HIV by Dr. Rakesh Prasad Sah

1. Retroviruses : HIV
By Dr. Rakesh
Prasad Sah
Assistant
Professor,
Microbiology

2. Introduction
• Are RNA viruses that belong to family Retroviridae.
• Possess reverse transcriptase (RNA directed DNA
polymerase) enzyme which prepares a DNA copy of the
RNA genome in host cell.

3. Classification
Subfamily Genus Virus Disease
Orthoretrovirinae Retrovirus Human T-cell Lymphotropic Virus
1 (HTLV-1)- oncogenic
Adult T-cell
leukaemia/lymphoma
Virus HTLV-2 Not associated with any
disease but prevalent in
intravenous drug users
Lentivirus Human Immunodeficiency Virus
type 1 (HIV-1)
AIDS
HIV-2 AIDS
Spumaretrovirinae Spumavirus Foamy virus Found in primary cell
culture but not
associated with disease

4. Human Immunodeficiency Virus
1

5. Introduction
• Etiologic agent of Acquired Immunodeficiency Syndrome (AIDS).
• AIDS was 1st recognised in USA in 1981 amonst homosexuals and
drug addicts in whom incidence of Kaposi’s sarcoma & P. carinii pneumonia was
high
• Discovered independently by Luc Montagnier of France and
Robert Gallo of the US in 1983.
• Former names of the virus include:
– Lymphadenopathy associated virus (LAV) (Luc Montagnier)
– Human T cell lymphotrophic virus (HTLV-III) (Robert Gallo)
– AIDS associated retrovirus (ARV)
• 1986, HIV name was given by International Committee on Virus nomenclature.

6. Introduction
• HIV-2 discovered in 1986, antigenically distinct virus endemic in West
Africa.
• 30 million worldwide are infected. 2 million deaths every year & 2.5 million
new cases every year.
• Leading cause of death of men aged 25-44 and 4th leading cause of death
of women in this age group.
• HIV-1 in humans was believed to be acquired from chimpanzee (Pan
troglodytes troglodytes) by cross species infections (simian
immunodeficiency virus or SIVcpz).
• HIV-2 through sooty mangabeys.

7. Characteristics of the virus
• Icosahedral (20 sided), enveloped virus of the lentivirus subfamily
of retroviruses.
• Retroviruses transcribe RNA to DNA.
• Two viral strands of RNA found in core surrounded by protein
outer coat.
• Outer envelope contains a lipid matrix within which specific viral
glycoproteins are imbedded.
• These knob-like structures responsible for binding to target cell.

8. Morphology
• The outer shell of the virus is known as the
Viral enevlope. Embedded in the viral
envelope is a complex protein known as env
which consists of an outer protruding cap
glycoprotein (gp) 120, and gp 41.
• Within the viral envelope is an HIV protein
called p17(matrix), and within this is the
viral core or capsid, which is made of
another viral protein p24(core antigen).

9. Structural Genes
Three main structuralgenes:
Group Specific Antigen(Gag)
Envelope (Env)
Polymerase (Pol)
6
Non-Structural Genes
 Tat (transcriptionaltransactivator)
 Nef (negativefactor)
 Rev (regulator of virusgene)
 Vif (viral infectivity factor)(destroys
apobec 3g protein)
 Vpu (increases viral release)(HIV-
1)
 Vpx (HIV-2)
 Vpr (promotes viralreplication)
 LTR (long terminalrepeat)
integration signals.

10. Group Specific Antigen (Gag)
• It encodes for core and shell proteins.
• Expressed as a precursor protein, p55.
• Cleaved into p15, p17 and p24.
• p 24 can be detected in serum during early
stages of infection till the appearance of antibodies.
• The decline of anti-p24 antibody from circulation indicates
progression of illness and is an indication of antiviral
treatment

11. Envelope (Env)
• Envelope (Env) gene codes for envelope protein
gp160; gp120 and gp41.
– gp160 cleaved to form gp120 and gp41.
– gp120 forms the 72 knobs which protrude
from outer envelope.
– gp41 is a transmembrane glycoprotein
antigen that spans the inner and outer
membranes and attaches to gp120.
– gp120 and gp 41 both involved with fusion
and attachment of HIV to CD4 antigen on host
cells.

12. Polymerase (Pol)
• Polymerase (Pol) codes for viral enzymes
such as reverse transcriptase.
• Expressed as precursor protein p100.
• Cleaved into p 31, p 51 and p 64.
• Located in the core, close to nucleic acids.
• Responsible for conversion of viral RNA
into DNA, integration of DNA into host cell
DNA and cleavage of protein precursors.

