Human RetrovirusesHuman Retroviruses

Dr. Medhat K. ShierDr. Medhat K. ShierVirology ConsultantVirology Consultant

RetrovirusesRetroviruses

RNA virusesRNA virusessingle stranded, positive sense, enveloped, single stranded, positive sense, enveloped,

icosahedral.icosahedral.Distinguished from all other RNA viruses by presence of an Distinguished from all other RNA viruses by presence of an unusual enzyme, reverse transcriptase.unusual enzyme, reverse transcriptase.Retro = reversalRetro = reversal

RNA is serving as a template for DNA synthesis.RNA is serving as a template for DNA synthesis.Two genera of human interestTwo genera of human interest

LentivirusLentivirus Lentus = slowLentus = slowHuman immunodeficiency viruses 1 & 2 (HIV-1 & Human immunodeficiency viruses 1 & 2 (HIV-1 &

-2)-2)Human T-cell lymphotropic virus-bovine leukemia virus Human T-cell lymphotropic virus-bovine leukemia virus group (HTLV-BLV)group (HTLV-BLV)

Human T-cell leukemia viruses 1 & 2 (HTLV-1 & -Human T-cell leukemia viruses 1 & 2 (HTLV-1 & -2)2)

StructureStructure

The viral envelope formed from host cell membrane; The viral envelope formed from host cell membrane; contains 72 spiked knobs. These consist of a contains 72 spiked knobs. These consist of a transmembrane protein TMtransmembrane protein TM ( (gp 41gp 41), which is linked ), which is linked to to surface protein SUsurface protein SU ( (gp 120gp 120) that binds to a cell ) that binds to a cell receptor during infection. The virion has cone-receptor during infection. The virion has cone-shaped, icosahedral core, containing the shaped, icosahedral core, containing the major major capsid protein CAcapsid protein CA ( (p24p24). Between capsid and ). Between capsid and envelope is an envelope is an outer matrix proteinouter matrix protein, , MAMA ( (p17p17).).

Two identical copies of positive sense ssRNA genome Two identical copies of positive sense ssRNA genome ((retroviruses are diploidretroviruses are diploid).).

Enzymes: Enzymes: reverse transcriptasereverse transcriptase, , integraseintegrase and and proteaseprotease..

HIV particlesHIV particles

HIV particlesHIV particles

HIV GenomeHIV Genome

Three major genesThree major genes

Gag geneGag gene codes for CA, MA and NC codes for CA, MA and NC proteinsproteins

Pol genePol gene codes for reverse transcriptase, codes for reverse transcriptase, protease, integrase and ribonuclease.protease, integrase and ribonuclease.

Env geneEnv gene codes for TM and SU codes for TM and SU

HIV ReplicationHIV Replication

The The first phasefirst phase of viral entery, reverse transcription and of viral entery, reverse transcription and integration into host genome is accomplished by integration into host genome is accomplished by viral proteinsviral proteins..

The The second phasesecond phase of synthesis and processing of viral genomes, of synthesis and processing of viral genomes, mRNAs and structural proteins, uses mRNAs and structural proteins, uses host cell machineryhost cell machinery..

AttachmentAttachment to specific cell surface receptor: to specific cell surface receptor: gp120 binds CD4gp120 binds CD4 molecule on the helper T cells, monocytes and dendritic cellsmolecule on the helper T cells, monocytes and dendritic cells

Viral enteryViral entery.. Reverse transcriptionReverse transcription of viral RNA into DNAof viral RNA into DNA. The resulting . The resulting

double stranded DNA is called provirus.double stranded DNA is called provirus. Integration of provirus into host cell DNAIntegration of provirus into host cell DNA. The viral integrase . The viral integrase

cleaves the chromosomal DNA and covalently inserts the cleaves the chromosomal DNA and covalently inserts the provirus. The insertion site is random.provirus. The insertion site is random.

Transcription and translation of viral DNA sequencesTranscription and translation of viral DNA sequences. The . The provirus is transcribed into a full length mRNA by the cell RNA provirus is transcribed into a full length mRNA by the cell RNA polymerase II.polymerase II.

Assembly and maturationAssembly and maturation of progeny virusof progeny virus..

HIVHIV

Acquired immunodeficiency syndrome Acquired immunodeficiency syndrome (AIDS) was first reported in US in (AIDS) was first reported in US in 19811981..

By By 19841984, AIDS was recognized as an , AIDS was recognized as an infectious disease caused by a virus.infectious disease caused by a virus.

Transmission of HIVTransmission of HIV

Sexual contactSexual contact: HIV is present in semen and : HIV is present in semen and vaginal secretions; either homoxesual or vaginal secretions; either homoxesual or heterosexual contactheterosexual contact

TransfusionsTransfusions: whole blood, plasma, clotting : whole blood, plasma, clotting factors or cellular fractions of blood.factors or cellular fractions of blood.

Contaminated needlesContaminated needles: accidentally or sharing : accidentally or sharing needles by drug users.needles by drug users.

PerinatalPerinatal: Transplacental, during delivery or via : Transplacental, during delivery or via breast milk.breast milk.

