thirty years have passed after discovering human immunodeficiency virus ( hiv ) , the etiological agent of the acquired immunodeficiency syndrome ( aids ) [ 14 ] .
two types of hiv are known : the most common hiv-1 , which is responsible to the worldwide aids epidemic , and the immunologically distinct hiv-2 , which is much less common and less virulent [ 6 , 7 ] but produces clinical findings similar to hiv-1 .
the hiv-1 type itself includes four groups m ( main ) , o ( outlier ) , n ( non - m , non - o ) , and p [ 912 ] , which have different geographic distributions but all produce similar clinical symptoms .
the m group further splits into 9 subtypes ( a through j ) [ 1315 ] , as well as at least 58 circulating recombinant forms ( crfs , http://www.hiv.lanl.gov/content/sequence/hiv/crfs/crfs.html , last accessed 06 may 2013 ) and multiple unique recombinant forms ( urfs ) . the vast majority of reports on drug resistance deal with hiv-1 subtype b infections in developed countries , and this is largely due to historical delays in access to antiretroviral therapy on a worldwide basis .
advances in antiretroviral therapy have revolutionized hiv management and the control of the spread of regional epidemics [ 1618 ] .
currently , a combination of several antiretroviral agents , termed highly active anti - retroviral therapy ( haart ) , has been highly effective in reducing the number of hiv particles in the blood stream ( as measured by a blood test called viral load ) and delaying disease progression .
clinical trials and observational studies have shown profound reductions in morbidity and mortality in patients infected with hiv as a result of combination antiretroviral therapy [ 16 , 1927 ] .
of relevance , advances in hiv treatment have had a positive impact on all the affected demographic and behavioral risk groups , with an expected longevity for hiv - infected patients that is now 73 years .
moreover , it should be considered that , thanks to the recent expansion in the number of antiretrovirals and antiretroviral classes , virological suppression has become achievable in most patients for whom numerous prior antiretroviral regimens had failed .
in addition , antiretroviral therapy results in efficacious treatment of hiv-1 , regardless of the viral subtype . however , despite advances in antiretroviral therapy , some treatments still fail .
a major cause of treatment failure is the development of drug resistance both in hiv-1 b and non - b subtypes [ 2834 ] . the extreme variability and the high evolution rate of hiv-1 favour the development of antiretroviral resistance .
indeed , hiv-1 infection is characterized by a high degree of genetic variability within infected persons .
this is explained by the fact that the virus population present at a certain time point within an infected person consists of a complex mixture of heterogeneous strains , termed 
the subsequent overgrowth or dominance of a certain viral strain over another is largely determined by its relative adaptation to a given intrahost environment , a factor particularly relevant to the emergence of drug resistant variants . indeed , the intrapatient virus population is a highly dynamic system , characterized by a high turnover rate and a high mutation rate [ 37 , 38 ] .
these evolutionary dynamics are the basis for a diversified population that can quickly generate drug - resistant variants in response to the therapy [ 3942 ] .
escape mutants that have a selective advantage under therapy become dominant in the population and lead to an increasing virus production and eventually to therapy failure .
the shifted population may be hit with a new drug combination , but finding such a potent regimen after treatment failure is challenging , since many accumulated mutations confer drug resistance not only to the administered drugs but also to structurally and functionally similar compounds [ 41 , 43 ] . identifying and understanding hiv-1 drug resistance therefore helps clinicians to avoid minimally active antiretrovirals in favor of newer drugs that are fully or nearly fully active [ 4446 ] .
for this reason , resistance testing has become an important diagnostic tool in the management of hiv infections [ 4752 ] . with the aid of hiv resistance tests
pharmacoeconomic studies have shown that these tests are also cost effective [ 53 , 54 ] . for several years
, national and international hiv treatment guidelines have been recommending the use of resistance testing both in drug - naive and drug - treated patients [ 4852 ] .
antiretroviral drug design , resistance research , and interpretation systems have been largely based on hiv-1 subtype b because of its predominance in the wealthy countries in which antiretroviral drugs were first introduced , as well as the availability of assays for drug resistance in such locations .
however , it should be noted that hiv-1 b subtype represents only about 10% [ 5557 ] of the overall subtypes in the world .
several studies showed that non - b subtype hiv-1 infections have been rapidly increasing over time in previously subtype b homogeneous areas such as europe and north america [ 5873 ] .
for example , in france , switzerland , and italy , it is estimated that non - b infections constitute roughly 15%35% of hiv-1 infections , with an increasing prevalence and complexity of intersubtype recombinants over the last years [ 59 , 64 , 66 , 7377 ] .
although non - b infections are infrequent in north america , a study in new york identified non - b infections in a few us citizens who never travelled abroad , suggesting that transmission of non - b subtype occurs in the united states independently of travel history .
moreover , new hiv-1 strains are constantly emerging [ 78 , 79 ] . due to the spread of hiv-1 non - b subtypes and the introduction of antiretroviral drugs in resource - limited settings ( known for the largest assortment of non - b subtypes ) , further knowledge concerning the responsiveness to antiretroviral therapy and hiv-1 drug resistance in non - b subtypes is required . in this regard ,
the development of specific mutations varies in different subtypes , and this can be explained mainly by the intrinsic properties of the virus and not only by a different pressure of antiretroviral drugs , as suggested in a recent study . to date , the improvement of the genotypic resistance test in terms of sensitivity , cost effectiveness and detection of drug resistance in non - b subtypes is a major topic . in order to achieve this goal , the introduction of new affordable assays is crucial .
group m subtype independent genotyping assays for detection of hiv-1 drug resistance were recently developed .
these assays could represent an alternative to commercial assays for hiv-1 drug resistance genotyping in routine diagnostics and for surveillance and monitoring of drug resistance in resource limited settings [ 8082 ] .
several discrepancies are still present in the interpretation of resistance patterns present in hiv-1 non - b subtypes by using different interpretation algorithms .
these discrepancies illustrate how hard it may be to reach an unambiguous conclusion , despite the efforts made over the last decade to interpret drug resistance in hiv-1 non - b subtypes [ 8387 ] .
the increasing prevalence of hiv-1 non - b subtypes could have implications not only for the development of resistance but also for diagnosis , vaccine design , and the clinical management of hiv infection [ 65 , 90 , 91 ] .
