Dr.

Maryam Virology
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Retroviridae &
AIDS

Retroviruses have many subfamilies in three basic groups.

1. Oncoretroviruses (cancer-causing retroviruses) include human T-lymphotropic virus (HTLV)
causing a type of leukemia in humans
2. Lentiviruses (slow viruses) include HIV-1 and HIV-2, the cause of acquired immune deficiency
syndrome (AIDS) in humans.
3. Spumaviruses (foamy viruses) are benign and not linked to any disease in humans or animals

Human Immunodeficiency Virus: Human immunodeficiency virus (HIV) 1 is the cause of

acquired immunodeficiency syndrome (AIDS).

Both HIV-1 and HIV-2 cause AIDS, but HIV-1 is found worldwide, whereas HIV-2 is found primarily in
West Africa. HIV is one of the two important human T-cell lymphotropic retroviruses (human T-cell
leukemia virus is the other). HIV preferentially infects and kills helper (CD4) T lymphocytes, resulting in
the loss of cell-mediated immunity and a high probability that the host will develop opportunistic
infections. Other cells (e.g., macrophages and monocytes) that have CD4 proteins on their surfaces can be
infected also.

HIV belongs to the lentivirus subgroup of retroviruses, which cause "slow" infections with long incubation
periods. HIV has a bar-shaped (type D) core surrounded by an envelope containing virus-specific
glycoproteins (gp120 and gp41). The genome of HIV consists of two identical molecules of single-stranded,
positive-polarity RNA and is said to be diploid. The HIV genome is the most complex of the known
20and env, which encode the structural
retroviruses. In addition to the three typical retroviral genes gag, pol,
proteins, the genome RNA has six regulatory genes (Table –1). Two of these regulatory genes, tat and rev,
are required for replication, and the other four, nef, vif, vpr, and vpu, are not required for replication and are
termed "accessory" genes.

Table –1 Genes and Proteins of Human Immunodeficiency Virus

Gene Proteins Encoded Function of Proteins

by Gene
I. Structural genes found in all retroviruses
gag p24, p7 Nucleocapsid

I
p17 Matrix
pol Reverse Transcribes RNA genome into DNA
transcriptase
Protease Cleaves precursor polypeptides
Integrase Integrates viral DNA into host cell DNA
env Gp120 Attachment to CD4 protein
Gp41 Fusion with host cell
II. Regulatory genes found in human immunodeficiency virus that are required for replication
tat Tat Activation of transcription of viral genes
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rev Rev Transport of late mRNAs from nucleus to cytoplasm
III. Regulatory genes found in human immunodeficiency virus that are not required for replication (accessory
genes)
nef Nef Decreases CD4 proteins and class I MHC proteins on surface of infected cells; induces
death of uninfected cytotoxic T cells; important for pathogenesis by SIV

y
vif Vif Enhances infectivity by inhibiting the action of APOBEC3G (an enzyme that causes
hypermutation in retroviral DNA)
vpr Vpr Transports viral core from cytoplasm into nucleus in nondividing cells
Vpu Vpu Enhances virion release from cell

The gag gene encodes the internal "core" proteins, the most important of which is p24, an antigen used in
serologic tests. The pol gene encodes several proteins, including the virion "reverse transcriptase," which
synthesizes DNA by using the genome RNA as a template, an integrase that integrates the viral DNA into
the cellular DNA, and a protease that cleaves the various viral precursor proteins. The env gene encodes
gp160, a precursor glycoprotein that is cleaved to form the two envelope (surface) glycoproteins, gp120 and
gp41.

Three enzymes are located within the nucleocapsid of the virion: reverse transcriptase, integrase, and
protease.

There are several important antigens of HIV:

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1. gp120 and gp41 are the type-specific envelope glycoproteins. gp120 protrudes from the surface and
interacts with the CD4 receptor on the cell surface. gp41 is embedded in the envelope and mediates

III
the fusion of the viral envelope with the cell membrane at the time of infection. Antibody against
gp120 neutralizes the infectivity of HIV, but the rapid appearance of gp120 variants will make
production of an effective vaccine difficult.
2. The group-specific antigen, p24, is located in the core and is not known to vary. Antibodies against
p24 do not neutralize HIV infectivity but serve as important serologic markers of infection.

