Human Immunodeficiency Virus

(HIV)
 Introduction
• Etiologic agent of Acquired Immunodeficiency
Syndrome (AIDS).
• Discovered independently by Luc Montagnier of
France and Robert Gallo of the US in 1983-84.
• Former names of the virus include:
Human T cell lymphotrophic virus (HTLV-III)
Lymphadenopathy associated virus (LAV)
AIDS associated retrovirus (ARV)
• HIV infects & destroys helper T cell (CD4)
leading to a no. of immunological deficiency
 Characteristics of the virus
• HIV occur in 2 main strains- HIV-1,HIV-2
• Size and shape 90 -120 nm in diam.
• Spherical, enveloped RNA virus
• Nucleocapsid consist of 2 identical copies of ssRNA
and also contain reverase transcriptase
• Envelope- outer envelope lipoprotein (lipid+ viral
protein) with spikes
Characteristics of the virus
• Genetic properties- HIV genome contains 9 genes-
• 3 structural gene (gag, pol & env)
1. Group Specific Antigen (Gag)
2. Envelope (Env)
3. Polymerase (Pol)
• 6 non structural gene (tat, rev, nef, vif, vpr & vpu
or vpx
• HIV1 contain vpu while HIV 2 contain vpx
• Gag and pol genes are processed by a viral
protease while env gene is processed by a cellular
protease
 Gag( encodes core & shell
protein)
↓
P55 ( precussor protein)

P17 P24 P15

which make up the viral core and shell
P24 which can detected in serum during the
early stage of infection
 env (synthesis of envelop
glycoprotein)
gp160

gp120 gp41(transmembrane
(forms surface spikes) anchoring
protein)
• gp 120 and gp41 are both involved with the
fusion and attachment of HIV to CD4 antigen on
host cells.
 Pol ( codes for
polymerase reverse transcriptase and
other viral enzymes protease and
endonuclease

P100 (precurssor
protein)

