antigen Screening of blood donors Screening of blood donors Western Blotting Western BlottingPCR Viral RNA or DNA provirus Viral RNA or DNA provirus Blood or tissue specimens Blood or tissue specimens Quantitative PCR (viral load): to determine disease stage and treatment follow up. Quantitative PCR (viral load): to determine disease stage and treatment follow up.

22. ELISA for HIV antibody Microplate ELISA for HIV antibody: coloured wells indicate reactivity

23. Western blot for HIV antibody There are different criteria for the interpretation of HIV Western blot results e.g. CDC, WHO, American Red Cross. There are different criteria for the interpretation of HIV Western blot results e.g. CDC, WHO, American Red Cross. The most important antibodies are those against the envelope glycoproteins gp120, and gp41 The most important antibodies are those against the envelope glycoproteins gp120, and gp41 p24 antibody is usually present but may be absent in the later stages of HIV infection p24 antibody is usually present but may be absent in the later stages of HIV infection

25. Treatment Anti-retroviral drugs Anti-retroviral drugs Reverse transcriptase inhibitors Reverse transcriptase inhibitors Protease inhibitors Protease inhibitors Multidrug therapy Multidrug therapy RT has no proofreading activity, resulting in production of many errors during viral DNA synthesis which leads to mutations in all HIV genes and accumulation of mutant viral strains. In presence of an antiviral drug, there is strong selection for mutations that confer resistance to that drug.Use multidrug therapy RT has no proofreading activity, resulting in production of many errors during viral DNA synthesis which leads to mutations in all HIV genes and accumulation of mutant viral strains. In presence of an antiviral drug, there is strong selection for mutations that confer resistance to that drug.Use multidrug therapy Early therapy Early therapy Viral load is a prognostic indicator of the rate of progression to AIDS. Infection should be treated as aggressively and as early as possible to minimize initial spread of the virus and give a lower chance for mutants to arise. Viral load is a prognostic indicator of the rate of progression to AIDS. Infection should be treated as aggressively and as early as possible to minimize initial spread of the virus and give a lower chance for mutants to arise.

26. Highly active antiretroviral therapy (HAART) Nucleoside analog reverse transcriptase inhibitors Act by serving as a chain terminator Act by serving as a chain terminator Zidovudine (AZT) Zidovudine (AZT) Didanosine (ddi) Didanosine (ddi) Lamivudine (3TC) Lamivudine (3TC) Non-nucleoside reverse transcriptase inhibitors Act by targeting the enzyme itself Act by targeting the enzyme itself Efavirenz Efavirenz Delaviridine Delaviridine Nevirapine Nevirapine Protease inhibitors Interfere with the processing of polyproteins in the budding virion, resulting in non-infectious particle. Interfere with the processing of polyproteins in the budding virion, resulting in non-infectious particle. Ritonavir Ritonavir Amprenavir Amprenavir Indinavir Indinavir Lopinavir Lopinavir

27. Drug resistance through viral mutations is a major problem in treatment of HIV patients

28. Prevention Vaccine: not yet available Vaccine: not yet available Blood supply screening Blood supply screening Perinatal transmission: AZT therapy Perinatal transmission: AZT therapy

29. HTLV HTLV-1 and -2 have 65% nucleotide sequence homology HTLV-1 and -2 have 65% nucleotide sequence homology Genetically and biologically similar Genetically and biologically similarHTLV-1 Adult T-cell leukemia Adult T-cell leukemia HTLV-associated myelopathy/tropical spastic paraparesis HTLV-associated myelopathy/tropical spastic paraparesisHTLV-2 Hairy cell leukemia Hairy cell leukemia

30. Transmission Vertical transmission Vertical transmission Sexual Sexual Blood products Blood products

31. Hairy cell leukemia Hairy cell leukemia a rare lymphocytic leukemia, of B cell origin; caused by HTLV-2. it is characterized by malignant cells that look ciliated. a rare lymphocytic leukemia, of B cell origin; caused by HTLV-2. it is characterized by malignant cells that look ciliated.

32. Laboratory diagnosis Screening of blood donors using ELISA Screening of blood donors using ELISA Confirmation by western blotting Confirmation by western blotting PCR PCR