Summary of Replicative Cycle

In general, the replication of HIV follows the typical retroviral cycle .The initial step in the entry of HIV
into the cell is the binding of the virion gp120 envelope protein to the CD4 protein on the cell surface. The
virion gp120 protein then interacts with a second protein on the cell surface, one of the chemokine
receptors. Chemokine receptors, such as CXCR4 and CCR5Rproteins, are required for the entry of HIV into
CD4-positive cells. The T cell–tropic strains of HIV bind to CXCR4, whereas the macrophage-tropic strains
bind to CCR5.Next, the virion gp41 protein mediates fusion of the viral envelope with the cell membrane,
and the virion core containing the nucleocapsid, RNA genome, and reverse transcriptase enters the
cytoplasm.

In the cytoplasm, reverse transcriptase transcribes the genome RNA into double-stranded DNA, which
``migrates to the nucleus where it integrates into the host cell DNA. The viral DNA can integrate at different
sites in the host cell DNA, and multiple copies of viral DNA can integrate. Integration is mediated by a
virus-encoded endonuclease (integrase). Viral mRNA is transcribed from the proviral DNA by host cell
RNA polymerase and translated into several large polyproteins. The Gag and Pol polyproteins are cleaved
by the viral-encoded protease, whereas the Env polyprotein is cleaved by a cellular protease.
e
The Gag polyprotein is cleaved to form the main core protein (p24), the matrix protein (p17), and several
a
smaller proteins. The Pol polyprotein is cleaved to form the reverse transcriptase, integrase, and protease.
The immature virion containing the precursor polyproteins forms in the cytoplasm, and cleavage by the viral
protease occurs as the immature virion buds from the cell membrane. It is this cleavage process that results
in the mature, infectious virion.
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Transmission & Epidemiology
a
Transmission of HIV occurs primarily by sexual contact and by transfer of infected blood. Perinatal
transmission from infected mother to neonate also occurs, either across the placenta, at birth, or via breast
milk. Infection occurs by the transfer of either HIV-infected cells or free HIV (i.e., HIV that is not cell-
associated). Although small amounts of virus have been found in other fluids, e.g., saliva and tears, there is
no evidence that they play a role in infection. In general, transmission of HIV follows the pattern of hepatitis
B virus, except that HIV infection is much less efficiently transferred, i.e., the dose of HIV required to cause
infection is much higher than that of HBV. People with sexually transmitted diseases, especially those with
ulcerative lesions such as syphilis, chancroid, and herpes genitalis, have a significantly higher risk of both
transmitting and acquiring HIV. Uncircumcised males have a higher risk of acquiring HIV than do
circumcised males.

Transmission of HIV via blood transfusion has been greatly reduced by screening donated blood for the
presence of antibody to HIV. However, there is a "window" period early in infection when the blood of an
infected person can contain HIV but antibodies are not detectable. Blood banks now test for the presence of
p24 antigen in an effort to detect blood that contains HIV.

Pathogenesis & Immunity

HIV infects helper T cells (CD4-positive cells) and kills them, resulting in suppression of cell-mediated
immunity. This predisposes the host to various opportunistic infections and certain cancers such as Kaposi's
sarcoma and lymphoma. However, HIV does not directly cause these tumors because HIV genes are not
found in these cancer cells. The initial infection of the genital tract occurs in dendritic cells that line the
mucosa (Langerhans' cells), after which the local CD4-positive helper T cells become infected. HIV is first
found in the blood 4 to 11 days after infection.

HIV infection also targets a subset of CD4-positive cells called Th17 cells. These cells are an important
mediator of mucosal immunity, especially in the GI tract. Th17 cells produce IL-17, which attracts
neutrophils to the site of bacterial infection. The loss of Th17 cells predisposes HIV-infected individuals to
blood stream infections by bacteria in the normal flora of the colon, such as E. coli.