P31 P51 P66

– 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
• Non structural regulatory gene-
1.Tat (trans activating gene) enhancing the expression
of viral gene.
2.Nef (negative factor gene) down regulatory viral
replication
3.Rev (regulator of virus gene) enhancing expression
of structural protein
4.Vif (viral infectivity factor gene) influencing
infectivity of virus particles
5.Vpu (only in HIV1 and vpx (HIV2) enhancing
maturation and release progeny virus from cells
6.Vpr stimulating promoter region of the virus
• Route of transmission-
1. Sexual contact
2. Perinatal
3. Large vol. of saliva
4. Blood
1.Sexual contact-
• main mode of spread
• 75% of all cases of HIV transmission
• Most cases of AIDS occur in homosexual or
bisexual male while heterosexual promiscuity
seem to be the dominant mode of HIV infection
 in Africa and Asia
• Other sexually transmitted disease may acts as a
cofactors for spread of HIV in particular
gonorrhoea and chlamydia
• Transmission from male to male and male to
female is a more potent route than that from female
to male
2. Parenteral transmission- next largest gp (25%) and
occur in 3gps of high-risk population
• Intravenous drug abusers by sharing needles,
syringes
• Haemophiliacs who have received large amount
of factor VIII concentrates
• Recipents of blood and blood products who have
received multiple transfusion of whole blood or
components like play platelets and plasma
3. Perinatal transmission- occur from infected
mother to new born during pregnancy
transplacentally or in immediate post-partum
period through contamination with maternal
blood, infected amniotic fluid or breast milk
• HIV isolated from– body fluids, semen, vaginal
secretions, cervical secretion, breast milk, CSF
synovial, pleural, peritoneal, pericardial and aminotic
fluid.
• Resistance-HIV is thermolabile, being inactivated in
10 min at 60C and in second at 100C and in dried
blood it may survive for upto 7 days
• HIV inactivated in 10 min by treatment with 50%
ethanol, 35% isopropanol, .5% lysol, .5%
paraformaldehyde, .3% hydrogen peroxide. .5%
iodine, 2% freshly prepared glutaraldehyde &
ethylene oxide
• Clinical features- Classified the clinical causes of
HIV infection into various gps
1. Group-I (acute HIV infection)- within 3-6 weeks of
infection with HIV, about 50% of person
characterised by low grade fever, malaise headache,
lymphadenopathy, skin rashes.
Spontaneous resolution occur within a weeks
Test of HIV antibodies are usually negative at the
onset of the illness but become positive during its
course. Hence this syndrome has been called
“seroconversion” illness.
2. Group-2 (Asymptomatic infection)- HIV (Ab)
present but no symptoms (infected person who
are usually well, but show +HIV Ab test and are
infectious)
The infecion progresses in course of time through
various stages, CD4 lymphocytopenia, minor
opportunistic infections, persistent generalised
lymphadenopathy, AIDS-related complex (ARC)
and malignancies
The median time b/w primary HIV infection and
development of AIDS has been stated as
approimately 10 years
• About 5-10% of infected appear to escape clinical
AIDS for 15 years or more. They have been termed
“long term survivors” or “long term nonprogressors”
• The CD4 + T cell count decrease steadily from over
1000 per micro liter to about 500 or less by the stage
acute infection. When count falls to 200 or less ,
clinical AIDS usually sets in.
3.Group-3 (persistent generalized lymphadenopathy)-
lymph nodes enlargement (more than 1 cm) that
persist for at least 3 months , in the absence of any
current illness
• or medication that may cause lymphadenopathy but
the cases may progress to ARC or AIDS.
4. Group 4 (AIDS & AIDS related complex (ARC)-
This gp includes patients with considerable
immunodeficiency suffering from various
symptoms or minor opportunistic infections.
The typical symptoms are fatigue, fever, persistent
diarrhea and weight loss
The common opportunistic infections are oral
candidiasis, herpes zoster, hairy cell leucoplakia,
salmonella or tuberculosis
• AIDS- the end stage of infection. The clinical
severity of AIDS varies with the type of infection. In
early AIDS, many patients are only during episodes
of infection, which may respond to treatment
• Oral manifestations- patients with AIDS are at greater
risk for bacterial, viral and fungal infections of the
mouth.
1. Bacterial infections- dental caries, necrotizing
gingivitis, ulcerative periodontitis
HIV gingivitis has a very destructive course leading
to periodontitis with loss of soft tissue
• and bone sequestrum formation and in extreme cases
tooth exfoliation.
• Fungal infections- candidiasis lesions are generally
bilateral, white soft hairy excrescences on the lateral
margin of tongue.
• Thrush- on palatal and buccal mucosae
• Angular chelitis- ulcers at the corner of the mouth
• Mucocutaneous candidiasis and asymptomatic
erythematous lesions of candidiasis – on palatal and
lingual mucosae
3. Viral infections- herpes simplex- nonhealing, deeper
more painful
• Oral hairy leukolpakia- greyish white adherent
plaques either lateral or bilateral margins of the
tongue
• Virus infection include herpatic stomatitis, herpes
zoster, koposi’s sarcoma
• Kaposi’s sarcoma- pink to red to purple to brown any
where on skin ,(1984) HHV 8 also called Kaposi’s
sarcoma herpes virus
• This is a multifocal systemic tumour due to
proliferating microvascular and fibroblastic processes
seen ussualy in sexual transmitted HIV infection
Oral Candidiasis (thrush)
Oral Hairy Leukoplakia
Kaposi’s sarcoma (KS)
Replication- Attachment of gp 120 to cell surface (CD4 receptor & specific hemokine
receptor)
↓
virus and cell memb. Fuse
↓
Nucleocapsid enter into host cell
↓
release RNA
↓
Viral RNA Converted into ds DNA by reverse transcriptase
↓
Viral ds DNA moves into the cell nucleus by enzyme integrase
↓
Viral DNA is integrated into ssDNA
↓
Produces latent infection
↓
Lytic infectic initiated releasing progeny virions
↓
Which infect other cell
• (a) HIV (red) attaches to two cell-surface receptors
(the CD4 antigen and a specific chemokine receptor).
• (b) The virus and cell membrane fuse, and the virion