13. Gene Products of HIV (HIV Antigens)
Gene Coding of HIV antigens Antigens
gag Core antigens p24 (Principal core antigens), p 15, p 55
Shell antigen p 18 (nucleocapsid protein)
env Envelope antigen gp 120, (principal envelope spike antigen)
gp 41 (transmembrane antigen)
pol Reverse transcriptase antigen p 31, p 51, p 64

14. Resistance
• Temperature: Inactivated at 56oC in 30 minutes and in seconds at 100oC.
• Disinfection:
– 35% Isopropyl alcohol: inactivation in 10 minutes.
– 70% ethanol
– 0.5% lysol
– 2% freshly prepared glutaraldehyde
– 0.5% sodium hypochlrite
– 3% hydrogen peroxide
• Extremes of pH (pH 1.0, pH 13.0)
• Resistant to Lyophilisation

15. Modes of transmission
• Sexual transmission, presence of STD increases
likelihood of transmission.
• Blood transfusion.
• Parenteral transmission.
• Exposure to infected blood or blood
products.
• Transplantation of infected tissues
or organs.
• Mother to fetus, perinatal transmission variable.

17. Types of Exposure and Relative Risk
S.N. Types of Exposure Relative risk per
exposure (%)
1. Sexual intercourse: anal, vaginal, oral 0.1-1.0
2. Transfusion of blood and blood
products
>90
3. Tissue and organ donations 50-90
4. Injection and injuries 0.5-1.0
5. Mother to baby 30

18. Pathogenesis
Provirus is integrated into genome of the infected cell  latent infection
In the cell, RNA is transcribed by reverse transcriptase into DNA (provirus)
After entry into blood stream, HIV comes into contact with CD4 lymphocyte

19. • a
HIV can be isolated from blood, lymphocytes, cell-free plasma, cervical
secretion, semen, saliva, urine, tears and breast milk.
From time to time, lytic infection is initiated and releases progeny
virions to infect other cells
Long and variable IP is due to latency

20. • a
Which leads to a marked damping effect on CMI
Viral infection can suppress the function of infected cells without
causing any structural damage
Infection causes damage to the T4 lymphocyte, T4 cells depleted in
numbers and the T4:T8 ratio is reversed.

21. • a
Dementia and other degenerative neurological lesions may also be seen
in AIDS
This renders the patient susceptible to life threatening opportunistic
infections and malignancies
Clinical manifestations in HIV infections are mainly due to failure of
immune responses.

22. Clinical Features: According to CDC, clinical course of
HIV infection
• Group I- Acute HIV infection: Acute onset of fever, malaise, sore
throat, myalgia, arthralgia, skin rash and lymphadenopathy. Viral nucleic acid or
viral p24 antigen may be detected. Antibodies to HIV usually negative. (3 to 6
months)
• Group II- Asymptomatic infection: Show positive HIV antibody tests
and are infectious. Person usually well.

23. Clinical Features: According to CDC, clinical course of
HIV infection
• Group III- Persistent generalised lymphadenopathy:
Enlarged nodes at two or more extragenital sites for at least 3 months.
• Group IV- Symptomatic HIV infection: CD4 T lymphocyte count
falls below 400 per cu. mm. Symptoms like diarrhea, fever, weight loss,
night sweats and opportunistic infection develops. Some patients develops
AIDS related complex or conditions.

24. Primary HIV Syndrome
• Cold or flu-like symptoms may occur 6 to 12 weeks after infection.
• Symptoms are relatively nonspecific.
• HIV antibody test often negative but becomes positive within 3 to 6 months
(window period), this process is known as seroconversion.
• Large amount of HIV in the peripheral blood.
• Primary HIV syndrome resolves itself and HIV infected person remains
asymptomatic for a prolonged period of time, often years (Clinical Latency).