HIV also infects brain monocytes and macrophages, producing multinucleated giant cells and significant
central nervous system symptoms. The death of HIV-infected cells is result of immunologic attack by
cytotoxic CD8 lymphocytes.

Persistent noncytopathic infection of T lymphocytes also occurs. Persistently infected cells continue to
produce HIV, which may help sustain the infection in vivo. Lymphoid tissue, e.g., lymph nodes, is the main
site of ongoing HIV infection.

A person infected with HIV is considered to be infected for life. This seems likely to be the result of
integration of viral DNA into the DNA of infected cells. Although the use of powerful antiviral drugs can
significantly reduce the amount of HIV being produced, latent infection in CD4-positive cells and in
immature thymocytes serve as a continuing source of virus.

The main immune response to HIV infection.

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HIV has three main mechanisms by which it evades the immune system: (1) integration of viral DNA into
host cell DNA, resulting in a persistent infection; (2) a high rate of mutation of the env gene; and (3) the
production of the Tat and Nef proteins that downregulate class I MHC proteins required for cytotoxic T cells
to recognize and kill HIV-infected cells. The ability of HIV to infect and kill CD4-positive helper T cells
further enhances its capacity to avoid destruction by the immune system.

Clinical Findings
The clinical picture of HIV infection can be divided into three stages: an early, acute stage; middle, latent
stage; and a late, immunodeficiency stage. In the acute stage, which usually begins 2 to 4 weeks after
infection, a mononucleosis-like picture of fever, lethargy, sore throat, and generalized lymphadenopathy
occurs. A maculopapular rash on the trunk, arms, and legs (but sparing the palms and soles) is also seen.
Leukopenia occurs, but the number of CD4 cells is usually normal. A high-level viremia typically occurs,
and the infection is readily transmissible during this acute stage. This acute stage typically resolves
spontaneously in about 2 weeks.

Antibodies to HIV typically appear 10 to 14 days after infection, and most will have seroconverted by 3 to 4
weeks after infection. Note that the inability to detect antibodies prior to that time can result in "false-
negative" serologic tests, i.e., the person is infected, but antibodies are not detectable at the time of the test.
This has important implications because HIV can be transmitted to others during this period. If the antibody
test is negative but HIV infection is still suspected, then a PCR-based assay for viral RNA in the plasma
should be done.

After the initial viremia, a viral set point occurs, which can differ from one person to another. The set point
represents the amount of virus produced, i.e., the viral load, and tends to remain "set," or constant, for years.
The higher the set point at the end of the initial infection, the more likely the individual is to progress to latent
symptomatic AIDS. It is estimated that an infected person can produce up to 10 billion new virions each
day. This viral load can be estimated by using an assay for viral RNA in the patient's plasma. (The assay
detects the RNA in free virions in the plasma, not cell-associated virions.)

In the middle stage, a long latent period, measured in years, usually ensues. In untreated patients, the latent
period usually lasts for 7 to 11 years. The patient is asymptomatic during this period. Although the patient is
asymptomatic and viremia is low or absent, a large amount of HIV is being produced by lymph node cells
but remains sequestered within the lymph nodes. This indicates that during this period of clinical latency, the
virus itself does not enter a latent state.

A syndrome called AIDS-related complex (ARC) can occur during the latent period. The most frequent
manifestations are persistent fevers, fatigue, weight loss, and lymphadenopathy. ARC often progresses to
AIDS.

The late stage of HIV infection is AIDS, manifested by a decline in the number of CD4 cells to below
400/µL and an increase in the frequency and severity of opportunistic infections. Table -2 describes some of
the common opportunistic infections and their causative organism seen in HIV-infected patients during the
late, immunocompromised stage of the infection.

