Download Impact of genetic diversity of HIV-1 on diagnosis

Indian J Med Res 121, April 2005, pp 287-314
Impact of genetic diversity of HIV-1on diagnosis, antiretroviral
therapy & vaccine development
Renu B. Lal, Sekhar Chakrabarti* & Chunfu Yang
Divisions of HIV/AIDS Prevention, National Center for HIV, STD & TB Prevention,
Centers for Disease Control & Prevention, Atlanta, USA &
*National Institute of Cholera & Enteric Diseases (ICMR), Kolkata, India
Accepted February 17, 2005
HIV-1 strains have diversified extensively through mutation and recombination since their
initial transmission to human beings many decades ago in central Africa. The high error rate of
HIV reverse transcriptase combined with the estimated in vivo HIV-1 replication rate of ten
billion new virions each day leads to extraordinary genetic diversity of HIV. Twenty seven
circulating genetic forms of the HIV-1 group M are presently recognized, including 11 subtypes
and sub-subtypes, and 16 circulating recombinant forms (CRF). Genotypic analyses have provided
a better understanding of the molecular diversity of HIV-1, enabling the detection of emerging
HIV-1 variants and improving the tracking of the epidemic worldwide. The rapid evolution of
HIV within infected hosts contributes significantly to the elusiveness of this pathogen from host
antiviral responses. The complex nature of HIV envelope glycoprotein that is inherently resistant
to neutralization, the selective infection, progressive destruction and impaired regeneration of
CD4+ T helper cells, generation of cytotoxic T lymphocyte (CTL) escape mutants, together with
high genetic diversity with continually evolving HIV variants worldwide, makes design of an
effective vaccine a formidable task. Given the rapidity and unpredictability with which HIV-1
genetic forms may propagate in future, a vaccine protective against all major HIV-1 circulating
genetic forms is desirable, which could require multivalent formulations. Understanding the
kinetics and directions of this continuing adaptation and its impact on viral fitness,
immunogenicity and pathogenicity are crucial to the successful design of effective HIV vaccines.
In this review, we focus on extensive diversity of HIV-1, emergence of recombinant forms and
their impact on diagnosis, antiretroviral therapy, disease progression, transmission, and vaccine
development.
Human immunodeficiency viruses (HIV-1 and
HIV-2) are the etiologic agents for AIDS in
humans 1-3. HIV-1 has spread to most parts of the
world, while HIV-2 has remained largely restricted
to West Africa1,3. AIDS was first recognized in the
1980s2, and is the leading cause of death in many
developing countries. UNAIDS estimates that 37.8
million individuals are infected with HIV, of which
about 70 per cent live in sub-Saharan Africa and
Asia 4. Despite extensive preventive programmes
worldwide, 4.8 million new infections occurred in
20034. Genetic variation is inherent to all RNA viruses
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Services, the Public Health Service, or the Centers for Disease Control and Prevention.
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but has been best characterized for HIV-1. The
extensive heterogeneity observed in the worldwide
epidemic of HIV-1 originates from the rapid viral
turnover (1010 viral particles/day) in an HIV infected
individual, and the high rate of incorrect nucleotide
substitutions during HIV reverse transcription (10-4/
nt) in the absence of proof-reading mechanisms5,6. It
is clear that zoonotic transmission of lentiviruses plays
an important role in the emergence of human
retroviruses 7,8.
HIV-1 continually evolves and migrates through
individual hosts, overcoming barriers to transmission,
avoiding different immune responses, and resisting
various antiretroviral regimens9-13. While a vaccine
(preventive or therapeutic) is the only hope to curtail
the epidemic, the diversity of HIV-114-16 provides an
extraordinary challenge in drug and vaccine
development. In the present review, we will focus on
extensive diversity of HIV-1, emergence of
recombinant forms and their impact on diagnosis,
antiretroviral therapy, disease progression,
transmission, and vaccine development.
Zoonotic transmission of the lentiviruses
HIV-1 and HIV-2 and the closely related simian
immunodeficiency viruses (SIV) belong to the
lentivirus subfamily of retroviruses17 (Fig.1). Humans
are exposed to a plethora of primate lentiviruses
through hunting and handling of primate bushmeat in
central Africa8,18,19. Both HIV-1 and HIV-2 are the
result of zoonotic transmission of SIVcpz in
chimpanzees (Pan troglodytes troglodytes) from
West central Africa20 and SIVsm in sooty mangabeys
(Cercocebus atys) from West Africa21, respectively.
Further, it is believed that at least two to three separate
zoonotic jumps from chimpanzees into humans led to
the disproportionate spread of HIV-1 groups M, O,
and N22-24. In addition, human have apparently picked
up as many as seven lineages of viruses from sooty
mangabeys resulting in HIV-2 subtypes A through G2527
. The possibility of additional zoonotic transfers of
primate lentiviruses from species other than
chimpanzees and sooty mangabeys and the
extraordinary impact that can result from such primate
lentiviral zoonotic transmission events remains a
continuim public health challenge7, 8,18,19.
Attempts to estimate the time of origin of HIV-1
using different methods of molecular clock analysis
showed that the origin of HIV-1 group M radiation
was in the 1930s. Korber et al28 have estimated that
the HIV-1 M group viruses last shared a common
ancestor between 1915 and 1941. Salemi et al29 used
a different method and arrived at a similar date for
the origin of the HIV-1 M group. They further
calculated that the common ancestor of the HIV-1
M group and the SIVcpz isolated from Pan
troglodytes troglodytes dated to the late 17th century
with a 99 per cent confidence interval for a date
between 1591 and 176122,29. The epidemic apparently
went unnoticed in central sub-Saharan Africa for
several decades22. The initial diversification of group
M may have occurred within or near the Democratic
Republic of Congo22, where the highest diversity of
group M strains has been observed30-33 and the earliest
known case of HIV-1 infection, dating from 1959,
has been documented34.
Genetic variants of HIV
One of the major characteristics of lentiviruses is
their extensive genetic variability, which is the result
of the high error rate, the recombinogenic properties
of the reverse transcriptase enzyme and the fast
turnover of virions in HIV infected individuals5,6,12,13.
HIV-1 has been divided into three groups, M, O and
N22-24,35. Within HIV-1 group M, which accounts for
the majority of infections worldwide, most sequences
fall into a limited number of discrete clades, allowing
the classification of HIV-1 group M strains into
subtypes and sub-subtypes (Fig.1). On the basis of
phylogenetic analysis of numerous isolates obtained
from diverse geographic origins, HIV is divided into
types, groups, subtypes, sub-subtypes, circulating
recombinant forms (CRFs) and unique recombinant
forms (URFs, Figs1, 2A, 2B)16,36-38. Currently nine
subtypes of HIV-1 group M exist: A-D, F-H, J and
K. Subtypes form clusters roughly equidistant from
each other in phylogenetic trees 12-15,39,40. Based on
such definition, it is clear that subtypes B and D would
be better considered as sub-subtypes of a single
subtype, however, it is difficult to change the
nomenclature now39,40. Within the subtypes A and F,
separate sub-clusters are distinguished, designated
LAL et al: IMPLICATIONS OF HIV GENETIC DIVERSITY
289
Fig.1. Phylogenetic relationship of primate lentivirus. The pol gene of primate lentivirus was used to generate the unrooted tree.
