1

Retroviruses

Maciej PrzybylskiChair and Department of Medical Microbiology

Retroviruses: classification

� Family: Retroviridae– Subfamily: Orthoretrovirinae

• Genus: Deltaretrovirus– HTLV-1-4(Human T-Lymphotropic Virus)

• Genus: Lentivirus– HIV-1, HIV-2(Human Immunodeficiency Virus)

2

HIV classification� HIV-1:

– Subtypes: • M (major)• N (new)• O (outlier)• P (pending)

� Subtype M:– clades A, B, C, D, F, G, H, J, K

– at least 15 CRF (circulating recombinant forms, e.g. CRF_01AE)

� HIV-2: – 8 groups of viruses, A and B are the

most important

Phenotype classification of HIV-1

� Syncytium inducing (SI) and� Non-syncytium inducing (NSI)(in CD4+ T lymphocytes cell line)

NSI type is detected in early stage of the infection, SI appears later (AIDS)

Most NSI utilizes CCR5 coreceptor (R5 strains), most SI - CXCR4 coreceptors (X4 strains). R5X4 recombinants are known

3

Retroviridae – basic characteristics

� genome: ss (+) RNA

� reverse transcriptase (RT)

� virion diameter: 100 nm

� structural proteins: Gag, Pol, Env

� regulatory proteins: Rev, Tat, Vif, Nef

2 identical ssRNA+ particles, length 7-11 kb, bound with hydrogen bridge

Structure of the mature HIV virion

4

� Adsorption (gp120/gp41 binds CD4/CCR5 /CXCR4)� Fusion� Release of RNA and reverse transcriptase to the

cytoplasm� Synthesis of ss cDNA (erratic transcription) and next,

ds cDNA� dsDNA/integrase/cellular transcriptional coactivator

(PIC complex) migrates to the nucleus� Integrase cuts cellular DNA and ligates vDNA� Integrated vDNA = provirus� Provirus may have episomal form (function unknown)� Expression and proteolysis of viral polyprotein� Assembly, maturation and egress of virions

Replication of HIV

PATHOGENESIS

Permisive cells

�CD4+ lymphocytes

�Macrophages/monocytes

�Langerhans cells

�Dendritic cells

�CNS cells (astrocytes, microglia)

5

PATHOGENESIS

� Dynamics of HIV proliferation:– massive replication (1010 of new virions a day,

half-life in serum: hours)– mutations and recombinations (quasispecies –

evolution in host organism)– some number of infected lymphocytes survives

long enough to establish resting form containing provirus

– „a library” of latently infected lymphocytes develops, carrying a lot of viral quasispecies, which are continuous source of reactivations

� Sources of CD4+ lymphopenia– Damaging effect of the virus– Apoptosis– Activity of cytotoxic cells– Nonspecific activation of infected

lymphocytes and their subsequent anergia

PATHOGENESIS

6

CLINICAL PICTURE

• Acute retroviral disease

• Asymptomatic period

• Persistent generalized lymphadenopathy

• Cachexia, pancytopenia, opportunistic infections

• AIDS (acquired immunodeficiency syndrome)

� incubation period: ~ 6 weeks� may last for months� symptoms (~50% of infected persons):

– body temperature >38°C– malaise– pharyngitis– rash– headache, muscle pain– arthritis– diarrhoea and vomiting– hepatosplenomegaly– opportunistic infections

Acute retroviral disease (ARD)

7

Asymptomatic period

� Few months – 2 years� In treated patients - years� „Residual” symptoms are observed� Duration depends on many factors:

– ARD severity– genetic characteristics of the virus– genetics of the patient– moment of treatment introduction

AIDS – CLINICAL PICTURE

• night sweats• eczema• opportunistic infections• cachexia• limfadenopathy• encephalopathy• nephropathy• neoplasms

CD4 < 350/µl

8

Indicative opportunistic infections in the course of AIDS

� Candida spp. infections : oral cavity, bronchia, lungs

� HPV-related invasive neoplasms

� systemic coccidioidomycosis� cryptococcosis� chronic (> 1 month)

cryptosporidiosis� cytomegalovirus disease� cytomegalovirus retinitis with

loss of sight� chronic ulcerative herpes

simplex� systemic histoplasmosis� Kaposi’s sarcoma

� Burkitt’s lymphoma� leukoplakia� multifocal infections with

Mycobacterium avium complex, M. kansasii or atypical mycobacteria

� multifocal or extrapulmonary tuberculosis

� Pneumocystis jiroveci (f. carinii) pneumonitis

� recurrent or atypical bacterial pneumonia

� recurrent sepsis due to Salmonella spp.