25. Clinical Latency Period
• HIV continues to reproduce, CD4 count gradually
declines from its normal value of 500-1200.
• Once CD4 count drops below 500, HIV infected
person at risk for opportunistic infections.
• The following diseases are predictive of the
progression to AIDS:
– Persistent Herpes-zoster infection
– Oral candidiasis (thrush)
– Oral hairy leukoplakia (Epstein Barr virus)
– Kaposi’s sarcoma 24 (KS) (Herpes Virus)
Candidiasis
Oral Hairy Leukoplakia

26. Oral Hairy Leukoplakia (OHL)
• Being that HIV reduces immunologic activity, the
intraoral environment is a prime target for chronic
secondary infections and inflammatory processes,
including OHL, which is due to the Epstein-Barr
virus under immunosuppressed conditions .
Kaposi’s sarcoma (KS)
• Kaposi’s sarcoma is a rare cancer of the blood vessels
that is associated with HIV caused due to Herpes virus. It
manifests as bluish-red oval-shaped patches that may
eventually become thickened. Lesions may appear singly
or in clusters.
OHL
Kaposi’s sarcoma

27. AIDS
• CD4 count drops below 200, person is considered to have advanced HIV disease
• If preventative medications not started the HIV infected person is now at risk for:
– Pneumocystis carinii pneumonia (PCP)
– Cryptococcal meningitis
– Toxoplasmosis
• If CD4 count drops below 50:
– Mycobacterium tuberculosis
– Cytomegalovirus
– Lymphoma
– Dementia
– Most deaths occur with CD4 counts below 50.

28. • When CD4+ cells fall below 200per mm3, the titre of virus
increases markedly and there is irreversible breakdown of
immune defence mechanisms, it is defined as AIDS.
• AIDS is the end stage of HIV infection.
• Most of patients with HIV die due to oppurtunistic
infections as given below :

29. Opportunistic infections and Malignancies commonly
associated with HIV infection
• Bacterial
– Mycobacterial Infection (TB
& NTB)
– M. avium complex
– Salmonellosis
• Viral
– CMV
– Herpes simplex
– Varicella-zoster
– Epstein-Bar (EB) virus
– Human herpes virus 6 (HHV6)
– Human herpes virus 8 (HHV8)

30. Opportunistic infections and Malignancies commonly
associated with HIV infection
• Mycotic
– Pnemocystis jiroveci penumonia
– Candidiasis
– Cryptococcosis
– Aspergillosis
– Histoplasmosis
– Coccidioidomycosis

31. Opportunistic infections and Malignancies commonly
associated with HIV infection
• Parasitic
– Toxoplasmosis
– Cryptosporidiosis
– Isosporiasis
– Generalised strongyloidiasis
• Malignancies
– Kaposi’s sarcoma
– B-cell lymphoma or non-Hodgkin’s
lymphoma

32. ‘Typical’ HIV-1 infection
symptoms
2 5 6 / 2 4 6 8 103 4
weeks years
Time following infection
HIV viral load
HIV- 1 p24 antigen
HIV proviral DNA
HIV antibodies
symptoms
‘window’
period
0 1
1° infection

33. Exposure
to HIV
Infection
No infection
Infection
Persistent
generalized
lymphadenopathy
(PGL)
Asymptomatic Neurological
disease
ARC AIDS
Acute infection
Clinical responses to HIV in Adults

34. Laboratory Diagnosis of HIV Infection
• Specific test for HIV
infections
– Antigen detection: p24 antigen
– Virus isolation
– Detection of viral nucleic acid
– Antibody detection
• Non-specific Tests
– Total and differential leucocyte
count
– T-lymphocyte subset assays
– Platelet count
– IgG and IgA levels
– Skin tests for CMI
Test for opportunistic infections and tumour

35. Laboratory Diagnosis of HIV Infection
• Non-specific Tests
– Total and differential
leucocyte count
– T-lymphocyte subset assays
– Platelet count
– IgG and IgA levels
– Skin tests for CMI

36. Specific Tests for HIV
• Antigen Detection
– Virus Ag (p24) & reverse transcriptase (RT) detected in blood after
two weeks.
– p24 Ag earliest marker to appear in blood.
– With seroconversion, Ab appears  p24 Ag disappears remains
absent during the long asymptomatic phase.
– Ap24 Agenemia reppears with the onset of clinical disease which
corresponds to loos of anti p24 Ab
– ELISA

37. Specific Tests for HIV
• Virus isolation
– Virus +nt in blood and body fluids mostly
within CD4 lymphocytes.
– Can be isolated from CD4 lymphocytes
of blood, B.M. and serum
– Patient’s lymphocytes are co-cultivated
with uninfected human lymphocytes in
the presence of IL-2.
– Same as above.