Two of the viruses presented in the tree (HIV-1 group N and SIVagm SAB have mosaic genomes. The small arrows in the tree
indicate where the sequences would branch in an env gene tree (data from Human Retroviruses and AIDS, 1999, edited by KuikenC,
Foley B, Hahn BH, Marx P, McCutchan F, Mellor,JH, Mullins J, Wolinsky, Korber B. Los Alamos National Laboratory, NM).
Source: http/www.hiv.lanl.gov (accessed on November 2004).
sub-subtypes (or sub-clusters) A1 and A2, and F1 and
F2, each pair of sub-subtypes being more closely
related to each other than with other subtypes41,42.
Some subtypes share a geographic localization,
whereas others appear to have a similar ancestry,
examples include subtype G viruses from Spain and
Portugal, and subtype C viruses from areas as
geographically diverse as India, Ethiopia and South
Africa 40,43 .
HIV-1 groups O and N, which are genetically very
divergent from group M, represent less than 5 per
cent of infections worldwide and have almost
exclusively been detected in West Central
Africa 23,24,35,44 . Elsewhere in the world, group O
viruses have been identified mainly from persons with
epidemiological links to West Central Africa, mainly
Cameroon and some neighboring countries 44,45 .
Further, HIV-1 group N infection has only been
identified in Cameroon24,36,46, a country endemic for
HIV-1, with all the major groups in
cocirculation 14,15,47 . Given that the conditions
favouring human exposure to chimpanzees have
increased in Cameroon and other parts of Africa
because of commercial logging and hunting practices,
it is possible that additional new transmissions of this
virus may have occurred7,8,19.
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Fig.2. Complex mosaic genomic structures of 16 circulating recombinant forms (CRFs). Letters and colours represent the different
subtypes of HIV-1 that comprise the CRFs. Data downloaded from HIV Sequence Database, Los Alamos, NM
Source: http//www/hiv/anl.gov/content/hiv-db/CRFs/CRFs.html (accessed on November 2004).
LAL et al: IMPLICATIONS OF HIV GENETIC DIVERSITY
HIV-1 intersubtype recombinants
In addition to the rapid accumulation of minor
genotypic changes, different HIV-1 strains can also
recombine at a high rate, generating large genetic
alterations 12,13,36,37 . Recombination requires the
simultaneous infection of a cell with two different
proviruses, allowing the encapsulation of one RNA
transcript from each provirus into a heterozygous
virion. After the subsequent infection of a new cell,
the reverse transcriptase generates a newly
synthesized retroviral DNA sequence that is
recombinant between the two parental genomes16,36.
These mosaic viruses display discrete breakpoints
between the genomic regions with different
phylogenetic associations 16,36. The fact that large
numbers of recombinant viruses exist clearly implies
that co-infection with divergent HIV-1 strains is more
frequent than previously thought 12-15. Indeed, dual
infections with different subtypes have been reported
in regions where multiple strains co-circulate 48.
Recent studies have documented cases of
superinfection with a virus from another subtype49,50
and provided evidence of recombination following
superinfection51,52. Dual infections with HIV-1 and
HIV-2 have frequently been reported in regions where
both viruses circulate, however, no recombinants
between these two viruses have yet been described16.
There are at least 16 different circulating
recombinant forms (CRFs) identified to date 16
(http://www.hiv.lanl.gov/content/hiv-db/CRFs/
CRFs.html). By definition, CRFs should resemble
each other over the entire genome, with similar
breakpoints reflecting common ancestry from the
same recombination event(s) (Fig.2A, 2B) 39 .
Presently, 16 CRFs of HIV-1 group M exits, each is
designated by an identifying number, with letters
indicating the subtypes involved, the letters are
replaced by “cpx”, denoting “complex” if more than
2 subtypes are involved16,39. A vast majority of the
CRF genomes are highly complex intersubtype
recombinants, with some being extremely stable and
others having a patchy appearance due to multiple
crossover points 16. For instance, CRF01_AE is
clearly E in env, U/A/E in the regulatory region, and
A in gag and pol, while the CRF04_cpx has an
extremely complex mosaic genome structure including
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subtypes A, G, H, K and U16. To date, CRFs have
been identified in nearly every region of the world
where two or more subtypes co-circulate and may
account for over 10 per cent of new HIV-1
infections 12-16.
CRF01_AE viruses are responsible for the
explosive epidemic in Southeast Asia, especially in
Thailand from where they have further spread to
surrounding countries like Vietnam, Cambodia,
Myanmar and China5 (Fig.3). The CRF01_AE have
been documented at low frequencies in several
Central African countries, like Central African
Republic, Cameroon and the Democratic republic of
Congo16. CRF02_AG, complex mosaic of alternating
subtype A and subtype G sequences, is the
predominant HIV-1 strain in West and West Central
Africa. These viruses have now also been introduced
in Europe, the US and South America 14,15,53-56 .
CRF03_AB is the predominant CRF among
intraveneous drug users (IDUs) in Kaliningrad,
Russia57,58. CRF04_cpx, found in Cyprus, is a complex
mosaic comprising subtypes A, G, H, K and unknown
fragments with multiple breakpoints 59,60 . All
CRF05_DF identified to date are linked to the
Democratic Republic of Congo, suggesting that the
original recombination event took place in central
Africa61. CRF06_cpx is a complex mosaic composed
of successive fragments of subtypes A, G, K and J62.
CRF06_cpx circulates in Senegal, Mali, Burkina Faso,
Ivory Coast, Niger and Nigeria62,63. Importantly, this
new variant is also present in other continents, Europe
(France) and Australia16.
CRF07_BC and CRF08_BC are two different BC
recombinants have been detected in IDUs in China64.
CRF07_BC is present in the northwestern part of
China 16,64-66 , whereas CRF08_BC is present in
Guangxi, southern China neighboring Myanmar 66.
CRF08_BC strains are mostly subtype C with
portions of the capsid and reverse transcriptase genes
from subtype B. Whereas the breakpoint in p24/p17
and the RT gene overlap with CRF08, CRF07_BC
strains have additional breakpoints in the p7/p6 genes,
the vpr/vpu, and in the 3' portion of nef16. The two
parental Thai-B and C subtypes have been reported
earlier to co-circulate among IDUs in southwestern
China, therefore clearly representing a potential
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Fig.3. Geographical distribution of HIV-1 subtypes, and circulating recombinant forms (CRFs) in different parts of the world.
Ten different epidemic patterns have been observed, as shown in different colours. (Data adopted from www.iavireport.org).
