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1.  HIV-1 Subtype B Protease and Reverse Transcriptase Amino Acid Covariation 
PLoS Computational Biology  2007;3(5):e87.
Despite the high degree of HIV-1 protease and reverse transcriptase (RT) mutation in the setting of antiretroviral therapy, the spectrum of possible virus variants appears to be limited by patterns of amino acid covariation. We analyzed patterns of amino acid covariation in protease and RT sequences from more than 7,000 persons infected with HIV-1 subtype B viruses obtained from the Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu). In addition, we examined the relationship between conditional probabilities associated with a pair of mutations and the order in which those mutations developed in viruses for which longitudinal sequence data were available. Patterns of RT covariation were dominated by the distinct clustering of Type I and Type II thymidine analog mutations and the Q151M-associated mutations. Patterns of protease covariation were dominated by the clustering of nelfinavir-associated mutations (D30N and N88D), two main groups of protease inhibitor (PI)–resistance mutations associated either with V82A or L90M, and a tight cluster of mutations associated with decreased susceptibility to amprenavir and the most recently approved PI darunavir. Different patterns of covariation were frequently observed for different mutations at the same position including the RT mutations T69D versus T69N, L74V versus L74I, V75I versus V75M, T215F versus T215Y, and K219Q/E versus K219N/R, and the protease mutations M46I versus M46L, I54V versus I54M/L, and N88D versus N88S. Sequence data from persons with correlated mutations in whom earlier sequences were available confirmed that the conditional probabilities associated with correlated mutation pairs could be used to predict the order in which the mutations were likely to have developed. Whereas accessory nucleoside RT inhibitor–resistance mutations nearly always follow primary nucleoside RT inhibitor–resistance mutations, accessory PI-resistance mutations often preceded primary PI-resistance mutations.
Author Summary
The identification of which mutations in a protein covary has played a major role in both structural and evolutionary biology. Covariation analysis has been used to help predict unsolved protein structures and to better understand the functions of proteins with known structures. The large number of published genetic sequences of the targets of HIV-1 therapy has provided an unprecedented opportunity to identify dependencies among mutations in these proteins that can be exploited to design inhibitors that have high genetic barriers to resistance. In our analysis, we identified many pairs of covarying drug-resistance mutations in HIV-1 protease and reverse transcriptase and organized them into clusters of mutations that often develop in a predictable order. Inhibitors that are active against early drug-resistant mutants are likely to be less prone to the development of resistance, whereas inhibitors that are active against fully evolved clusters of mutations may be useful drugs for salvage therapy.
doi:10.1371/journal.pcbi.0030087
PMCID: PMC1866358  PMID: 17500586
2.  Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis 
Rhee, Soo-Yon | Blanco, Jose Luis | Jordan, Michael R. | Taylor, Jonathan | Lemey, Philippe | Varghese, Vici | Hamers, Raph L. | Bertagnolio, Silvia | de Wit, Tobias F. Rinke | Aghokeng, Avelin F. | Albert, Jan | Avi, Radko | Avila-Rios, Santiago | Bessong, Pascal O. | Brooks, James I. | Boucher, Charles A. B. | Brumme, Zabrina L. | Busch, Michael P. | Bussmann, Hermann | Chaix, Marie-Laure | Chin, Bum Sik | D’Aquin, Toni T. | De Gascun, Cillian F. | Derache, Anne | Descamps, Diane | Deshpande, Alaka K. | Djoko, Cyrille F. | Eshleman, Susan H. | Fleury, Herve | Frange, Pierre | Fujisaki, Seiichiro | Harrigan, P. Richard | Hattori, Junko | Holguin, Africa | Hunt, Gillian M. | Ichimura, Hiroshi | Kaleebu, Pontiano | Katzenstein, David | Kiertiburanakul, Sasisopin | Kim, Jerome H. | Kim, Sung Soon | Li, Yanpeng | Lutsar, Irja | Morris, Lynn | Ndembi, Nicaise | NG, Kee Peng | Paranjape, Ramesh S. | Peeters, Martine | Poljak, Mario | Price, Matt A. | Ragonnet-Cronin, Manon L. | Reyes-Terán, Gustavo | Rolland, Morgane | Sirivichayakul, Sunee | Smith, Davey M. | Soares, Marcelo A. | Soriano, Vincent V. | Ssemwanga, Deogratius | Stanojevic, Maja | Stefani, Mariane A. | Sugiura, Wataru | Sungkanuparph, Somnuek | Tanuri, Amilcar | Tee, Kok Keng | Truong, Hong-Ha M. | van de Vijver, David A. M. C. | Vidal, Nicole | Yang, Chunfu | Yang, Rongge | Yebra, Gonzalo | Ioannidis, John P. A. | Vandamme, Anne-Mieke | Shafer, Robert W.
PLoS Medicine  2015;12(4):e1001810.
Background
Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes.
Methods and Findings
We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling.
Conclusions
Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.
In this individual patient and sequence-level meta-analysis, Soo-Yon Rhee and colleagues measure regional trends in HIV-1 transmitted drug resistance prevalence and investigate the specific mutations responsible for TDR in different regions and in different virus subtypes.
Editors' Summary
Background
About 35 million people are currently infected with HIV, the virus that causes AIDS by destroying immune system cells and leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-infected individuals died within ten years of infection. Then, in 1996, effective antiretroviral (ARV) therapy—drug combinations that suppress HIV replication by inhibiting reverse transcriptase and other essential viral enzymes—became available. For people living in affluent countries, HIV/AIDS became a chronic condition, but because ARV therapy was expensive, HIV/AIDS remained fatal in low- and middle-income countries (LMICs). In 2003, the international community began to work towards achieving universal access to ARV therapy. Now, more than 10 million HIV-positive individuals in LMICs receive ARV therapy, usually as a fixed-dose combination of two nucleoside reverse transcriptase inhibitors (NRTIs), such as tenofovir and lamivudine, plus a non-nucleoside reverse transcriptase inhibitor (NNRTI), such as efavirenz or nevirapine.
Why Was This Study Done?
The global scale-up of ARV therapy has reduced deaths from HIV/AIDS and the incidence of HIV infection in LMICs, but the development of resistance to ARV therapy is threatening these advances. HIV rapidly accumulates genetic changes (mutations), some of which make HIV resistant to ARV therapy. Up to 30% of patients receiving a fixed-dose NRTI/NNRTI combination develop virological failure, and a high proportion of these patients develop mutations associated with resistance to the ARVs in their regimen. Moreover, the proportion of newly infected, ARV-naïve individuals with transmitted drug resistance (TDR) is also increasing. Organizations involved in HIV/AIDS control need to understand the regional and temporal mutational patterns of TDR to inform the development of guidelines for first-line ARV therapy and of inexpensive resistance mutation assays for use in LMICs. Here, using a statistical approach called meta-analysis to combine information from individual patients about the resistance mutations they carry, the researchers investigate the molecular epidemiology of TDR (the patterns of molecular changes underlying TDR in populations) and identify the HIV drug-resistance mutations most responsible for TDR in different world regions.
What Did the Researchers Do and Find?
The researchers identified 287 studies published between 2000 and 2013 from 111 countries that included the reverse transcriptase sequences of HIV viruses from 50,870 ARV-naïve, HIV-positive individuals. The researchers analyzed each virus sequence for the presence of 93 surveillance drug-resistance mutations (SDRMs) previously shown to be specific indicators of TDR. Meta-analysis of these data indicated that the average overall prevalence of TDR (the proportion of ARV-naïve, HIV-positive individuals infected with a virus carrying one or more SDRMs) ranged from 2.8% in sub-Saharan Africa to 11.5% in North America. In sub-Saharan Africa, the odds (chance) of TDR increased 1.09-fold per year following national ARV scale-up; this increase was attributable to an increase in NRTI- and NNRTI-associated resistance. By contrast, in LMICs in south/southeast Asia, the odds of TDR remained unchanged following ARV scale-up. In Latin America/Caribbean, North America, Europe, and upper-income Asian countries, the odds of TDR have increased by around 1.10-fold per year since 1995, mainly as a result of increased NNRTI resistance. Four NNRTI-associated and 16 NRTI-associated SDRMs accounted for most NNRTI- and NRTI-associated TDR, respectively, in all regions. Notably, in sub-Saharan Africa and south/southeast Asia, most of the NNRTI-associated SDRMs detected were associated with high-level resistance to nevirapine or efavirenz. Finally, the researchers report that 95% of TDR viruses in sub-Saharan Africa and south/southeast Asia were unrelated and had therefore arisen independently.
What Do These Findings Mean?
