Laboratory Activity 17 – Determination of Anti-HIV 1/2 Flashcards
(AIDS); human retrovirus
HIV-1 and 2
(+) ss-RNA
Retroviruses
reverse transcriptase
Retroviruses
This enzyme enables the virus to convert viral RNA to DNA in contrast to the normal process of transcription, where DNA is converted to RNA
reverse transcriptase
HIV infects
CD4+ T cells
macrophages
dendritic cells
HIV Mode of Transmission
Sexual contact
Mother to infant
Needle sharing by drug users
Blood or blood product transfusion Transplantation of tissues or organs
Contact with contaminated blood/body fluids
Human T cell lymphotropic virus type III (HTLV-III)
Lymphadenopathyassociated virus (LAV)
AIDS-associated retrovirus (ARV)
HIV-1
Endemic in West Africa; less pathogenic and has a lower rate of transmission
HIV-2
Group-specific antigen
gag
Polymerase
pol
Envelope
env
Transactivator
tat
Regulator of viral expression
rev
Viral infectivity
vif
Viral protein R
vpr
Viral protein U
vpu
Negative-regulation factor
nef
Core proteins and matrix proteins
gag
Reverse transcriptase, protease, and integrase enzymes
pol
Transmembrane glycoproteins
env
Positive regulator of transcription
tat
Allows export of unspliced and partially spliced transcripts from the nucleus
rev
Affects particle infectivity
vif
Transport of DNA to the nucleus; augments virion
roduction; arrests cell cycle
vpr
Promotes intracellular degradation of CD4 and
nhances the release of virus from the cell membrane
vpu
Augments viral replication in vivo and in vitro; down- regulates CD4 and MHC class II
nef
codes for gp160/120 and gp41
HSV 1
codes for gp140 and gp34
HSV 2
codes for p 55 → p15, p17, and p24
Note: sor codes for p24
HSV 1
codes for p26
HSV 2
Transcribes RNA to DNA
Reverse transcriptase
inserts viral DNA to host cell’s DNA
Integrase
cleaves structural protein
RNase and Protease
May last from many months to years
Primary stage
also known as AIDS-related complex (ARC)
Intermediate stage
Infected persons may experience an initial lu-like illness, and then remain symptomatic or exhibit only chronic ymphadenopathy for many years.
Primary stage
There are physical symptoms, but no opportunistic infections
Intermediate stage
is usually seen within 2 to 10 years after initial infection with the HIV
Final stage
Pneumocystis jiroveci, Mycobacterium tuberculosis, and fungal infections
Pneumonia
oral candidiasis; thrush
Candida albicans
diarrhea and weight loss
Cryptosporidia and microsporidia
Enteric bacterial infection
Salmonella and Campylobacter
Toxoplasma gondii
cysts in the brain
Cryptococcus neoformans
meningitis
Cytomegalovirus
inflammation of retina, brain and spinal cord
multi-focal tumor of endothelial cells
Kaposi’s sarcoma
The CD4:CD8 ratio
reduced from normal
CD4-positive T-cell counts
decreased
CD4-positive T-cell function
impaired
B-cell activation
abnormal
Macrophage function
impaired
NK cell function
impaired
Viral antigen is coated on a solid support → patient serum added → after incubation and washing, enzyme-labeled antihuman globulin (AHG) is added followed by substrate
Enzyme-linked immunosorbent assay (ELISA)
HIV antigen is adsorbed onto carrier particles → the particles agglutinate in the presence of antibody
Slide agglutination test
Anti-HIV bound to a solid support is incubated with serum → after washing, enzyme-labeled anti-HIV-1 is added followed by substrate
p24 antigen test (HIV-1 Ag)
p24 antigen precedes antibody by several weeks; positives must be confirmed by neutralization assay
p24 antigen test (HIV-1 Ag)
a. Screening Tests
Enzyme-linked immunosorbent assay (ELISA)
Slide agglutination test
p24 antigen test (HIV-1 Ag)
b. Confirmatory Tests
Western blot testing
Immunofluorescence assay (IFA)
Radioimmunoprecipitation Assay (RIPA)
A lysate of HIV antigen is separated into components by electrophoresis and transferred to nitrocellulose paper → the resulting blot is cut into strips and reacted with serum → labeled antihuman globulin is added
Western blot testing
HIV infection is indicated by reactivity in two (2) of the following bands:
o Antibody test: Serum is incubated with virally infected cells on a glass slide → fluorescence-labeled antihuman globulin is added
o Antigen test: Patient cells are fixed to a slide and incubated with HIV-specific antiserum
Immunofluorescence assay (IFA)
Employs a soluble second antibody to precipitate the bound antigen
Radioimmunoprecipitation Assay (RIPA)
o IgM antibody against the p24 core protein usually develops within [?] after infection
6 to 8 weeks
o Within weeks, IgG antibodies against p24 appear, as do antibodies against [?] (i.e., gp160, gp120, gp41) and [?] (i.e., p31, p51, p66)
envelope gene products
polymerase gene products
o The HIV isolation technique can detect antigens before antibodies develop and can be used to monitor antiviral treatment
• The patient’s monocytes are stimulated and grown in culture ® the culture supernatant can be tested for the presence of antigens
• A modified enzyme-linked immunosorbent assay (ELISA) can be performed on plasma, serum, cerebrospinal fluid (CSF), and culture fluids to detect the presence of HIV antigens
• Immunofluorescence assay (IFA) can be used to detect HIV antigens
• Virus grown on culture
• Expensive, time-consuming and hazardous; not routinely performed
can be used to detect viral RNA
In situ hybridization and filter, hybridization
o Cells from peripheral blood and lymph nodes can be used
In situ hybridization and filter, hybridization
can be used to detect viral DNA in infected cells
Southern blot hybridization
can be used to amplify both RNA and DNA, which provides extremely sensitive systems for RNA and DNA detection
Polymerase chain reaction (PCR) techniques
o Viral DNA from infected cells is amplified, then identified using labeled probes
Polymerase chain reaction (PCR) techniques
o Extremely sensitive technique
Polymerase chain reaction (PCR) techniques
Tests to Stage and Monitor HIV
• CD4 T cell count
• HIV-1 viral load assay
o Purposes: Used to predict disease progression, determine when to start antiretroviral therapy, monitor response to therapy
Tests to Stage and Monitor HIV
o Test 2-8 weeks after the start of therapy and then every 3-4 months (the same assay should be used to assess changes)
Tests to Stage and Monitor HIV
Tests to Stage and Monitor HIV
o Methods include:
• Reverse transcriptase-polymerase chain reaction (RT-PCR)
• Branched DNA (bDNA)
• Nucleic acid sequence-based amplification (NASBA)
