Challenges of HIV Diversity:

A phylodynamic perspective

Abstract
The current HIV pandemic is a major global public health challenge and many challenges

remain to HIV control. HIV has a high rate of evolutionary change leading to an enormous amount

of genetic diversity at both intra- and inter-host levels. This provides many challenges not only to

the control of HIV but also for phylodynamic studies of the virus. At the intra-host level selection

and recombination provide challenges to current phylodynamic methods based on coalescent

theory, whereas at the inter-host level, the complexity of current epidemics and differences in

evolutionary rate between HIV lineages present challenges to phylodynamic studies. The diversity

of HIV also poses many challenges for its control. Different HIV types, groups, and subtypes

require different antiretroviral treatment regimens, and suboptimal regimens are believed to be the

main source of HIV drug resistance. The development of an effective vaccine against HIV also

remains a challenge due to the generation of escape mutants to both host antibody and cytotoxic

T cell responses. Whilst, many challenges still remain to both the control and study of HIV,

developments are being made in overcoming these challenges, driving research and our

understanding further forwards.

Introduction
The Human Immunodeficiency Virus (HIV) is a retrovirus that infects various cells and tissues of

the human immune system leading to the development of Acquired Immunodeficiency Syndrome

(AIDS). AIDS is characterised by a gradual failure of the immune system leading to death, usually

from opportunistic infections or cancers. The HIV pandemic is a major global public health

challenge with no cure for HIV infection, and around 37 million people living with HIV worldwide

(WHO, 2015). Combination antiretroviral therapy (cART), using 3 or more antiretroviral drugs,

effectively controls viral replication ands helps prevent further transmission (Deeks et al., 2012).

HIV originates from the cross-species transmission of different Simian Immunodeficiency Viruses

(SIV) to humans. There are two known types of HIV, HIV-1 and HIV-2. HIV-1 is the cause of

the majority of HIV infections worldwide, whilst HIV-2 is largely confined to West Africa and is

less virulent and infectious (Campbell-Yesufu and Gandhi, 2011). HIV-1 consists of four groups:

M, N, O, and P, each representing a different cross-species transmission from non-human

primates. Groups M and N originate from SIVcpz from chimpanzees in West-Central Africa (Keele

et al., 2006), whilst groups O and P originate from SIVgor transmitted from Western Lowland

Gorillas in Cameroon (Darc et al., 2015). HIV-2 has its origins in SIVsmm from Sooty Mangabeys

in West Africa. HIV group M is responsible for the global HIV pandemic and has been divided

into nine subtypes as the virus has diversified.

The rapid evolution of RNA viruses such as HIV, means that their ecological and evolutionary

dynamics occur on the same timescale. The shape of a pathogens phylogeny is determined by a

combination of evolutionary, immunological and evolutionary processes and the study of these

interactions is termed phylodynamics (Pybus and Rambaut, 2009, Grenfell et al., 2004). HIV

evolves rapidly as a result of its high rate of viral replication and the high mutation and

recombination rates of its reverse transcriptase enzyme, which lacks a proof-reading mechanism

(Hemelaar, 2012). This rapid evolution has lead to an enormous amount of genetic diversity in

HIV with diversity arising not only at intra-host but also at inter-host levels. This provides many

challenges to both the control of HIV and the study of the virus itself.
Intra-host Dynamics
The high rate of evolutionary change of HIV means that its viral evolution can be studied

within a single host for the duration of the infection. During infection, the HIV population is

targeted by host immune responses providing strong diversifying selection on the HIV genome.

This selection is seen clearly in the env gene of HIV-1, which encodes a glycoprotein found on the

outside of the HIV viral envelope (Choisy et al., 2004). Alongside selection, viral genetic diversity

is also generated through recombination events between strains within a singe host. Recombination

events can occur both between strains from a single infection event or, in the case of coinfection

and superinfection, between strains from different infection events. Many cases of superinfection,

where an individual has been infected with a second strain following infection from a first, have

been identified and the presence of circulating recombinant forms, HIV variants formed from

recombination between two different strains found in more than three individuals, further

highlights the role of recombination in the generation of HIV diversity (Burke, 1997).