moreover , patients infected by certain hiv-1 non - b subtypes present accelerated disease progression [ 9297 ] and higher cognitive impairment . in the light of the above
mentioned , the aim of this review is to provide a complete picture about hiv-1 genetic diversity and its implications .
mutational escape results in a remarkable degree of viral diversity within hiv-1 and in its adaptation to both immune activity and antiretroviral therapy .
however , not all escape mutations are advantageous to the virus since some of them can severely affect viral fitness [ 99 , 100 ] .
the extensive genetic diversity of hiv-1 is due to its high replication rate , the error - prone reverse transcriptase , and recombination events that may occur during virus replication [ 101 , 102 ] .
the molecular basis of hiv-1 variability is a highly error - prone reverse transcriptase enzyme .
the activity of this enzyme , essential for viral replication , is specifically required for the conversion of single - stranded genomic rna into double - stranded viral dna , which is later integrated into the host genomic dna .
for this reason , hiv-1 reverse transcriptase inhibitors are powerful inhibitors of hiv-1 replication and represent an important class of antiretroviral agents .
hiv-1 reverse transcriptase is a multifunctional enzyme that possesses rna - dependent and dna - dependent dna polymerase activities as well as an rnase h activity that specifically degrades the rna strand of rna / dna hybrids . as an intrinsic property , and in contrast to other dna polymerases , hiv-1 reverse transcriptase lacks a proofreading function .
this error - prone nature of reverse transcriptase , together with the high rate of virus production sustained by hiv-1 infection in vivo , strongly contributes to the continuous generation of new viral variants [ 13 , 106 , 107 ] .
the rate of nucleotide substitutions introduced by reverse transcriptase is approximately 10 per nucleotide per cycle of replication , which is equal to one nucleotide substitution per genome during a single replication cycle .
hiv-1 has a rapid turnover , and it is estimated that approximately 10 virions per day are generated in an infected individual .
the composite lifespan of plasma virus and virus - producing cells is very short with a half - life of approximately two days , and an almost complete replacement of wild - type strains by drug resistant virus occurs in plasma within 24 weeks . during antiretroviral treatment ,
rapid viral turnover in combination with a high mutation rate is a primary factor behind the emergence of hiv variants with antiretroviral drug resistance .
each retroviral particle contains two copies of single - stranded rna , and template switches occur frequently during reverse transcription , thus generating mutations and recombination by intramolecular and intermolecular jumps .
recombination may link drug resistant mutations in hiv-1 , leading to increased resistance to a particular drug , or the generation of multidrug resistant variants .
in addition , recombination may lead to the acquisition of mutations that compensate for a loss in viral fitness or replicative capacity due to previous acquisition of resistance mutations .
since recombination can create a multiple drug resistant virus out of two single drug resistant strains , it is generally believed that the capacity of the virus to recombine facilitates the evolution of drug resistance [ 9 , 110 , 113115 ] .
this rapid evolution of drug resistance in hiv remains a major obstacle for hiv therapy .
recombination is a strategy for viral rejuvenation , and it is likely that recombination between hiv strains may lead to the evolution of fitter forms and viral strains acquiring drug resistance to all major classes of hiv-1 inhibitors . a different scenario could be that a fitter virus can be generated by recombining parts of two parental genomes with lesser fitness , or alternatively a less fit virus can be generated by breaking up favourable combinations of mutations in the parental genomes .
this interaction between recombination , mutations , and viral fitness is highly intricate , but nonetheless , recombination and its mechanisms , especially at the level of diverse subtypes , warrant further investigation .
the potential for genetic differences among subtypes to yield different patterns of resistance - conferring mutations is supported by natural variation among hiv subtypes in genetic content ( 40% variation in the env gene , and 810% variation in the pol / gag genes ) .
this issue acquires special relevance in view of the fact that the hiv pol gene is the major target for all major classes of anti - hiv drugs and most hiv strains show hotspots for recombination in gag - pol and env regions .
on the basis of genetic homology , hiv-1 has been classified into four groups : m ( main ) , o ( outlier ) , n ( non - m , non - o ) , and the recently identified p [ 912 ] . the m group further splits into 9 subtypes ( a through j ) [ 1315 ] , as well as at least 58 circulating recombinant forms ( crfs , http://www.hiv.lanl.gov/content/sequence/hiv/crfs/crfs.html , last accessed 06 may 2013 ) and multiple unique recombinant forms ( urfs )
. in general genetic variation is of 2535% and 1520% between and within subtypes , respectively .
for example , subtypes a and f have been subdivided into five ( a1 , a2 , a3 , a4 , a5 ) and two subtypes ( f1 , f2 ) , respectively .
crfs are intersubtype recombinant hiv-1 genomes that have infected three or more subjects who are not epidemiologically related , so they can be assumed to make an epidemiologically relevant contribution to the hiv-1 m group epidemic .
the crfs are labelled with numbers rather than letters , and numbered in the order they were first adequately described as the first time .
urf variants are widely distributed worldwide , with recombination breakpoints different from those found in crfs .
very few isolates of hiv-1 n group have been identified and sequenced from humans , while the diversity of sequences within the o group is nearly as great as the diversity of sequences in the m group .
today , the hiv-1 m group , the cause of the worldwide pandemic , has a near global distribution , whereas the n and o groups are restricted to individuals of west african origin .
the m and n hiv-1 groups have a common ancestor , one that is most closely related to the siv strain found in chimpanzees ( sivcpz ; pan troglodytes troglodytes ) [ 117 , 118 ] that live mainly in gabon , cameroon , and the republic of the congo .
this hiv-1 progenitor probably was passed from chimpanzees to human hunters through blood borne transmission [ 119 , 120 ] .
recent evidence suggests that the hiv-1 o and p groups may have originated in wild gorillas , in which the closest relatives of these two groups have been identified [ 10 , 121 ] .
phylogenetic analysis of hiv-1 and related viruses from nonhuman primates suggests that three independent transmission events , which happened in early 20th century , spawned the hiv-1 m , n , and o groups [ 119 , 120 ] .
in particular , the earliest direct evidence of hiv infection in humans was found retrospectively in a serum sample and in a lymph node biopsy specimen stored in 1959 and 1960 , respectively , in kinshasa in the democratic republic of the congo [ 122 , 123 ] .