Source: IAVI Report online (http://www.iavireport.org) accessed on November 2003.
reservoir for recombination16,64. CRF09_cpx has been
described in Senegal and a US military seroconvertor 67. CRF10_CD was recently identified in
Dar-es-Salaam, Tanzania68. In this country, subtypes
A, C and D co-circulate in equal proportions and many
samples with discordant subtype designations
between 2 or more genomic regions have already
been documented68,69. CRF11_cpx, involving subtypes
A, G, J and E is observed in Republic of Congo,
Cameroon and the Central African Republic
(CAR) 70,71 . CRF12_BF has been identified in
Argentina and Uruguay with most of the genome
sequence originated from subtype F and with small
patches from subtype B72.
CRF13_cpx is a complex recombinants comprising
subtypes A, G, J, and CRF01_AE and was identified
in Cameroon 73. Likewise, CRF14_BG has been
identified in Spain where the most prevalent subtype
is B74. It is interesting to note that some of the CRFs
have recombined with other subtypes and given rise
to new CRFs like CRF15_01B found in Thailand75.
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The newest CRF, CRF16_A2D was identified in
Kenya, South Korea and Argentina76.
The pattern of mosaic recombinant viruses is
becoming even more complex, since recombination
involving viruses that are recombinant is already
occurring16. In some cases, unique recombinant form
(URF) viruses seem to have originated by secondary
recombination of a CRF 73,77,78. Mosaics involving
CFR02_AG have been observed in various African
countries, and some of the established CRFs, like
CRF11 and CRF13, contain sequences that are
derived from CRF01_AE77. Recombinations between
two CRFs have been described in studies from China77
and Niger78, in the latter case, three full-length genome
sequences revealed the presence of complex and
diverse CRF02/CRF06 recombinants. Various other
complex recombinants including even small or large
fragments from unclassified sequences have been
reported from Africa where all subtypes cocirculate16.
The majority of CRFs have only been documented
in local epidemics. This is the case for CRF03_AB,
CRF04_cpx, CRF05_DF, CRF07_BC, CRF08_BC,
and CRF10_CD. Some are spreading into different
countries, but actually their prevalence seems to be
low, like CRF06_cpx, CRF09_cpx and CRF11_cpx.
However, CRF01_AE and CRF02_AG account for
large numbers of HIV-1 infections worldwide, and
play a major role in the global epidemic, in southeast
Asia and Africa,4,38,40, respectively, and they are also
introduced to other continents 53,55,79. While the
emergence of these recombinant viruses has
important implications for both monitoring HIV-1
genetic diversity and developing effective vaccines,
predicting which recombinant strains will lead to future
expansion of the epidemic is difficult.
The subtypes of most HIV-1 strains can be
determined by sequence analysis of any of the major
regions of the genome. Intersubtype nucleotide
sequence divergence may exceed 20, 15 and 25 per
cent for gag, pol, and env, respectively80. Subtyping
from a single gene region should be done cautiously,
as recombinant strains comprising two or more
subtypes may be missed by this approach. In order
to define a new subtype, sub-subtype or CRF,
representative strains must be identified in at least
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three individuals with no direct epidemiological
linkage. Only full-length sequencing can determine
the exact pattern of mosaics within an isolate that is
recombinant 16,39 . A variety of complementary
approaches have been developed to identify
sequences that are recombinants, and to map the
positions of breakpoints within mosaic sequences. The
links to several websites in which different
programmes for sequence analyses are available:
http://hiv-web.lanl.gov/ at the Los Alamos HIV
database website, http://grinch.zoo.ox.ac.uk/
RAPlinks.html at David Robertson’s site, http://
sray.med.som.jhmi.edu/RaySoft/ at Stuart Ray’s
website and http://evolution.genetics.washington.edu/
at Joseph Felsenstein’s website.
HIV intergroup recombinants
Recombination between strains from distant
lineages may contribute substantially to new HIV-1
strains and could have important consequences.
Recombination between two highly divergent groups
M and O of HIV-1 have been reported from
Cameroon 81,82 . Group M/O mosaic viruses can
replicate well in vivo and in vitro, and can even
become the predominant variant within the patient’s
viral population81. If recombinant inter-group viruses
have a better fitness than the parental group O viruses,
their prevalence may increase rapidly with
consequences on serological and molecular diagnosis,
and treatment since differences among susceptibilities
to certain antiretroviral drugs have been observed in
vitro 83.
Likewise, detailed phylogenetic analysis of two
group N viruses have suggested that group N viruses
are the result of a recombination event between an
SIVcpz like and an HIV-1 like virus 84 . Using
sequences from the 5’end of the genome, group N
forms an independent lineage most closely related to,
but still distant from, group M, whereas sequences
from the 3’ end of the genome cluster more closely
with a chimpanzee virus (SIVcpzUS) 16,84 . This
observation offers further substantiation of a
chimpanzee-human zoonosis7-9. These observations
also open the hypothesis that distant SIVs and HIVs
can potentially recombine, particularly in individuals
who are exposed to SIV by cross-species
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transmission 18,19 . The HIV-1 group M pandemic
provides compelling evidence for the overwhelming
impact that can result from even a single event of
primate lentiviral zoonotic transmission7.
phylogenies of most non-African HIV-1 strains can
be traced to central African isolates 14,15,17,30 .
Increased fitness has likely played a key role in the
predominance and extreme variation of HIV-1 group
M over group O or N isolates9,10,90.
HIV-2 strains
Both the epidemiology and the molecular
phylogenies of HIV-2 viruses are less well understood
than those of the HIV-1 M group 12,17,26,85,86. The
diverse HIV-2 lineages are thought to have arisen in
sooty mangabeys, with several cross-species transfers
from sooty mangabeys into humans, one for each
subtype of HIV-2 17,27 (Fig.1). Thus far, seven
subtypes (A-G) of HIV-2 have been described, with
subtypes A and B being the predominant strains16,27.
The nucleotide and amino acid sequence diversity of
HIV-2 viruses is greater than the diversity of the HIV1 M group viruses, but roughly equivalent to the
diversity within the HIV-1/SIVcpz clade (Fig.1). The
subtypes of HIV-2 are thus analogous to the groups
(M, N and O) of HIV-1, both in terms of sequence
diversity and in terms of the cross-species transfer
events thought to have created them17,25-27.
Subtype A is most frequent in western part of
West Africa (Senegal and Guinea-Bissau) and subtype
B predominates in Ivory Coast3,26,27,86-88. The other
subtypes have been documented only in few
individuals, with subtypes C, D, E and F found in rural
areas in Sierra Leone and Liberia and subtype G from
Ivory Coast3,27,86. While dual infections with HIV-1
and HIV-2 have frequently been reported in regions
where both viruses circulate 16,85,86 , as yet no
recombinants between them have been described. In
this case, the level of genetic divergence may be too
high for successful recombination, although its
possibility cannot be entirely excluded.
