Download MICR 454L Lec 13 HIV-1EP - Cal State LA

MICR 454L
Emerging and Re-Emerging
Infectious Diseases
Lecture 13: HIV
Dr. Nancy McQueen & Dr. Edith Porter
Overview
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Brief history
Nomenclature
Morphology and nature of the genome
Viral replication cycle
Pathogenesis and clinical symptoms
Diagnosis
Treatment
Prevention
Threat
Brief History
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Late 1970’s physicians in L.A., San Francisco, and
New York observe clusters of patients with similar
symptoms
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Severe fungal pneumonia (Pneumocystis jirovecii – formerly
P. carinii)
Sudden weight loss
History of recurrent infections
Bluish-purple spots on the skin (Kaposi’s sarcoma) caused by human herpesvirus type 8
All of the patients were
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Young
Male
Homosexual
Pneumocystis jirovecii
DIF of P. jirovecii
Kaposi’s sarcoma
Brief History
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The term AIDS (Acquired Immune Deficiency Syndrome)
was first coined in 1981 and used in a CDC report in
1982:
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A condition characterized by a marked depletion of CD4+ T
lymphocytes
Opportunistic infections
Due to a transmissible agent acquired though
 Sexual contact
 Blood exchange
In 1982-83, a retrovirus was isolated from the blood of
individuals by groups in Paris and Maryland, lead by Luc
Montagnier and Robert Gallo, respectively.
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The virus, subsequently named human immunodeficiency virus type I (HIV-1), was demonstrated to be the cause of AIDS.
Nomenclature
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There are two types of HIV
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HIV-1 - is the most common cause of AIDS worldwide
HIV-2 - is the most common cause of AIDS in West Africa and is
rarely found outside this region
Both types have many subtypes (clades)
The infection caused by HIV-2 is less severe than that caused by
HIV-1.
HIV belongs to the family Retroviridae, sub-family Lentiviridae
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Retroviruses
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The name comes from “backward” nucleic acid synthesis
Retroviruses convert genomic viral (+)RNA to dsDNA that then
integrates into the host cell DNA (provirus)
Use RT (reverse transcriptase), RNA-dependent, DNA
polymerase for making DNA from an RNA template
Percentage of Population
Infected with HIV in Africa
Where did AIDS come from?
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There is no definitive answer to this question
Evidence indicates HIV-1 and HIV-2 arose from
different evolutionary lines
HIV-2 appears to have arisen from a mutation in a
simian immunodeficiency virus (SIV) of mangabey
monkeys in West Africa
HIV-1 is genetically similar to SIV carried by
chimpanzees in Central Africa
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The chimpanzee virus appears to be a hybrid of two
mangabey SIVs.
Where did AIDS come from?
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It has been suggested that HIV-1 jumped the
species barrier in small villages around 1930 by
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Humans eating infected monkeys
Humans being bitten by infected monkeys
The end of European colonialism and the
subsequent disruption of the social structure of subSaharan Africa resulted in
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Urbanization
Increased prostitution
Growth of highway transportation
Increased sexual promiscuity
Spread of the disease
Morphology and Nature of the
Genome
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Enveloped with glycoprotein spikes
Icosahedral
2 identical strands of + single stranded RNA
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Although the RNA is of the positive sense, expression of
the genome requires that the virus is copied into cDNA and
that the cDNA is integrated into the host cell DNA
gp 120
gp 41
Fusion at the
plasma membrane
Note that
reverse
transcription
takes place
inside
preintegration
complex in the
cytoplasm
Budding from
plasma
membrane
mRNA
synthesis
and
genomic
RNA
synthesis
take place
inside
nucleus
using host
cell
enzymes
HIV Attachment is Mediated by
gp120 and gp41
Fusion peptide
gp41
gp120
CD4 receptor
CXCR4 (lymphocytes) or
CCR5 (macrophages) coreceptor
HIV Attaches to Chemokine
Co-Receptors
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Chemokine receptors are receptors of chemical messages
involved in cellular communication resulting in chemotaxis.
