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ABSTRACT: In the past decade, tissue engineering has evolved from a promising technology to an established scientific field. Large attention has focussed on developing scaffolds from both biodegradable and nondegradable polymers to be cultivated with cells, to replace human body defects. The major drawback of most polymers is however their limited cell-interactive properties. An additional complication when developing a surface modification protocol for those materials is the transferability of protocols from 2D substrates to 3D scaffolds. In the present work, we therefore report on possible biological effects originating from the transfer of a double protein coating protocol, involving gelatin type B and fibronectin, from 2D poly-ε-caprolactone (PCL) films to 3D PCL scaffolds produced by rapid prototyping. A variety of techniques including scanning electron microscopy, X-ray photoelectron spectroscopy and confocal fluorescence microscopy confirmed a successful and homogeneous protein-coating on both 2D and 3D substrates. Interestingly, the biological performance of the double protein-coated PCL substrates, reflected by the initial cell adhesion, proliferation, and colonization was superior compared to the other surface modification steps, independent of the material dimension.
Journal of Biomedical Materials Research Part A 04/2012; 100(7):1783-91. · 2.63 Impact Factor
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Retrovirology. 01/2010; 7:60-74.
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ABSTRACT: The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells. Mammalian viruses encode IFN antagonists to counteract the IFN response in infected cells. A number of IFN antagonists are also capable of blocking RNAi in infected cells and therefore serve as RNA-silencing suppressors. Virus replication in infected cells is restricted by these innate antiviral mechanisms, which may kick in earlier than the viral antagonistic or suppressor protein can accumulate. The yield of virus vaccines and viral gene delivery vectors produced in mammalian producer cells may therefore be suboptimal. To investigate whether blocking of the innate antiviral responses in mammalian cells leads to increased viral vector production, we expressed a number of immunity suppressors derived from plant and mammalian viruses in human cells. We measured that the yield of infectious human immunodeficiency virus-1 particles produced in these cells was increased 5- to 10-fold. In addition, the production of lentiviral and adenoviral vector particles was increased 5- to 10-fold, whereas Sindbis virus particle production was increased approximately 100-fold. These results can be employed for improving the production of viral gene transfer vectors and viral vaccine strains.
Gene therapy 05/2008; 15(7):545-52. · 4.75 Impact Factor
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ABSTRACT: Lichen planus (LP) is a common inflammatory skin disease of unknown aetiology. Viral causes have been suggested.
To find candidate viruses associated with LP.
Lesional and nonlesional skin samples, peripheral blood mononuclear cells and serum were obtained from patients with LP. Ultrastructural, viral DNA, immunohistochemical and serological analyses were performed, and comparisons were made with psoriatic and normal skin.
Electron microscopy revealed typical 120-200-nm enveloped particles with a 100-nm nucleus resembling human herpesvirus (HHV) virions both in dermis and in epidermis of lesional LP tissue. HHV-7 DNA was found in 11 of 18 lesional LP samples, as opposed to only one of 11 nonlesional LP samples (P =0.06), two of 11 lesional psoriasis samples (P = 0.05) and none of four normal skin samples. No relation was found between LP skin and DNA of other known HHVs (HHV-1-6 and 8). With immunohistochemistry, significantly more HHV-7+ cells were found in lesional LP epidermis than in normal epidermis. Lesional LP dermis contained significantly more HHV-7+ cells than nonlesional LP, psoriatic or normal dermis. Moreover, LP skin contained overwhelmingly and consistently more plasmacytoid dendritic cells (upregulated in virally induced conditions) than nonlesional LP samples.
We conclude that HHV-7 replicates in LP lesions, but not in psoriasis, another inflammatory skin condition. HHV-7 is possibly involved in the pathogenesis of LP. These preliminary data make further research on this topic of interest.