� toxoplasmosis of CNS� molluscum contagiosum

Kaposi’s sarcoma

9

Oportunistic infections – zoster, Pneumocystis jiroveci pneumonia, oral hairy leukoplakia, oral HPV

Opportunistic infections – oral candidiasis

10

AIDS – HIV neuroinfection (HAND), cachexia

HIV-1: epidemiology

� routes of transmission: sexual contact, parenteral, vertical

� contagious material: blood, sperm, cervical mucus, vaginal lubrication, breast milk

� HIV is not transmitted with: fecal-oral route, respiratory route, by arthropod vectors and direct contact

� Blood-free saliva, urine, tears and sweat are considered non-infectious

11

HIV: world epidemiology

12

EPIDEMIOLOGY: POLAND

• first case of HIV infection 1985• first case of AIDS: 1986

Cumulative data 1985 – 2018:

Number of HIV infected persons (31.12.2014): 21 148

– intravenous drugs: 6299– homosexual contact: 3033– heterosexual contact: 1620

AIDS developed in 3441 person (1360deaths)

www.pzh.gov.pl/epimeld

POLAND: EPIDEMIOLOGY

www.pzh.gov.pl/epimeld

13

Poland: Sources of HIV infections - 2012

www.pzh.gov.pl/epimeld

Poland: Sources of HIV infections - 2016

www.pzh.gov.pl/epimeld

14

POLAND: AIDS PREVALENCE PER 100 000 - 2012

www.pzh.gov.pl/epimeld

POLAND: AIDS PREVALENCE PER 100 000 - 2017

www.pzh.gov.pl/epimeld

15

POLAND: PREVALENCE OF HIV INFECTIONS PER 100 000 - 2017

www.pzh.gov.pl/epimeld

HIV: LABORATORY DIAGNOSIS

� Screening serological assays – simultaneous detection of:– anti-HIV-1/2 antibodies (anti-GAG, POL, ENV)– anti-HIV-2 antibodies– HIV-1 p24 antigen

� 4th generation ELISA test� Confirmatory serological assays:

– detection of specific anti-HIV-1 and anti-HIV-2 antibodies– Detection of HIV-1/2 RNA

• NAT (qPCR, NASBA, TMA)• bDNA (branched DNA)

16

HIV infections diagnostics

� Congenital/perinatal infections:– Confirmation/exclusion of infection in

mother– In vitro cultivation of peripheral blood

lymphocytes– Detection of p24 antigen and HIV RNA– Detection of provirus DNA– Testing in 5th day of life, re-test after 3

months

HIV infections diagnostics

� Monitoring of treatment– Quantitative detection of HIV RNA– HIV-1 genotyping– Identification of HIV-1 CCR5 and CXCR4

mutations– Detection of virus mutations associated with

resistance to antivirals

– Assessment of the number of CD4+ lymphocytes– Detection of HLA-B5701 gene– Monitoring: 2-4 times a year (adults) and 4 times a

year (children)

17

HIV-related parameters in untreated patient

Antiretroviral therapy (ART)

� (HA)ART or cART: highly active ART, combined ART

Never use any drug in monotherapyfor the treatment of HIV infection

18

AI FI

NRTINSTI

NNRTI

PI

Anti-HIV agents, antiretrovirals (ARV)

� NRTI (nucleoside reverse transcriptase inhibitors)� NtRTI (nucleotide reverse transcriptase inhibitors)� NNRTI (non-nucleos(t)ide reverse transcriptase inhibitors)� PI (protease inhibitors)� Inhibitors of cytochrome metabolizing protease inhibitors

(PI boosters)� INSTI (integrase inhibitors)� FI (fusion inhibitors)

19

Anti-retroviral drugs� NRTIs:

– 3TC lamivudine– ABC abacavir– d4T stavudine– FTC emtricitabine– TDF tenofovir (disoproxil fumarate)

– ZDV zidovudine– ddI didanosine

� NNRTIs:– EFV efavirenz– DLV delavirdine– ETV etravirine– NVP nevirapine– RPV rilpivirine

� INSTIs– DTG dolutegravir– EVG elvitegravir– RAL raltegravir– ENF enfuvirtide

� PIs:– ATV atazanavir– DRV darunavir– IDV indinavir– LPV lopinavir– SQV saquinavir– TPV tipranavir– FPV fosamprenavir (prodrug)

