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FUNDAMENTALS OF HIV

HIV virology, testing and


monitoring

crossed species to man, the second most common being HIV-2,


which derives from SIVsm, a strain of SIV found to affect a
West African monkey known as the sooty mangabey.

Life cycle

Temi Lampejo

The primary targets of HIV-1 are T lymphocytes and macrophages, including macroglial cells located within the central
nervous system. Through initial engagement of host cell receptors, a complex cascade of processes is set into motion that
ultimately results in the generation of further infectious virus
particles. The process by which HIV-1 replicates within a host
cell is illustrated in Figure 1.

Deenan Pillay

Abstract
Human immunodeficiency virus (HIV), the cause of AIDS, is widely
accepted as a cross-species infection descended from monkey simian immunodeficiency virus (SIV). Although originating in Africa, this virus has
spread to cause a worldwide pandemic and, along the way, has evolved
a number of mechanisms that enable HIV to evade host immune responses. It is hoped that vaccine developments may in the future provide
a means to effective immune control. Despite increasing global awareness
of HIV, in approximately 20e25% of individuals in the UK and USA the
infection remains undiagnosed, with higher observed rates in regions of
Asia and sub-Saharan Africa. As diagnostic methods continue to evolve,
the onus lies on medical practitioners to promote and facilitate increased
use of currently available tests. The most recent immunoassay technology
includes combined antigen (p24) and antibody techniques (4th generation tests) and rapid, portable point-of-care tests, which provide the opportunity for earlier diagnosis, initiation of therapy and reduced
transmission of HIV. Molecular techniques (detection of viral RNA or proviral DNA) are vital in the diagnosis of early infection and vertical transmission in addition to HIV monitoring and detection of drug resistance.
Along with current genotypic and phenotypic drug resistance assays,
readily available web-based systems aid in the clinical interpretation of
detected drug resistance mutations, and should ultimately facilitate
choice of therapy.

Structure of HIV
The outermost structure of the spherical HIV-1 particle is the
host-cell derived envelope, which accommodates the transmembrane envelope proteins gp120 and gp41,2 appearing as
spikes, up to 72 in number, projecting outwards from the virion
(Figure 2). The gp120 protein, which binds the CD4 receptor, is
highly immunogenic and represents the target for most host
antibodies.3 These mostly strain-specific antibodies occupy the
CD4 binding site on gp120 blocking their interaction.3 Some
recently recognized broader-acting gp120 antibodies have very
high potency; they are considered a natural template for effective
antibody design.4,5
Lying beneath the outer lipid bilayer is the matrix, primarily
consisting of the Gag protein p17 (a cleavage product of the viral
Gag protein). The central area of the HIV-1 virion is occupied by
the cone-shaped core (or capsid), which consists of a shell of p24
protein (also a product of the Gag gene), and a core structure
consisting of two single strands of RNA and a third Gag
protein, p7.

Keywords CD4; HIV transmission; resistance assay; tropism; viral

Tropism

replication

Cell entry requires binding to a co-receptor; for HIV-1 there are


two co-receptors, CCR5 or CXCR4. The viral specificity is determined by the V-3 loop of the gp120 molecule.6 Most HIV variants
use CCR5; a smaller proportion use CXCR4 and some use either
(dual tropic). Within an HIV-infected individual, strains of both
tropisms may co-exist. Advanced disease is associated with an
increased prevalence of CXCR4 tropic strains. CCR5 antagonists
such as maraviroc prevent interaction between gp120 and the
CCR5 co-receptor. They are therefore active against CCR5-tropic
strains but not against exclusively CXCR4 variants. Hence, HIV-1
tropism is routinely tested prior to starting these agents. Tropism
can be determined either by phenotypic assays with recombinant
viruses or by genotypic (sequence-based) methods.7

Introduction
A number of controversial theories regarding the origins of the
human immunodeficiency virus (HIV) have been described. It is
now clear that it originates from simian immunodeficiency virus
(SIV), a virus that infects monkeys. SIV shares many genomic,
phylogenetic, and epidemiological characteristics with HIV,
strongly supporting the idea of cross-species transmission.1 The
precise origins of the pandemic HIV strain, HIV-1, have been
difficult to define but it is now generally accepted that HIV-1
derives from chimpanzee SIV (SIVcpz). Other SIVs have also

Within-host and between-host evolution


Evolution of HIV-1 occurs at such a rapid rate that individually
infected people have been shown to harbour genetically distinct
strains.8 Isolation of similar HIV-1 nucleotide sequences from
different individuals suggests they share a common source of
infection. However, the viral genome can change in chronic
HIV-1 infection by up to 1% per year: if an individual transmits
HIV several years after acquiring the infection, the transmitted
strain is likely to be distinctly different from the primary variant.8

Temi Lampejo MBBS BSc(Hons) is a Virology Registrar at University College


London Hospitals NHS Foundation Trust, UK. Competing interests: none
declared.
Deenan Pillay MBBS PhD FRCPath is Professor of Virology, UCL, and Head
of Department of Infection, UCL, UK. Competing interests: none
declared.