Individual HIV strains are classified as being either lymphotropic
or macrophageotropic based on which chemokine receptor the
virus recognizes.
Rare individuals who are resistant to HIV may have genetic
defects (32-nucleotide deletion) in their R5 chemokine receptors
(1% of whites of European descent are homozygous for the
defect and 20% are heterozygous).
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Protection from plague?
Defect only protects against HIV-1, subtype B that is transmitted
sexually and is prevalent in the U.S. and Europe.
HIV Penetration
HIV Structure
Viral coresite of RT
activity
Model for HIV Uncoating
HIV Reverse Transcription
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All retroviruses have an RNA dependent, DNA polymerase =
reverse transcriptase (RT). These RT enzymes:
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Can use either RNA or DNA as a template
Have no proofreading and 1-10 errors/provirus are made
Require a primer to prime synthesis
Synthesize only in the 5’ to 3’ direction
Have RNAse H activity which degrades RNA in an RNA/DNA
hybrid
Use a jumping strategy to make full length DNA copies of the
linear RNA template.
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The synthesis occurs within the core of the virus
The product is called the preintegration complex
The DS DNA than moves into the nucleus for integration into the
host cell DNA
HIV - Latent versus Productive
Infection
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After integration of proviral DNA into the host cell
genome, there are two possible outcomes
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Productive infection in which new viruses are made and
released
Latent infection in which no new virus is made and released
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Latent infections do not contribute to disease, but they they
represent a significant hurdle to treatment and virus eradication
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They are not recognized by the immune system because they do not
express viral proteins
Latently infected T cells and macrophages can start producing virus
following
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Activation of T cells via the T cell receptor
Virus infections
Treatment with cytokines
HIV Infection of T cells
HIV Infection of
Macrophages
An infected macrophage. Viral particles are within the vacuoles.
How do you get AIDS?
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Sexual contact
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Homosexual
Heterosexual
Anal sex causes more damage with a greater chance of
transmission
Transfusions with infected blood or blood products
(hemophiliacs)
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Sharing blood contaminated needles and syringes
Mother to child
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Placental
During birth
Through infected breast milk
Transmission Modes
Male versus Female
Transmission Modes
Progression of HIV Infection
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HIV-1 strains are sub-classified into CCR5-requiring (R5) and
CXCR4-requiring (X4) depending upon their co-receptor
requirements for infection.
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A single point mutation can change an R5 virus into an X4 virus
R5 viruses are regarded as the actual transmitters of infection, while
disease progression is associated with the appearance of X4
viruses.
Recent studies suggest that in females R5 viruses infect CD4+
CCR5+ Langerhans cells (a type of dendritic cell) of the vaginal
epidermis
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Circulating monocytes then transport the virus to CD4+ T cells in lymph
nodes where active viral replication of X4 virus occurs.
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X4 variants are generated via reverse transcriptase mistakes made
during the RT activity of the virus in the macrophage
Progression of HIV Infection
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Others studies suggest that the initial target of HIV
infection at mucosal surfaces appears to be dendritic
cells containing a DC-SIGN receptor (a lectin).
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Thus, the appearance of X4 viruses is early in infection and HIV
spreads systemically to seed in various organs within a few days
of sexual exposure.
Following attachment to DC-SIGN on dendritic cells, HIV is
internalized, escapes lysosomal degradation and subsequent
MHC presentation to the immune system.
When the dendritic cell migrates to a lymph node, the endocytic
vesicle functions in facilitating the transfer of intact infectious
virus to naïve CD4+ T cells.
No matter what the initial dendritic cells of infection are,
the take-home message here is that systemic spread
occurs very early in the infection.