Because many drug-resistance mutations reduce HIV’s fitness and tend to be lost rapidly in individuals not exposed to ARV therapy, differences among the datasets used in this meta-analysis with respect to how long each ARV-naïve patient had been infected with HIV before virus sampling may limit the accuracy of these findings. Nevertheless, the finding that most of the TDR strains detected in sub-Saharan Africa and south/southeast Asia arose independently suggests that improved patient adherence to ARV therapy and the use of ARV regimens that contain drugs to which HIV rarely develops resistance (regimens with a high genetic barrier to resistance) should reduce the generation of new ARV-resistant strains and mitigate TDR increases. In addition, the finding that a few NNRTI-resistance mutations were responsible for most cases of transmitted high-level resistance suggests that an inexpensive assay that detects these specific mutations may be useful for pre-therapy screening in LMICs with high TDR levels.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001810.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, summaries of recent research findings on HIV care and treatment, and personal stories about living with HIV/AIDS
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on antiretroviral drugs and on universal access to ARV therapy; Avert also provides personal stories about living with HIV/AIDS
The World Health Organization provides information on all aspects of HIV/AIDS (in several languages), including its guidelines on the use of antiretroviral therapy for treating and preventing HIV infection
The UNAIDS World AIDS Day Report 2014 provides up-to-date information about the AIDS epidemic and efforts to halt it, including progress towards universal access to antiretroviral therapy
The Stanford University HIV Drug Resistance Database includes information about surveillance drug-resistant mutations (SDRMs) and an interactive map displaying HIV drug resistance in ARV-naïve populations
doi:10.1371/journal.pmed.1001810
PMCID: PMC4388826  PMID: 25849352
3.  A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism 
PLoS Medicine  2007;4(1):e36.
Background
HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors.
Methods and Findings
We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy.
Conclusions
HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.
Changes in the cleavage site of the Gag substrate for the HIV protease can convey resistance to protease inhibitors and might contribute to virologic failure during therapy that includes these drugs.
Editors' Summary
Background.
Twenty-five years ago, infection with the human immunodeficiency virus (HIV)—the causative agent of AIDS—was a death sentence. However, drugs that attack various stages of the HIV life cycle were soon developed that, although not curing the infection, kept it in check when used in combination and greatly increased the life expectancy of people infected with HIV. Unfortunately, viruses resistant to these drugs have rapidly emerged and antiviral therapy now fails in many patients. The use of HIV protease inhibitors (PIs) in combination therapies, for example, has led to the stepwise selection of viral variants resistant to these drugs. Resistance is first acquired when the viral protease changes so that PIs no longer bind to it and inhibit it efficiently. These changes often reduce the efficiency with which the protease binds its substrates—polyproteins called Gag and GagPol that it chops up into smaller proteins to make new viral particles. So the next step is the accumulation of changes elsewhere in the protease that make it work better, and sometimes changes in its substrate that make it easier to cut; these compensatory changes do not directly affect viral resistance to PIs.
Why Was This Study Done?
To prevent viruses with resistance to PIs emerging, drug doses are kept high in patients and new PIs are being developed with high potency against known PI-resistant HIV variants. Both approaches set a “high genetic barrier” to the development of PI resistance by ensuring that HIV has to incorporate many changes in its protease to become resistant. But, the HIV genome naturally changes—mutates—very rapidly, so novel HIV variants could emerge that are less susceptible to the new potent PIs without the virus having to leap this high genetic barrier. In this study, the researchers have investigated whether HIV can find an alternative route to PI resistance that does not involve the introduction of multiple changes into its protease.
What Did the Researchers Do and Find?
The researchers took wild-type HIV and treated it in the laboratory with a new PI regimen that has a high genetic barrier. By gradually increasing its concentration, the researchers selected three viral populations that were able to grow in 4- to 8-fold higher concentrations of the PI than wild-type virus. None of these populations had mutations in the viral protease. Instead, they all had mutations near one of the sites—the NC/p1 site—where the protease normally cuts the Gag polyprotein. These mutations, the researchers report, enhanced the overall efficiency with which the wild-type protease cleaved the polyprotein, and a selection experiment with another PI showed that the development of PI resistance through alterations near the NC/p1 cleavage site was not unique to one PI. The researchers also investigated the potential clinical significance of this new drug resistance mechanism by looking for the same mutations in nearly 30,000 patient samples. Many of the samples did indeed have these mutations. Finally, they showed that mutations at the NC/p1 cleavage site were associated with virological failure (increased viral replication) during PI therapy in an ongoing clinical trial.
What Do These Findings Mean?
These results suggest that increased polyprotein processing because of mutations in the natural substrate of the HIV protease might be a new mechanism by which HIV can become resistant to PIs. This strategy, which occurs in the laboratory and in patients, allows HIV to develop PI resistance without the need for multiple changes in its protease and so avoids the high genetic barrier to resistance that new PIs provide. Clinical studies are now needed to test which of the mutations seen in this study contribute to virological failure, whether the degree of this failure is clinically relevant, and whether these substrate mutations enhance the effect of protease mutations. If the clinical importance of the new mechanism is confirmed, genetic examination of both the polyprotein and the protease will be needed when trying to figure out why a PI-containing therapy is failing in individual patients. Furthermore, it will be necessary to test whether this mechanism can contribute to the development of resistance when evaluating new drugs.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040036.
US National Institute of Allergy and Infectious Diseases factsheet on HIV infection and AIDS
US Department of Health and Human Services information on AIDS
US Centers for Disease Control and Prevention information on HIV/AIDS
Aidsmap information on HIV and AIDS provided by the charity NAM
BioAfrica, Bioinformatics for HIV Research, information on HIV-1 protease cleavage sites
doi:10.1371/journal.pmed.0040036
PMCID: PMC1769415  PMID: 17227139
4.  Distinguishing Functional Amino Acid Covariation from Background Linkage Disequilibrium in HIV Protease and Reverse Transcriptase 
PLoS ONE  2007;2(8):e814.
Correlated amino acid mutation analysis has been widely used to infer functional interactions between different sites in a protein. However, this analysis can be confounded by important phylogenetic effects broadly classifiable as background linkage disequilibrium (BLD). We have systematically separated the covariation induced by selective interactions between amino acids from background LD, using synonymous (S) vs. amino acid (A) mutations. Covariation between two amino acid mutations, (A,A), can be affected by selective interactions between amino acids, whereas covariation within (A,S) pairs or (S,S) pairs cannot. Our analysis of the pol gene — including the protease and the reverse transcriptase genes — in HIV reveals that (A,A) covariation levels are enormously higher than for either (A,S) or (S,S), and thus cannot be attributed to phylogenetic effects. The magnitude of these effects suggests that a large portion of (A,A) covariation in the HIV pol gene results from selective interactions. Inspection of the most prominent (A,A) interactions in the HIV pol gene showed that they are known sites of independently identified drug resistance mutations, and physically cluster around the drug binding site. Moreover, the specific set of (A,A) interaction pairs was reproducible in different drug treatment studies, and vanished in untreated HIV samples. The (S,S) covariation curves measured a low but detectable level of background LD in HIV.
doi:10.1371/journal.pone.0000814
PMCID: PMC1950573  PMID: 17726544
5.  Dispersion of the HIV-1 Epidemic in Men Who Have Sex with Men in the Netherlands: A Combined Mathematical Model and Phylogenetic Analysis 
PLoS Medicine  2015;12(11):e1001898.
Background
The HIV-1 subtype B epidemic amongst men who have sex with men (MSM) is resurgent in many countries despite the widespread use of effective combination antiretroviral therapy (cART). In this combined mathematical and phylogenetic study of observational data, we aimed to find out the extent to which the resurgent epidemic is the result of newly introduced strains or of growth of already circulating strains.
Methods and Findings
As of November 2011, the ATHENA observational HIV cohort of all patients in care in the Netherlands since 1996 included HIV-1 subtype B polymerase sequences from 5,852 patients. Patients who were diagnosed between 1981 and 1995 were included in the cohort if they were still alive in 1996. The ten most similar sequences to each ATHENA sequence were selected from the Los Alamos HIV Sequence Database, and a phylogenetic tree was created of a total of 8,320 sequences. Large transmission clusters that included ≥10 ATHENA sequences were selected, with a local support value ≥ 0.9 and median pairwise patristic distance below the fifth percentile of distances in the whole tree. Time-varying reproduction numbers of the large MSM-majority clusters were estimated through mathematical modeling. We identified 106 large transmission clusters, including 3,061 (52%) ATHENA and 652 Los Alamos sequences. Half of the HIV sequences from MSM registered in the cohort in the Netherlands (2,128 of 4,288) were included in 91 large MSM-majority clusters. Strikingly, at least 54 (59%) of these 91 MSM-majority clusters were already circulating before 1996, when cART was introduced, and have persisted to the present. Overall, 1,226 (35%) of the 3,460 diagnoses among MSM since 1996 were found in these 54 long-standing clusters. The reproduction numbers of all large MSM-majority clusters were around the epidemic threshold value of one over the whole study period. A tendency towards higher numbers was visible in recent years, especially in the more recently introduced clusters. The mean age of MSM at diagnosis increased by 0.45 years/year within clusters, but new clusters appeared with lower mean age. Major strengths of this study are the high proportion of HIV-positive MSM with a sequence in this study and the combined application of phylogenetic and modeling approaches. Main limitations are the assumption that the sampled population is representative of the overall HIV-positive population and the assumption that the diagnosis interval distribution is similar between clusters.