moreover , they have been instrumental in the validation and extrapolations of molecular clock computer programs used to estimate the time to the most recent common ancestors ( tmrcas ) and evolutionary rates of various hiv lineages [ 123 , 124 ] .
the hiv-1 m group appears to be the oldest hiv lineage in humans , with an estimated time to the tmrca in the first two or three decades of the 1900s [ 123 , 124 ] . in particular ,
the tmrca shared between the m group and sivcpz is estimated at 1853 ( 17991904 ) .
cross - species transfer is therefore inferred to have taken place sometime between 1853 and the early 1900s , although there is some uncertainty , given the size of the confidence intervals .
hiv-1 m group subtypes can be considered as a result of the high error rate of reverse transcriptase enzyme during virus replication and selective pressure exerted by the immune system . since their introduction into humans ,
hiv-1 m group subtypes have expanded rapidly in west and central africa and established multiple epidemics around the world .
regional expansion of hiv-1 has come in waves with the rapid emergence of specific subtypes due in part to specific modes and routes of transmission .
for example , intravenous drug use in southeast asia in the mid-1980s and in eastern europe and russia during the early 1990s led to the rapid spread of crf01_ae and of subtype a , respectively [ 126 , 127 ] . a similar expansion of subtype b hiv-1 transmission occurred among men who have sex with men in north america and europe in the early 1980s
however , hiv-1 subtype c ( the most dominant subtype in the world responsible for more than 50% of overall infections ) appears to have slowly emerged throughout the world over the past 10 to 15 years as a consequence of multiple introductions [ 126 , 128 ] . in recent years a substantial increase of recombinant forms has been observed as a consequence of the increased genetic complexity of the global epidemic [ 57 , 59 , 64 , 66 , 7377 , 129131 ] .
however , the prevalence of recombinant forms ( estimated to be around the 20% ) is still underestimated .
indeed , genetic complexity is not always detected , and this is mainly due to the subtyping of only one genetic region and not of the full genome .
consequently , specimens previously considered  pure  variants may be classified as recombinants when additional viral genes are analyzed .
the drugs currently approved for clinical use by the us food and drug administration ( fda ) and used to treat hiv infection belong to 6 distinct classes ( http://www.fda.gov/forconsumers/byaudience/forpatientadvocates/hivandaidsactivities/ucm118915.htm , last accessed 06 may 2013 ) : ( 1 ) seven nucleoside and one nucleotide reverse transcriptase inhibitors ( nrtis ) ; ( 2 ) five nonnucleoside reverse transcriptase inhibitors ( nnrtis ) ; ( 3 ) nine protease inhibitors ( pis ) ; ( 4 ) one fusion inhibitor ( fi ) ; ( 5 ) one cc chemokine receptor 5 ( ccr5 ) antagonist ; ( 6 ) two integrase inhibitors ( inis ) .
each of these drug classes act at different steps in the hiv replication cycle ( figure 1 ) .
the nnrtis and nrtis block the reverse transcription of the viral rna genome in cdna , which is catalysed by the reverse transcriptase .
the inis block the integration of viral genome into the dna of the host cell .
although it seems that combination antiretroviral regimens are effective against all hiv-1 group m subtypes , there is emerging evidence of subtype differences in drug resistance relevant to antiretroviral strategies in different parts of the world . for this purpose
, extensive sampling of hiv genetic diversity and ad hoc analyses are required to tailoring of antiretroviral therapies in different parts of the world .
hiv-1 non - b subtypes present clade - specific substitutions in positions related to drug resistance   that could accelerate the emergence of drug - resistant viruses , change or induce alternative pathways of resistance , influence viral replicative capacity in vitro , impair the interpretation of genotypic resistance algorithms [ 70 , 77 , 134137 ] , and affect drug - binding affinity [ 138 , 139 ] .
specific mutation development varies with different subtypes because of the intrinsic properties of the virus as well as the different pressures of antiretroviral drugs .
in particular , three factors are involved in this phenomenon.intersubtype differences in codon usage : subtype differences in nucleotide and mutational motifs , which are defined as the number of transitions or transversions needed to develop resistance to different classes of antiretroviral drugs , may affect the genetic barrier for resistance , as shown in the development of some mutations in different hiv-1 proteins , such as ( i ) mutations associated with resistance to nnrtis at the codon 106 of the reverse transcriptase in c and b subtypes ( see section 4.2.2 ) [ 140 , 141 ] ; ( ii ) mutations at the protease codons 74 in c subtypes , 82 in g subtypes , and 89 in several non - b subtypes such as c and crf02_ag ( see section 4.2.3 ) [ 87 , 142145 ] ; ( iii ) mutations in the integrase gene at codons 140 , 148 , 151 , 157 , and 160 ( see section 4.2.4 ) [ 146 , 147].intersubtype amino acid differences involved in minor structural changes in the targets of therapy : in this situation , different mutations emerge under the same drug pressure ( e.g. , the protease mutation d30n ; see section 4.2.3).intersubtype differences in sequence motifs favoring nucleotide substitutions involved in drug resistance ( e.g. , the reverse transcriptase mutation k65r ; see section 4.2.1 ) .
intersubtype differences in codon usage : subtype differences in nucleotide and mutational motifs , which are defined as the number of transitions or transversions needed to develop resistance to different classes of antiretroviral drugs , may affect the genetic barrier for resistance , as shown in the development of some mutations in different hiv-1 proteins , such as ( i ) mutations associated with resistance to nnrtis at the codon 106 of the reverse transcriptase in c and b subtypes ( see section 4.2.2 ) [ 140 , 141 ] ; ( ii ) mutations at the protease codons 74 in c subtypes , 82 in g subtypes , and 89 in several non - b subtypes such as c and crf02_ag ( see section 4.2.3 ) [ 87 , 142145 ] ; ( iii ) mutations in the integrase gene at codons 140 , 148 , 151 , 157 , and 160 ( see section 4.2.4 ) [ 146 , 147 ] .
intersubtype amino acid differences involved in minor structural changes in the targets of therapy : in this situation , different mutations emerge under the same drug pressure ( e.g. , the protease mutation d30n ; see section 4.2.3 ) .
intersubtype differences in sequence motifs favoring nucleotide substitutions involved in drug resistance ( e.g. , the reverse transcriptase mutation k65r ; see section 4.2.1 ) .