HIV’s Favorite Cellular Targets
Infection of several types of brain
cells probably contributes to lethargy
and HIV dementia
Infection of gut intestinal
epithelium and lymphoid
tissue creating memory cells
carrying CCR5+ and CD4+
receptors
Naïve CD4+ inflammatory cells
are normally responsible for
macrophage activation and cellmediated immunity through CD8+
cytotoxic T cell activation
Dendritic cells- Are relatives of macrophages
that reside throughout the tissues and are
responsible for processing foreign matter
and presenting it to lymphocytes
CD4+ macrophages, dendritic and
other antigen-presenting cells
harbor HIV, but are not usually
killed by it. The cells are a
continuing source of the virus and
can carry it to the brain
Naïve CD4+ helper cells
that normally control
antibody production by
B cells. Some of these
become CCR5+ CD4+
memory cells
Stages of HIV Infection
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The CDC has classified HIV infection into four
stages
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Prodromal stage (primary, acute infection)
Latency period (asymptomatic stage)
Persistent generalized lymphadenopathy (early and
medium stage HIV symptomatic disease)
Full blown AIDS (Late-stage HIV infection)
Prodromal stage (primary, acute
infection) of HIV Infection
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Characterized by fever, rash, diarrhea, aches,
headaches, lymphadenopathy, and fatigue (flu-like
symptoms)
The CD4+ cell count drops, but not enough to impair
immune function
HIV actively replicates and releases new viruses into
the bloodstream so the viral load is high
Seroconversion and appearance of anti-HIV
antibodies occurs at the end of this stage
Latency period (asymptomatic
stage) of HIV infection
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No symptoms or only swollen lymph nodes
Virus is hiding in inactive T cells, including T memory cells
in lymphoid tissues
Antibody against HIV can be found
CD4+ cells increase, but do not reach pre-HIV infected
level
Viral load in the blood decreases and stabilizes
Persistent Generalized
Lymphadenopathy (early and medium
stage HIV symptomatic disease)
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Swollen lymph nodes, recurrent fevers, skin rashes,
night sweats, persistent diarrhea, persistent cough,
and extreme fatigue occur
Neurological symptoms of memory loss, confusion,
and depression may occur.
Opportunistic infections, including oral and vaginal
candidiasis (thrush) and herpesvirus outbreaks occur
CD4+ count decreases
Viral load increases
Full blown AIDS (Late-stage
HIV infection)
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CD4+ T cell count decreases to 200/mm3 or less
Lymph nodes degenerate
Severe, recurring opportunistic infections
Viral load
CD4+
count
HIV Disease Progression
Rates
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How quickly the disease progresses varies from individual to
individual
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Progression rate is determined by
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The nature of the immune response
 Slower progression occurs in those who develop a good
CD8 T-cell response or those who recognize a diversity of
epitopes
Level of basal RNA during latent stage
HIV subtype
Host genetic susceptibility
Co-infections - may enhance viral replication
Pathogenesis- Contributions
to Clinical Immunodeficiency
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Lysis of CD4+ T cells caused by
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viral budding
gp120 cross-linking of CD4 receptors priming
cells for apoptosis
down regulation of MHC class I molecules cause
NK cells to destroy the infected cell
syncytium formation (giant, multinucleated cells
formed by the fusion of the membranes of
adjacent cells)
Syncytia formation
Pathogenesis- Contributions
to clinical immunodeficiency
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Functional impairment of immune system
 Soluble gp120 blocks interaction of CD4+ T cells with Class II
MHC on antigen presenting cells (APCs)
 Endogenous gp120 prevents CD4 from being transported to the
cell surface.
 Nef and Vpu viral proteins downregulate CD4
 Impaired macrophage and natural killer cell function
 Destruction of architecture of lymph nodes
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Central nervous system damage
 Release of inflammatory cytokines from HIV-infected CNS
macrophages
 Soluble gp120 may interfere with neurotransmitter action on
neurons
Diagnosis of HIV Infection
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ELISA – used for first screening
Western blot – used to confirm a positive ELISA
result
RT-PCR – used for following viral load (serum viral
concentration) during treatment
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Detects the presence of viral RNA by making a cDNA copy
of the RNA and amplifiying that copy by PCR.