Worldwide distribution of HIV-1 variants
Divergent inter-patient HIV-1 evolution coupled
with new introductions into susceptible human
populations has lead to the current HIV-1
diversification from the original zoonotic transmission
in central Africa 7-9,12,13. Although a founder effect
results in further HIV-1 spread and divergent
evolution in different regions in the world10,12,89, the
Genotypic analysis has provided a better
understanding of the molecular epidemiology of HIV1, enabling the detection of emerging HIV-1 variants
and improving the tracking of the epidemic
worldwide12-16. The predominant viral forms in the
global epidemic are subtypes A and C, followed by
subtype B and the recombinants CRF01_AE and
CRF02_AG38,40. The map (Fig.3) summarizes current
understanding of the global distribution of HIV-1
strains4. Ten different epidemic patterns have been
observed, as indicated by the different colours in the
map which gives an overview of HIV-1 subtypes and
recombinants in any given location. In the Americas,
Western Europe and Australia, subtype B
predominates, but in eastern South America, there is
a substantial proportion of BF recombinants in
addition to subtype B. In eastern Europe, subtypes
A, B, and AB recombinant strains dominate the
epidemic. Three different patterns have been
observed in Asia: subtype C (India), a mixture of B,
C, and BC Recombinants (China), and a mixture of
subtype B and CRF01_AE (Thailand). Africa shows
the greatest diversity. Subtype C dominates the South
and East Africa, except for significant foci of subtypes
A and D, as shown. West and West Central Africa
harbour mainly CRF02_AG, alongside a complex
array of other recombinants each present at low
frequency. The most complex epidemic is in Central
Africa, where rare subtypes and a wide variety of
recombinant forms circulate without any discernible
predominant strains30-33. There is paucity of subtype
information in Northern Africa, the Middle East, and
Central Asia (gray).
HIV subtypes in India
Molecular epidemiologic studies carried out in
different parts of India have indicated that HIV-1
subtype C is the most prevalent subtype in India91-101
(Fig.4). Few HIV-2 infections have been identified,
almost all of these represent infections with HIV-2
subtype A102,103. The distribution of the different HIV-
Fig.4. HIV-1 subtype distribution, with predominance of subtype C in India. Note emergence of Thai subtype B and dual
infections with subtype C and Thai subtype B.
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1 group M variants in India based on published studies,
which often used different molecular techniques
including the heteroduplex mobility assay (HMA), and
partial or full-length genome sequencing, are
summarized in Table I. Initial analysis of small
fragments of V3-V5 envelope region of subtype C
sequences from India, along with sequences from
other countries with predominance of subtype C, has
revealed that Indian C sequences form a
phylogenetically distinct lineage within subtype C,
designated as C3 (Indian). Additionally while majority
of subtype C infections reveal maximum homology
to the C3-Indian reference strain (IND868), almost
a third of the infections show homology to C2Zambian strain (ZM18) and a minor percentage
shows close resemblance to C1-Malawi strain
(MA959)91,97. Overall, C3 lineage sequences are more
closely related to each other (10% divergence) than
to subtype C sequences from Botswana, Burundi,
South Africa, Tanzania, and Zimbabwe (range, 1521%). The prevalence of three positions identified as
signature amino acid substitution sites for C3
subcluster sequences (K340E, K350A, and G429E)
are high among Indian sequences (56-87%) than nonIndian Cs (<22%)95,97,99. To further examine if Indian
sequences formed a geographic cluster within subtype
C, a larger envelope sequence analysis using neighborjoining method was carried out for several subtype C
sequences (Fig.5). The Indian sequences usually
clustered as separate groups, alongwith a single
sequence from Mynamar and China. A similar
observation of distinct cluster was observed for
sequences from South Americas (Brazil, Argentina,
Urguay). In contrast, sequences from south and east
Africa do not form discrete cluster (Fig.5), indicating
that African subtype C sequences are almost no
closer to each other than they are to all other subtype
C sequences. The distinct Indian cluster observed in
subgenomic envelope analysis has further been
substaintiated using full-length analysis. A recent
study which analysed 40 full-length sequences of
subtype C available in the data base in 2003, revealed
that Indian sequences form a distinct geographic
cluster supported by very high boot-strap
values104. These data further support the notion that
the Indian sequences are distinct from other subtype
C, possibly the result of a single introduction followed
by rapid spread 97,105. Because of such geographic
clustering, India’s current vaccine efforts have
focussed on developing vaccine comprising Indian
subtype C in hopes to minimize any impact of
diversity on vaccine efficacy. Indeed, clinical trends
on modified vaccinia Ankara (MVA) recombinant
using Indian subtype C are developed jointly by
ICMR and IAVI and Phase I clinical trials started in
February, 2005.
Limited studies have provided evidence for non-C
subtypes, including some unique recombinant forms,
in different parts of India (Table I). The first A/C
recombinant was detected in India after full length
genomes of 6 seroconverter from an STD cohort in
Pune92. Additional studies from various parts of the
country have identified infection with subtype A, Thai
B and CRF01_AE. More importantly, Thai B is
recognized as the second major subtype circulating
in northeastern regions95,99. The classification of HIV
strains is evolving and depends on the sensitivity of
techniques used in molecular epidemiology. Because
most of the studies in India were based on a single
gene region analysis, or used the techniques that are
not sensitive enough to discriminate CRFs from unique
recombinants, the role played by CRFs may be
underestimated in these studies. For example, many
studies used HMA, which needs less sophisticated
equipment but is not sensitive enough to discriminate
between pure subtypes and CRFs. Systematic studies
involving better sampling and uniform technologies
are needed to better understand the evolving HIV-1
epidemic in the country, especially in high risk groups
from northeastern region of India.
Dual infections with subtype C and Thai B have
recently been observed in Manipur, a region in
northeast India with very high prevalence of HIV-1
among IDUs93,99,106. In addition, recent unpublished
data have revealed discordant subtypes in gag and
env regions of 4 (14%) of the 28 IDU studied from
Manipur (Chakrabarty, unpublished), suggesting the
presence of potential C/Thai B recombinants. Studies
are in progress to ascertain if these infections resemble
the CRF07/08 BC recombinants identified in
China 64-66 or represent unique recombinant forms.
Overland heroin export routes have been associated
with dual epidemics of injecting drug use and HIV
infections in Asian countries 106 . Whether drug
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297
Fig.5. Phylogenetic analysis of envelope sequences of diverse HIV-1 subtype C. A neighbor-joining tree was generated the stability
of tree topography was tested by bootstrapping and only significant bootstrap values that define geographic clusters are shown. Two
geographic clusters with high bootstrap values include Asia (IN: India; CN: China; MM: Mynamar) and South America (BR: Brazil;
AR: Argentina; UR: Uraguay), whereas east and South African specimens did not form a cluster (ET: Ethiopia; ZA: South Africa;
TZ:Tanzania; BW: Botswana; KE: Kenya; BI: Burundi; DJ Dijoundi) are shown .