� FI: ENF enfuvirtide

� CCR5 antagonist : MVC maraviroc

� Cytochrome inhibitors– RTV ritonavir– COBI cobicistat

When to stART?� without diagnosis of AIDS:

– serum HIV RNA increases 0,6 log10/ml– serum HIV RNA > 30,000 copies/ml– CD4 count ≤350 µl

– do not delay therapy if CD4 count is close to that value– CD4 count ≤500 if there is coinfection with HBV or HCV

� in case of:– AIDS diagnosis

– HIV-associated nephropathy– HIV-associated encephalopathy– HIV-associated opportunistic cancers

� with patient’s full and self-imposed consent, directly observed therapy is recommended

20

First line ART in naïve patients

� two NRTI� and (select one):

– ritonavir-boosted PI� or

– NNRTI� or

– integrase inhibitor

� single detection of 50-400 HIV copies/ml (blip) is not an indication of ART failure

� when blips repeat, one should start investigation for resistance to used antivirals

� persisting low level viraemia (over 400 copies/ml) may indicate ART failure

Specific situations

� coinfections:– HIV/TB– HIV/HBV – HIV/HCV– HIV/malaria

� HIV-related cancers� HIV-associated neurocognitive impairment� cardiovascular disease� pregnant women

21

PROPHYLAXIS

• Post-exposition prophylaxis (PEP)

• Perinatal chemoprophylaxis (starting from 12. week of pregnancy)

• High-level Ig human serum

• Vaccine - ?

• education and preventive measures• sexual contacts• drug abusers• blood/graft donors• vertical transmission• health care workers

protection• patient protection

PEP in Poland = 2 NRTI + PI, 1 month, cost ~4000 zł, not refunded

HIV STABILITY

� Virus inactivation:– temperature (autoclaving)– chemical disinfectants (room temp., 10-minute

exposition):• 2% glutaraldehyde• sodium hypochlorite (10 000 ppm available chlorine)

• 70% ethanol

• 35% propanol

• 0,3% hydrogen peroxide

– at 60ºC there is 100-fold decrease per hour of infectious virus titer

22

After parenteral exposition to HIV containing material

• do not stop bleeding• do not squeeze the wound• wound routine disinfection• assessment of risk• post-exposition chemoprophylaxis (tenofovir +

lamivudine/emtricitabine + lopinavir) - ASAP• HIV testing (source and exposed person)• safe behaviour – protection of other people

„Natural immunity” to HIV

� Defective virus (e.g. vif gene mutations) –delay in disease progression

� Genetic background:– ∆32 CCR5 mutation (absence of HIV coreceptor)– duplicated CCL3L1 genes, encoding CCR5 ligand – presence of HLA-B5701 gene (superreactive

limphocytes)– individual differences in chemokine system

23

HTLV-1� Genetic organization typical for retroviruses� STLV (simian) and HTLV (human) form

Primate T-Lymphotropic Virus species� There is 7 subtypes of HTLV-1 (a-g)� In contrast to HIV, HTLV has relatively stable

genome and is present in population for thousands of years

� HTLV-1 is responsible for human disease, pathogenic potential of HTLV-2, -3 and -4 is unknown

HTLV-1: epidemiology� Causes infections on all continensts, but is typically

endemic virus� Endemic countries harbour most of the HTLV-1

cases: Western Africa, Japan, Carribbean region, Southern America

� Seroprevalence in endemic regions varies from 0,5% to 17%

� In non-endemic countries prevalence is very low (1/50 000 – 1/1 000 000)

� Transmission requires direct cell-to-cell contact, major route of infection is breastfeeding from infected mother, infections are described also in i.v. drugs abusers and in transplant/whole blood transfusion recipients

24

HTLV-1: diseases

� 90-95% of infections is asymptomatic� HTLV-1 associated leukemia/lymphoma

(adult T-cell leukemia/lymphoma -ATLL) – 55-75% of symptomatic cases

� HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP)

� Oncogenic virus

ATLL� Risk factors: high proviral load, advanced age, family

history of ATLL� Incubation period – years� Symptoms depend on involved organ:

– Skin– Gastrointestinal tract– Central nervous system

� High mortality, survival time from 3 to 16 months� Diagnosis: blood picture (characteristic „flower cells”),

viral RNA detection in blood� Treatment: IFN-alpha + AZT

25

HAM/TSP

� Demyelination and inflammation of the central nervous system, leading to a slowly progressive spastic paraparesis

� Total motor disability of lower limbs in 50% of patients

� There is no recommended therapy