Both selection and recombination provide a challenge to phylodynamic methods when applied

to HIV. In its simplest form coalescent theory assumes that there is no selection or recombination

(Wakeley, 2013). These assumptions are generally not applicable for HIV, and care must therefore

be taken when interpreting results from such studies. Extensions of coalescent theory accounting

for both selection and recombination do exist (Wakeley, 2013), however their use remains limited

to small datasets. Most studies employ a multiple loci model which assumes free recombination

between a set sequence regions but no recombination within them (Lemey et al., 2006). While

recombination between HIV strains does not always occur at the same points in a sequence, there

are regions where sequence breaks for recombination are most likely to occur, such as the constant

region of the env gene (Archer et al., 2008).

Upon infection the genetic diversity of HIV is relatively low, increasing over the course of the

infection. Shankarappa et al. (1999) identified three distinct phases in the evolution of the C2-V5

region of the HIV-1 env gene during the asymptomatic period that follows the initial acute HIV
infection. The early phase of infection is of variable length and during this period genetic diversity

and divergence increase in a linear fashion. The intermediate phase follows this, lasting 1.8 years

on average, and is characterised by a continued increase in divergence but a stabilisation or decline

in genetic diversity. Last, comes the late phase, in which divergence slows or stabilises, with

diversity continuing to stabilise or declining. Therefore, the earlier cART is begun the less HIV

diversity will be available for selection to act upon. Drug resistance may therefore take longer to

arise in such individuals as resistance mutations will have to occur de novo.

During infection the rapid process of within-host evolution produces strains that evade host

cytotoxic T cell responses, known as T cell escape mutants (Goonetilleke et al., 2009). Alongside

cytotoxic T cell responses, the host B cell response produces neutralising antibodies against HIV.

Initially these antibodies recognise only a very narrow range of epitopes and so mutants that evade

such responses are rapidly generated (Wei et al., 2003). Broadly neutralising antibodies that

recognise conserved regions of the env gene are sometimes generated however this is rare and only

appears to occur several years into chronic infection (McMichael and Haynes, 2012). Escape

mutants also provide a major challenge to vaccine development. A vaccine will be rendered

ineffective if escape mutants emerge against responses primed by vaccination and these strains are

then transmitted to new individuals, especially as such genes may spread rapidly through

recombination. An effective vaccine will therefore have to prime a response against multiple

conserved epitopes to control the virus before escape mutants against all targeted epitopes are

generated.

Antiretroviral therapies suppress but do not eradicate HIV-1 infection such that a persistent

but low level of HIV-1 can be detected in blood plasma and cellular reservoirs even after several

years of therapy (Deeks et al., 2012). During antiretroviral therapy HIV-1 can remain in cellular

reservoirs and it is believed that the predominant reservoir of HIV-1 is CD4+ T cells, more

specifically memory CD4+ T cells (Palmer et al., 2011). These reservoirs mean that cART cannot

fully eradicate the infection and failure of cART due to drug resistance leads to a resurgence in viral

load and the progression to AIDS. Antiretroviral therapies do slow within-host evolution to very

low rates with viral sequences from patients after years of therapy being similar to those obtained
before therapy (Josefsson et al., 2013), although recently it has been found that virus evolution

continues during cART, even when the virus is undetectable in the blood (Lorenzo-Redondo et

al., 2016). Whilst this may be the case the limited correlation of genetic divergence with time

suggests that during antiretroviral therapy there is little substantial viral evolution (Josefsson et al.,

2013). Providing cART is begun as soon as possible following infection it may be possible to reduce

viral evolution, leaving recombination the only major source of generating viral diversity.

Inter-host Dynamics
The inter-host dynamics of HIV varies greatly to that seen at the intra-host level. In contrast

to within-host evolution, there appears to be little selection during inter-host transmission. Unlike

the ladder-like phylogenies produced from a single infected individual inter-host phylogenies

display multiple lineages persisting through time. This agrees with the finding that there is little

genetic variation in the ability of different HIV lineages to infect new individuals, which is to be

expected if there is little to no selection at the inter-host level (Lemey et al., 2006). One exception

to this is HIV-1 subtype C, which may be more sexually transmissible than other subtypes (John-

Stewart et al., 2005) but this remains to be confirmed. Whilst a relatively large amount of diversity

is created within an individual during infection, transmission to a new individual represents a

genetic bottleneck with a large proportion of new infections being initiated by a single infectious

unit (Hemelaar, 2012).