the following subsections describe the impact of hiv-1 subtypes on the different classes of antiretrovirals so far available .  
table 1 provides an overview of the resistance associated mutations which are related to subtype diversity .
the impact of subtype in terms of emergence of resistance in the nrti antiretroviral class is mainly due to the more rapid selection of primary resistance mutations in hiv-1 subtype c infected patients than in those infected by a subtype b virus . some of the differences in rates of acquisition of the mutation k65r or thymidine analogue mutations ( tams ) are doubtless due to treatment regimens and disease stage , as well as access to viral load testing in many developing countries .
however , one study suggests that increased rates of k65r acquisition in subtype c may be due to the nature of the subtype c  rna template .
the lysine to arginine mutation at position 65 ( k65r ) is a major mutation that confers broad high - level resistance to most nrtis , except zidovudine .
the nucleotide sequence at codons 64 - 65 - 66 ( containing a homopolymeric stretch of adenine bases ) differs between subtypes b and subtype c viruses .
this leads to reverse transcriptase pausing during the synthesis of double - stranded dna from the single - stranded dna intermediate template , a process that is template - specific but independent of the reverse transcriptase enzyme [ 14 , 15 , 148 ] .
subsequent misalignment of the subtype c template and primer leads to the aag to agg change responsible for the k65r mutation   ( figure 2 ) . on the contrary ,
subtype b templates are prone to frequent pausing at codon 67 , facilitating the generation of mutation d67n and tams instead of k65r ( figure 2 , table 1 ) [ 148 , 150 , 151 ] .
a recent study showed a very high rate ( > 65% ) of k65r for patients infected by subtype c who failed tenofovir - based first - line regimens in south africa .
furthermore , a rate of 7 to 15% of k65r and/or k70e mutations was observed in subtype c endemic area in patients failing regimens including stavudine , didanosine , or zidovudine .
studies from israel reported a high frequency of k65r with treatment failure in subtype c viruses from ethiopian immigrants , while in indian patients a rate of k65r around 1012% at failure of first line combination therapy including stavudine , lamivudine , and nevirapine was observed . by using ultrasensitive pyrosequencing , variants carrying k65r mutation showed a higher frequency in subtype c infected patients than those infected by subtype b ( 1.04% versus 0.25% ) .
in addition to the recruitment for monitoring k65r prevalence in patients with subtype c hiv infection , also the tam pathway ( 67n/70r/215y ) deserves further attention .
indeed , in patients infected by subtype c from botswana , india , south africa , and malawi tams were also observed at treatment failure with zidovudine and didanosine [ 153 , 155 , 157 , 158 ] .
larger numbers of patients and followups will be required to determine whether any consistent effect of the emergence of k65r in subtype c is clinically relevant .
the emergence of mutations associated with resistance to nnrtis occurs after a single dose of nevirapine ( sdnvp ) [ 159161 ] , a procedure recommended for the prevention of mother - to - child hiv transmission ( pmtct ) by initial guidelines from the world health organization ( who ) [ 162 , 163 ] .
replacement of sdnvp may be necessary to reduce infant hiv infection risk and drug resistance development .
indeed , high frequency of drug resistance was observed in 69% of pregnant women infected with subtype c , 36% of those with subtype d , 19% of those with subtype a , and 21% of those with the crf02_ag , despite the absence of resistance before the administration of antiretroviral therapy [ 163167 ] .
moreover , by using ultrasensitive sequencing techniques , several studies revealed that in patients infected by subtype c a higher prevalence of nevirapine - resistance mutations ( k103n and y181c ) was found , reaching 70% to 87% , as compared with 42% of patients with subtype a with resistant viruses [ 168 , 169 ] .
these findings underscore the role of viral subtype in facilitating the development of drug resistance , which is exacerbated by the fact that resistance to nnrtis can also be transmitted through breast feeding .
indeed , the v106 m mutation is commonly selected in subtype c viruses ( in approximately 30% of patients ) after exposure to nevirapine or efavirenz , whereas a v106a substitution is only rarely selected ( in approximately 5% of patients ) by these two nnrtis in other subtypes ( table 1 ) .
this peculiarity of subtype c results from the fact that v106 is encoded by gta in subtype b viruses and gtg in subtype c viruses [ 141 , 171 ] . a single g - to - a transition at the first position of codon 106 in subtype c viruses results in v106 m , which confers high - level resistance to efavirenz and nevirapine . in contrast , in subtype b viruses , v106 m requires two nucleotide substitutions ( gta - atg ) and therefore occurs infrequently [ 140 , 141 ] .
k103n and y181c mutations remain important in both subtypes b and c , with k103n occurring in 40% of subtype b and 29% of subtype c viruses and y181c occurring in 23% of subtype b and 12% of subtype c viruses . in a recent italian study ,
the prevalence of mutation k103n in nnrti - treated patients was lower in subtype c infected patients compared with patients infected by b , f , or crf02_ag subtypes ( table 1 ) .
conversely , the mutations y181c and y188l were found with higher frequency in patients infected with subtype c compared to patients infected with subtype b .
another substitution that is seen more frequently among patients with a subtype c virus is g190a , which is also a result of naturally occurring g190a
reverse transcriptase polymorphisms at residue 98 are common in subtype g , but they are also associated with nnrti resistance in subtype b ( table 1 ) [ 15 , 173 ] .
the second - generation nnrti etravirine has demonstrated good efficacy across subtypes . a recent study highlighted the fact that hiv-1 genetic variant does not significantly influence the genotypic prediction of etravirine susceptibility in infected individuals failing efavirenz containing regimens .
however , among the etravirine resistance associated mutations , some polymorphic substitutions resulted in drug - naive individuals infected with non - b subtypes , especially crf02_ag .
noteworthy , in a recent study it was found that the mutation e138k was the first mutation to emerge in either subtype b , c , or crf02_ag clinical isolates under etravirine pressure ( table 1 ) .
another study confirmed this finding for subtype c , while a preferential selection of y181c for the subtype b virus was observed ( table 1 ) .
a novel mutation in the c - terminal domain of reverse transcriptase ( n348i ) has recently been reported to reduce susceptibility to etravirine in subtypes a , b , and c ( table 1 ) . in particular , in a south - african clinical trial , the n348i mutation was observed in 24% of subtype c infected patients failing a first - line nnrti regimen ( most commonly with nevirapine ) .
