| 1. |
- Brodin, Johanna, et al.
(författare)
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PCR-Induced Transitions Are the Major Source of Error in Cleaned Ultra-Deep Pyrosequencing Data
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ultra-deep pyrosequencing (UDPS) is used to identify rare sequence variants. The sequence depth is influenced by several factors including the error frequency of PCR and UDPS. This study investigated the characteristics and source of errors in raw and cleaned UDPS data. Results: UDPS of a 167-nucleotide fragment of the HIV-1 SG3Denv plasmid was performed on the Roche/454 platform. The plasmid was diluted to one copy, PCR amplified and subjected to bidirectional UDPS on three occasions. The dataset consisted of 47,693 UDPS reads. Raw UDPS data had an average error frequency of 0.30% per nucleotide site. Most errors were insertions and deletions in homopolymeric regions. We used a cleaning strategy that removed almost all indel errors, but had little effect on substitution errors, which reduced the error frequency to 0.056% per nucleotide. In cleaned data the error frequency was similar in homopolymeric and non-homopolymeric regions, but varied considerably across sites. These site-specific error frequencies were moderately, but still significantly, correlated between runs (r = 0.15-0.65) and between forward and reverse sequencing directions within runs (r = 0.33-0.65). Furthermore, transition errors were 48-times more common than transversion errors (0.052% vs. 0.001%; p<0.0001). Collectively the results indicate that a considerable proportion of the sequencing errors that remained after data cleaning were generated during the PCR that preceded UDPS. Conclusions: A majority of the sequencing errors that remained after data cleaning were introduced by PCR prior to sequencing, which means that they will be independent of platform used for next-generation sequencing. The transition vs. transversion error bias in cleaned UDPS data will influence the detection limits of rare mutations and sequence variants.
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| 2. |
- Casper, C, et al.
(författare)
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Coreceptor usage of primary HIV type 1 isolates obtained from different lymph node subsets
- 2005
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Ingår i: AIDS Research and Human Retroviruses. - : Mary Ann Liebert Inc. - 1931-8405 .- 0889-2229. ; 21:12, s. 1003-1010
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Tidskriftsartikel (refereegranskat)abstract
- Biological characteristics of virus quantitatively rescued from different cell types present in lymph nodes of HIV-1-infected individuals in various stages of their disease were determined, not including patients with AIDS defining illness. Viruses were obtained by cocultivation with donor monocyte-derived macrophages and T-lymphocytes and their biological phenotype compared to viruses obtained from the peripheral blood mononuclear cells of the same patient. The biological phenotype was determined on established cell lines (U937-2, CEM, and MT-2) and on the U87.CD4 coreceptor indicator cell lines and variable region 3 (V3) of the envelope was subjected to direct sequencing. All isolates obtained from lymph node subsets used CCR5 as coreceptor. Furthermore, these viruses were also sensitive to inhibition by beta-chemokines as analyzed for viruses of one patient. All 12 V3 regions showed a unique sequence indicating compartmentalization within each patient. The biological phenotype of CCR5-dependent (R5) HIV-1 isolates obtained from PBMC resembles the phenotype of viruses isolated from different lymph node cell subsets.
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| 3. |
- Ederth, Josefine, et al.
(författare)
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Molecular characterization of HCV in a Swedish county over 8 years (2002-2009) reveals distinct transmission patterns.
- 2016
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Ingår i: Infection ecology & epidemiology. - : Informa UK Limited. - 2000-8686. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis C virus (HCV) is a major public health concern and data on its molecular epidemiology in Sweden is scarce. We carried out an 8-year population-based study of newly diagnosed HCV cases in one of Sweden's centrally situated counties, Södermanland (D-county). The aim was to characterize the HCV strains circulating, analyze their genetic relatedness to detect networks, and in combination with demographic data learn more about transmission.
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| 4. |
- Esbjörnsson, Joakim, et al.
(författare)
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HIV-1 Molecular Epidemiology in Guinea-Bissau, West Africa: Origin, Demography and Migrations
- 2011
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:2
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Tidskriftsartikel (refereegranskat)abstract
- The HIV-1 epidemic in West Africa has been dominated by subtype A and the recombinant form CRF02_AG. Little is known about the origins and the evolutionary history of HIV-1 in this region. We employed Maximum likelihood and Bayesian methods in combination with temporal and spatial information to reconstruct the HIV-1 subtype distribution, demographic history and migration patterns over time in Guinea-Bissau, West Africa. We found that CRF02_AG and subsubtype A3 were the dominant forms of HIV-1 in Guinea-Bissau and that they were introduced into the country on at least six different occasions between 1976 and 1981. These estimates also corresponded well with the first reported HIV-1 cases in Guinea-Bissau. Migration analyses suggested that (1) the HIV-1 epidemic started in the capital Bissau and then dispersed into more rural areas, and (2) the epidemic in Guinea-Bissau was connected to both Cameroon and Mali. This is the first study that describes the HIV-1 molecular epidemiology in a West African country by combining the results of subtype distribution with analyses of epidemic origin and epidemiological linkage between locations. The multiple introductions of HIV-1 into Guinea-Bissau, during a short time-period of five years, coincided with and were likely influenced by the major immigration wave into the country that followed the end of the independence war (1963-1974).