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Table I. Distribution of HIV-1/HIV-2 subtypes in India
City/State
Risk group
Pune 91
STD clinic
attendee
Pune 92
Recent
seroconverter
Pune, Delhi94
No.tested
Gene region
Subtype
Subcluster
V3V5 HMA 2
96% C
2% A
2%B
66% C3
34%C2
6
Full length
sequencing
83% C
17% A/C
1992-1996
28
LTR
100%C
New Mumbai 96
Heterosexual
21
V3/ Vpr
B and C
Mumbai 101
Drug naïve cases
128
Protease/RT
96% C
2% A/C
0.8% A
0.8% AE
Delhi 98
Seropositive
patients in
AIIMS, Delhi
125
V3V5
78% C
11% B’
2.4% A
2% E
Manipur 93
IDU
233
Peptide assay
71% C
29% B’
Kolkata 95
FSW
40
V3V5 HMA
95% C
5% Untypeable
Kolkata 97
FSW
21
V3V5
95% C
5% A
Kolkata 99
FSW
54
V3V4
92% C
8% untypable
Manipur 99
IDU
25
V3V4
68% C
20% ThaiB
12% dual
(ThaiB/C)
South India100
Seropositivefrom 256
Hospital/, Pvt.
Clinic
LTR
98.8% C
0.4% A
0.4% B
0.4% B/C
South India102
Seropositive
V3 region
HIV-2 subtype
Kolkata 103
Patients
attending
clinic
46
18
68% C3
29%C2
3%C1
50% C3
13% C2
2% C1
27% C-untypable
A
STD, Sexually transmitted disease; HMA, heteroduplex mobility assay; IDU, intraveneous drug user; FSW, female sex worker;
RT, reverse transcriptase.
Superscript numerals represent reference numbers
LAL et al: IMPLICATIONS OF HIV GENETIC DIVERSITY
trafficking across the Burma-India border to Manipur,
with predominant subtype C, B and CRF01_AE, will
result in new waves of recombinant infections in the
bordering cities needs to be analyzed extensively. The
recent findings of 40 per cent prevalence in high-risk
groups in Kolkata, together with an estimated annual
incidence of 7 per cent, provide a fertile enouncement
for any recombinants that have better replicative or
fitness advantage over the existing strains to emerge
as the predominant strain in this area107. Emerging
trends of unique recombinants in neighbouring
northeastern countries, including Bangladesh 108 ,
Mynamar 109 , Thailand 110 and China 64,66 , can
significantly impact the landscape of the epidemic,
including vaccine design strategies in India65,111,112.
Impact of HIV variability on diagnosis
HIV diversity has enormous impact on diagnostics,
as commercial assays must detect all known variants
of HIV-1 113,114 . Serologic assays are based on
detection of antibodies to structural proteins, p24gag
and gp41env, which are conserved among most HIV1 isolates80. Earlier studies had shown that persons
infected with highly divergent HIV-1 group O, failed
to be diagnosed accurately by some serologic tests
due to gp41 variation45,113. This problem was overcome
by addition of group O lysate/peptide in the standard
antigen mixture. Although most of current enzyme
immuno assays (EIA) and rapid tests are sensitive
and specific for diagnosing persons with chronically
established infections with HIV-1 group M
subtypes 113,116,117, some difficulties still remain in
diagnosing persons with recent infections with nonB subtypes primarily because antigens used for the
assays were based on HIV-1 subtype B strains 118.
Continued analysis of commercial assays is needed
to ensure that newly emerging variants do not escape
detection by these tests113,119.
While genetic variability has had minimal impact
on serologic detection of HIV-1 (due to amino acid
conservation of the immunodominant epitopes on
structural proteins), molecular diagnostic assays have
been more challenging113,114. Several generic primers
that can identify all subtypes of HIV-1, groups M, N
and O and SIVcpz have been developed as tools for
detection and confirmation of HIV/SIVcpz infections
299
worldwide120. These primers provide a tool that not
only detects infection with divergent viruses, but also
allows subsequent phylogenetic analysis to identify
subtypes for molecular epidemiologic studies120,121.
The utility of molecular detection of plasma viremia
has increased considerably in clinical settings,
especially for monitoring patients on antiretroviral
(ARV) therapy. Four commercial viral-load assays
are available: Amplicor HIV-1 Monitor v1·5 (Roche
Molecular Diagnostics), nucleic-acid-sequence-based
amplification (NASBA, Organon Teknika), HIV-1
Quantiplex branched DNA (bDNA, Chiron
Diagnostics), and the LCx HIV-RNA quantitative
assay (LCx, Abbott). The ability of these assays to
quantify viral load from non-B subtype infections has
been examined in several studies 122-127. Although,
much progress has been made to improve the
sensitivity of nucleic acid-based assays to quantify
viral load across all subtypes of HIV-1, some RNA
viral load assays still produce erroneous
results 128-130 . Thus continued monitoring of viral
load assays against multiple subtypes of HIV-1
is needed to ensure accurate measurement of
viral load in diverse populations infected with various
subtypes of HIV-1.
Impact of HIV-1 subtypes on antiretroviral
therapy
Antiretroviral drug resistance is the major obstacle
for clinical management of ARV therapy 131-133 .
Current antiretroviral drugs comprise at least 16 drugs
targeted against two pol gene enzymes, the reverse
transcriptase (RT) and protease133. The development
of drug resistance to the nucleoside RT inhibitors
(NRTIs), non-nucleoside RT inhibitors (NNRTIs) and
protease inhibitors (PIs), is both a cause and a result
of virologic treatment failure with incomplete virus
suppression 133,134 . Our understanding of drug
resistance is largely limited to developed countries
with predominant HIV-1 subtype B infections132-136.
However, with introduction of ARV in other parts of
the world, emerging data indicate that viral subtype
may influence the effectiveness of antiretroviral
treatment and that pre-existing mutations could reduce
the effectiveness of ARV 137-139. In the absence of
any drug exposure, RT and protease sequences from
B and non-B HIV-1 are polymorphic among about
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INDIAN J MED RES, APRIL 2005
40 per cent of the first 240 RT amino acids and 30
per cent of the 99 protease amino acids 138,139. The
genetic baseline diversity of protease and RT from
non-B subtype HIV-1 infected persons not exposed
to antiretroviral therapy is summarized in Table II.
Some of these amino acid substitutions occur at high
rates in non-subtype B viruses at positions associated
with drug resistance in subtype B140,141.
The HIV-1 protease is a dimeric and symmetric
molecule made up of two 99-residue polypeptide
chains. The substrate cleft of the protease is formed
by residues at positions 8, 23, 25-27, 28-30, 32, 47-52
and 53, 80, 82 and 84134,142. These predicted amino
acid residues appear to be highly conserved across
subtypes (with the exception of V82I mutation in
subtype G) in drug naïve persons136,139,143-147. There
are a number of amino acid substitutions that occur
at high rates in certain non-subtype B viruses at
positions associated with subtype B drug resistance
(Table II). The V82I mutation occurs in about 1 per
cent of untreated individuals infected with subtype
B, it has been identified at 6 per cent in subtype C,
9 per cent in subtype F, and is the consensus (>50%)
in subtype G isolates from untreated persons139. This
mutation alone confers minimal resistance to the
available PIs in subtype B, however amino acid
changes at V82 resulting in V82A, F, S or T are
associated with high level resistance to most PIs144,145.