Conclusions
The resurgent HIV epidemic amongst MSM in the Netherlands is driven by several large, persistent, self-sustaining, and, in many cases, growing sub-epidemics shifting towards new generations of MSM. Many of the sub-epidemics have been present since the early epidemic, to which new sub-epidemics are being added.
Daniela Bezemer and colleagues investigate the extent to which the resurgent HIV epidemic in the Netherlands is the result of newly introduced strains, or of growth of already circulating strains.
Editors' Summary
Background
Since the first recorded case of AIDS in 1981, the number of people infected with HIV, the virus that causes AIDS, has risen steadily. Now, three and a half decades later, about 35 million people (more than half of whom are women) are infected with HIV, the virus that causes AIDS. HIV is most often spread by having unprotected sex with an infected partner, and, globally, most sexual transmission of HIV occurs during heterosexual sex. Nevertheless, many new HIV infections still occur in men who have sex with men (MSM; homosexual, bisexual, and transgender men, and heterosexual men who sometimes have sex with men), and, in some countries, HIV/AIDS still predominantly affects the MSM community. In the US, for example, 78% of new HIV infections occurred among MSM in 2010 although MSM represent only 4% of the total population, and, in 2011, 54% of all people living with HIV were MSM. Indeed, despite HIV-positive individuals being diagnosed earlier these days and having access to effective combination antiretroviral therapy (cART), which both halts disease progression and reduces the risk of HIV transmission, the HIV epidemic among MSM is resurgent (growing again) in many Western countries.
Why Was This Study Done?
To control this resurgent epidemic, it is important to know as much as possible about HIV transmission among MSM so that effective prevention strategies can be designed. Here, the researchers use phylogenetic analysis and mathematical modeling to ask whether the introduction of new strains or the spread of already circulating strains is responsible for the resurgent HIV-1 subtype B epidemic occurring among MSM in the Netherlands. Viral phylogenetic analysis infers evolutionary relationships between viral strains by examining their genetic relatedness and can be used to identify HIV transmission clusters. HIV-1 viruses are classified into subtypes based on their genetic sequence and geographical distribution. HIV-1 subtype B is a common subtype that is found in west and central Europe, the Americas, and several other regions.
What Did the Researchers Do and Find?
The researchers built a phylogenetic tree for the HIV epidemic in MSM in the Netherlands by analyzing HIV-1 subtype B polymerase gene sequences found in 5,852 participants (73% of whom were MSM) in the ATHENA cohort, an observational cohort of all HIV-1-infected patients in care in the Netherlands since 1996 (when cART became available). Examination of this tree identified 106 large transmission clusters (groups of ten or more closely related subtype B HIV-1 strains). Half of the HIV-1 polymerase sequences from HIV-1-positive MSM registered in the ATHENA cohort in the Netherlands were included in 91 MSM-majority clusters: large transmission clusters in which more than half the related sequences originated from MSM. At least 54 of the MSM-majority clusters were circulating before 1996 and have persisted until the present day. Moreover, about a third of new HIV infections diagnosed among MSM since 1996 involve viruses included in these long-lived clusters. The researchers then used mathematical modeling to estimate that the effective reproduction number (the number of secondary infections per primary infection) for all the MSM-majority clusters was around one for the whole study period. Thus, these clusters were self-sustaining and not contracting. Notably, MSM-majority clusters (particularly the newer clusters) tended to have higher reproduction numbers in recent years. Moreover, although the average age at diagnosis within each of the MSM-majority clusters increased over the study period at a rate of 0.45 years/year, the average age at diagnosis was lower at initiation of new clusters and only increased by 0.28 years/year.
What Do These Findings Mean?
These findings suggest that several large, persistent, and self-sustaining sub-epidemics, many of which have been present since early in the AIDS epidemic, are driving the resurgent HIV epidemic among MSM in the Netherlands, despite the widespread availability of treatment, increasing rates of diagnosis, and earlier treatment initiation. Importantly, however, these findings also suggest that some sub-epidemics have emerged more recently and that some sub-epidemics, particularly the newer ones, are growing and may be preferentially affecting younger MSM. The accuracy of these findings may be limited by some aspects of the study. For example, the reproduction number estimates assume that the time from diagnosis of a case to the diagnoses of secondary cases is similar across clusters. Nevertheless, the new insights provided by this study should help guide the development of strategies to curb the resurgent HIV epidemic that is currently affecting MSM in the Netherlands and elsewhere.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001898.
The US Centers for Disease Control and Prevention provides information on all aspects of HIV/AIDS, including information on HIV/AIDS among MSM (in English and Spanish)
NAM/aidsmap provides basic information about HIV/AIDS and summaries of recent research findings, including information on HIV and MSM and personal stories from MSM living with HIV
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including MSM and HIV; Avert also provides personal stories about living with HIV/AIDS
The World Health Organization provides information on all aspects of HIV/AIDS, including HIV/AIDS and MSM (in several languages)
The UNAIDS Fast-Track Strategy to End the AIDS Epidemic by 2030 provides up-to-date information about the AIDS epidemic and efforts to halt it
A 2011 World Bank Report The Global HIV Epidemics among Men Who Have Sex with Men is available
A PLOS Computational Biology Topic Page (a review article that is a published copy of record of a dynamic version of an article in Wikipedia) about viral phylodynamics is available
doi:10.1371/journal.pmed.1001898
PMCID: PMC4631366  PMID: 26529093
6.  An Evolutionary-Network Model Reveals Stratified Interactions in the V3 Loop of the HIV-1 Envelope 
PLoS Computational Biology  2007;3(11):e231.
The third variable loop (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope is a principal determinant of antibody neutralization and progression to AIDS. Although it is undoubtedly an important target for vaccine research, extensive genetic variation in V3 remains an obstacle to the development of an effective vaccine. Comparative methods that exploit the abundance of sequence data can detect interactions between residues of rapidly evolving proteins such as the HIV-1 envelope, revealing biological constraints on their variability. However, previous studies have relied implicitly on two biologically unrealistic assumptions: (1) that founder effects in the evolutionary history of the sequences can be ignored, and; (2) that statistical associations between residues occur exclusively in pairs. We show that comparative methods that neglect the evolutionary history of extant sequences are susceptible to a high rate of false positives (20%–40%). Therefore, we propose a new method to detect interactions that relaxes both of these assumptions. First, we reconstruct the evolutionary history of extant sequences by maximum likelihood, shifting focus from extant sequence variation to the underlying substitution events. Second, we analyze the joint distribution of substitution events among positions in the sequence as a Bayesian graphical model, in which each branch in the phylogeny is a unit of observation. We perform extensive validation of our models using both simulations and a control case of known interactions in HIV-1 protease, and apply this method to detect interactions within V3 from a sample of 1,154 HIV-1 envelope sequences. Our method greatly reduces the number of false positives due to founder effects, while capturing several higher-order interactions among V3 residues. By mapping these interactions to a structural model of the V3 loop, we find that the loop is stratified into distinct evolutionary clusters. We extend our model to detect interactions between the V3 and C4 domains of the HIV-1 envelope, and account for the uncertainty in mapping substitutions to the tree with a parametric bootstrap.
Author Summary
The third variable loop (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope is a principal determinant of viral growth characteristics and an important target for the immune system. Interactions between residues of V3 allow the virus to shift between combinations of residues to escape the immune system while retaining its structure and functions. Comparative study of HIV-1 V3 sequences can detect such interactions by the covariation of sites in the sequence, which can then be used to inform vaccine development, but current methods for detecting such associations rely on biologically unrealistic assumptions. We demonstrate that these assumptions cause an excessive number of spurious associations, and present a new approach that couples phylogenetic and Bayesian network models, and greatly reduces this number while retaining the ability to detect real associations. Our analysis reveals that the V3 loop is stratified into discrete layers of interacting residues, suggesting a partition of functions along this viral structure with implications for vaccine development.
doi:10.1371/journal.pcbi.0030231
PMCID: PMC2082504  PMID: 18039027
7.  Minimal effect of MDR1 and CYP3A5 genetic polymorphisms on the pharmacokinetics of indinavir in HIV-infected patients 
What is already known about this subject
Before this study, few data were available on the potential effect of genetic variants of P-glycoprotein or the CYP3A5 enzyme on the pharmacokinetic variability of protease inhibitors (PI).MDR1 C3435T polymorphism was often linked with the pharmacokinetic variability of nelfinavir. CYP3A5*3 polymorphism was linked with the pharmacokinetic variability of calcineurin inhibitors and was therefore strongly suspected of being one of the key factors in the pharmacokinetic variability of other CYP3A susbtrates.