British Journal of Dermatology 03/2006; 154(2):361-4. · 3.67 Impact Factor
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ABSTRACT: Semen samples from a donor who seroconverted for human immunodeficiency virus type 1 (HIV-1) during the period that he was donating at our clinic were stored before and after infection. Semen analysis was done on all of these samples before cryopreservation. Retrospectively, both qualitative and quantitative HIV-1 testing was performed on the cryopreserved semen samples to determine the time of primary HIV-1 infection. After HIV-1 infection, semen volume, sperm motility and the percentage of spermatozoa with normal morphology were reduced compared with the same parameters before HIV-1 infection. HIV-1 RNA was intermittently detectable in semen. HIV-1 infection led to a reduction in semen volume, sperm motility and normal sperm morphology in this donor. However, the clinical significance of these findings is unclear. A longitudinal cohort study on the effects of HIV-1 infection on semen quality is necessary to confirm these findings.
Human Reproduction 01/2005; 19(12):2845-8. · 4.47 Impact Factor
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ABSTRACT: BACKGROUND: Multicentric Castleman's disease (MCD) is a rare disease, but is more frequent in AIDS patients. MCD has only been reported twice before in patients receiving immunosuppressive therapy after renal transplantation, and never in patients receiving immunosuppressive therapy without transplantation. About half of the cases of MCD are human herpesvirus 8 (HHV8) - related, in contrast to Kaposi's sarcoma, a more common complication arising after immunosuppression, where the virus is found in virtually all cases. CASE PRESENTATION: We report a HIV-1 negative, non-transplant patient who developed HHV8-associated multicentric Castleman's disease and Kaposi's sarcoma after 17 years of immunosuppressive treatment with cyclosporin A for a minimal change nephropathy. Chemotherapy with liposomal doxorubicin resolved both symptoms of multicentric Castleman's disease and Kaposi's sarcoma in this patient. A concomitant decline in the HHV8 viral load in serum/plasma, as determined by a quantitative real-time PCR assay, was observed. CONCLUSIONS: Multicentric Castleman's disease can be a complication of cyclosporin A treatment. Both multicentric Castleman's disease and Kaposi's sarcoma in this patient were responsive to liposomal doxorubicin, the treatment of choice for Kaposi's sarcoma at the moment, again suggesting a common mechanism linking both disorders, at least for HHV8-positive multicentric Castleman's disease and Kaposi's sarcoma.HHV8 viral load measurements can be used to monitor effectiveness of therapy.
BMC Blood Disorders 01/2004; 3(1):3.
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ABSTRACT: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed
We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort.
For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples.
These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.
BMC Infectious Diseases 10/2002; 2:18. · 3.12 Impact Factor
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ABSTRACT: Abstract
Background
Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed
Methods
We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort.
Results
For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10<sup>7</sup> molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples.
Conclusion
These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.
BMC Infectious Diseases. 01/2002;
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ABSTRACT: To examine the epidemiological factors influencing the distribution and spread of HIV-1 subtypes among heterosexuals in the Netherlands.
A nationwide serosurveillance in 21 HIV/AIDS centres from 1997 to 1999 involved 200 individuals for whom the mode of HIV transmission was heterosexual contact or unknown. HIV-1 subtypes were determined by phylogenetic analysis of env V3 sequences and correlated with sociodemographic characteristics of the subjects and their sexual partners.
HIV-1 subtype B infection occurred in 121 subjects (60%). Non-B subtypes were identified in 31 (A), 24 (C), 10 (D), six (E), four (F) and three (G) individuals; one had an unclassified subtype. The proportion of subtype B was about 60% in four of the six regions of the Netherlands, but in the Northwest and Southwest regions these proportions were 76% and 46%, respectively. The Surinamese and Antilleans, large immigrant groups, were all infected with subtype B, as were almost all individuals with an unknown source. The proportions of non-B viruses did not change significantly over time in Amsterdam, where subtyping was available from 1988 onward, but a shift in the various subtype B strains was observed, suggesting introductions of new subtype B strains in Amsterdam.
To date, HIV-1 non-B subtypes in the Netherlands are still found predominantly among heterosexuals with an epidemiological link with sub-Saharan Africa. Despite continuing introductions of non-B subtypes, the B/non-B distribution has been stable over time, most likely as a result of introductions of subtype B strains from Caribbean and South American countries.