In subtypes F and G, the M46L or I substitution, which
leads to resistance to most of the PIs in subtype B,
has been identified in 4 and 7 per cent of sequences
from untreated individuals, respectively139.
Sequence comparison from untreated and treated
persons, has identified mutations at protease positions
20, 36, 63, 71, 77 and 93 as “secondary protease
mutations” in subtype B. While mutations at these
positions do not cause high level drug resistance
themselves, they contribute to drug resistance when
present together with certain primary protease
mutations, or have been shown to compensate for
the decrease in catalytic efficiency caused by PIselected primary protease mutations134,136,138,144. The
higher frequency of protease polymorphism in non-B
isolates, including protease positions 20, 36, 63, 82
and 93, has raised concern that PI treatment of nonsubtype B infected persons could be less effective
than that of subtype B 132,137,138 . The enzymatic
efficiency of protease has a potential role in explaining
drug resistance differences among subtypes 142,144.
In vitro studies reporting susceptibility of clinical
HIV-1 isolates of different subtypes to PIs have
shown either a lack of or only small differences in
susceptibility between subtypes146-148. Although the
majority of phenotypic data point to similarities in
susceptibility between B and non-B subtypes, there
is increasing evidence that V82I and related
polymorphism in protease may play a role in subtype
specific susceptibility to some PIs133,136,138,139.
Analysis of RT region has indicates that none of
the residues involved in polymerase activity (43, 65,
72, 110, 113, 115, 116, 151, 160, 183, 184, 185, 186,
and 219) are polymorphic (in the absence of
antiretroviral drugs) in drug naïve persons131,133,134.
Comparison of non-B RT consensus sequences to the
subtype B RT consensus shows differences in overall
23 residues, of which 1 (position 179) is in a position
related to subtype B drug resistance (Table
II) 135,137,139. Polymorphism in non-B RT positions,
which are associated with subtype B NRTI
resistance137-139 include codons 69 (6% in subtype F),
75 (2% in CRF01_AE) and 118 (5, 2 and 6% in
subtypes A, B and D, respectively). Mutations at
position 69 (T69D/ N/S/A) have been reported with
exposure to all NRTIs, and contribute to NRTI
resistance. Polymorphism at positions associated with
subtype B non-nucleoside RT inhibitor (NNRTI)
resistance is seen at codons 98 (in subtypes B, C, G),
106 (in subtypes G, CRF01_AE and CRF02_AG) and
179 (polymorphic in subtypes B, C, D, F, G,
CRF02_AG, and is the consensus position in subtype
A) (Table II). A98G, V106A and V179D are residues
in the NNRTI binding region of the p66 subunit
associated with different levels of resistance to the
NNRTIs. There are accruing reports of K103N and
additional major NNRTI-related drug resistance
mutations in untreated persons132,148-151. In addition,
mutations at RT positions 98 and 179, which are
considered secondary NNRTI associated mutations
in subtype B, and specific adjacent polymorphisms
(RT positions 177 and 178), are frequently identified
among drug naive non-B infected persons137,139. The
LAL et al: IMPLICATIONS OF HIV GENETIC DIVERSITY
301
Table II. Comparison of consensus B sequences in protease (A) and Reverse Transcriptase (B) with non-B subtypes. Columns denote
subtypes and circulating recombinant forms (CRFs), and rows denote protein positions in respective protein. Sites with known drug
resistance in subtype B are in Bold. Open squares are similar amino acids to consensus B sequence and shaded boxes represent
polymorphism in the position compared to consensus subtype B sequence
impact of such primary and secondary mutations in
RT of non-B subtypes in the developing world for
clinical management of HIV-1 infection remains to
be determined.
Several web-accessible sites which review
antiretroviral drug resistance include the International
AIDS society (IAS), USA (http://www.iasusa.org);
Los Alamos Database (http://hiv-web.lanl.gov);
Stanford HIV Drug Resistance Database (http://
hivdb.stanford.edu); and HIV Resistance Web
(http://www.hivresistanceweb.com).
Impact of HIV-1 subtypes on HIV transmission
and disease progression
It has been postulated that viral sequence
variability may dictate biologic differences that
partially explain the different epidemic patterns seen
in different regions of the world. Indeed, several
reports have documented that HIV-1 subtypes may
differ with respect to viral load 152, chemokine coreceptor usage153, transcriptional activation levels154
and antiretroviral drug susceptibility137-139. While the
exact functional consequences of these subtype-
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INDIAN J MED RES, APRIL 2005
related differences have not been delineated, there is
some evidence that such differences may result in
altered rates of disease progression 155 , sexual
transmission (unpublished data), and mother-to-child
transmission (MTCT) 156,157.
Limited studies on subtypes and transmissibility
have yielded conflicting results. A previous study in
Tanzania suggested that subtypes A, C and
recombinants are more likely to be perinatally
transmitted than subtype D 156. A study in Uganda
reported no difference in rates of perinatal
transmission for subtypes A and D, whereas a large
perinatal transmission study from Kenya provided a
strong evidence that women infected with subtype D
were more likely to transmit virus to their infants than
those infected with subtype A157. Additional studies
are needed to better understand the role of subtypes
in perinatal transmission.
HIV-1 subtype-specific differences in disease
progression have been studied in several natural
history cohorts. For instance, two studies based on
incident cases have found subtype-specific
differences in disease progression. A study in Senegal
with limited number of patients reported that women
infected with non-A subtypes were 8 times more likely
to develop AIDS than those infected with subtype
A155. A large cohort study in Uganda based on more
than 1000 patients demonstrated that subtype D was
associated with faster progression to death and with
a lower CD4 cell count during follow-up, compared
with subtype A158. However, no difference in disease
progression have been found between patients
infected with subtypes B and C in Israel159 , among
patients infected with subtypes A, B, C and D in
Sweden160, subtypes B and CRF01_AE in Thailand161
or subtypes CRF02_AG or other subtypes in
Cameroon162. Several studies suggest faster disease
progression among HIV-1 infected persons from
Africa than those infected in developed countries155,163
and may presumably be due to difference in
environmental factors or presence of other copathogens that may serve as cofactors for faster
disease progression among patients in the developing
world. Large cohort-based studies are needed to
further confirm role of subtypes in HIV-1 transmission
and disease progression.
Implications of HIV-1 variation on vaccine
design
The importance of immune responses relevant for
vaccines against the range of HIV-1 genetic variants
has been the subject of intense discussion38,164-167.