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| 5. |
- Hedskog, Charlotte, et al.
(författare)
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Dynamics of HIV-1 Quasispecies during Antiviral Treatment Dissected Using Ultra-Deep Pyrosequencing
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:7, s. e11345-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. Principal Findings: Here, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180-220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406-35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07-0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. Conclusions: With this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.
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| 6. |
- Karlsson, Annika, et al.
(författare)
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Low Prevalence of Transmitted Drug Resistance in Patients Newly Diagnosed with HIV-1 Infection in Sweden 2003-2010.
- 2012
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 7:3
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Tidskriftsartikel (refereegranskat)abstract
- Transmitted drug resistance (TDR) is a clinical and epidemiological problem because it may contribute to failure of antiretroviral treatment. The prevalence of TDR varies geographically, and its prevalence in Sweden during the last decade has not been reported. Plasma samples from 1,463 patients newly diagnosed with HIV-1 infection between 2003 and 2010, representing 44% of all patients diagnosed in Sweden during this period, were analyzed using the WHO 2009 list of mutations for surveillance of TDR. Maximum likelihood phylogenetic analyses were used to determine genetic subtype and to investigate the relatedness of the sequences. Eighty-two patients showed evidence of TDR, representing a prevalence of 5.6% (95% CI: 4.5%-6.9%) without any significant time trends or differences between patients infected in Sweden or abroad. Multivariable logistic regression showed that TDR was positively associated with men who have sex with men (MSM) and subtype B infection and negatively associated with CD4 cell counts. Among patients with TDR, 54 (68%) had single resistance mutations, whereas five patients had multi-drug resistant HIV-1. Phylogenetic analyses identified nine significantly supported clusters involving 29 of the patients with TDR, including 23 of 42 (55%) of the patients with TDR acquired in Sweden. One cluster contained 18 viruses with a M41L resistance mutation, which had spread among MSM in Stockholm over a period of at least 16 years (1994-2010). Another cluster, which contained the five multidrug resistant viruses, also involved MSM from Stockholm. The prevalence of TDR in Sweden 2003-2010 was lower than in many other European countries. TDR was concentrated among MSM, where clustering of TDR strains was observed, which highlights the need for continued and improved measures for targeted interventions.
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| 7. |
- Mild, Mattias, et al.
(författare)
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Differences in molecular evolution between switch (R5 to R5X4/X4-tropic) and non-switch (R5-tropic only) HIV-1 populations during infection.
- 2010
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Ingår i: Infection, Genetics and Evolution. - : Elsevier BV. - 1567-7257 .- 1567-1348. ; 10, s. 356-364
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Tidskriftsartikel (refereegranskat)abstract
- The recent introduction of entry inhibitors in the clinic as components of antiretroviral treatment has heightened the interest in coreceptor use of human immunodeficiency virus type 1 (HIV-1). Viruses using CCR5 as coreceptor (R5 viruses) are generally present over the entire course of infection whereas viruses using the CXCR4 coreceptor (R5X4/X4 viruses) emerge in about 50% of infected individuals during later stages of infection. The CCR5-to-CXCR4 switch represents a concern because CCR5 inhibitors, while suppressing R5 viruses, may allow the emergence of CXCR4-tropic viruses. In this study, HIV-1 populations that maintained CCR5 usage during infection were compared with populations that switched coreceptor usage to include CXCR4 later during infection, with the aim to find molecular properties of the virus populations associated with the CCR5-to-CXCR4 switch. We amplified and molecularly cloned the V1-V3 region of the HIV-1 envelope from 51 sequential HIV-1 isolates derived from 4 to 10 serial samples for each of the patients. Four of the patients had virus populations that switched coreceptor usage to include CXCR4 (switch populations: SP) during infection and four patients had viral populations that maintained exclusive CCR5 usage (non-switch populations: nSP). Coreceptor usage was determined experimentally on individual clones from dualtropic R5X4 isolates. In nSP we found that the number of potential N-linked glycosylation sites (PNGS) increased over time, whereas no pattern of change was observed in SP. We also found differences in V2 length and V3 charge between R5 viruses of nSP and R5 viruses of SP before the switch. The V2 region was significantly longer in R5 viruses of SP compared to viruses of nSP throughout the course of infection, and the V3 charge increased with time in R5 populations from SP, while it remained unchanged or decreased in nSP. These molecular properties could prove important for understanding the evolution of coreceptor usage in HIV-1 populations, and maybe even for predicting an upcoming coreceptor switch at early stages after primary infection.
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| 8. |
- Mild, Mattias, et al.
(författare)
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Frequent intrapatient recombination between HIV-1 R5 and X4 envelopes: Implications for coreceptor switch.