Can also be used to detect specific resistant variants
Indirect Western Immunoblot
for HIV diagnosis
Indirect Western Immunoblot
for HIV Diagnosis
Patient antibodies
Need to have 2
or more bands
showing up to
confirm a
positive ELISA
Treatment of HIV infection
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All anti-HIV drugs have numerous side effects
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Reverse transcriptase inhibitors
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Nucleoside analogues such as AZT
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Prevent successful synthesis of cDNA by RT; act as chain terminators
when incorporated in place of a normal nucleoside
Possibly the best application of AZT is its use during pregnancy as a
way to reduce the risk of HIV being passed from mother to child.
Non-nucleoside analogue RT inhibitors
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Bind non-competitively to RT to block its polymerization function
Nucleotide analogues
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Common side effects include nausea, vomiting, headache, fatigue,
weakness, and/or muscle pain.
Other side effects included changes in body fat distribution
(lipodystrophy), inflammation, insomnia, and kidney disorders.
Work similar to the nucleoside analogues
Protease inhibitors
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Prevent cleavage of polyproteins (by viral protease) required to make
mature virus
Examples for Lipodystrophy in
AIDS Patients
Treatment of HIV infection
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Fusion inhibitors – available to people with
limited treatment options.
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Integrase inhibitors??
Combination therapy – helps prevent the
development of resistant strains
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HAART - highly active anti-retroviral therapy (3 or more
different classes of drugs in combination)
 There are serious long-term side effects, such as an
accumulation of lactic acid in the bloodstream and physical
and metabolic changes that cause changes in fat
distribution as well as cholesterol and glucose abnormalities
that can lead to a risk of heart disease.
 Long-term use of the drugs can also promote the
development of drug-resistant strains of HIV.
HIV Anti-Viral Therapies
Nucleoside Analogues
Protease Inhibiters
Protease Inhibiters
•This picture shows the HIV protease (purple and green) complexed with the inhibitor
(spacefill). This prevents the normal viral substrate from reacting with the protease and thus,
the viral polypeptides are not cleaved.
Antiviral Therapy - in the
future?
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Antisense therapy – the mechanism of action is
similar to that of hybrid arrested translation.
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A single stranded RNA or DNA molecule that is
complementary to a viral mRNA is made.
It will combine, by complementary base-pairing, with
the mRNA to block translation of the mRNA into a
protein product
Hence an essential viral protein is not made
Antiviral Therapy - in the
future?
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Antisense RNA
Antiviral Therapy - in the
future?
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si RNA = smalling interfering RNAs.
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These are small double stranded RNAs that are
21-22 nucleotides in length and that are
homologous to an mRNA that you wish to silence
(prevent it from being translated).
The siRNA complexes with a cellular
endonuclease and the complex will target
homologous mRNA for degradation.
Action of siRNA
Other Treatments of HIV
Infection
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Anti-inflammatories are being considered
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Prevent destruction of the lymphatic architecture
• Therapeutic HIV vaccines
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Designed to boost the body’s immune response to
HIV in individuals already infected with HIV
Clinical trials are in progress
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IL-2 treatment is being tried to purge HIV from the
reservoirs of long-living immune cells which appear to
be persistently infected despite long periods of potent
anti-HIV therapy.
Preventative HIV Vaccines
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Three main types of vaccines are being
studied
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Subunit vaccines
Recombinant-vector vaccines
DNA vaccines
Combination vaccination via a prime-boost
strategy is also being studied
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One type of vaccine is given (prime)
This is followed later by a different type of vaccine
(boost)
Prevention - what can you do?
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Abstinence
Use of condoms
Education
No sharing or reuse of needles
Threats
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Development of drug resistant strains
Despite evidence that prevention programs instituted some time ago
are beginning to have an impact in some countries, the HIV/AIDS
epidemic continues to grow.