Table –2 Common Opportunistic Infections in AIDS Patients

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Site of Infection Disease or Symptom Causative Organism
Lung 1. Pneumonia Pneumocystis carinii, cytomegalovirus
2. Tuberculosis Mycobacterium tuberculosis
Mouth 1. Thrush Candida albicans
2. Hairy leukoplakia Epstein-Barr virus
3. Ulcerations Herpes simplex virus-1, Histoplasma capsulatum
Esophagus 1. Thrush Candida albicans
2. Esophagitis Cytomegalovirus, herpes simplex virus-1
Intestinal tract Diarrhea Salmonella sp., Shigella sp, cytomegalovirus,
Cryptosporidium parvum, Giardia lamblia
Central nervous 1. Meningitis Cryptococcus neoformans
system
2. Brain abscess Toxoplasma gondii
3. Progressive multifocal JC virus
leukoencephalopathy
Eye Retinitis Cytomegalovirus
Skin 1. Kaposi's sarcoma Human herpesvirus 8
2. Zoster Varicella-zoster virus
3. Subcutaneous nodules Cryptococcus neoformans
Reticuloendothelial Lymphadenopathy or Mycobacterium avium complex, Epstein-Barr
system splenomegaly virus

The two most characteristic manifestations of AIDS are Pneumocystis pneumonia and Kaposi's sarcoma.
Many AIDS patients have severe neurologic problems, e.g., dementia and neuropathy, which can be caused
by either HIV infection of the brain or by many of these opportunistic organisms.

Laboratory Diagnosis

The presumptive diagnosis of HIV infection is made by the detection of antibodies by ELISA. Because
there are some false-positive results with this test, the definitive diagnosis is made by Western blot analysis.

HIV can be grown in culture from clinical specimens, but this procedure is available only at a few medical
centers. The polymerase chain reaction (PCR) is a very sensitive and specific technique that can be used to
detect HIV DNA within infected cells. Some individuals who do not have detectable antibodies have been
shown by this test to be infected. As already mentioned, the amount of viral RNA in the plasma (i.e., the
viral load) can also be determined using PCR-based assays.

During the first month after infection, antibody tests may be negative. These false-negative tests are due to
insufficient antibody being made early in infection to be detected in the ELISA test. The average time for
seroconversion is 10 to 14 days and most, but not all, will have seroconverted by 4 weeks.

Treatment
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The treatment of HIV infection has resulted in a remarkable reduction in mortality and improvement in the
quality of life of infected individuals. The specific goals of treatment are to restore immunologic function
that reduces the incidence of both opportunistic infections and certain malignancies as well as to reduce viral
load, which reduces the chance of transmission to others.

Treatment of HIV infection typically involves multiple antiretroviral drugs. The use of a single drug
(monotherapy) for treatment is not done because of the high rate of mutation to drug resistance.

The choice of drugs is complex and depends on several factors, e.g., whether it is an initial infection or an
established infection, the number of CD4 cells, the viral load, the resistance pattern of the virus, and whether
the patient is pregnant or is coinfected with HBV or HCV.

In general, initial antiretroviral therapy consists of one of two regimens, each one of which consists of three
drugs. One regimen has two nucleoside reverse transcriptase inhibitors (NRTI), such as lamivudine and
zidovudine plus a protease inhibitor, such as fosamprenavir. The second regimen has the same two NRTI
plus a nonnucleoside reverse transcriptase inhibitors (NNRTI), such as Nevirapine.

These combinations are known as HAART, which is an acronym for "highly active antiretroviral therapy."
It is very effective in prolonging life, improving quality of life, and reducing viral load but does not cure the
chronic HIV infection, i.e., replication of HIV within CD4-positive cells continues indefinitely.
Discontinuation of HAART almost always results in viremia (a return of the viral load to its pretreatment set
point) and a fall in the CD4 count.

Enfuvirtide (Fuzeon) is the first of a new class of anti-HIV drugs known as fusion inhibitors, i.e., they
prevent the fusion of the viral envelope with the cell membrane. Enfuvirtide is a synthetic peptide that binds
to gp41 on the viral envelope, thereby blocking the entry of HIV into the cell. It must be administered by
injection and is quite expensive.