What this study adds Our results showed that both MDR1 C3435T and CYP3A5*3 polymorphisms are involved in the pharmacokinetic variability of the absorption or elimination of indinavir, but probably jointly with other factors.The potent CYP3A inhibitory effect of ritonavir may hide the variability linked to genetic differences in the CYP3A5 gene, thereby reducing the overall pharmacokinetic variability of the boosted protease inhibitor.Genotyping MDR1 and/or CYP3A5 does not appear to be a clinically relevant factor in optimizing protease inhibitor boosted regimens.
Aims
The protease inhibitor indinavir is characterized by an important interindividual pharmacokinetic variability, which results from the actions of the metabolizing enzymes cytochrome P450 (CYP) 3A and the multidrug efflux pump P-glycoprotein (P-gp), encoded by MDR1. Using a population pharmacokinetic approach, we investigated the effect of several MDR1 and CYP3A5 polymorphisms on the pharmacokinetic parameters of indinavir in HIV-infected patients.
Methods
Twenty-eight patients receiving indinavir alone or together with ritonavir were included. Indinavir pharmacokinetics were studied over a 12 h interval. Genetic polymorphisms were assessed by real-time PCR assays and direct sequencing for MDR1 and by PCR-SSCP analysis for CYP3A5.
Results
The pharmacokinetics of indinavir were best described by a one-compartment model with first-order absorption. In the final model, the MDR1 C3435T genotype and ritonavir were identified as statistically significant covariates (P ≤ 0.001) for the absorption rate constant (95% confidence interval on the difference between CC and CT genotype 0.37, 5.53) and for clearance (95% confidence interval on the difference 5.8, 26.2), respectively. Patients with the CYP3A5*3/*3 genotype receiving indinavir alone had a 31% decrease in the indinavir clearance rate compared with patients carrying the CYP3A5*1/*3 genotype.
Conclusions
The MDR1 C3435T genotype affects the absorption constant of indinavir suggesting that P-gp may be implicated in its pharmacokinetic variability. Through its inhibition of CYP3A and P-gp, ritonavir could attenuate the pharmacokinetic variability linked to genetic differences, reducing significantly the interindividual variability of indinavir. However, genotyping MDR1 and/or CYP3A5 to optimize protease inhibitor boosted regimens does not seem clinically relevant.
doi:10.1111/j.1365-2125.2007.02903.x
PMCID: PMC2000655  PMID: 17517050
CYP3A5; indinavir; MDR1; P-glycoprotein; pharmacokinetics; single nucleotide polymorphism
8.  Relative Fitness and Replication Capacity of a Multinucleoside Analogue-Resistant Clinical Human Immunodeficiency Virus Type 1 Isolate with a Deletion of Codon 69 in the Reverse Transcriptase Coding Region▿  
Journal of Virology  2007;81(9):4713-4721.
Deletions, insertions, and amino acid substitutions in the β3-β4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Δ69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Δ69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Δ69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Δ69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.
doi:10.1128/JVI.02135-06
PMCID: PMC1900151  PMID: 17314158
9.  Mutation Patterns and Structural Correlates in Human Immunodeficiency Virus Type 1 Protease following Different Protease Inhibitor Treatments 
Journal of Virology  2003;77(8):4836-4847.
Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease—including 22 not previously associated with drug resistance—were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 Å of each other—many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.
doi:10.1128/JVI.77.8.4836-4847.2003
PMCID: PMC152121  PMID: 12663790
10.  Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal 
PLoS Medicine  2015;12(9):e1001880.
Background
The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents.
Methods and Findings
We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937–1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974–1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988–1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe.
Conclusions
In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.
Editors' Summary
Background
Tuberculosis (TB)—a contagious bacterial disease that usually infects the lungs—is a global public health problem. Every year, about 9 million people develop active TB disease, and 1.5 million people die from the disease. Mycobacterium tuberculosis, the organism that causes TB, is spread in airborne droplets when people with TB cough. The symptoms of TB include cough, weight loss, and fever. Diagnostic tests for the disease include sputum smear microscopy (microscopic analysis of mucus coughed up from the lungs) and chest X-rays. TB can be cured by taking a regimen of multiple antibiotics daily for 6 mo. However, the emergence of multidrug-resistant tuberculosis (MDR-TB, TB with resistance to both isoniazid and rifampicin) and extensively drug-resistant tuberculosis (XDR-TB, MDR-TB with additional resistance to both quinolones and second-line injectable agents), together with the spread of HIV (which increases susceptibility to TB), is now threatening TB control efforts. MDR-TB is caused by M. tuberculosis strains that have acquired mutations (genetic changes) that make them resistant to isoniazid, rifampicin, and sometimes other anti-TB drugs; XDR-TB is caused by bacteria that are resistant to isoniazid, rifampicin, one or more fluoroquinolones (for example, ofloxacin), and at least one injectable second-line drug (for example, kanamycin).
Why Was This Study Done?
A better understanding of the origins of drug-resistant TB is essential for effective control of TB. Public health experts need to know whether the emergence of drug-resistant TB is caused by inadequacies in TB control or related to other factors such as the spread of HIV and whether new resistant strains of M. tuberculosis repeatedly emerge during XDR-TB outbreaks or whether the transmission of a single drug-resistant strain drives these outbreaks. Here, the researchers use whole genome sequencing and dating analysis to investigate the origin and evolution of an XDR-TB outbreak identified in 2005 in Tugela Ferry, KwaZulu-Natal, South Africa. The predominant strain of XDR M. tuberculosis isolated during this large XDR-TB outbreak belongs to a subfamily called LAM4. Since the outbreak began, XDR-TB has also been reported in hospitals across KwaZulu-Natal, and some of these outbreaks have been caused by bacterial strains not falling within the LAM4 spoligotype (“spoligotyping” characterizes M. tuberculosis strains based on the presence of unique DNA sequences in a specific region of the bacterial genome).
What Did the Researchers Do and Find?
The researchers tested the antibiotic susceptibility of 337 clinical isolates of M. tuberculosis collected in KwaZulu-Natal between 2008 and 2013 and of three historical isolates—two collected in the province in the mid-1990s and a third from the Tugela Ferry XDR outbreak. They sequenced the whole genome of these isolates and used comparative techniques to assess the isolates’ relatedness and to investigate the acquisition of drug resistance. This analysis revealed a 50-member clone of XDR bacteria among the isolates collected across KwaZulu-Natal that was highly related to the LAM4 strain (a clone is defined here as a set of strains in which each member differs by no more than ten single nucleotide polymorphisms [SNPs] from at least one other member; an SNP is a type of genetic variant). Mutations that conferred isoniazid resistance in this clone were acquired in about 1957; MDR and XDR strains emerged in about 1984 and 1995, respectively. The analysis also indicates that MDR and XDR evolved de novo 56 times and nine times, respectively, and that isoniazid resistance nearly always evolved before rifampicin resistance.
What Do These Findings Mean?
These findings provide new information about the ordering and timing of the acquisition of drug-resistance mutations by M. tuberculosis in KwaZulu-Natal but do not necessarily represent the evolution of XDR-TB in other settings. Most notably, these findings indicate that the ancestral precursor of the Tugela Ferry XDR outbreak strain gained resistance to first-line antibiotics shortly after these antibiotics became available for clinical use. Subsequent stepwise accumulation of additional resistance mutations that occurred over decades led to the emergence of MDR and XDR strains. Importantly, the emergence of these strains occurred before the explosion of HIV in KwaZulu-Natal. Thus, these findings highlight the dire repercussions of the failure of historic attempts to control resistance to first-line anti-TB drugs and draw attention to the need for new anti-TB drugs to be used prudently to prevent early fixation of resistance and to protect the useful lifespan of these agents. Finally, the finding that isoniazid resistance is a key initiation event for progression to MDR and XDR suggests that TB control programs should test routinely for both isoniazid and rifampicin resistance to ensure early detection of drug-resistant TB.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001880.