AIDS 12/2001; 15(17):2277-86. · 6.24 Impact Factor
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ABSTRACT: Most HIV-1 subtype F viruses described so far have been isolated from individuals originating in South America, Romania, or Central Africa. Previous studies have shown that subtype F viruses from these three areas can be distinguished by phylogenetic tree analysis of various parts of the HIV genome. Subtype F strains circulating in Central Africa and classified as subgroup F2 and F3 have relatively large nucleotide distances from strains of subgroup F1, which includes some African strains, along with strains from Romania and South America. Subtype F strains have now appeared in Europe. In this study, we analyzed the complete gag gene and a large fragment of the pol gene of seven strains of African origin that represent the three F subgroups. At least five of the seven strains appear to be intersubtype recombinants. Of four strains circulating in Belgium and the Netherlands, three were F/D mosaics and the fourth harboured a G(gag)/GH(pol)/F3(env) recombinant structure. Two of the three F/D mosaics showed identical breakpoints and were independently introduced in Belgium and the Netherlands. At least two of the mosaics were further transmitted. The remaining three strains of the seven we studied were isolated from individuals in Cameroon. Two included large or smaller F1 fragments in gag and pol. The third strain was subtype D along the entire gag and pol fragment. A parental African subtype F that showed no evidence for recombination was not found.
Virology 06/2000; 270(2):267-77. · 3.35 Impact Factor
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ABSTRACT: We have shown previously that human herpesvirus 8 (HHV8) seroconversion for antibodies to the latency-associated nuclear antigen encoded by ORF73 and/or the lytic capsid antigen (vp19) encoded by ORF65 is associated with orogenital contact and is strongly linked to the development of Kaposi's sarcoma among HIV-infected individuals in the Amsterdam Cohort Studies. Here, we investigate the relationship between seroconversion to these antigens and primary HHV8 infection. Between 1984 and 1997, 215 HHV8 seroconversions to ORF73 (106 cases or 49%) and/or to ORF65 (159 cases or 74%) were recorded in the cohort of homosexual men. The HHV8 seroconversion rate among HIV-infected homosexual men (6.2 per 100 person years) was consistently higher than among HIV-uninfected men (2.6 per 100 person years). In HIV-infected but not in uninfected individuals, seroconversion to ORF73/latency-associated nuclear antigen precedes that to ORF65/vp19. Antibody levels to both ORF65- and ORF73-encoded antigens were higher in HIV-infected than in HIV-uninfected men, and among HIV-seropositives, antibody levels to ORF65/vp19 rise even higher with declining CD4 cell counts and peak with Kaposi's sarcoma development, suggesting continuing and increasing viral replication. In 10.3% of HHV8 seroconversions, transient serum viremia could be demonstrated before or at seroconversion. Together with the previously reported link between unprotected orogenital sex and HHV8 seroconversion, our observations suggest that HHV8 seroconversions result from primary infections.
Proceedings of the National Academy of Sciences 05/2000; 97(9):4838-43. · 9.68 Impact Factor
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ABSTRACT: We studied sequence differences in regulatory elements of the long terminal repeat (LTR) and primer-binding site (PBS) among various human immunodeficiency virus type 1 (HIV-1) subtypes. Phylogenetic sequence analysis of a fragment of 729 base pairs (bp) covering the Gag-coding region for half of p24 and all of p17 revealed the gag subtype of all 60 viruses included in the study: A (n = 20), B (n = 12), C (n = 7), D (n = 10), E (n = 3), F (n = 4), G (n = 3), and H (n = 1). The subtype was also determined by analysis of a 689-bp fragment comprising the LTR and the PBS motif. Comparison of the LTR versus gag sequences showed a mosaic genome for seven isolates. After analysis of all sequences, we could describe subtype-specific differences in sequences encompassing the regulatory elements of the LTR and the PBS motif.