38. Specific Tests for HIV
• Detection of Viral Nucleic acid
– PCR, useful in window period
• DNA PCR  peripheral lymphocytes are lysed and proviral DNA
is amplified. Highly sensitive & specific.
• RNA PCR (RT-PCR)  used for diagnosis and also for
monitoring level of viraemia
RT-PCR

39. Specific Tests for HIV
• Antibody detection
– Simplest and most commonly employed technique for diagnosis.
– Several weeks to months for Abs to appear.
– IgM3-4 weeks followed by IgG Abs
– IgM Abs disappear in 8-10 weeks while IgG Abs remains
throughout life.
– With severe immunodeficiency in AIDS, some components of anti-
HIV Ab (e.g. anti-p24) may disappear.

40. Specific Tests for HIV
– HIV infected persons remain negative for Abs during
window period, when initial viral replication takes place
for about 2-3 weeks.

41. Specific Tests for Lab Diagnosis of HIV Infection
Test Window period Acute infection Asymptomatic inf ARC and AIDS
Antigen: p24, RT +* + - +
Virus isolation ++ ± - +
Antibody
ELISA test - + + +
Western blot test - + + +
Partial p24 and/or
gp 120
(Full pattern) (Absence of p24
antibody)
* Positive in less than 50% cases.

42. Laboratory Tests for Detection of Specific Antibodies in HIV
infection
1. Screening (E/R/S) tests
a) ELISA
b) Rapid tests
i. Dot blot assay
ii. Lateral flow assay (Immunochromatography)
iii. Particle agglutination (latex, gelatin)
iv. HIV spot and comb tests
c) Simple tests
a) These are based on ELISA principle
2. Supplemental tests
a) Western blot test
b) Indirect
imunofluorescence test
c) Radio Immuno
Precipitation assay

43. Western Blot
• Most popular confirmatory test.
• Antibodies to p24 and p55 appear earliest but decrease
or become undetectable.
• Antibodies to gp31, gp41, gp 120, and gp160 appear
later but are present throughout all stages of the disease.
• Interpretation of results.
• No bands, negative.
• In order to be interpreted as positive a minimum of 3
bands directed against the following antigens must be
present: p24, p31, gp41 or gp120/160.
• CDC criteria require 2 bands of the following: p24, gp41
or gp120/160.

44. Non-specific Tests
• Total and differential leucocyte count
– In AIDS, leucopenia with a lymphocyte count less than 400 per mm3.
• T-lymphocyte subset assays
– CD4:CD8 T-cell ration of 2:1, is reversed to 0.5:1 in cases of AIDS. The count of
CD4 lymphocytes falls below 200 per mm3.
• Platelet count
– Thrombocytopenia in AIDS patients
• IgG and IgA levels
– Levels of both are raised
• Skin test for CMI
– Diminished as evident from tuberculin test or other skin tests for CMI

46. Nucleoside reverse
transcriptase
inhibitors (NRTIs)
Non-nucleoside
reverse
transcriptase
inhibitors
(NNRTIs)
Protease
inhibitors
Fusion
inhibitors
Integrase
Inhibitors
Entry
inhibitors
Zidovudine (AZT) Nevirapine Ritonavir Enfuvirtide Raltergravir Maraviroc
Lamivudine Efavirnez (sustiva) Lopinavir
Stavudine Delaviridine Indinavir
Didanosine Nelfinavir
Abacavir Saquinavir
Zalcitabine Amprenavir
Emitricitabine Darunavir

47. Postexposure Prophylaxis (PEP)
Category of exposure Status of Source
HIV positive and
clinically
asymptomatic (Low
risk)
HIV positive and
clinically
symptomatic (High
risk)
HIV status
not known
(Unknown)
Mild exposure (Mucous membrane/ non-intact
skin with small volume)
Consider two drug
PEP regimen
Start two drug PEP
regimen
UsuallynoPEPor
considertwoPEP
regimen
Moderate exposure (Mucous membrane/non –
intact skin with large volume or percutaneous
superficial exposure with solid needle)
Start two drug PEP
regimen
Startthreedrug
PEPregimen
Severe exposure (Percutaneous with large
volume)
Start three drug PEP
regimen

48. • Zidovudine 300mg BD and Lamivudine 150mg BD are used in basic two drug
regimen.
• Expanded three drug PEP regimen, a protease inhibitor
– Lopinavir 400mg BD or 800mg OD or ritonavir 100 mg BD or 200 mg OD are
preferred as third drug.
• To be effective these drugs must be started within the first 72 hrs and ideally within
2 hrs.
• PEP should be continued for a period of 4 weeks.

49. a