The World Health Organization (WHO) provides information (in several languages) on TB and on MDR-TB; the Global Tuberculosis Report 2014 provides information about TB around the world; a supplement to the report entitled “Drug-Resistant TB—Surveillance and Response” is available
The Stop TB Partnership is working towards TB elimination and provides personal stories about TB (in English and Spanish)
The United States Centers for Disease Control and Prevention provides information about TB and about drug-resistant TB (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases also has detailed information on TB, including a drug-resistant TB visual tour
TB & Me, a collaborative blogging project run by patients being treated for MDR-TB and Mèdecins sans Frontiéres, provides more patient stories
The not-for-profit organization Global Health Education provides information about TB in South Africa
MedlinePlus has links to further information about TB (in English and Spanish)
doi:10.1371/journal.pmed.1001880
PMCID: PMC4587932  PMID: 26418737
11.  Deep Sequencing of Protease Inhibitor Resistant HIV Patient Isolates Reveals Patterns of Correlated Mutations in Gag and Protease 
PLoS Computational Biology  2015;11(4):e1004249.
While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle.
Author Summary
Understanding the structure of HIV proteins and the function of drug-resistant mutations of these proteins is critical for the development of effective HIV treatments. Selected gag mutations have been shown to provide compensatory functions for protease resistance mutations and may directly contribute to the development of drug resistance. To determine associations between protease inhibitor mutations and gag, we utilized deep sequencing of HIV gag and protease from a collection of viral isolates from patients treated with highly active retroviral protease inhibitors. Deep sequencing allows for accurate measurement of mutation frequencies at each position, allowing estimation, using a novel method we developed, of the covariation between any two residues on gag. Using this information, we characterize the variation within gag and protease and identify the most strongly correlated pairs of inter- and intra-protein residues. Our results suggest that matrix and p1/p6 mutations form the core of a network of strongly correlated gag mutations and contribute to recurrent treatment failure. Extracting gag residue covariation information from the deep sequencing of patient viral samples may provide insight into structural aspects of the Gag polyprotein as well new areas for small molecule targeting to disrupt Gag function.
doi:10.1371/journal.pcbi.1004249
PMCID: PMC4404092  PMID: 25894830
12.  Episodic Sexual Transmission of HIV Revealed by Molecular Phylodynamics 
PLoS Medicine  2008;5(3):e50.
Background
The structure of sexual contact networks plays a key role in the epidemiology of sexually transmitted infections, and their reconstruction from interview data has provided valuable insights into the spread of infection. For HIV, the long period of infectivity has made the interpretation of contact networks more difficult, and major discrepancies have been observed between the contact network and the transmission network revealed by viral phylogenetics. The high rate of HIV evolution in principle allows for detailed reconstruction of links between virus from different individuals, but often sampling has been too sparse to describe the structure of the transmission network. The aim of this study was to analyze a high-density sample of an HIV-infected population using recently developed techniques in phylogenetics to infer the short-term dynamics of the epidemic among men who have sex with men (MSM).
Methods and Findings
Sequences of the protease and reverse transcriptase coding regions from 2,126 patients, predominantly MSM, from London were compared: 402 of these showed a close match to at least one other subtype B sequence. Nine large clusters were identified on the basis of genetic distance; all were confirmed by Bayesian Monte Carlo Markov chain (MCMC) phylogenetic analysis. Overall, 25% of individuals with a close match with one sequence are linked to 10 or more others. Dated phylogenies of the clusters using a relaxed clock indicated that 65% of the transmissions within clusters took place between 1995 and 2000, and 25% occurred within 6 mo after infection. The likelihood that not all members of the clusters have been identified renders the latter observation conservative.
Conclusions
Reconstruction of the HIV transmission network using a dated phylogeny approach has revealed the HIV epidemic among MSM in London to have been episodic, with evidence of multiple clusters of transmissions dating to the late 1990s, a period when HIV prevalence is known to have doubled in this population. The quantitative description of the transmission dynamics among MSM will be important for parameterization of epidemiological models and in designing intervention strategies.
Using viral genotype data from HIV drug resistance testing at a London clinic, Andrew Leigh Brown and colleagues derive the structure of the transmission network through phylogenetic analysis.
Editors' Summary
Background.
Human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS), is mainly spread through unprotected sex with an infected partner. Like other sexually transmitted diseases, HIV/AIDS spreads through networks of sexual contacts. The characteristics of these complex networks (which include people who have serial sexual relationships with single partners and people who have concurrent sexual relationships with several partners) affect how quickly diseases spread in the short term and how common the disease is in the long term. For many sexually transmitted diseases, sexual contact networks can be reconstructed from interview data. The information gained in this way can be used for partner notification so that transmitters of the disease and people who may have been unknowingly infected can be identified, treated, and advised about disease prevention. It can also be used to develop effective community-based prevention strategies.
Why Was This Study Done?
Although sexual contact networks have provided valuable information about the spread of many sexually transmitted diseases, they cannot easily be used to understand HIV transmission patterns. This is because the period of infectivity with HIV is long and the risk of infection from a single sexual contact with an infected person is low. Another way to understand the spread of HIV is through phylogenetics, which examines the genetic relatedness of viruses obtained from different individuals. Frequent small changes in the genetic blueprint of HIV allow the virus to avoid the human immune response and to become resistant to antiretroviral drugs. In this study, the researchers use recently developed analytical methods, viral sequences from a large proportion of a specific HIV-infected population, and information on when each sample was taken, to learn about transmission of HIV/AIDS in London among men who have sex with men (MSM; a term that encompasses gay, bisexual, and transgendered men and heterosexual men who sometimes have sex with men). This new approach, which combines information on viral genetic variation and viral population dynamics, is called “molecular phylodynamics.”
What Did the Researchers Do and Find?
The researchers compared the sequences of the genes encoding the HIV-1 protease and reverse transcriptase from more than 2,000 patients, mainly MSM, attending a large London HIV clinic between 1997 and 2003. 402 of these sequences closely matched at least one other subtype B sequence (the HIV/AIDS epidemic among MSM in the UK primarily involves HIV subtype B). Further analysis showed that the patients from whom this subset of sequences came formed six clusters of ten or more individuals, as well as many smaller clusters, based on the genetic relatedness of their HIV viruses. The researchers then used information on the date when each sample was collected and a “relaxed clock” approach (which accounts for the possibility that different sequences evolve at different rates) to determine dated phylogenies (patterns of genetic relatedness that indicate when gene sequences change) for the clusters. These phylogenies indicated that at least in one in four transmissions between the individuals in the large clusters occurred within 6 months of infection, and that most of the transmissions within each cluster occurred over periods of 3–4 years during the late 1990s.
What Do These Findings Mean?
This phylodynamic reconstruction of the HIV transmission network among MSM in a London clinic indicates that the HIV epidemic in this population has been episodic with multiple clusters of transmission occurring during the late 1990s, a time when the number of HIV infections in this population doubled. It also suggests that transmission of the virus during the early stages of HIV infection is likely to be an important driver of the epidemic. Whether these results apply more generally to the MSM population at risk for transmitting or acquiring HIV depends on whether the patients in this study are representative of that group. Additional studies are needed to determine this, but if the patterns revealed here are generalizable, then this quantitative description of HIV transmission dynamics should help in the design of strategies to strengthen HIV prevention among MSM.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050050.
Read a related PLoS Medicine Perspective article
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including a list of organizations that provide information for gay men and MSM
The US Centers for Disease Control and Prevention provides information on HIV/AIDS and on HIV/AIDS among MSM (in English and Spanish)
Information is available from Avert, an international AIDS charity, on HIV, AIDS, and men who have sex with men
The Center for AIDS Prevention Studies (University of California, San Francisco) provides information on sexual networks and HIV prevention
The US National Center for Biotechnology Information provides a science primer on molecular phylogenetics
UK Collaborative Group on HIV Drug Resistance maintains a database of resistance tests
HIV i-Base offers HIV treatment information for health-care professionals and HIV-positive people
The NIH-funded HIV Sequence Database contains data on genetic sequences, resistance, immunology, and vaccine trials
doi:10.1371/journal.pmed.0050050
PMCID: PMC2267814  PMID: 18351795
13.  Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity 
Journal of Virology  2004;78(6):3123-3132.
The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (Å) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease “flaps” stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 Å. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1′, S3, and S3′ pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher koff rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide kon and koff data (Kd = koff/kon) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.
doi:10.1128/JVI.78.6.3123-3132.2004
PMCID: PMC354404  PMID: 14990731
14.  Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases 
Journal of Virology  2001;75(19):9458-9469.