AIDS Research and Human Retroviruses 04/2000; 16(5):499-504. · 2.25 Impact Factor
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ABSTRACT: We studied the phylogeny of HIV-1 subtype F viruses from children and adults in Romania in order to (1) clarify whether the Romanian subtype F epidemic was caused by one or several virus introductions and (2) gain insight into the route of spread of the HIV-1 subtype F virus among children and adults in Romania. env (V3), gag (p17/half p24), and pol (prot/half RT) sequences were obtained from three districts in Romania: Tirgu Mures (n = 9, children), Craiova (n = 15, children), and Bucharest (n = 13, adults). Of 37 HIV V3 sequences from Romania, 35 belonged to the genetic subtype F in the neighbor-joining tree, whereas 2 sequences from adults clustered with subtypes A and C. Within the subtype F cluster, no bootstrap-supported subclusters were observed according to geographic area in Romania. Two of the adult V3 sequences that clustered with the children were obtained from individuals who tested HIV seropositive in 1989 and 1990, showing that the subtype F virus was present among adults when the HIV epidemic began among children in Romania. The HIV-1 subtype F viruses obtained from children showed a mean pairwise V3 nucleotide distance of 7.9% and maximum distances of between 18 and 19%; both are higher than previously described. The mean V3 distances (overall, synonymous, and nonsynonymous) were significantly higher for adults than for children. One V3 sequence from the Democratic Republic of Congo clustered within the Romanian sequences, suggesting that the subtype F virus in Romania may originate from this area. Our data also suggest that HIV-1 subtype F was present among Romanian adults before it appeared in 1989 among institutionalized children. The juvenile population was most likely infected with the HIV-1 subtype F virus on more than one occasion, presumably through HIV-contaminated blood (products) obtained from adults.
AIDS Research and Human Retroviruses 04/2000; 16(4):327-36. · 2.25 Impact Factor
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ABSTRACT: To identify new subtype G human immunodeficiency virus type 1 (HIV-1) strains and AG recombinant forms, we collected 28 serum samples from immigrants to the Netherlands from 12 countries throughout Africa. Based on the gag sequences 22 isolates were identified as subtype A or G. Phylogenetic analysis of discontinuous regions of the gag (726 nt), pol (1176 nt) and env (276 nt) genes revealed 13 AG recombinants with the mosaic structure A(gag)/G(pol)/A(env), three with A(gag)/G(pol)/G(env) and one other with A(gag) /G(pol)/G(env), in addition to 'pure' subtypes A(gag)/A(pol)/A(env) (n=1) and G(gag)/G(pol)/G(env) (n=4). To analyse the crossover points in more detail, a new RT-PCR was developed resulting in a large contiguous sequence of 2600 nt from the gag region to half the pol region. All the 13 A(gag)/G(pol)/A(env) recombinants appeared to belong to the circulating recombinant form (CRF) AG (IbNG). The three A(gag)/G(pol) /G(env) recombinants differed from the CRF AG (IbNG) subtype, suggesting the identification of a new CRF subtype. The recovery of AG recombinants from African countries a thousand miles apart indicates the active spread of new recombinants.
Journal of General Virology 03/2000; 81(Pt 2):515-23. · 3.36 Impact Factor
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AIDS Research and Human Retroviruses 12/1998; 14(16):1483-6. · 2.25 Impact Factor
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ABSTRACT: To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4+ T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 delta32). Three of these individuals (all nonrecipients) had a CCR5 delta32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 delta32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.
Journal of Virology 02/1998; 72(1):218-24. · 5.40 Impact Factor
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A M de Roda Husman,
M Koot, M Cornelissen,
I P Keet,
M Brouwer,
S M Broersen,
M Bakker,
M T Roos,
M Prins,
F de Wolf,
R A Coutinho,
F Miedema,
J Goudsmit,
H Schuitemaker
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ABSTRACT: Heterozygosity for a 32-nucleotide deletion in the C-C chemokine receptor 5 gene (CCR5 delta 32) is associated with delayed disease progression in persons infected with HIV-1.
To compare the predictive value of CCR5 genotype with that of established markers in the clinical course of HIV-1 infection.
Retrospective longitudinal study and nested case-control study. The latter included only long-term survivors, who were individually matched with progressors.
Amsterdam, the Netherlands.
364 homosexual men with HIV-1 infection.
Polymerase chain reaction was used for CCR5 genotyping. Univariate and multivariate Cox proportional hazard analyses were done for disease progression with CCR5 genotype, CD4+ T-lymphocyte counts, T-lymphocyte function, HIV-1 biological phenotype (syncytium-inducing or non-syncytium-inducing HIV-1), and viral RNA load in serum as covariates.