core enters the cell.
• (c) The viral RNA and core proteins are released from
the virion core and are then actively transported to the
nucleus.
• (d) The viral RNA genome is converted into double-
stranded DNA through an enzyme unique to viruses,
reverse transcriptase (red dot).
• (e) The double-stranded viral DNA moves into the cell
nucleus.
• (f) Using a unique viral enzyme called integrase, the
viral DNA is integrated into the cellular DNA.
• (g) Viral RNA is synthesized by the cellular enzyme
RNA polymerase II using integrated viral DNA as a
template. Two types of RNA transcripts shorter spliced
RNA (h) and full-length genomic RNA (j) are
produced.
• (h) Shorter spliced RNAs are transported to the
cytoplasm and used for the production of several viral
proteins that are then modified in the Golgi apparatus
of the cell (i).
• (j) Full-length genomic RNAs are transported to the
cytoplasm (k).
• (l) New virion is assembled and then buds off.
• (m) Mature virus is released.
• Pathogensis- virus enters the blood or tissue and come into
contact with a suitable host cell
↓
Local cell infection (macrophage or lymphocytes)
↓
Replication
↓
Viraemia
↓
Action CD8 lymphocytes, NK, ADCC on infected cells
↓
Asymptomatic period
↓
Virus multiplication in lymph nodes
↓
Fall in CD4 lymphocytes below 400 per cubic mm
↓
Virion spill over in blood from degenerating
lymphocytes
↓
Further fall in CD4 lymphocytes
↓
Susceptibility to other infections
Lab diagnosis of HIV-
1. Immunological test- fallowing parameters help to
establish immunodeficiency in HIV infection
• Total leucocyte and lymphocyte count to
demonstrate leucopenia and lymphocyte count
usually below 2000/mm3
• T cell subset assay absolute CD4 T cell count will
be usually less than 200/mm T4:T8 cell ratio is
reversed
• Platelets count will show thromocytopenia
• Raised IgG and IgA level
• DiminishedCMI as indicated by skin tests
• Lymph node biopsy show profound abnormalities
B- Specific tests-
1. Isolation of virus- it can be isolated from the peripheal
lymphocytes, bone marrow and serum.
2. Detection of HIV Ag- it is a single massive infection
as by blood transfusion the virus Ag may be detectable
in blood after about 2 weeks. The major core Ag P24
is the earliest virus to appear in blood ELISA can be
used for detection of this Ag
3. Detection of nucleic acid- the most sensitive and
specific test. It is done by PCR and hybridisation
4. Detection of Ab- simplest and most widely used. After
infection, Ab are appear 2-8 weeks to months during
this period , the individuals may be highly infectious
• The seronegative infective stage is k/a the window period.
Infection can be detected during the window period by p24
assay
• serological tests for Anti HIV Abs are of 2 types-screening
confirmatory test
• Highly sensitive -positive reaction should
be confirmed
• Simple to perform -highly specific
• Large no. of samples at a time- highly specific
• May give few false positive test
• Eg-ELISA -WESTERN BLOT
• Antibodies detected in ELISA include those directed
against: p24, gp120, gp160 and gp41, detected first in
infection and appear in most individuals
• Different types of ELISA techniques used:
– indirect
– competitive
– sandwich
• ELISA are for screening only, false positives do
occur and may be due to AI disease, alcoholism,
syphilis, and immunoproliferative diseases.
• ELISA tests useful for:
– Screening blood products.
– Diagnosing and monitoring patients.
– Determining prevalence of infection.
– Research investigations
• Western blot- in this test, strips of
nitrocellulose paper are used which are blotted
with proteins of HIV separated by
polyacrylamide gel electrophoresis. Strip is
reacted with sera, enzyme is conjugated with
antihuman globulin and then substrate is added
which produces colour band where Ab reacts
with specific Ag. Position of band indicates
Ag with which it reacted positive test show
presence of at least 2 bands of following Ag,
24, gp41, gp120/160
• 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.
• Treatment- Azidothymidine also k/a
zydovudine, Protease inhibitors like ritonavir,
indinavir which have been used as
monotherapy or in various combination.
 Immunologic Manifestations
• Early stage slight depression of CD4
count, few symptoms, temporary.
• Window of up to 6 weeks before antibody
is detected, by 6 months 95% positive.
• During window p24 antigen present, acute
viremia and antigenemia.
 Immunologic Manifestations
• Antibodies produced to all major antigens.
– First antibodies detected produced against
gag proteins p24 and p55.
– Followed by antibody to p51, p120 and gp41
– As disease progresses antibody levels
decrease.
 Immunologic Manifestations
• Immune abnormalities associated with increased
viral replication.
– Decrease in CD4 cells due to virus budding from
cells, fusion of uninfected cells with virally infected
cells and apoptosis.
– B cells have decreased response to antigens possibly
due to blockage of T cell/B cell interaction by binding
of viral proteins to CD4 site.
– CD8 cells initially increase and may remain elevated.
– As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and susceptibility of
patient to opportunistic infections.
– Death comes due to immuno-incompetence.
 Immunologic Manifestations
• Immune abnormalities associated with increased
viral replication.
– Decrease in CD4 cells due to virus budding from
cells, fusion of uninfected cells with virally infected
cells and apoptosis.
– B cells have decreased response to antigens possibly
due to blockage of T cell/B cell interaction by binding
of viral proteins to CD4 site.
– CD8 cells initially increase and may remain elevated.
– As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and susceptibility of
patient to opportunistic infections.
– Death comes due to immuno-incompetence.
 Truvada
• Truvada is made up of HIV drugs from a
class called nucleoside/nucleotide reverse
transcriptase inhibitors (NRTIs), also
known as “nukes.”
• The NRTIs block reverse transcriptase, a
protein that HIV needs to make more
copies of itself. This may slow down HIV
disease
 ‘typical’ primary HIV-1 infection
symptoms symptom
s
HIV proviral DNA