We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.
doi:10.1128/JVI.75.19.9458-9469.2001
PMCID: PMC114513  PMID: 11533208
15.  Computational Analysis of HIV-1 Protease Protein Binding Pockets 
Mutations that arise in HIV-1 protease after exposure to various HIV-1 protease inhibitors have proved to be a difficult aspect in the treatment of HIV. Mutations in the binding pocket of the protease can prevent the protease inhibitor from binding to the protein effectively. In the present study, the crystal structures of 68 HIV-1 proteases complexed with one of the nine FDA approved protease inhibitors from the Protein Data Bank (PDB) were analyzed by (a) identifying the mutational changes with the aid of a developed mutation map and (b) correlating the structure of the binding pockets with the complexed inhibitors. The mutations of each crystal structure were identified by comparing the amino acid sequence of each structure against the HIV-1 wild type strain HXB2. These mutations were visually presented in the form of a mutation map to analyze mutation patterns corresponding to each protease inhibitor. The crystal structure mutation patterns of each inhibitor (in vitro) were compared against the mutation patterns observed in in vivo data. The in vitro mutation patterns were found to be representative of most of the major in vivo mutations. We then performed a data mining analysis of the binding pockets from each crystal structure in terms of their chemical descriptors to identify important structural features of the HIV-1 protease protein with respect to the binding conformation of the HIV-1 protease inhibitors. Data mining analysis is performed using several classification techniques: Random Forest (RF), linear discriminant analysis (LDA), and logistic regression (LR). We developed two hybrid models, RF-LDA and RF-LR. Random Forest is used as a feature selection proxy, reducing the descriptor space to a few of the most relevant descriptors determined by the classifier. These descriptors are then used to develop the subsequent LDA, LR, and hierarchical classification models. Clustering analysis of the binding pockets using the selected descriptors used to produce the optimal classification models reveals conformational similarities of the ligands in each cluster. This study provides important information in understanding the structural features of HIV-1 protease which cannot be studied by other existing in vivo genomic datasets.
doi:10.1021/ci100200u
PMCID: PMC2981608  PMID: 20925403
16.  The Role of Viral Introductions in Sustaining Community-Based HIV Epidemics in Rural Uganda: Evidence from Spatial Clustering, Phylogenetics, and Egocentric Transmission Models 
PLoS Medicine  2014;11(3):e1001610.
Using different approaches to investigate HIV transmission patterns, Justin Lessler and colleagues find that extra-community HIV introductions are frequent and likely play a role in sustaining the epidemic in the Rakai community.
Please see later in the article for the Editors' Summary
Background
It is often assumed that local sexual networks play a dominant role in HIV spread in sub-Saharan Africa. The aim of this study was to determine the extent to which continued HIV transmission in rural communities—home to two-thirds of the African population—is driven by intra-community sexual networks versus viral introductions from outside of communities.
Methods and Findings
We analyzed the spatial dynamics of HIV transmission in rural Rakai District, Uganda, using data from a cohort of 14,594 individuals within 46 communities. We applied spatial clustering statistics, viral phylogenetics, and probabilistic transmission models to quantify the relative contribution of viral introductions into communities versus community- and household-based transmission to HIV incidence. Individuals living in households with HIV-incident (n = 189) or HIV-prevalent (n = 1,597) persons were 3.2 (95% CI: 2.7–3.7) times more likely to be HIV infected themselves compared to the population in general, but spatial clustering outside of households was relatively weak and was confined to distances <500 m. Phylogenetic analyses of gag and env genes suggest that chains of transmission frequently cross community boundaries. A total of 95 phylogenetic clusters were identified, of which 44% (42/95) were two individuals sharing a household. Among the remaining clusters, 72% (38/53) crossed community boundaries. Using the locations of self-reported sexual partners, we estimate that 39% (95% CI: 34%–42%) of new viral transmissions occur within stable household partnerships, and that among those infected by extra-household sexual partners, 62% (95% CI: 55%–70%) are infected by sexual partners from outside their community. These results rely on the representativeness of the sample and the quality of self-reported partnership data and may not reflect HIV transmission patterns outside of Rakai.
Conclusions
Our findings suggest that HIV introductions into communities are common and account for a significant proportion of new HIV infections acquired outside of households in rural Uganda, though the extent to which this is true elsewhere in Africa remains unknown. Our results also suggest that HIV prevention efforts should be implemented at spatial scales broader than the community and should target key populations likely responsible for introductions into communities.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
About 35 million people (25 million of whom live in sub-Saharan Africa) are currently infected with HIV, the virus that causes AIDS, and about 2.3 million people become newly infected every year. HIV destroys immune system cells, leaving infected individuals susceptible to other infections. HIV infection can be controlled by taking antiretroviral drugs (antiretroviral therapy, or ART) daily throughout life. Although originally available only to people living in wealthy countries, recent political efforts mean that 9.7 million people in low- and middle-income countries now have access to ART. However, ART does not cure HIV infection, so prevention of viral transmission remains extremely important. Because HIV is usually transmitted through unprotected sex with an infected partner, individuals can reduce their risk of infection by abstaining from sex, by having one or a few partners, and by using condoms. Male circumcision also reduces HIV transmission. In addition to reducing illness and death among HIV-positive people, ART also reduces HIV transmission.
Why Was This Study Done?
Effective HIV control requires an understanding of how HIV spreads through sexual networks. These networks include sexual partnerships between individuals in households, between community members in different households, and between individuals from different communities. Local sexual networks (household and intra-community sexual partnerships) are sometimes assumed to be the dominant driving force in HIV spread in sub-Saharan Africa, but are viral introductions from sexual partnerships with individuals outside the community also important? This question needs answering because the effectiveness of interventions such as ART as prevention partly depends on how many new infections in an intervention area are attributable to infection from partners residing in that area and how many are attributable to infection from partners living elsewhere. Here, the researchers use three analytical methods—spatial clustering statistics, viral phylogenetics, and egocentric transmission modeling—to ask whether HIV transmission in rural Uganda is driven predominantly by intra-community sexual networks. Spatial clustering analysis uses the geographical coordinates of households to measure the tendency of HIV-infected people to cluster spatially at scales consistent with community transmission. Viral phylogenetic analysis examines the genetic relatedness of viruses; if transmission is through local networks, viruses in newly infected individuals should more closely resemble viruses in other community members than those in people outside the community. Egocentric transmission modelling uses information on the locations of recent sexual partners to estimate the proportions of new transmissions from household, intra-community, and extra-community partners.
What Did the Researchers Do and Find?
The researchers applied their three analytical methods to data collected from 14,594 individuals living in 46 communities (governmental administrative units) in Rakai District, Uganda. Spatial clustering analysis indicated that individuals who lived in households with individuals with incident HIV (newly diagnosed) or prevalent HIV (previously diagnosed) were 3.2 times more likely than the general population to be HIV-positive themselves. Spatial clustering outside households was relatively weak, however, and was confined to distances of less than half a kilometer. Viral phylogenetic analysis indicated that 44% of phylogenetic clusters (viruses with related genetic sequences found in more than one individual) were within households, but that 40% of clusters crossed community borders. Finally, analysis of the locations of self-reported sexual partners indicated that 39% of new viral transmissions occurred within stable household partnerships, but that among people newly infected by extra-household partners, nearly two-thirds were infected by partners from outside their community.
What Do These Findings Mean?
The results of all three analyses suggest that HIV introductions into communities are frequent and are likely to play an important role in sustaining HIV transmission in the Rakai District. Specifically, within this rural HIV-endemic region (a region where HIV infection is always present), viral introductions combined with intra-household transmission account for the majority of new infections, although community-based sexual networks also play a critical role in HIV transmission. These findings may not be generalizable to the broader Ugandan population or to other regions of Africa, and their accuracy is likely to be limited by the use of self-reported sexual partner data. Nevertheless, these findings indicate that the dynamics of HIV transmission in rural Uganda (and probably elsewhere) are complex. Consequently, to halt the spread of HIV, prevention efforts will need to be implemented at spatial scales broader than individual communities, and key populations that are likely to introduce HIV into communities will need to be targeted.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001610.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV and AIDS in Uganda and on HIV prevention strategies (in English and Spanish)
The UNAIDS Report on the Global AIDS Epidemic 2013 provides up-to-date information about the AIDS epidemic and efforts to halt it
The Center for AIDS Prevention Studies (University of California, San Francisco) has a fact sheet about sexual networks and HIV prevention
Wikipedia provides information on spatial clustering analysis (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A PLOS Computational Biology Topic Page (a review article that is a published copy of record of a dynamic version of the article as found in Wikipedia) about viral phylodynamics is available
Personal stories about living with HIV/AIDS are available through Avert, NAM/aidsmap, and Healthtalkonline
doi:10.1371/journal.pmed.1001610
PMCID: PMC3942316  PMID: 24595023
17.  Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection. 