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FUNDAMENTALS OF HIV

HIV-1 replication cycle


13

Attachment
Binding to the CD4 receptor and one of the co-receptors (CXCR4 or CCR5)
Entry inhibitors

Fusion
Fusion of the virus with the host cell membrane

Uncoating
Uncoating of the viral genomic RNA

Reverse transcription
Viral RNA is converted to DNA by the enzyme reverse transcriptase
Nucleoside and non-nucleoside reverse transcriptase inhibitors

Nuclear import
The pre-integration complex is transported into the nucleus

Integration
The enzyme integrase catalyses the integration of proviral DNA into the
host genome
Integrase inhibitors

Transcription
Proviral DNA is transcribed to yield messenger RNA

Nuclear export
Export of viral messenger RNA from the nucleus

Translation
Viral structural proteins are synthesized

12
1
11

CD4

CCR5
CXCR4
Gag

10

3
9
2

Nucleus
7

10 Assembly

Association with the matrix protein Gag and assembly of the virus
particle
11 Budding

The immature virus particle buds from the membrane of the infected cell
12 Release

The virus particle is released from the host cell


13 Maturation

Classes of antiretroviral agents blocking a particular stage


of the cycle are shown in red.

The protease enzyme mediates maturation to an infectious virus particle


Protease inhibitors

Figure 1

Phylogenetics is the study of the evolutionary descent of different


strains through nucleic acid sequence analysis. It has been
useful in identifying the sources of transmission in outbreaks
and in demonstrating routes of geographical spread, but its utility
in uncovering specific inter-individual transmission remains
limited.8

Structure of the HIV-1 virion particle


Lipid membrane

Gag p17
matrix protein

gp120

HIV diagnosis

RNA

gp41

Reverse transcriptase

HIV testing has significant benefits both for the individual and for
public health. Establishing an early diagnosis enables prompt to
access to care and treatment, resulting in improved patient
outcome. Testing is also important for the purposes of disease
prevention and reduction of transmission rates within a population. Early screening tests for HIV diagnosis, based on immunoassays for immunoglobulin G (IgG) antibodies to HIV, detected
HIV antibodies only about 45e60 days after infection. The current standard-of-care tests (fourth-generation assays) can detect
IgM and IgG antibodies against both HIV-1 and HIV-2 (which
usually appear within 20e30 days of infection9) as well as the
viral antigen p24. Viral p24 antigen can be detected approximately 5e7 days before the appearance of antibodies thereby
reducing the window period between infection and a positive
test result (Figure 3).
A patient being tested for HIV should have an initial combined
HIV antigen/antibody screening test. If positive or indeterminate,
subsequent tests are performed for confirmation and also to

p24 capsid
protein

Figure 2

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FUNDAMENTALS OF HIV

Detectable markers of early HIV infection and the relative sensitivities of


first-, second-, third- and fourth-generation immunoassays used in HIV diagnosis
HIV RNA
HIV IgG Ab

HIV p24 Ag
HIV IgM Ab

20

40

60

80

Days post-infection

4th generation
Ag/Ab assay
3rd generation
Ab assay
2nd generation
Ab assay

1st generation
Ab assay

Ab, antibody; Ag, antigen; HIV, human immunodeficiency virus; Ig, immunoglobulin.

Figure 3

differentiate HIV-1 from HIV-2. The patient is then asked to


provide a second blood sample in order to confirm the diagnosis.
Additional testing for viral RNA and/or proviral DNA may be
required in specific situations including:
 indeterminate antigen/antibody test results
 suspected HIV seroconversion
 high-risk exposure to HIV
 neonatal HIV testing.
Near-patient tests, also referred to as point-of-care tests
(POCTs), are now widely available. The benefits of these simple
and rapid screening tests are most evident in settings where
almost immediate results are needed to help clinical decisionmaking (such as following an exposure incident), in the community without direct access to laboratory services, and in
circumstances where an individual declines a test or is unlikely
to return for their results.6 A result is available within 1e30
minutes of obtaining a capillary-blood fingerprick or mouth-swab
sample. Establishing POCT in a particular setting requires quality
assurance measures and accessible accredited laboratory testing.
Despite the commercial availability of point-of-care assays able
to detect HIV antibody and p24 antigen and to differentiate between HIV-1 and HIV-2, they remain less sensitive and specific
than laboratory screening tests. All patients with positive and
indeterminate POCT results and those suspected of acute HIV
infection therefore require laboratory testing to confirm HIV
status.