In 2007, the FDA approved the use of maraviroc—a drug that blocks the binding of the gp120 envelope
protein of HIV to CCR-5, which is an important coreceptor on the cell surface. It should be used in
combination with other antiretroviral drugs in patients infected with CCR-5 tropic strains of HIV.

Also, in 2007, the FDA approved the use of raltegravir (Isentress), the first drug to inhibit the HIV-
encoded integrase. It is recommended for use in patients who have been treated with other antiretroviral
drugs but continue to produce significant levels of HIV.

"Immune reconstitution syndrome" may occur in HIV-infected patients who are treated with a HAART
regimen and who are coinfected with other microbes such as hepatitis B virus, hepatitis C virus,
Mycobacterium avium complex, Cryptococcus neoformans, and Toxoplasma gondii. In this syndrome, an
exacerbation of clinical symptoms occurs because the antiretroviral drugs enhance the ability to mount an
inflammatory response. HIV-infected patients with a low CD4 count have a reduced capacity to produce
inflammation, but HAART restores the inflammatory response and, as a result, symptoms become more
pronounced. To avoid immune reconstitution syndrome, the coinfection should be treated prior to instituting
HAART whenever possible.

Prevention
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No vaccine is available. Prevention consists of taking measures to avoid exposure to the virus, e.g., using
condoms, not sharing needles, and discarding donated blood that is contaminated with HIV. Postexposure
prophylaxis, such as that given after a needle-stick injury or a high-risk nonoccupational exposure, consists
of two drugs, e.g., lamivudine and zidovudine for low-risk exposures and the same two drugs plus
lopinavir/ritonavir for high-risk exposures. Two steps can be taken to reduce the number of cases of HIV
infection in children: ZDV or nevirapine should be given perinatally to HIV-infected mothers and neonates,
and HIV-infected mothers should not breast feed. In addition, the risk of neonatal HIV infection is lower if
delivery is accomplished by cesarean section rather than by vaginal delivery. Circumcision reduces HIV
infection.

Several drugs are commonly taken by patients in the advanced stages of AIDS to prevent certain
opportunistic infections. Some examples are trimethoprim-sulfamethoxazole to prevent Pneumocystis
pneumonia, fluconazole to prevent recurrences of cryptococcal meningitis, ganciclovir to prevent
recurrences of retinitis caused by cytomegalovirus, and oral preparations of antifungal drugs, such as
clotrimazole, to prevent thrush caused by Candida albicans.

Why is it difficult to develop a vaccine for HIV and AIDS?

There are many reasons for this, including:

 Nobody has ever recovered from HIV infec5on, so there is no natural mechanism to imitate
 HIV destroys the immune system cells that are meant to :ght against it
 Soon a=er infec5on, HIV inserts its gene5c material into human cells, where it remains hidden from the
immune system .
 HIV occurs in several subtypes, each of which is very diAerent from the others
 Even within each subtype, HIV is highly variable and constantly changing
 There are no good animal models to use in experiments although the use of non human primate (NHP)
models could become a more signi:cant model for HIV vaccine design and tes5ng in the future.

Human T-Cell Lymphotropic Virus

Human T-cell lymphotropic virus-1 (HTLV) causes two distinctly different diseases: a cancer called adult T-
cell leukemia/lymphoma and a neurologic disease called HTLV-associated myelopathy (also known as
tropical spastic paraparesis or chronic progressive myelopathy). HTLV-2 also appears to cause these
diseases, but the association is less clearly documented. (All information in this section refers to HTLV-1
unless otherwise stated.)

HTLV and HIV are the two medically important members of the retrovirus family. Both are enveloped
viruses with reverse transcriptase in the virion and two copies of a single-stranded, positive-polarity RNA
genome. However, HTLV does not kill T cells, whereas HIV does. In fact, HTLV does just the opposite; it
causes malignant transformation that "immortalizes" the infected T cells and allows them to proliferate in an
uncontrolled manner.