Antimicrobial Agents and Chemotherapy  1996;40(11):2535-2541.
Patient human immunodeficiency virus type 1 (HIV-1) isolates that are resistant to protease inhibitors may contain amino acid substitutions L10I/V, M46L/I, G-48V, L63P, V82A/F/T, I84V, and L90M in the protease gene. Substitutions at positions 82 and/or 90 occur in variants that display high levels of resistance to certain protease inhibitors. Nucleotide substitutions at these two sites also lead to the loss of two HindII restriction enzyme digestion sites, and these changes make possible a rapid procedure for the detection of drug-resistant variants in patients on protease inhibitor therapy. This procedure was used to detect the emergence of mutated viruses at various times after the initiation of therapy with the HIV-1 protease inhibitor indinavir. The method includes viral RNA isolation from plasma and reverse transcription PCR amplification of the protease gene with fluorescence-tagged primers. The PCR product is digested with HindII, the cleavage products are separated on a urea-acrylamide gel in a DNA sequencer, and the extent of cleavage is automatically analyzed with commercially available software. In viruses from 34 blood samples from four patients, mutations leading to an amino acid change at residue 82 appeared as early as 6 weeks after the start of therapy and persisted throughout the course of the study period (48 weeks). Mutations leading to double substitutions at residues 82 and 90 were seen at a lower frequency and appeared later than the change at position 82. The changes detected by restriction enzyme cleavage were confirmed by DNA sequencing of the cloned protease genes by reverse transcription PCR amplification of viral RNA from isolates in plasma. In addition to the changes at positions 82 and 90, we have identified M46L/I, G48V, and I54V substitutions in isolates derived from indinavir-treated patients. HindII analysis of uncloned, PCR-amplified DNA offers a rapid screening procedure for the detection of virus isolates containing mutations at amino acid residues 82 and 90 in the HIV-1 protease gene. By using other restriction enzymes, the same method can be used to detect additional protease drug-resistant variants and is generally applicable for the detection of mutations.
PMCID: PMC163570  PMID: 8913459
18.  Statistical correlation of nonconservative substitutions of HIV gp41 variable amino acid residues with the R5X4 HIV-1 phenotype 
Virology Journal  2016;13:28.
Background
The interaction of the envelope glycoprotein of HIV-1 (gp120/gp41) with coreceptor molecules has important implications for specific cellular targeting and pathogenesis. Experimental and theoretical evidences have shown a role for gp41 in coreceptor tropism, although there is no consensus about the positions involved. Here we analyze the association of physicochemical properties of gp41 amino acid residues with viral tropism (X4, R5, and R5X4) using a large set of HIV-1 sequences. Under the assumption that conserved regions define the complex structural features essential for protein function, we focused our search only on amino acids in the gp41 variable regions.
Methods
Gp41 amino acid sequences of 2823 HIV-1 strains from all clades with known coreceptor tropism were retrieved from Los Alamos HIV Database. Consensus sequences were constructed for homologous sequences (those obtained from the same patient and having the same tropism) in order to avoid bias due to sequence overrepresentation, and the variability (entropy) per site was determined. Comparisons of hydropathy index (HI) and charge (Q) of amino acid residues at highly variable positions between coreceptor groups were performed using two non-parametrical tests and Benjamini-Hochberg correction. Pearson’s correlation analysis was performed to determine covariance of HI and Q values.
Results
Calculation of variability per site rendered 58 highly variable amino acid positions. Of these, statistical analysis rendered significantly different HI or Q only for the R5 vs. R5X4 comparison at twelve positions: 535, 602, 619, 636, 640, 641, 658, 662, 667, 723, 756 and 841. The largest differences in particular amino acid frequencies between coreceptor groups were found at 619, 636, 640, 641, 662, 723 and 756. A hydrophobic tendency of residues 619, 640, 641, 723 and 756, along with a hydrophilic/charged tendency at residues 636 and 662 was observed in R5X4 with respect to R5 sequences. HI of position 640 covariated with that of 602, 619, 636, 662, and 756.
Conclusions
Variability and significant correlations of physicochemical properties with viral phenotype suggest that substitutions at residues in the loop (602 and 619), the HR2 (636, 640, 641, 662), and the C-terminal tail (723, 756) of gp41 may contribute to phenotype of R5X4 strains.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-016-0486-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12985-016-0486-6
PMCID: PMC4754869  PMID: 26879054
gp41; gp41 hydrophobicity; gp41 loop; gp41 variability; HIV-1; HIV-1 coreceptor; HR2; R5X4 phenotype
19.  Genetic diversity and molecular epidemiology of multidrug-resistant Mycobacterium tuberculosis in Minas Gerais State, Brazil 
BMC Infectious Diseases  2015;15:306.
Background
We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil.
Methods
One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions).
Results
Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB.
Conclusions
Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20–30 % of the isolates we analyzed might be MDR-TB transmission cases.
doi:10.1186/s12879-015-1057-y
PMCID: PMC4521345  PMID: 26231661
MDR-TB; Molecular epidemiology; Spoligotyping; MIRU-VNTR; IS6110-RFLP; Minas Gerais; Brazil
20.  Genetic Diversity of Mycobacterium tuberculosis in Peru and Exploration of Phylogenetic Associations with Drug Resistance 
PLoS ONE  2013;8(6):e65873.
Background
There is limited available data on the strain diversity of M tuberculosis in Peru, though there may be interesting lessons to learn from a setting where multidrug resistant TB has emerged as a major problem despite an apparently well-functioning DOTS control programme.
Methods
Spoligotyping was undertaken on 794 strains of M tuberculosis collected between 1999 and 2005 from 553 community-based patients and 241 hospital-based HIV co-infected patients with pulmonary tuberculosis in Lima, Peru. Phylogenetic and epidemiologic analyses permitted identification of clusters and exploration of spoligotype associations with drug resistance.
Results
Mean patient age was 31.9 years, 63% were male and 30.4% were known to be HIV+. Rifampicin mono-resistance, isoniazid mono-resistance and multidrug resistance (MDR) were identified in 4.7%, 8.7% and 17.3% of strains respectively. Of 794 strains from 794 patients there were 149 different spoligotypes. Of these there were 27 strains (3.4%) with novel, unique orphan spoligotypes. 498 strains (62.7%) were clustered in the nine most common spoligotypes: 16.4% SIT 50 (clade H3), 12.3% SIT 53 (clade T1), 8.3% SIT 33 (LAM3), 7.4% SIT 42 (LAM9), 5.5% SIT 1 (Beijing), 3.9% SIT 47 (H1), 3.0% SIT 222 (clade unknown), 3.0% SIT1355 (LAM), and 2.8% SIT 92 (X3). Amongst HIV-negative community-based TB patients no associations were seen between drug resistance and specific spoligotypes; in contrast HIV-associated MDRTB, but not isoniazid or rifampicin mono-resistance, was associated with SIT42 and SIT53 strains.
Conclusion
Two spoligotypes were associated with MDR particularly amongst patients with HIV. The MDR-HIV association was significantly reduced after controlling for SIT42 and SIT53 status; residual confounding may explain the remaining apparent association. These data are suggestive of a prolonged, clonal, hospital-based outbreak of MDR disease amongst HIV patients but do not support a hypothesis of strain-specific propensity for the acquisition of resistance-conferring mutations.
doi:10.1371/journal.pone.0065873
PMCID: PMC3691179  PMID: 23826083
21.  Inferring within-patient HIV-1 evolutionary dynamics under anti-HIV therapy using serial virus samples with vSPA 
BMC Bioinformatics  2009;10:360.
Background
Analysis of within-patient HIV evolution under anti-HIV therapy is crucial to a better understanding the possible mechanisms of HIV drug-resistance acquisition. The high evolutionary rate of HIV allows us to trace its evolutionary process in real time by analyzing virus samples serially collected from the same patient. However, such studies are still uncommon due to the lack of powerful computational methods designed for serial virus samples. In this study, we develop a computational method, vSPA (viral Sequential Pathway Analysis), which groups viral sequences from the same sampling time into clusters and traces the evolution between clusters over sampling times. The method makes use of information of different sampling times and traces the evolution of important amino acid mutations. Second, a permutation test at the codon level is conducted to determine the threshold of the correlation coefficient for clustering viral quasispecies. We applied vSPA to four large data sets of HIV-1 protease and reverse transcriptase genes serially collected from two AIDS patients undergoing anti-HIV therapy over several years.
Results
The results show that vSPA can trace within-patient HIV evolution by detecting many amino acid changes, including important drug-resistant mutations, and by classifying different viral quasispecies coexisting during different periods of the therapy.