HIV-1 readily mutates to escape the immune
response, evolving in ways that allow it to persist in
the host 10,11,38,168-171. While correlates of immune
protection against HIV-1 are not clearly understood,
it is known that neutralizing antibodies (nAb)
correlates with protection from infection, cellmediated immune responses correlates with protection
from disease and that the effectiveness of antiviral
memory T cells is dependent upon both the quality
and the quantity of antigen-specific T cells11,172. It is
widely agreed that both cell-mediated (polyfunctional
CD4+ and CD8+ T cell) responses and humoral
responses (specifically induction of virus-neutralizing
antibodies (nAbs), conferring broad and long-lasting
protection against diverse HIV variants, are needed
for an efficient vaccine against HIV-111,166,172. An
ideal vaccine should include both a component to elicit
CMI (polyfunctional CD4 and CD8 responses with
ability to induce both gamma-interferon and
interleukin-2) and one to stimulate nAbs166,167,172. The
most likely design of such a vaccine could be the use
of DNA plasmid and/or a live, recombinant virus
vector to evoke the former response along with a
subunit protein for the latter167,173-175.
Given the enormous worldwide diversity of HIV
variants, the design and choice of antigen identities
for inclusion in candidate AIDS vaccines is a daunting
challenge 38,174,175. Reported preferential intraclade
over cross-clade cellular and humoral responses and
the existence of subtype-specific epitopes support the
use of vaccines based on immunogens that genetically
match the viruses circulating in the targeted
population in which it is tested 175 . Use of such
vaccines is recommended by a WHO-UNAIDS
Vaccine Advisory Committee and is being
LAL et al: IMPLICATIONS OF HIV GENETIC DIVERSITY
303
implemented in several current trials166,174. However,
given the rapidity and unpredictability with which
HIV-1 genetic forms may propagate in future, a
vaccine protective against all major HIV-1 circulating
genetic forms is desirable, which could require
multivalent formulations164,175. Indeed, emerging data
suggest that HIV-1 evolutionary relationships may be
more useful than regional considerations for vaccine
designs164,172. Thus, the use of consensus or ancestor
sequences for a specific-subtype has been proposed
in order to minimize the genetic differences between
vaccine strains and contemporary isolates112,176. This
strategy could potentially enhance cross-reactivity and
breadth of a vaccine response relative to any single
strain. In regions where several subtypes co-circulate,
inclusion of each prevalent subtype in the vaccine
may improve coverage 164,167,175.
The data on the link between genetic subtypes and
HIV-specific immune responses remains
controversial11,165,166. Various studies have identified
both subtype-specific 185-189 as well as cross-clade
epitopes in HIV-1 proteins 189-194. However, intrasubtype CTL responses are usually stronger and more
frequently
detected
than
inter-subtype
187,194,195
. This is more prominent for those
responses
responses targeted to the more variable env gene,
but also to gag, pol, and nef 195 . Cross-clade
recognition of various CTL epitopes have led to the
idea that a single subtype immunogen could elicit CTL
responses protecting against other subtypes165,166,196.
This may suggest that matching HIV vaccine
candidates to the prevalent HIV-1 strains might be
less important for vaccines targeted at induction of
T-cell responses to conserved proteins (for instance,
gag and pol) 164,176.
While the role of neutralizing antibodies in
sterilizing immunity against HIV-1 has clearly been
recognized 164,172, the induction of such response is
difficult due to the envelope’s conformational
complexity and sequence diversity169,172,177. Most of
the neutralization antibody sensitivity studies have
shown that neutralizing serotypes do not correlate with
HIV-1 genetic subtypes 178 . Few studies have
examined the elucidation of cross-clade neutralization
by HIV-1 vaccines, since antibody responses with
neutralizing capacity against primary isolates have not
usually been obtained 177,179-181 . While broadly
neutralizing activities have been detected frequently
only in long-term non-progressors, the cross-clade
recognition of neutralizing activities have shown
relatively restricted repertoire182. Given the relative
importance of neutralizing antibodies in protection
against HIV-1 166,169 , attempts are being made to
devise immunogen that can potentially induce
neutralizing immune responses 175,177. New findings
reveal that HIV-1 protects itself from antibodies by
putting up a shield of constantly shifting sugar
moieties169,183. This shield may be contributing to the
poor performance of candidate HIV-1 vaccines. A
recent study showing preferential heterosexual
transmission of unique monophyletic viral strains that
are sensitive to neutralization provides new hope for
vaccine design strategies184.
Further, there is evidence that the genetic identities
of circulating strains of HIV may continue to evolve
at the population level in response to HLA-restricted
immune pressures 10,197. CTL escape mutants also
need to be considered for successful vaccine
strategies11,170,171. It has been proposed that mutations
that ultimately result in diminished recognition of a
given epitope by a host’s CD8+ T cells via loss of
epitopes binding to MHC class I molecules, or
interference with antigenic processing, will increase
in prevalence as a result of HIV infection of
individuals that share common HLA alleles10,197-199.
Thus, HIV variants may evolve in such a way that
they are not recognized by the CTL responses
restricted by the HLA alleles that predominate in a
given human population199. The implication of cladespecific epitopes and their role in vaccine
development is presented elsewhere200.
Are there intersubtype HIV-1 variations in host
adaptation and viral fitness?
The proportions of subtypes in defined populations
are not stable but are in constant flux due to new
introductions of HIV-1 subtypes, changes in human
behaviour, therapeutic intervention, mode of
transmission, and possibly, subtype fitness12,89,90. Over
the past decade, there has been a considerable shift
in the epicenter of the HIV-1 epidemic from sub-
304
INDIAN J MED RES, APRIL 2005
Saharan Africa to Southern Africa, India, and
Southeast Asia4. Subtype C has now emerged as the
predominant clade in the world due to these regional
pandemics and accounts for at least half of all
infections worldwide 4. Although subtype B likely
preceded subtype C as a founder clade in India and
China, most of the new infections in these countries
are attributable to subtype C isolates or intersubtype
B/C recombinants 64,66. Similar trends have been
observed in Kenya, Tanzania, and South America
(e.g., Brazil and Argentina)38,40. This rapid insurgence
of subtype C may be due to a founder effect or to
intersubtype fitness differences.
Any difference in ex vivo fitness likely reflects
genotypic or phenotypic variations between
subtypes201,202. For example, most subtype C isolates
appear to have a third NF-kB element in the long
terminal repeat (LTR) as compared to the two sites
found in most subtypes, which augments transcription
in the presence of HIV-1 Tat protein94,203-205. Direct
binding of tat to NF-kB in TAR independent fashion
further suggests that extra NF-kB site in subtype C
may differentially regulate transcription206. Moreover,
a recent study indicated that subtype-specific LTRbased viral replication rates in vivo are strongly
influenced by the host environment207. Recently, the
tat protein of HIV-1 subtype C has been shown to be
defective in chemotactic activity 208, suggesting
possible biological differences in the subtype C viruses.