Pathogenesis and clinical Pathogenesis and clinical significance (1)significance (1)

Initial infectionInitial infection: : genital tract macrophages genital tract macrophages HIV disseminates via bloodHIV disseminates via blood Dendritic cells in lymphoid tissueDendritic cells in lymphoid tissue CD4+ lymphocytesCD4+ lymphocytes

Acute phase viremiaAcute phase viremia: several weeks after the initial infection, 1/3 : several weeks after the initial infection, 1/3 – 2/3 of individuals experience an acute disease syndrome – 2/3 of individuals experience an acute disease syndrome similar to infectious mononucleosis. Circulating antibody appears similar to infectious mononucleosis. Circulating antibody appears in 1 – 10 weeks after the initial infection (in 1 – 10 weeks after the initial infection (seroconversionseroconversion).).

Latent periodLatent period: lasts from months to many years (average 10 : lasts from months to many years (average 10 years). During this period, 90% of HIV proviruses are years). During this period, 90% of HIV proviruses are transcriptionally silent. Although there is continous loss of CD4+ transcriptionally silent. Although there is continous loss of CD4+ cells in which HIV is replicating, active replacement through cells in which HIV is replicating, active replacement through stem cell multiplication is occurring. The infection remains stem cell multiplication is occurring. The infection remains clinically asymptomatic as long as the immune system is clinically asymptomatic as long as the immune system is functional.functional.

Pathogenesis and clinical Pathogenesis and clinical significance (2)significance (2)

Clinical complications during the latent periodClinical complications during the latent period: there are : there are multiple non-specific conditions such as persistent multiple non-specific conditions such as persistent generalized lymadenopathy, diarrhea, chronic fevers, night generalized lymadenopathy, diarrhea, chronic fevers, night sweats and weight loss. The more common opportunistic sweats and weight loss. The more common opportunistic infections such as herpes zoster and candidiasis may occur infections such as herpes zoster and candidiasis may occur repeatedly during this period. The CD4+ cell count remains repeatedly during this period. The CD4+ cell count remains normal or gradually declines but is greater than 200 / ul. normal or gradually declines but is greater than 200 / ul. Progression from asymptomatic infection to AIDS is not Progression from asymptomatic infection to AIDS is not sudden.sudden.

AIDSAIDS: Coinfection with HHV-6 can transactivate transcription : Coinfection with HHV-6 can transactivate transcription from the silent HIV provirus, increasing HIV replication. Any from the silent HIV provirus, increasing HIV replication. Any stimulation of an immune response causing activation of stimulation of an immune response causing activation of resting T cells also activates HIV replication. Appearance of resting T cells also activates HIV replication. Appearance of HIV mutants with altered antigenic specificity which are not HIV mutants with altered antigenic specificity which are not recognized by the existing humoral antibody or cytotoxic T recognized by the existing humoral antibody or cytotoxic T lymphocytes; also contributes to progression with CD4+ lymphocytes; also contributes to progression with CD4+ count falling below 200 / ul and appearance of serious count falling below 200 / ul and appearance of serious diseases and opportunistic infections.diseases and opportunistic infections.

Opportunistic Infections (1)Opportunistic Infections (1)

Bacteria:Bacteria: Mycobacterium avium complexMycobacterium avium complex

Disseminated miliary diseaseDisseminated miliary disease Chronic bronchitis, pneumoniaChronic bronchitis, pneumonia Chronic osteomyelitis, renal infectionChronic osteomyelitis, renal infection

Mycobacterium tuberculosisMycobacterium tuberculosis Chronic pneumonitis, osteomyelitisChronic pneumonitis, osteomyelitis Meningitis, miliary diseaseMeningitis, miliary disease

Streptococcus pneumoniaeStreptococcus pneumoniae Salmonella spp.Salmonella spp. Haemophilus influenza (pneumonia)Haemophilus influenza (pneumonia) Campylobacter spp. (diarrhea)Campylobacter spp. (diarrhea) Shigella spp. (diarrhea)Shigella spp. (diarrhea)

Opportunistic Infections (2)Opportunistic Infections (2)

Fungi:Fungi: Candida spp. Oral, vaginal or systemic Candida spp. Oral, vaginal or systemic

candidiasiscandidiasis Histoplasma capsulatum (disseminated disease)Histoplasma capsulatum (disseminated disease) Cryptococcus neoformans (meningitis)Cryptococcus neoformans (meningitis)

Pneumocystis cariniiPneumocystis carinii Unicellular eukaryoteUnicellular eukaryote Taxonomic status is uncertainTaxonomic status is uncertain Most common opportunistic pathogen in AIDS Most common opportunistic pathogen in AIDS

patientspatients Fatal pneumoniaFatal pneumonia

Opportunistic Infections (3)Opportunistic Infections (3)

Parasites:Parasites: Toxoplasma gondii (focal encephalitits)Toxoplasma gondii (focal encephalitits)