HIV antibodies
‘window’
period

HIV viral load

HIV-1 p24 antigen

0 1 2 3 4 5 6 / 2 4 6 8 10
1° infection weeks years
Time following infection
ELISA Sandwich
 Other Screening Tests
• Agglutination tests using latex particles, gelatin
particles or microbeads are coated with HIV
antigen and will agglutinate in the presence of
antibody.
• Dot-Blot Testing utilizes paper or nitrocellulose
impregnated with antigen, patient serum is
filtered through, and anti-antibody is added with
enzyme label, color change is positive.
– A rapid, cost-effective and may become an alternative
to standard ELISA and Western blot testing.
Particle Agglutination
 Western Blot
• Most popular confirmatory test.
– Utilizes a lysate prepared from HIV virus.
– The lysate is electrophoresed to separate out the HIV
proteins (antigens).
– The paper is cut into strips and reacted with test sera.
– After incubation and washing anti-antibody tagged
with radioisotope or enzyme is added.
– Specific bands form where antibody has reacted with
different antigens.
– Most critical reagent of test is purest quality HIV
antigen.
– The following antigens must be present: p17, p24,
p31, gp41, p51, p55, p66, gp120 and gp160.
 Western Blot
• 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.
 Western Blot
• 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.
 gp160
gp120