- 2007
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Ingår i: Journal of Virology. - 1098-5514. ; 81:7, s. 3369-3376
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Tidskriftsartikel (refereegranskat)abstract
- Emergence of human immunodeficiency virus type 1 (HIV-1) populations that switch or broaden coreceptor usage from CCR5 to CXCR4 is intimately coupled to CD4(+) cell depletion and disease progression toward AIDS. To better understand the molecular mechanisms involved in the coreceptor switch, we determined the nucleotide sequences of 253 V1 to V3 env clones from 27 sequential HIV-1 subtype B isolates from four patients with virus populations that switch coreceptor usage. Coreceptor usage of clones from dualtropic R5X4 isolates was characterized experimentally. Sequence analysis revealed that 9% of the clones from CXCR4-using isolates had originated by recombination events between R5 and X4 viruses. The majority (73%) of the recombinants used CXCR4. Furthermore, coreceptor usage of the recombinants was determined by a small region of the envelope, including V3. This is the first report demonstrating that intrapatient recombination between viruses with distinct coreceptor usage occurs frequently. It has been proposed that X4 viruses are more easily suppressed by the immune system than R5 viruses. We hypothesize that recombination between circulating R5 viruses and X4 viruses can result in chimeric viruses with the potential to both evade the immune system and infect CXCR4-expressing cells. The broadening in cell tropism of the viral population to include CXCR4-expressing cells would gradually impair the immune system and eventually allow the X4 population to expand. In conclusion, intrapatient recombination between viruses with distinct coreceptor usage may contribute to the emergence of X4 viruses in later stages of infection.
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| 9. |
- Mild, Mattias, et al.
(författare)
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High intrapatient HIV-1 evolutionary rate is associated with CCR5-to-CXCR4 coreceptor switch
- 2013
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Ingår i: Infection, Genetics and Evolution. - : Elsevier BV. - 1567-7257 .- 1567-1348. ; 19, s. 369-377
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Tidskriftsartikel (refereegranskat)abstract
- In approximately 70% of individuals infected with HIV-1 subtype B, the virus switches coreceptor use from exclusively CCR5 use (R5 virus) to either inclusion of or exclusively CXCR4 use (X4 virus) during infection. This switch is associated with an accelerated loss of CD4(+) T-cells and a faster progression to AIDS. Despite intensive research, the mechanisms responsible for coreceptor switch remains elusive. In the present study, we investigated associations between viral evolutionary rate and selection pressure versus viral coreceptor use and rate of disease progression in eight patients with longitudinally sampled HIV-1 env V1-V3 sequences. By employing a Bayesian hierarchical phylogenetic model, we found that the HIV-1 evolutionary rate was more strongly associated with coreceptor switch than with rate of disease progression in terms of CD4(+) T-cell decline. Phylogenetic analyses showed that X4 variants evolved from R5 populations. In addition, coreceptor switch was associated with higher evolutionary rates on both the synonymous and non-synonymous substitution level, but not with dN/dS ratio rates. Our findings suggest that X4 viruses evolved from pre-existing R5 viral populations and that the evolution of coreceptor switch is governed by high replication rates rather than by selective pressure. Furthermore, the association of viral evolutionary rate was more strongly associated with coreceptor switch than disease progression. This adds to the understanding of the complex virus-host interplay that influences the evolutionary dynamics of HIV-1 coreceptor use. (C) 2013 Elsevier B. V. All rights reserved.
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| 10. |
- Mild, Mattias
(författare)
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Intrapatient evolution of HIV-1 in the context of coreceptor usage
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The variable region 1 to 3 (V1-V3) of the HIV-1 envelope plays an important role in coreceptor usage. Early in infection HIV- 1 is using CCR5 as coreceptor to enter target cells (R5 viruses) whereas viruses using CXCR4 as coreceptor (X4 viruses) may appear later in infection. This broadening or switch in coreceptor usage is associated with progression to AIDS. In my thesis work, we have compared the molecular evolution of V1-V3 between virus populations that maintained CCR5 coreceptor usage (non-switch populations, nSP) with virus populations that used CXCR4 as coreceptor (switch populations, SP). We also developed a novel biological cloning system. We found an increase in the number of potential N-linked glycosylation sites in V1 -V3 over time in nSP, while SP showed no change. Since glycans are an important defense against neutralizing antibodies, we hypothesize that the antibody response differed between patients with SP and nSP. We found that V2 length and evolution of V3 charge differed between R5 viruses from SP and nSP, already before coreceptor switch in SP. Therefore, these molecular properties could prove important for understanding, and maybe even for predicting, the evolution of coreceptor usage in HIV-populations. Due to the presence of the X4 subpopulations, SP evolved faster compared to nSP. In addition, R5 and X4 from SP were subjected to different selective pressures. We showed that R5 and X4 viruses recombine frequently. We hypothesize that such rearrangements may affect antibody recognition of X4 and allow for antibody escape and expansion of X4 subpopulations.
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