combination antiretroviral therapy (cART). In treated individuals, it is a marker of treatment efficacy and indicates potential drug resistance or adherence issues. The primary tool for
measuring HIV RNA in disease monitoring is quantitative
reverse transcription-polymerase chain reaction (RT-PCR).
Follow-up monitoring should use the same laboratory assay.
Viral load assays are calibrated for blood, but can be adapted for
use in other body fluids such as CSF. However, there is currently
insufficient evidence to suggest that this contributes usefully to
ongoing monitoring in HIV.6
All patients with newly diagnosed infection should have a
baseline (pre-treatment) HIV viral load. Early or primary HIV
infection is characterized by high rates of viral replication and
correspondingly high levels of HIV RNA. Immune responses
subsequently suppress the virus and a decline in viraemia occurs
after a period of 4e6 months. An equilibrium is eventually
established between viral replication and the host immune system, reaching a pseudo-steady-state with viral loads that gradually increase over time without therapy. The rate of disease
progression in the absence of therapy can be predicted by this
steady-state viral load. Clinically stable patients not having antiretroviral therapy should have HIV viral load measurements
every 6 months.6 A minority of HIV-infected individuals control
viral replication spontaneously in the absence of ART at low (HIV
RNA <2000 copies/ml; viraemic controllers) or undetectable
(<50 copies/ml; elite controllers) viral loads.11 Elite and viraemic controllers have heightened immunoregulatory responses
to HIV and slower rates of CD4 count decline, resulting in
reduced rates of disease progression and improved clinical
outcome.11 In patients who progress to advanced disease, a
dramatic rise in viral load occurs and correlates with clinical
complications such as opportunistic infections.

HIV monitoring
The best way to monitor the rate of viral replication is to
measure the number of virions in blood, the viral load. Viral
load predicts the long-term risk of disease progression.10 In
untreated patients, it guides the timing and choice of

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FUNDAMENTALS OF HIV

Monitoring therapy
Antiretroviral therapy aims to achieve suppression of HIV RNA
below the limit of quantification. The widely used limit, as
determined by current commercially used PCR assays, has been
50 copies/ml. Novel assays are available providing lower limits
of quantification (as low as 20 copies/ml) but there is insufficient
evidence to support reliable interpretation of results at such low
levels. After commencing ART, viral load should be measured at
3- to 6-monthly intervals (depending on clinical status, adverse
effects and adherence) to confirm full HIV suppression below 50
copies/ml, which is the goal of therapy. The viral load serves as a
key marker for predicting survival and sustained HIV RNA suppression is associated with a slower progression to AIDS.12
A rebound in viral load (greater than 50 copies/ml) may
indicate treatment failure. A confirmed rebound (through testing
of a second sample) should prompt further evaluation of the
patients clinical condition and relevant markers including CD4
counts. However, a rebound in viral load usually occurs before
clinical deterioration or falling CD4 counts and serves as an early
indicator of possible failure of therapy.

resistance mutations) occurs within weeks following cessation of


antiretroviral therapy, as wild-type strains re-emerge from cells
harbouring the virus.14 Hence, resistance tests are best performed on
samples taken whilst the patient is still taking the failing regimen.
Viral resistance tests are either phenotypic, where viruses
are grown in drug-containing media, or genotypic, based on
interpretation of the RNA sequence. Genotypic assays are used
most widely, as they are simpler, cheaper and provide faster
results (1e2 weeks) compared to phenotypic testing (2e3
weeks). However, standard genotypic techniques underestimate drug resistance mutation prevalence, as only 20% of
minority species (variants present at very low titres) are
detected. These minority variants, although present at low
level, may still be of clinical significance. Despite their limitations, both genotypic and the less commonly used phenotypic
assays are predictive of virological response to antiretroviral
therapy. A number of website-based algorithms are available
for interpretation of sequence-based resistance tests.7,15e17 A

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HIV resistance
Drug resistance is a major cause of treatment failure and disease
progression in HIV. The high rate of HIV-1 replication coupled
with the marked infidelity of HIV-1 reverse transcriptase results
in the generation of a vast array of mutations. In the presence of a
drug-selection pressure, mutations will be selected that enable
continued replication.
Primary resistance
Primary resistance occurs when an individual becomes infected
with a drug-resistant strain of HIV transmitted from another individual. Currently, in the UK, 11% of treatment-nave individuals demonstrate the presence of at least one mutation
conferring resistance to antiretroviral therapy.13 For this reason,
resistance testing should always be performed before starting
treatment to guide selection of a regimen likely to be successful.
This should be performed on a sample as close to the time of
diagnosis as possible (even if treatment is not yet indicated) to
maximize the likelihood of detecting drug resistance mutations,
as wild-type virus is likely to outgrow resistant strains in a newly
infected untreated host.
Secondary resistance
Secondary resistance occurs when viral loads rebound despite
continuing treatment. Usually this results from periods of poor
adherence; active replication in the presence of suboptimal drug
concentrations selects for resistant strains, which then come to
predominate. This is usually an indication to switch treatment, as
continuing therapy is likely to result in further resistance mutations and a more extensively resistant virus, which will be more
difficult to treat. Antiretroviral drugs have a differing genetic
barrier to resistance e the number of mutations required to
enable virus to replicate in the presence of that drug. Drugs with
a higher barrier are more robust in patients with poor adherence.
In this setting, resistance assays can guide choice of second-line
treatment. If there has been a period without ART in patients with
secondary resistance, reversion to the wild-type virus (with no

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FUNDAMENTALS OF HIV

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