this mutation is not included in standard mutation lists or algorithms , but more data are urgently required to determine its clinical relevance . to date , the efficacy and the resistance patterns of the novel second - generation nnrti rilpivirine seem to be similar across the different subtypes [ 181 , 182 ] .
a study focused on hiv-1 subtype crf01_ae showed that the presence of mutations associated with resistance to rilpivirine are uncommon in patients infected by this subtype and failing a first - line nnrti - containing regimen .
however , cross - resistance between rilpivirine and etravirine was high . moreover , approximately 60% of patients had a high level of etravirine resistance , thus suggesting that the role of etravirine in hiv-1 subtype crf01_ae infected patients in second - line therapy is limited in late nnrti failure settings .
nnrti resistance associated mutations such as y181i , y188l , g190a , k101a , v106i , and v179i are natural polymorphisms in hiv-2 , thus hiv-2 proves to be naturally resistant to all nnrtis .
nonpolymorphic substitutions in the protease gene have a greater impact on baseline susceptibility to pis than polymorphic mutations . however , intersubtype amino acid differences can create subtle structural differences in the targets of therapy .
for example , subtype b infected patients receiving nelfinavir are more likely to develop d30n than those with viruses belonging to subtypes c , f , g and crf01_ae , which are more likely to develop l90 m or n88s ( table 1 ) [ 186 , 187 ] . more specifically , by comparing b and g subtypes , even in cases where the first mutation is l90 m in both these subtypes ,
indeed , in subtype b , l63p is the second mutation and occurs in almost all cases , suggesting that the progression of resistance is dependent on the emergence of this mutation , followed by the selection of v77i and other mutations .
differently , in subtype g , in almost 100% of cases , l89i follows the emergence of l90 m , suggesting a role in subtype g similar to that of l63p in subtype b. in this regard , findings indicate that the association of l89i with l90 m may further increase the pi resistance of subtype g viruses when compared with l90 m alone .
although subtype c isolates from ethiopian immigrants to israel favoured the l90 m pathway , patients with subtype c viruses in botswana did have d30n , suggesting that subtype c viruses from ethiopia and southern africa might be different [ 186 , 189 ] .
the basis for the higher preponderance of d30n in subtype b than in other subtypes may be related to the flexibility of the protease flap region and destabilization of the pi complex in subtype b , whereas an accessory n83 t mutation may be needed to rescue fitness and confer resistance in subtype c [ 142 , 190 ] .
recent evidence has suggested that the polymorphism at codon 36 in the protease gene ( m36 in subtype b and i36 in most other non - b subtypes ) affects both the patterns of resistance that emerge under drug pressure and viral replication capacity ( table 1 ) .
similarly , the m89 polymorphism in subtypes a , c , and crf01_ae ( l89 in subtype b ) preferentially leads to the emergence under drug pressure of the m89 t mutation , which confers high - level resistance to nelfinavir , atazanavir , and lopinavir .
there is also in vitro evidence that crf2_ag viruses with the 17e/64 m polymorphisms demonstrate hypersusceptibility to certain pis ( nelfinavir , atazanavir , and indinavir ) .
differences in polymorphisms in the protease gene have been reported among several non - b subtypes ; these include the following protease residues : 10 , 20 , and 63 in subtype a ; 20 , 53 , 63 , 74 , and 82 in subtype c ; 13 and 20 in subtype d ; 10 , 14 , 20 , and 77 in subtype f ; 20 , 67 , 73 , 82 , and 88 in subtype g ; 20 , 63 , 82 , and 89 in the crf01_ae ; 20 and 89 in the crf02_ag ( table 1 ) . in particular , in patients treated with pis , l89v was predominantly found in crf02_ag , while the tipranavir resistance mutation t74p was predominantly found in the c subtype in comparison to b subtype .
the mutation v82 m was mainly found in subtype g while the mutations v82a / f / s were mainly present in other subtypes ; the mutation n88s was found in subtypes c and crf02_ag , while n88d was present in subtype b . in southern brazil , scientists reported a lower relative frequency of primary resistance to pis in subtype c rather than in subtype b . despite clear evidence for preferential emergence ,
diminished susceptibilities among wild - type isolates have been found for crf02_ag recombinant viruses in three different studies of nelfinavir and atazanavir [ 138 , 173 , 194 ] .
a study suggested that distortions in the k26 pocket of the a / g protease may be responsible for a lower binding energy of nelfinavir and lower susceptibility of a / g viruses .
the protease and gag genes coevolve as a functional unit when hiv is subjected to drug pressure from pis .
mutations in gag can act as compensatory substitutions that can increase both rates and levels of resistance to pis , as well as viral replication capacity .
no genotypic system for the determination of drug resistance to pis currently monitors gag , despite the fact that relevant mutations in gag can increase resistance by a factor of 2 to 2.5 , depending on the subtype .
it is likely that different subtypes could develop compensatory gag mutations at different rates ; this might ultimately justify the genotyping of gag in resistance testing .
the primary mutations in the integrase gene associated with ini resistance are e92q , y143r / c , q148k / r / h , and n155h . the residues associated with primary resistance seem to be highly conserved across subtypes , but polymorphisms at the sites of secondary resistance mutations are more common in non - b subtypes [ 147 , 198200 ] .
signature subtype differences in integrase at codons 140 , 148 , 151 , 157 , and 160 among hiv subtypes may affect the genetic barrier for resistance .
these variations predict higher genetic barriers to the development of g140s and g140c mutations in subtypes c , crf02_ag , and a / crf01_ae , as well as higher genetic barriers to v151i in subtypes crf02_ag and crf01_ae [ 146 , 147 ] . regarding the impact of subtype on primary resistance development , it was recently observed that the subtype b integrase enzyme with the n155h mutation ( e92q ) exhibited increased resistance to raltegravir compared to the subtype c enzyme ( table 1 ) . in addition a new potential resistance pathway for raltegravir might include the mutation g118r , a substitution associated with lower susceptibility to raltegravir in crf_02ag subtypes .
notably , polymorphisms such as t97a , v151i , and g163r , already known as raltegravir secondary resistance mutations , showed a considerable prevalence ( 9% ) in several recent studies also analyzing non - b subtype infected patients [ 203 , 204 ] .