Viruses: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, JCV (progressive multifocal leukoencephalopathy,

affect the white matter of the brain)affect the white matter of the brain) CMV (Chorioretinitis, encephalitis, enterocolitis, CMV (Chorioretinitis, encephalitis, enterocolitis,

gastritis)gastritis)

Malignancies associated with Malignancies associated with AIDSAIDS

Kaposi’s sarcomaKaposi’s sarcoma HHV-8HHV-8 LymphomasLymphomas EBVEBV

Laboratory DiagnosisLaboratory Diagnosis

Antigen / antibody detectionAntigen / antibody detection ELISA, serumELISA, serum HIV-1 & -2 antibodies, HIV-1 CA (p24) antigenHIV-1 & -2 antibodies, HIV-1 CA (p24) antigen Screening of blood donorsScreening of blood donors Western BlottingWestern Blotting

PCRPCR Viral RNA or DNA provirusViral RNA or DNA provirus Blood or tissue specimensBlood or tissue specimens Quantitative PCR (viral load): to determine Quantitative PCR (viral load): to determine

disease stage and treatment follow up.disease stage and treatment follow up.

ELISA for HIV antibodyELISA for HIV antibody

Microplate ELISA for HIV antibody: coloured wells Microplate ELISA for HIV antibody: coloured wells indicate reactivityindicate reactivity

Western blot for HIV antibodyWestern blot for HIV antibody

There are different There are different criteria for the criteria for the interpretation of HIV interpretation of HIV Western blot results e.g. Western blot results e.g. CDC, WHO, American CDC, WHO, American Red Cross.Red Cross.

The most important The most important antibodies are those antibodies are those against the envelope against the envelope glycoproteins gp120, and glycoproteins gp120, and gp41gp41

p24 antibody is usually p24 antibody is usually present but may be present but may be absent in the later stages absent in the later stages of HIV infectionof HIV infection

TreatmentTreatment

Anti-retroviral drugsAnti-retroviral drugs Reverse transcriptase inhibitorsReverse transcriptase inhibitors Protease inhibitorsProtease inhibitors

Multidrug therapyMultidrug therapy RT has no proofreading activity, resulting in production RT has no proofreading activity, resulting in production

of many errors during viral DNA synthesis which leads to of many errors during viral DNA synthesis which leads to mutations in all HIV genes and accumulation of mutant mutations in all HIV genes and accumulation of mutant viral strains. In presence of an antiviral drug, there is viral strains. In presence of an antiviral drug, there is strong selection for mutations that confer resistance to strong selection for mutations that confer resistance to that drug.that drug. Use multidrug therapyUse multidrug therapy

Early therapyEarly therapy Viral load is a prognostic indicator of the rate of Viral load is a prognostic indicator of the rate of

progression to AIDS. Infection should be treated as progression to AIDS. Infection should be treated as aggressively and as early as possible to minimize initial aggressively and as early as possible to minimize initial spread of the virus and give a lower chance for mutants spread of the virus and give a lower chance for mutants to arise.to arise.

Highly active antiretroviral therapy Highly active antiretroviral therapy (HAART)(HAART)

Nucleoside analog reverse transcriptase inhibitorsNucleoside analog reverse transcriptase inhibitors Act by serving as a chain terminatorAct by serving as a chain terminator

Zidovudine (AZT)Zidovudine (AZT) Didanosine (ddi)Didanosine (ddi) Lamivudine (3TC)Lamivudine (3TC)

Non-nucleoside reverse transcriptase inhibitorsNon-nucleoside reverse transcriptase inhibitors Act by targeting the enzyme itselfAct by targeting the enzyme itself

EfavirenzEfavirenz DelaviridineDelaviridine NevirapineNevirapine

Protease inhibitorsProtease inhibitors Interfere with the processing of polyproteins in the Interfere with the processing of polyproteins in the

budding virion, resulting in non-infectious particle.budding virion, resulting in non-infectious particle. RitonavirRitonavir AmprenavirAmprenavir IndinavirIndinavir LopinavirLopinavir

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

PreventionPrevention

VaccineVaccine: not yet available: not yet available

Blood supply screeningBlood supply screening

Perinatal transmissionPerinatal transmission: AZT therapy: AZT therapy

HTLVHTLV

HTLV-1 and -2 have 65% nucleotide HTLV-1 and -2 have 65% nucleotide sequence homologysequence homology

Genetically and biologically similarGenetically and biologically similar

HTLV-1HTLV-1 Adult T-cell leukemiaAdult T-cell leukemia HTLV-associated myelopathy/tropical spastic HTLV-associated myelopathy/tropical spastic

paraparesisparaparesis

HTLV-2HTLV-2 Hairy cell leukemiaHairy cell leukemia

TransmissionTransmission

Vertical transmissionVertical transmission

SexualSexual

Blood productsBlood products

Hairy cell leukemiaHairy cell leukemia a rare lymphocytic a rare lymphocytic

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

LaboratoryLaboratory diagnosisdiagnosis

Screening of blood donors using Screening of blood donors using ELISAELISA

Confirmation by Confirmation by western blottingwestern blotting

PCRPCR