Studies on HIV-1 protease activity showed increased
cleavage of peptide substrates by HIV-1 subtype C
versus subtype B protease144,209,210. This phenotypic
data suggest that dominance of subtype C in the HIV1 epidemic could be due to increased replicative
capacity of subtype C isolates over other HIV-1
subtype isolates.
In addition to these phenotypic differences,
subtype C appears to have preferential usage of
CCR5 co-receptor and that subtype C isolates rarely
switch from a NSI/R5 phenotype to an SI/CXCR4
phenotype during late disease153,211. Analysis of coreceptor usage during disease progression has
delineated that SI/X4 HIV-1 isolates dominate late in
disease, whereas NSI/R5 variants are typically
present during the asymptomatic phase 212. Further,
in vivo findings suggest that NSI/R5 HIV-1 isolates
out-compete the SI/X4 variants at the site of primary
infection. The reasons for this are not completely
understood. Recent studies have provided evidence
that subtype C virus can use CXCR4213,214, whether
antiretroviral therapy is driving the emergence of
CXCR4 variants remains to be determined215.
The emerging data on viral fitness have indicated
that subtype C may have altered fitness, thus resulting
in their longer survival201,202. Recent studies analyzing
the relative fitness in a pair-wise competition indicate
that subtype C isolates are likely less fit than subtype
B isolates 201 . Decreased fitness together with
persistence of NSI/R5 HIV-1 isolates linked to slower
disease progression could result in increased
transmission time. It is evident that to survive, viruses
must continue to propagate in a living host and that a
low/attenuated level of pathogenesis represents a
tradeoff between virulence and transmissibility10,199.
In the case of HIV-1, the continual spread of this
virus is a consequence of long asymptomatic but
transmissible periods following initial infection.
Individuals infected with a more pathogenic (high
replication) strain will progress faster to HIV-1
disease, decreasing the probability of viral
transmission. In contrast, attenuated HIV-1 strains
(i.e., lower replicative capacity) would in theory delay
disease progression and increase the likelihood of
transmission. Thus, it is likely that the poor relative
fitness of subtype C isolates can fit into model for
subtype C dominance in the epidemic. Whether such
decreased fitness alone can account for such an
explosive subtype C epidemic around the globe
remains to be determined.
Conclusion and perspective
HIV-1 pandemic strains have diversified
extensively through mutation and recombination since
their initial zoonotic transmission to human beings
many decades ago in central Africa. The high error
rate of HIV reverse transcriptase combined with the
estimated in vivo HIV-1 replication rate of ten billion
new virions each day leads to extraordinary genetic
diversity of HIV. Twenty seven circulating genetic
forms of the HIV-1 group M are presently recognized,
including 11 subtypes and sub-subtypes, and 16
circulating recombinant forms. In addition, many
LAL et al: IMPLICATIONS OF HIV GENETIC DIVERSITY
unique recombinant forms (URFs) including
secondary recombinant mosaic patterns also have
been identified around the world. Over the past
decade, there is a shift in the epicenter of the HIV-1
epidemic from sub-Saharan Africa to Southern Africa,
India, Southeast Asia and China. Subtype C has now
emerged as the predominant subtype in the HIV
epidemic and accounts for at least half of all infections
worldwide. Genotypic analyses have provided a better
understanding of the molecular diversity of HIV-1,
enabling the detection of emerging HIV-1 variants
and improving the tracking of the epidemic worldwide.
New genetic forms, including recombinants, are
being introduced in different areas of the world,
changing the molecular epidemiology of the infection.
Viruses resulting from recombination may be more
virulent than nonrecombinant viruses due to altered
fitness or cellular tropism and may have different
antiretroviral drug susceptibilities. Additionally,
recombinant viruses could accelerate disease
progression and increase the likelihood of mother-tochild transmission and sexual transmission by
increasing viral load in the blood and genital tract;
however, epidemiologic evidence for such an
association has not yet been established.
The rapid replication rate of HIV and its inherent
genetic variation have led to the identification of many
HIV variants that exhibit altered drug susceptibility.
Maintaining low-to-undetectable plasma HIV RNA
levels prevents progression to AIDS and minimizes
the possible emergence of drug resistant HIV-1
variants. Although most clinical outcome and
phenotypic reports indicate similarity between B and
non-B isolates, a few studies point to potential
differences. As an increasing number of individuals
with non-subtype B infection access ARV therapy,
these genotypic differences in non-B consensus
(wild-type) sequences may result in new drug
resistant forms, as well as differences in the outcomes
of antiretroviral therapy. Genotypic and biochemical
studies provide an explanation for potential intersubtype differences in susceptibility; however, larger
and more rigorous prospective studies are required
to translate the observed inter-subtype differences in
resistance patterns to more effective, long-term drug
treatment strategies.
305
The rapid evolution of HIV within infected hosts
contributes significantly to the elusiveness of this
pathogen from host antiviral responses. The complex
nature of HIV envelope glycoprotein that is
inherently resistant to neutralization, the selective
infection, progressive destruction and impaired
regeneration of CD4+ T helper cells, generation of
CTL escape mutants, together with high genetic
diversity with continually evolving HIV variants
worldwide, makes design of an effective vaccine a
formidable task. The diverse HIV subtypes, CRFs,
the inter-subtype mosaic genomes further
exacerbate the problem of designing of relevant
vaccine immunogens. Moreover, given the recent
observation that HIV may exhibit a frequency of
recombination diversification that is up to an order
of magnitude greater than previously appreciated,
the complexities of confronting HIV diversity with
effective vaccine design will remain an ongoing
challenge. Given the rapidity and unpredictability
with which HIV-1 genetic forms may propagate in
future, a vaccine protective against all major HIV-1
circulating genetic forms is desirable, which could
require multivalent formulations. Indeed emerging
data suggest that use of consensus or ancestor
sequences for a specific subtype could potentially
enhance cross-reactivity and breadth of a vaccine
response relative to any single strain.
High mutation rate of HIV-1 together with
selection and adaptation to most environmental
changes result in the generation of viral quasispecies.
HIV-1 quasispecies are evolutionary and clinically
relevant since this genetic variability can respond to
selective pressure (e.g., host immune system and
antiretroviral therapy), including those mediated by
virus specific cytotoxic T lymphocytes. Understanding
the kinetics and directions of this continuing adaptation
and its impact on viral fitness, immunogenicity and
pathogenicity will be crucial to the successful design
of effective HIV vaccines. Further, there is evidence
that circulating strains of HIV may continue to evolve
at the population level in response to HLA-restricted
immune pressures. Such global evolution of HIV
might potentially require periodic re-assessment of
the composition of the best immunogens for inclusion
in AIDS vaccines.
306
INDIAN J MED RES, APRIL 2005
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Reprint requests: Dr Renu B. Lal, Division of HIV/AIDS Prevention, National Center for HIV, STD, and TB Prevention
Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA
e-mail: [email protected]