By the end of 2005, 40.3 million people were living with HIV/AIDS,
including 17.5 million women and 2.3 million children under the age
of 15.
In 2005 alone, a total of 3.1 million people died of HIV/AIDS-related
causes.
Globally, new infections among women, especially young women,
continued to outpace those among men, - a stark reminder that
gender inequity and violence against women fuel the epidemic.
Life-saving drugs to prevent mother-to-child transmission of HIV
have not been available to the hundreds of thousands of infants who
have become needlessly infected at birth or through breastfeeding in
the last year.
World-wide, only one in ten persons infected with HIV has been
tested and knows his/her HIV status.
Threats - Africa
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Ninety-six percent of people with HIV live in the developing world,
most in sub-Saharan Africa.
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The epidemic continues to grow in this region, with nearly a million new
infections between 2003 and 2005.
In some African countries, three quarters of those infected are
women - many of whom have not had more than one sexual partner.
In six African countries, (Botswana, Lesotho, Namibia, South Africa,
Swaziland and Zimbabwe), more than one in five of all pregnant
women have HIV/AIDS.
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In Swaziland, nearly 40% of pregnant women are HIV-positive.
Without prevention efforts, 35% of children born to an HIV positive
mother will become infected with HIV.
At least a quarter of newborns infected with HIV die before age one, and
up to 60% will die before reaching their second birthdays.
Take Home Message
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AIDS is caused by the retroviruses HIV-1 and HIV-2
The genome of HIV consists of 2 identical strands of + RNA
The receptor and coreceptors for HIV are CD4, and chemokine
receptors, respectively.
The two main types of cells infected by HIV are CD4+ T cells, and
macrophages.
HIV is an enveloped virus that enters the host cell by fusing its
envelope with the plasma membrane of the host cell.
Reverse transcriptase makes a cDNA copy of the viral RNA. The
cDNA is then integrated into the host cell DNA.
HIV is transmitted through sexual contact, through contact with
infected blood or its products, or from mother to child.
The virus is disseminated early on in the infection.
Take Home Message
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As the diseases progresses there is a decrease in CD4+ T cells
with an increase in viral load.
As the CD4+ T cell count decreases, the patient gets recurrent
opportunistic infections.
Treatment includes RT inhibitors, protease inhibitors, and fusion
inhibitors.
HAART uses 3 or more drugs in combination.
Drug resistance is a problem.
Despite ongoing research to develop a vaccine, no effective
vaccine has been developed.
The world-wide threat is enormous, with over 40 million people
infected.
Countries in Africa and India have the highest percentage of the
population infected.
Resources
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The Microbial Challenge, by Krasner, ASM Press, Washington DC, 2002.
Brock Biology of Microorganisms, by Madigan and Martinko, Pearson
Prentice Hall, Upper Saddle River, NJ, 11th ed, 2006.
Microbiology: An Introduction, by Tortora, Funke and Case; Pearson
Prentice Hall; 9th ed, 2007.
Fundamentals of Molecular Virology, by Nicholas Acheson; Wiley and Sons;
2007
AIDS Update, 2007 by Stine, Gerald J. ; McGraw-Hill Higher Education,
2007
www.nlm.nih.gov
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Resources
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http://www.kcom.edu/faculty/chamberlain/website/lectures/lecture/ai
ds.htm&h=292&w=301&sz=92&hl=en&start=20&um=1&tbnid=eSWT
0UZKO1aU6M:&tbnh=113&tbnw=116&prev=/images%3Fq%3DKap
osi%2527s%2Bsarcoma%26svnum%3D10%26um%3D1%26hl%3D
en%26lr%3Dlang_en%26sa%3DN
http://www.maxillofacialcenter.com/images/KaposiCtitle.jpg
http://www.primeboost3.org/images/map_hiv_e.gif
http://content.answers.com/main/content/wp/en/9/90/Africa_HIVAIDS_300px.png
http://www.med.upenn.edu/bmcrc/morph/images/Fig-11-HIV.jpg
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