the additional ini - resistance associated mutations k156n and s230n mutations were more frequent in b subtype while v72i , l74i , t125a , v201i , and t206s were more frequent in certain non - b subtypes [ 200 , 205 ] .
no differences in phenotypic raltegravir resistance were found in several non - b isolates tested .
dolutegravir is a second - generation ini which recently entered the battery of antiretrovirals against hiv-1 infection .
it showed a higher genetic barrier to resistance and retains activity against viruses with resistance to raltegravir or elvitegravir .
interestingly , among the mutations associated with in vitro resistance to dolutegravir , the mutations l101i and t124a were polymorphic and significantly more prevalent in patients with non - b subtypes .
in addition , mutation r263k seems to be preferentially selected in subtype b under dolutegravir pressure ( table 1 ) . among the eis today available in clinics ,
both enfuvirtide ( targeting gp41 hiv-1 protein ) and maraviroc ( targeting cellular ccr5 receptors ) have shown antiviral activity against hiv-1 b and non - b subtypes [ 207 , 208 ] .
however , the high level of diversity ( 20 to 40% ) in the env region predicts that this class of drugs will probably have higher potential for natural and emergent drug resistance .
clinical data have shown that the fi enfuvirtide is active against non - b subtypes owing to a highly conserved binding domain , although hiv-2 and hiv-1 class o viruses have natural resistance against this drug [ 209 , 210 ] .
so far , some studies suggest that there are significant differences in baseline susceptibility to hiv eis among the predominant hiv-1 subtypes [ 211 , 212 ] . for instance
, it was found that in drug - naive patients many more mutations associated with resistance to eis occur in subtype c compared with subtype b strains ( table 1 ) .
due to the worldwide spread of non - b viruses and the introduction of antiretroviral drugs in developing countries ( known for the largest assortment of non - b subtypes ) , further knowledge concerning responsiveness hiv-1 drug resistance in non - b subtypes is required .
moreover , it should be taken into account that the increasing prevalence of hiv-1 non - b subtypes could have several implications not only for the development of resistance but also for other topics such as the response to antiretroviral therapy , disease progression , and transmission .
there is a solid body of evidence indicating that the type and degree of hiv-1 resistance to nrtis , nnrtis , and pis vary between different subtypes [ 136 , 186 , 213 , 214 ] .
the development of nelfinavir resistance in subtypes b and g represents a classic example of this phenomenon . a different level of resistance has been observed among different subtypes . indeed , the recombinant form crf02_ag is more susceptible to nelfinavir and ritonavir than subtypes c and f ; subtype g is more sensitive to tipranavir and lopinavir than other subtypes , and the subtype c has accelerated risk in developing resistance to tenofovir [ 189 , 215 , 216 ] .
an explanation for the extreme variability of hiv-1 subtypes in the response to antiretrovirals can be given by the presence of some polymorphisms that can influence both the emergence of drug - resistance mutations and the response to drugs .
for example , polymorphisms at residues 20 and 36 of hiv-1 protease decrease the genetic barrier to tipranavir resistance in subtypes a , c , f , and g , while nucleotide heterogeneity at 64 and 65 positions in the reverse transcriptase accelerates development of k65r in subtype c [ 150 , 189 ] . in some cases ,
drug exposure may lead to amplification of such polymorphisms as a98g / s in reverse transcriptase and m36i , k20i , and l89 m in protease , leading to a potential for resistance .
it is commonly accepted that , as a result of a high degree of polymorphism in the protease gene , drug - naive patients infected with non - b subtypes present a certain degree of resistance to the pis . however , the data which support this assumption are largely controversial , as highlighted by two studies that showed conflicting results . indeed , in a study   analysing the hiv-1 phenotype from 42 patients ( 19 , subtype g ; seven , subtype c ; six , subtype f ; 10 , crf02_ag ) by the antivirogram assay ( virco bvba ) , no differences in baseline susceptibility to any pi were found , with the exception of an unexpected hypersusceptibility to nelfinavir with crf02_ag . on the contrary ,
a second study , investigating drug susceptibility of 39 isolates from treatment - naive ghanaian patients , mostly infected with crf02_ag , showed reduced susceptibility to several pis , especially nelfinavir .
this reduced in vitro susceptibility is not in agreement with the available clinical data , which show no difference in time to undetectable viral load among different subtypes [ 217219 ] .
continuous research on the role of polymorphisms in the development of drug resistance is therefore necessary .
studies are needed to assess genotypes both before and after therapy in the context of possible associations between polymorphisms and drug resistance .
this area of research could include polymorphism variability in variants of the same subtype in different geographic regions .
this information might improve the efficacy of certain drug combinations over others in the context of second- or third - line therapeutic strategies .
polymorphisms should improve current algorithms for interpretation of genotyping results in a subtype - independent way .
as access to antiretroviral therapy in resource limited settings increases , where non - b subtypes are predominant , it remains imperative to establish appropriate treatment strategies for long - term clinical benefit that limits the emergence of drug resistance .
the use of nontoxic , effective antiretroviral drugs should yield excellent clinical responsiveness , regardless of the viral subtype .
subtype differences , suboptimal therapies , and deficiencies in health care delivery systems can create conditions for accelerated development of resistance .
urgent recruitment for low - cost viral - load monitoring is needed to prevent and detect drug resistance , as well as to avoid unnecessary treatment switches [ 220 , 221 ] .
unfortunately , pooling of resistance data often masks the role of regional differences in viral subtypes and antiretroviral therapies in the development of drug resistance . in resource - poor settings ,
such studies have used different nrti backbones ( e.g. , combinations of zidovudine and didanosine , zidovudine and lamivudine , or stavudine and lamivudine ) . in parts of africa ,
treatment failure has been reported in as many as 40% of patients after 2 years . of note ,
resistance rates in india to two drug classes were observed in more than 80% of patients who failed their first - line regimen using combinations of nrtis and nnrtis .
larger longitudinal studies are necessary to determine the response to first - line combinations of antiretroviral drugs .
the availability of genotypic resistance testing needs to be expanded to include all countries in which antiretroviral drugs are used .
cross - resistance among drugs is important , especially in settings where treatment options may be limited .