In the case-control study, 48% of long-term survivors were heterozygous for CCR5 delta 32 compared with 9% of progressors (odds ratio, 6.9 [95% CI, 1.9 to 24.8]). In the total study sample, CCR5 delta 32 heterozygotes had significantly delayed disease progression (P < 0.001; relative hazard, 0.4 [CI, 0.3 to 0.6]), a 1.5-fold slower decrease in CD4+ T-lymphocyte count (P = 0.01), and a 2.6-fold lower viral RNA load (P = 0.01) at approximately 2.3 years after seroconversion compared with CCR5 wild-type homozygotes. At the end of the study, both groups showed the same prevalence of syncytium-inducing HIV-1, but CCR5 delta 32 heterozygotes had a delayed conversion rate. The protective effect of CCR5 delta 32 heterozygosity was stronger in the presence of only non-syncytium-inducing HIV-1. The CCR5 genotype predicted disease progression independent of viral RNA load, CD4+ T-lymphocyte counts, T-lymphocyte function, and HIV-1 biological phenotype.
The addition of CCR5 genotype to currently available laboratory markers may allow better estimation of the clinical course of HIV-1 infection.
Annals of internal medicine 11/1997; 127(10):882-90. · 16.73 Impact Factor
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ABSTRACT: 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.
Journal of Virology 10/1997; 71(9):6348-58. · 5.40 Impact Factor
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ABSTRACT: Disease progression in HIV-1-infected individuals is strongly associated with persistent and high numbers of HIV-1 RNA copies. We previously reported a markedly lower viral RNA load in eight long-term asymptomatics (LTAs) compared to seven matched progressors (at 1 year after seroconversion or entry in the study, p < 0.001) (Hogervorst E, et al.: J Infect Dis 1995;171:811-821). Here we extend our study to examine whether a difference in viral load can be attributed to infection by viruses having distinct vpr and vpu genes. Sequencing of vpr and vpu genes from serum samples collected at seroconversion from both long-term asymptomatics and progressors showed full-length and intact open reading frames of both genes in all subjects. At the protein level, no difference was discerned in domains of putative functional importance within Vpr and Vpu between the two groups. Phylogenetic analysis showed no clustering of LTA sequences, which interdigitated with sequences from progressors. We therefore concluded that nonprogression is not likely to be explained by deletion of vpr and vpu, or by gross sequence abnormality in these genes.
AIDS Research and Human Retroviruses 02/1997; 13(3):247-52. · 2.25 Impact Factor
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ABSTRACT: Genetic subtypes of human immunodeficiency virus type 1 can be distinguished on the basis of phylogenetic analysis of their envelope (env) gene. A significant proportion of human immunodeficiency virus type 1 strains was retrospectively shown to result from recombination events between viruses belonging genetically to distinct subtypes (D. L. Robertson, P. M. Sharp, F. E. McCutchan, and B. H. Hahn, Nature [London] 374:124-126, 1995). To establish the frequency of natural infections with recombinant viruses and to exclude tissue culture artifacts, we analyzed plasma samples from the UNAIDS sample collection. The collection includes samples from 53 individuals infected with subtype A (n = 9), subtype B (n = 15), subtype C (n = 1), subtype D (n = 13), and subtype E (n = 15) on the basis of V3 region analysis. Phylogenetic analysis of the gag gene fragment showed intersubtype recombinant genomes in 23 cases: 3 of 9 (33%) of subtype A, 2 of 15 (13%) of subtype B, 3 of 13 (23%) of subtype D, and all of subtype E. Of the 23 recombinant viruses, 19 had a gag gene from one subtype and env from another (B(env)/C(gag), A(env)/C(gag), D(env)/A(gag), and E(env)/A(gag)). Phylogenetic analysis clustered the A(gag) of subtype E viruses as an outgroup of subtype A, suggesting that these viruses may belong to a distinct A' cluster. The remaining four recombinant viruses (B(env)/B(p17)F(p24), A(env)/A(p17)D(p24), A(env)/A(p17)C(p24), and D(env)/ D(p17)A(p24)) had breakpoint crossover sites in the proximity of the p17-p24 protein processing site. We conclude that recombination in the gag gene is highly frequent among the major env subtypes and that selection of recombinants is apparently based on particularly beneficial combinations of gag and env gene products.
Journal of Virology 12/1996; 70(11):8209-12. · 5.40 Impact Factor