Conclusion
Given that many new anti-HIV drugs will be available in the near future, vSPA may be useful for quickly providing information on the acquisition of HIV drug-resistant mutations by monitoring the within-patient HIV evolution under anti-HIV therapy as a computational approach.
doi:10.1186/1471-2105-10-360
PMCID: PMC2776027  PMID: 19863822
22.  Amino Acid Preferences of Retroviral Proteases for Amino-Terminal Positions in a Type 1 Cleavage Site▿ ‡  
Journal of Virology  2008;82(20):10111-10117.
The specificities of the proteases of 11 retroviruses were studied using a series of oligopeptides with amino acid substitutions in the P1, P3, and P4 positions of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr↓Pro-Ile-Val-Gln) in human immunodeficiency virus type 1 (HIV-1). Previously, the substrate specificity of the P2 site was studied for the same representative set of retroviral proteases, which included at least one member from each of the seven genera of the family Retroviridae (P. Bagossi, T. Sperka, A. Fehér, J. Kádas, G. Zahuczky, G. Miklóssy, P. Boross, and J. Tözsér, J. Virol. 79:4213-4218, 2005). Our enzyme set comprised the proteases of HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus (AMV), Mason-Pfizer monkey virus, mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, human T-lymphotropic virus type 1, bovine leukemia virus, walleye dermal sarcoma virus, and human foamy virus. Molecular models were used to interpret the similarities and differences in specificity between these retroviral proteases. The results showed that the retroviral proteases had similar preferences (Phe and Tyr) for the P1 position in this sequence context, but differences were found for the P3 and P4 positions. Importantly, the sizes of the P3 and P4 residues appear to be a major contributor for specificity. The substrate specificities correlated well with the phylogenetic tree of the retroviruses. Furthermore, while the specificities of some enzymes belonging to different genera appeared to be very similar (e.g., those of AMV and MMTV), the specificities of the primate lentiviral proteases substantially differed from that observed for a nonprimate lentiviral protease.
doi:10.1128/JVI.00418-08
PMCID: PMC2566267  PMID: 18701588
23.  pol gene diversity of five human immunodeficiency virus type 1 subtypes: evidence for naturally occurring mutations that contribute to drug resistance, limited recombination patterns, and common ancestry for subtypes B and D. 
Journal of Virology  1997;71(9):6348-6358.
Naturally occurring mutations in the polymerase gene of human immunodeficiency virus type 1 (HIV-1) have important implications for therapy and the outcome of clinical studies. Using 42 virus isolates obtained from the UNAIDS sample collection, we analyzed the protease (99 amino acids [aa]) and the first 297 aa of reverse transcriptase (RT) coding regions. Based on the V3 sequence analysis, the collection includes subtype A (n = 5), subtype B (n = 12), subtype C (n = 1), subtype D (n = 11), and subtype E (n = 13) viruses. Of the 42 protease genes, 37 contained naturally occurring mutations at positions in the gene that contribute to resistance to protease inhibitors (indinavir, saquinavir, ritonavir, and nelfinavir) in clade B isolates. The phenotypic effect of these substitutions in non-B isolates is unclear. The The 5'half RT coding region of the 42 isolates was found to be less variable, although 19 of the 42 RT sequences contained amino acid substitutions known to contribute to nucleoside and/or nonnucleoside drug resistance. Since the virus isolates were obtained in 1992, it is unlikely that the infected subjects received protease inhibitors, but we found evidence that one subject acquired a zidovudine (AZT)-resistant HIV-1 strain from a contact who had received AZT. Phylogenetic analysis identified five subtype pol clusters: A, B, C, D, and A'. Comparison of env and pol sequences of the same viruses showed no more recombination events than were already identified on the basis of gag/env comparison (M. Cornelissen, G. Kampinga, F. Zorgdrager, J. Goudsmit, and the UNAIDS Network for HIV Isolation and Characterization, J. Virol. 70:8209-8212, 1996). In one of the known recombinants, a crossover site between subtypes A and C could be identified, and in another, a crossover site could not be identified due to lack of a reference subtype F pol sequence. We analyzed the ds/da ratio of gag, pol, and env sequences of 35 isolates, excluding the recombinants. Our analysis showed that gag and pol are subjected to purifying selection with an average ds/da ratio above 1, independent of the subtype and in contrast with V3 (ds/da approximately 1). Based on the low ds/da ratio of the intergroup analysis of A/E and B/D gag and pol sequences, we analyzed the evolutionary relation between subtypes B and D in more detail by constructing separate phylogenetic trees for synonymous and nonsynonymous substitutions. Our analysis suggests a common ancestry for subtypes B and D that is distinct from that of subtypes A and E.
PMCID: PMC191908  PMID: 9261352
24.  Global analysis of sequence diversity within HIV-1 subtypes across geographic regions 
Future virology  2012;7(5):505-517.
Aims
HIV-1 sequence diversity can affect host immune responses and phenotypic characteristics such as antiretroviral drug resistance. Current HIV-1 sequence diversity classification uses phylogeny-based methods to identify subtypes and recombinants, which may overlook distinct subpopulations within subtypes. While local epidemic studies have characterized sequence-level clustering within subtypes using phylogeny, identification of new genotype – phenotype associations are based on mutational correlations at individual sequence positions. We perform a systematic, global analysis of position-specific pol gene sequence variation across geographic regions within HIV-1 subtypes to characterize subpopulation differences that may be missed by standard subtyping methods and sequence-level phylogenetic clustering analyses.
Materials & methods
Analysis was performed on a large, globally diverse, cross-sectional pol sequence dataset. Sequences were partitioned into subtypes and geographic subpopulations within subtypes. For each subtype, we identified positions that varied according to geography using VESPA (viral epidemiology signature pattern analysis) to identify sequence signature differences and a likelihood ratio test adjusted for multiple comparisons to characterize differences in amino acid (AA) frequencies, including minority mutations. Synonymous nonsynonymous analysis program (SNAP) was used to explore the role of evolutionary selection witihin subtype C.
Results
In 7693 protease (PR) and reverse transcriptase (RT) sequences from untreated patients in multiple geographic regions, 11 PR and 11 RT positions exhibited sequence signature differences within subtypes. Thirty six PR and 80 RT positions exhibited within-subtype geography-dependent differences in AA distributions, including minority mutations, at both conserved and variable loci. Among subtype C samples from India and South Africa, nine PR and nine RT positions had significantly different AA distributions, including one PR and five RT positions that differed in consensus AA between regions. A selection analysis of subtype C using SNAP demonstrated that estimated rates of nonsynonymous and synonymous mutations are consistent with the possibility of positive selection across geographic subpopulations within subtypes.
Conclusion
We characterized systematic genotypic pol differences across geographic regions within subtypes that are not captured by the subtyping nomenclature. Awareness of such differences may improve the interpretation of future studies determining the phenotypic consequences of genetic backgrounds.
doi:10.2217/fvl.12.37
PMCID: PMC3400699  PMID: 22822410
geography; HIV-1; pol gene sequences; protease; reverse transcriptase; subtyping
25.  Transmission Cluster of Multiclass Highly Drug-Resistant HIV-1 Among 9 Men Who Have Sex With Men in Seattle/King County, WA, 2005−2007 
Background
From 2005 through 2007, Seattle health care providers identified cases of primary multiclass drug-resistant (MDR) HIV-1 with common patterns of resistance to antiretrovirals (ARVs). Through surveillance activities and genetic analysis, the local Health Department and the University of Washington identified phylogenetically linked cases among ARV treatment–naive and -experienced individuals.
Methods
HIV-1 pol nucleotide consensus sequences submitted to the University of Washington Clinical Virology Laboratory were assessed for phylogenetically related MDR HIV. Demographic and clinical data collected included HIV diagnosis date, ARV history, and laboratory results.
Results
Seven ARV-naive men had phylogenetically linked MDR strains with resistance to most ARVs; these were linked to 2 ARV-experienced men. All 9 men reported methamphetamine use and multiple anonymous male partners. Primary transmissions were diagnosed for more than a 2-year period, 2005−2007. Three, including the 2 ARV-experienced men, were prescribed ARVs.
Conclusions
This cluster of 9 men with phylogenetically related highly drug-resistant MDR HIV strains and common risk factors but without reported direct epidemiologic links may have important implications to public health. This cluster demonstrates the importance of primary resistance testing and of collaboration between the public and private medical community in identifying MDR outbreaks. Public health interventions and surveillance are needed to reduce transmission of MDR HIV-1.
doi:10.1097/QAI.0b013e318185727e
PMCID: PMC2586929  PMID: 18769347
HIV; HIV-1; multiple drug resistance; disease clustering; highly active antiretroviral therapy

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