relatively few in vitro comparative data are available for pis in non - b subtypes , even if such data may be crucial for an understanding of cross - resistance to this class of antiretrovirals [ 191 , 224 ] .
such data are important , since pis are often the only available option for drug sequencing in resource - limited settings after the failure of first- or second - line treatment .
this means that resistance to pis usually requires the presence of large numbers of resistance mutations .
for this reason , there is an advantage of using pis to avoid early resistance development .
therefore , differences among subtypes with regard to drug resistance are likely to be more important for nrtis , nnrtis , and inis than for pis .
our knowledge of the impact of hiv-1 subtypes on responses to antiretroviral therapy is very limited .
most studies have been small or poorly designed , and only a few have had an evaluation period longer than 48 weeks .
an important limitation of these studies was to compare subtype b with non - b subtypes grouped together , a necessary oversimplification when dealing with small numbers of patients infected with non - b subtypes [ 217219 , 225227 ] .
a study by the paediatric european network for treatment of aids ( penta 5 trial )   investigated the response to therapy of 113 children infected with 7 different subtypes ( a , 15% ; b , 41% ; c , 16% ; d , 9% ; f , 5% ; g , 2% ; h , 1% ) and the two crfs crf01_ae ( 5% ) and crf02_ag ( 7% ) .
however , these results should be used with caution because the study did not have the statistical power to rule out minor differences between subtypes . moreover , only four drugs were used ( zidovudine , lamivudine , abacavir , and nelfinavir ) . although polymorphisms at resistance - associated positions were highly prevalent in non - b subtypes , no negative impact of the mutations in virological responses was found ; therefore , the authors concluded that polymorphisms at resistance - associated positions in subtype b could not necessarily be interpreted as conferring resistance in non - b subtypes .
retrospectively analysed the virological response in 362 patients : 265 europeans infected with subtype b and 97 africans infected with non - b subtypes .
subtype was presumed from ethnic and epidemiological data , with confirmation further extrapolated from genotyping the samples from 60% of the africans and 30% of the european patients .
there was a significant imbalance between the two groups in several important parameters , including gender , transmission - risk groups , cd4 cell counts , and antiretroviral regimens used .
results showed no difference in time to undetectable viral load or recovery of cd4 count ; however , a significant difference was observed in viral load over time , with a continuous increase in the african group after 9 months , suggesting a higher rate of therapy failure .
based on indirect evidence ( i.e. , the number of resistance mutations at time of failure ) , the authors concluded that the difference in virological response was mainly due to poorer adherence in the african group rather than the infecting viral subtype .
as the study was not controlled for adherence , its conclusions remain controversial . a study which analysed retrospectively b and non - b subtypes ( mainly focused on pure subtype a or recombinants with a subtype a protease ) showed no differences in the time to undetectable viral load or in the viral load measured over time . however , the gain in cd4 count was significantly lower in the non - b group , which showed an increase of 161 cells / mm , compared with 236 cells / mm in the subtype b group .
these results were consistent with those of camacho , who compared retrospectively the pathways to nelfinavir resistance in 101 patients ( 46 subtype b , 55 subtype g ) failing a first - line regimen including this pi . at the time of failure , the only parameter significantly different between the two groups was the cd4 count ( 423 cells / mm in the subtype b group and 360 cells / mm in the subtype g group ; p < 0.001 ) .
assessed virologic and immunologic responses to starting haart in a large cohort of 2116 patients , with the specific objective of comparing outcomes in patients with subtype b infection and those with subtype c , subtype a , crf02_ag , and subtype d infection , the predominant non - b subtypes circulating in the united kingdom .
overall , 1906 ( 90% ) patients achieved viral load undetectability within 12 months after they started haart , of whom 335 ( 18% ) subsequently experienced virologic rebound . in adjusted analyses ,
viral load suppression occurred more rapidly in patients infected with subtype c ( hazard ratio ( 95% confidence interval , ci ) 1.16 [ 1.011.33 ] , p = 0.04 ) and subtype a ( 1.35 [ 1.041.74 ] , p = 0.02 ) relative to subtype b infection .
the virologic rebound occurred marginally faster in patients with subtype c infection ( 1.40 [ 1.001.95 ] , p = 0.05 ) , but the hazard of virologic rebound was similar with other subtypes .
although patients infected by subtype b viruses showed higher baseline cd4 cell counts and maintained the advantage throughout therapy , cd4 cell count recovery occurred at similar rates with all subtypes .
the effect of pretreatment hiv-1 drug resistance on the response to first - line combination antiretroviral therapy in sub - saharan africa has recently been evaluated in a large prospective cohort .
pretreatment drug resistance results were available for 2579 ( 94% ) of 2733 participants . among them , 123 ( 5% ) had pretreatment drug resistance to at least one prescribed drug , and 52 ( 2% ) had pretreatment drug resistance and received fully active antiretroviral therapy .
more than 50% of participants for whom it was possible to determine the subtype were infected by hiv-1 subtype c ( 1405 patients , 54% ) , while 638 ( 25% ) harboured subtype a , 296 ( 11% ) d , 117 ( 5% ) a / g recombinant , 68 ( 3% ) g , 48 ( 2% ) other recombinants , seven ( < 1% ) other subtypes , and five ( < 1% ) b. compared with participants without pretreatment drug resistance , the odds ratio ( or ) for virological failure ( or ( 95%  ci ) : 2.13 [ 1.443.14 ] , p < 0.0001 ) and acquired drug resistance ( 2.30 [ 1.553.40 ] ; p <
0.0001 ) was increased in participants with pretreatment drug resistance to at least one prescribed drug but not in those with pretreatment drug resistance and fully active antiretroviral therapy .
cd4 cell count increased less in participants with pretreatment drug resistance than in those without ( 35 cells per l difference after 12 months ; 95%  ci 1358 ; p = 0.002 ) .
further ad hoc studies are needed to better evaluate the therapeutic implications of specific non - b subtype differences in terms of long - term efficacy of different antiviral regimens .
several studies on disease progression showed that , among non - b subtypes , subtypes c and d were found to be more aggressive , followed by g , ae , ag , and a , the least aggressive of all hiv-1 subtypes [ 9297 ] . in this regard
, it should be noted that because the different hiv-1 subtypes are not uniformly dispersed , comparisons of virulence and transmissibility are hampered by potential confounders , such as ethnic , socioeconomic , and other epidemiological factors .