Whilst recombination is a challenge for phylodynamic studies of intra-host dynamics it does

not appear to be as big of a challenge for studies of inter-host dynamics for several reasons. Firstly,

estimated rates of co-infection and superinfection are generally very low and vary among risk

groups, with superinfection expected to be greater in high-risk groups such as commercial sex

workers and people who inject drugs. Secondly recombinants that are generated from more diverse

parental strains are the easiest to identify with current programs and can be excluded from genetic

analysis (Lemey et al., 2006). This seems to be supported by several studies of HIV-1 where

phylogenetic reconstructions of transmission that do not take recombination into account are
consistent with those produced from epidemiological data (Pybus and Rambaut, 2009).

Recombination can also be accounted for at the inter-host level in ancestral recombination graph

models (Ward et al., 2013).

Within-Country/Region Dynamics

With the exception of countries in West-Central Africa, within-country epidemics are usually

caused predominantly by the transmission of a single HIV-1 subtype. For example, the HIV-1

epidemic in Europe is dominated by subtype B (Fig. 1). The diversity of HIV-1 within-countries

and regions is generated largely from its relative isolation within groups. Phylodynamic methods

can be used to reconstruct transmission histories and identify transmission routes that may be

misclassified or unidentified by patient history. For example, a study in North Carolina found 12%

of Latino men were identified in phylogenetic clusters with only men or men who have sex with

men but reported heterosexual risk (Dennis et al., 2015). Several studies of HIV-1 clusters have

shown close agreement between phylogenetic reconstructions and the true history of transmission,

although phylogenetic methods alone cannot always identify all transmission chains (Bezemer et

al., 2015).

Within the HIV-1 subtype B epidemic in Europe epidemiological data suggested that most new

infections occurred locally however phylogeographic methods suggested frequent migration of

lineages across countries (Paraskevis et al., 2009). This apparent discrepancy between data sources

suggests that the viral dispersion identified here may date from earlier in the epidemic and whilst

understanding previous migrations is useful, more recent transmission histories are more helpful

for directing public health decisions. Because of the complexity of the European epidemic and lack

of comprehensive sampling across countries, the sampling density was not high enough inn this

study to capture transmission chains within countries. This remains a challenge for phylodynamic

approaches as sampling densities need to be sufficiently high to capture such transmission

dynamics at country and district scales. As the cost of sequencing continues to fall it will become

more economical to sample larger proportions of epidemics. However, as larger and larger data

sets are produced it will become increasingly challenging to work with them computationally.
 HIV-1 transmission between risk groups varies but seems to occur much less frequently than

within risk groups. For example, in the Netherlands HIV-1 subtype B epidemic a large transmission

cluster consisting mainly of people who inject drugs, showed links to heterosexual transmission,

and were almost completely separated from transmission clusters of men who have sex with men

(Bezemer et al., 2015). HIV-1 lineages can also be relatively compartmentalized by social groupings

such as ethnicity. For example, immigrant Latinos in North Carolina were significantly more likely

to be in transmission clusters with other Latinos (Dennis et al., 2015). Human mobility also affects

within-country epidemics. Within Europe, Greece, Portugal, Serbia, and Spain are apparent sources

of new viral migration, with Austria, Belgium, and Luxembourg acting as virus sinks. The majority

of European countries however show bidirectional migration within the epidemic (Paraskevis et

al., 2009).