p68
p55
p53

gp41-45

Spectrum p40

p34
of anti-HIV p24

testing p18

p12

early recent / established advanced

DNA PCR
RNA PCR
p24 Ag
3rd gen ELISA
1st gen ELISA
Detuned ELISA
1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr
Western Blot
• Expensive – $ 80 - 100
• technically more difficult
• visual interpretation
• lack standardisation
– - performance
– - interpretation
– - indeterminate reactions –
resolution of ??
• ‘Gold Standard’ for
confirmation
 Western Blot
• Indeterminate results are those samples that produce
bands but not enough to be positive, may be due to the
following:
– prior blood transfusions, even with non-HIV-1 infected blood
– prior or current infection with syphilis
– prior or current infection with malaria
– autoimmune diseases (e.g., diabetes, Grave’s disease, etc)
– infection with other human retroviruses
– second or subsequent pregnancies in women.
– run an alternate HIV confirmatory assay.
• Quality control of Western Blot is critical and requires
testing with strongly positive, weakly positive and
negative controls.
 Indirect immunofluorescence
• Can be used to detect both virus and
antibody to it.
• Antibody detected by testing patient serum
against antigen applied to a slide,
incubated, washed and a fluorescent
antibody added.
• Virus is detected by fixing patient cells to
slide, incubating with antibody.
 Detection of p24 HIV antigen
• The p24-antigen screening assay is an EIA
performed on serum or plasma.
• P24 antigen only present for short time,
disappears when antibody to p24 appears.
• Anti-HIV-1 bound to membrane, incubated with
patient serum, second anti-HIV-1 antibody
attached to enzyme label is added (sandwich
technique), color change occurs.
• Optical density measured, standard curve
prepared to quantitate results.
 Detection of p24 HIV antigen
• Positive confirmed by neutralizing
reaction, preincubate patient sample with
anti- HIV, retest, if p24 present immune
complexes form preventing binding to HIV
antibody on membrane when added.
• Test not recommended for routine
screening as appearance and rate of rise
are unpredictable.
• Sensitivity lower than ELISA.
 Detection of p24 HIV antigen
• Most useful for the following:
– early infection suspected in seronegative
patient
– newborns
– CSF
– monitoring disease progress
Polymerase Chain Reaction (PCR)
• Looks for HIV DNA in the WBCs of a person.
• PCR amplifies tiny quantities of the HIV DNA present,
each cycle of PCR results in doubling of the DNA
sequences present.
• The DNA is detected by using radioactive or biotinylated
probes.
• Once DNA is amplified it is placed on nitrocellulose
paper and allowed to react with a radiolabeled probe, a
single stranded DNA fragment unique to HIV, which will
hybridize with the patient’s HIV DNA if present.
• Radioactivity is determined.
 Virus isolation
• Virus isolation can be used to definitively
diagnose HIV.
• Best sample is peripheral blood, but can use
CSF, saliva, cervical secretions, semen, tears or
material from organ biopsy.
• Cell growth in culture is stimulated, amplifies
number of cells releasing virus.
• Cultures incubated one month, infection
confirmed by detecting reverse transcriptase or
p24 antigen in supernatant.
 Viral Load Tests
• Viral load or viral burden is the quantity of
HIV-RNA that is in the blood.
• RNA is the genetic material of HIV that
contains information to make more virus.
 Viral Load Tests
• Viral load tests measure the amount of HIV-RNA
in one milliliter of blood.
• Take 2 measurements 2-3 weeks apart to
determine baseline.
• Repeat every 3-6 months in conjunction with
CD4 counts to monitor viral load ant T-cell count.
• Repeat 4-6 weeks after starting or changing
antiretroviral therapy to determine effect on viral
load.
 Testing of Neonates
• Difficult due to presence of maternal IgG
antibodies.
• Use tests to detect IgM or IgA antibodies,
IgM lacks sensitivity, IgA more promising.
• Measurement of p24 antigen.
• PCR testing may be helpful but still not
detecting antigen soon enough: 38 days to
6 months to be positive.
 References
• http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivlc.html#translat

• http://pathmicro.med.sc.edu/lecture/HIV3.htm
• http://www.avert.org/hivstages.htm
• http://www.aidsinfo.nih.gov/guidelines/
• http://www.hopkins-aids.edu/publications/pocketguide/pocketgd0105.pdf
• http://www.modares.ac.ir/sci/saman_h/Pages/applications.htm
• http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02
• http://www.hivandhepatitis.com/recent/test/realtime/061604_f.html