the report by kiwanuka et al .   concerning this topic provided a good opportunity to compare rates of disease progression associated with these different subtypes within a similar population , thanks to the cocirculation of hiv-1 subtypes a and d , and several intersubtype recombinants in the rakai district of uganda .
in particular , kiwanuka et al . compared rates of progression among hiv-1 seroconverters who were followed as part of the rakai health sciences program .
subjects identified during 19972002 were followed through 2004 , when antiretroviral therapy became available in rakai district .
the primary end point was the time to achievement of a cd4 cell count of 250 cells / mm or death due to aids .
progression to a cd4 cell count of 250 cells / mm was significantly less common among subjects infected with hiv-1 subtype a ( 20% ) , compared with subjects infected with subtype d ( 40% ) , recombinant forms ( 40% ) , or multiple subtypes ( 53% ) ( p = 0.03 ) .
death from aids was also less common among subjects infected with hiv-1 subtype a. these differences were reflected in the longer time to aids onset for subjects infected with hiv-1 subtype a ( 8.05 years ) compared with those infected with non - a subtypes ( d = 6.49 years ; recombinant forms = 5.57 years ; multiple subtypes = 5.80 years ) . by multivariable models
( adjusting for viral load , age , and sex ) , subjects infected with non - a subtypes more likely progressed to aids compared with those infected with hiv-1 subtype a. similarly , subjects infected with non - a subtypes had an approximately 68-fold greater risk of death from aids .
viral factors that could explain some of the hiv-1 pathogenicity are higher ex vivo replicative capacity , higher genetic diversity , and cxcr4 coreceptor usage [ 231233 ] . in this regard , a less common emergence of cxcr4-using ( x4 ) variants was found in hiv-1 subtype a infection compared with hiv-1 subtype d infection , thus explaining the apparent lower virulence of hiv-1 a [ 234 , 235 ] .
a european study showed how subtype d has a fourfold higher rate of cd4 count decline , in the absence of antiretroviral therapy , compared with other subtypes in the study ( a , b , c , and crf02_ag , which had similar rates of cd4 loss ) , even when adjusted for baseline cd4 count .
subtype d was also associated with higher rates of dementia in individuals with advanced immunosuppression if compared with subtype a . a study by kiwanuka et al .
showed how in uganda subtype a viruses have a significantly higher rate of heterosexual transmission than subtype d viruses .
the faster disease progression and the lower rate of heterosexual transmission of subtype d compared with subtype a can explain the changes in the proportions of subtypes a and d observed over time in uganda and kenya [ 238 , 239 ] . in particular , a significant decrease in the prevalence of subtype d with a concurrent increase of subtype a was observed . despite the importance of viral characteristics in determining the rate of hiv-1 disease progression , recent findings from genomic studies show that host genetic factors also play a crucial role .
the genetic determinants that influence susceptibility to hiv-1 and limit aids vary in different populations and among individuals .
meta - analyses of large cohort studies have identified several genetic variants that regulate hiv cell entry ( particularly chemokine coreceptors and their ligands with copy number variations ) , acquired and innate immunity ( major histocompatibility complex ( mhc ) , killer immunoglobulin - like receptors ( kirs ) , and cytokines ) , and others ( trim5- and apobec3 g ) that influence the outcome of hiv infection [ 240242 ] . of the various genes that contribute toward host genetic propensity ,
mhc turns out to be the major contributor because it is responsible both for restriction of cytotoxic t lymphocyte ( ctl ) epitopes and for the emergence of ctl escape mutants .
leukocyte antigen ( hla ) alleles have been shown to be associated with the rate of disease progression in africans and caucasians [ 243245 ] .
the interplay of viral , immune , and host genetic factors in the control of hiv-1 replication has been recently evaluated in hiv controllers [ 246 , 247 ] .
similar efforts should be undertaken in diverse human populations infected with a variety of hiv-1 subtypes in order to understand fully the complex interplay of virus and host in aids pathogenesis .
hiv diversity plays a central role in the hiv pandemic ; for this reason , it is today imperative that global molecular epidemiology surveillance is continued and improved using rigorous sampling strategies .
the improved knowledge of the significance of non - b subtypes for resistance evolution and interpretation , in response to antiretroviral therapy , disease progression and vaccine design are becoming mandatory today not only because antiretroviral therapy is being introduced in countries where non - b subtypes are driving the epidemic but also because the number of infections by these variants is increasing sharply in previously subtype b homogeneous areas such as europe and north america [ 5769 , 7173 , 248 ] .
it is reassuring that current antiretroviral strategies appear to be effective against a broad spectrum of hiv subtypes .
however , further ad hoc studies in larger and homogenous populations infected by different specific non - b subtypes are required to better evaluate their therapeutic implications in terms of long - term efficacy of different antiviral regimens .
further elucidation of viral polymorphisms and properties associated with transmission , viral load set point , and disease progression may lead to new approaches to disease prevention and treatment .
efforts should be undertaken in diverse human populations infected with a variety of hiv-1 subtypes in order to fully understand the complex interplay of hiv and host in aids pathogenesis .
 despite advances in antiretroviral therapy that have revolutionized hiv disease management , effective control of the hiv infection pandemic remains elusive . beyond the classic non - b endemic areas , 
 hiv-1 non - b subtype infections are sharply increasing in previous subtype b homogeneous areas such as europe and north america . 
 as already known , several studies have shown that , among non - b subtypes , subtypes c and d were found to be more aggressive in terms of disease progression . luckily , the response to antiretrovirals against hiv-1 seems to be similar among different subtypes , but these results are mainly based on small or poorly designed studies . on the other hand , differences in rates of acquisition of resistance among non - b subtypes are already being observed . 
 this different propensity , beyond the type of treatment regimens used , as well as access to viral load testing in non - b endemic areas seems to be due to hiv-1 clade specific peculiarities . 
 indeed , some non - b subtypes are proved to be more prone to develop resistance compared to b subtype . 
 this phenomenon can be related to the presence of subtype - specific polymorphisms , different codon usage , and/or subtype - specific rna templates . 
 this review aims to provide a complete picture of hiv-1 genetic diversity and its implications for hiv-1 disease spread , effectiveness of therapies , and drug resistance development . 