Figure 1 Global distribution of HIV-1 subtypes and recombinants. In the main figure, pie charts representing
the distribution of HIV-1 group M subtypes and recombinants from 2004 to 2007 in each region are
superimposed on the regions. The relative surface area of the pie charts correspond to the relative numbers of
people living with HIV in the regions. Figure from Hemelaar, 2012.
Global Dynamics

Originating in Western-Central Africa Group M subtypes have spread across the globe and are

the predominant cause of HIV-1 infection worldwide. Subtype B is the predominant subtype in

Europe and the Americas, however globally subtype C has the highest prevalence causing just

under half of all HIV-1 infections (Fig. 1). The majority of HIV-1 infections in West-Central Africa

are now caused by circulating and unique recombinant forms, HIV strains generated by

recombination between two different strains. Whilst group M is responsible for the global

pandemic, group O is responsible for only a few tens of thousands of infections in West-Central

Africa, whilst Group N and P are responsible for only a handful of infections in Cameroon

(Hemelaar, 2012). Similarly HIV-2 has largely been confined to West Africa however increasing

numbers of cases are being reported in Europe, India and the USA (Campbell-Yusuf and Gandhi,

2011).

Such high levels of diversity globally present many challenges for HIV control. Antiretroviral

drugs were originally developed against HIV-1 group M subtype B however they have been found

to be as affective against other group M subtypes (Geretti et al., 2009). The susceptibility of group

O to antiretroviral drugs varies and different types of drugs are more effective than others (Tebit

et al., 2016). HIV-2 varies greatly in its susceptibility to antiretroviral drugs with certain types having

almost no effect on HIV-2 infection, requiring different optimal drug regimens (Adj-Tour et al.,

2003, Chiara et al., 2010, Harries et al., 2010). It is therefore important that HIV types, groups and

subtypes can be differentiated, yet most current HIV tests in Sub-Saharan Africa cannot provide

this information and so incorrect treatments are often prescribed. This also presents challenges to

cART as the prescription of suboptimum treatment regimens can lead to the emergence of drug

resistance (Siliciano and Siliciano, 2013).

Another challenge for current phylodynamic methods is the timing of the most recent

common ancestor (MRCA) of HIV lineages due to the differences in the rate of evolution between

lineages. The inferred timing of the MRCA for a group, using molecular clock analysis, can be used

to estimate when an infection first appeared within a country or region. For example, from the
inference of the MRCA it has been found that HIV-1 subtype B likely moved from Africa to Haiti

around 1966 (Gilbert et al., 2007). Strict molecular clock methods assume constant evolutionary

rates across phylogenetic lineages with this assumption being loosened in more recent

developments of relaxed molecular clock methods (Drummond et al., 2006). Current relaxed

molecular clock methods however may be insufficient for dealing with the type of variation in

substitution rate seen in HIV-1, with results using all group M subtypes together producing

different estimates from those obtained considering each subtype separately (Wertheim et al., 2012,

Lunar et al., 2015).

Conclusions
The diversity of HIV has presented many challenges to phylodynamic methods and HIV

control, whilst many of these challenges have been overcome in various ways some challenges still

remain. Current challenges to phylodynamic studies include recombination and selection, the

requirement for high sampling densities, and rate heterogeneity between lineages. At the intra-host

level recombination and selection still present a challenge. However, the development of

extensions to coalescent theory mean that the inclusion of recombination and selection into models

is now largely a challenge in the application of these to sequence data. Similarly, the requirement

for high sampling densities due to the complexity of HIV epidemics is becoming less of a challenge

in phylodynamic studies as the cost of sequencing continues to fall. New nanopore DNA

sequencing instruments mean that viral sequences can be sequenced in 15-60 minutes in the field

increasing the potential for high sampling densities and improving surveillance capabilities (Quick

et al., 2016), although some computational challenges do still remain when working with large data

sets.

As well as challenges to phylodynamic studies the diversity of HIV also presents challenges

to HIV control such as the generation of escape mutants, the existence of cellular reservoirs, and

resistance to antiretroviral drugs. The rapid generation of escape mutants against both antibody

and T cell responses provides a major challenge to the effectiveness of a potential vaccine, requiring
the priming of responses targeting multiple conserved epitopes. The existence of cellular reservoirs

would also not present a problem to vaccination, however they do to cART. Cellular reservoirs

mean that cART cannot cure HIV as the virus remains in these reservoirs. Globally challenges to

HIV control include the need for different treatment regimens HIV types and groups, as well as

immigration. Both of these challenges can be largely overcome through increased awareness of

such challenges, along with the use of effective testing and prescription of optimal treatment

regimens, and early diagnosis and treatment to reduce HIV transmission in populations and

geographic areas at high risk of infection.

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