Continual human immunodeficiency virus type 1 (HIV-1) evolution and expansion within the human population have led to unequal distribution of HIV-1 group M subtypes. In particular, recent outgrowth of subtype C in southern Africa, India, and China has fueled speculation that subtype C isolates may be more fit in vivo. In this study, nine subtype B and six subtype C HIV-1 isolates were added to peripheral blood mononuclear cell cultures for a complete pairwise competition experiment. All subtype C HIV-1 isolates were less fit than subtype B isolates (P < 0.0001), but intrasubtype variations in HIV-1 fitness were not significant. Increased fitness of subtype B over subtype C was also observed in primary CD4؉ T cells and macrophages from different human donors but not in skin-derived human Langerhans cells. Detailed analysis of the retroviral life cycle during several B and C virus competitions indicated that the efficiency of host cell entry may have a significant impact on relative fitness. Furthermore, phyletic analyses of fitness differences suggested that, for a recombined subtype B/C HIV-1 isolate, higher fitness mapped to the subtype B env gene rather than the subtype C gag and pol genes. These results suggest that subtype B and C HIV-1 may be transmitted with equal efficiency (Langerhans cell data) but that subtype C isolates may be less fit following initial infection (T-cell and macrophage data) and may lead to slower disease progression.
Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID 50 ). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles. However, a direct comparison of these surrogate assays and actual infectious HIV-1 titers from TCID 50 assays has not been performed with even the most conserved laboratory strains, let alone the highly divergent primary HIV-1 isolates of different subtypes. This study indicates that endogenous RT activity, not p24 content or viral RNA load, is the best surrogate measure of infectious HIV-1 titer in both cell-free supernatants and viruses purified on sucrose cushions. Sequence variation between HIV-1 subtypes did not appear to affect the function or activity of the RT enzyme in this endogenous assay but did affect the detection of p24 capsid by both enzyme immunoassays and Western blots. Clear groupings of non-syncytiuminducing (NSI), CCR5-tropic (R5), and SI/CXCR4-tropic (X4) HIV-1 isolates were observed when we compared the slopes derived from correlations of RT activity with infectious titers. Finally, the replication efficiency or fitness of both the NSI/R5 and SI/X4 HIV-1 isolates was not linked to the titers of the virus stocks.
Retroviral recombination results from strand switching, during reverse transcription, between the two copies of genomic RNA present in the virus. We analysed recombination in part of the envelope gene, between HIV-1 subtype A and D strains. After a single infection cycle, breakpoints clustered in regions corresponding to the constant portions of Env. With some exceptions, a similar distribution was observed after multiple infection cycles, and among recombinant sequences in the HIV Sequence Database. We compared the experimental data with computer simulations made using a program that only allows recombination to occur whenever an identical base is present in the aligned parental RNAs. Experimental recombination was more frequent than expected on the basis of simulated recombination when, in a region spanning 40 nt from the 5′ border of a breakpoint, no more than two discordant bases between the parental RNAs were present. When these requirements were not fulfilled, breakpoints were distributed randomly along the RNA, closer to the distribution predicted by computer simulation. A significant preference for recombination was also observed for regions containing homopolymeric stretches. These results define, for the first time, local sequence determinants for recombination between divergent HIV-1 isolates.
Background: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population.
Human immunodeficiency virus type 1 (HIV-1) isolates derived from HIV-infected, treatment-naive Ugandan infants were propagated and tested for sensitivity to antiretroviral (ARV) drugs. Although most subtype A and D isolates displayed inhibition profiles similar to those of subtype B strains, a subtype D isolate identified as D14-UG displayed high-level resistance to nevirapine in peripheral blood mononuclear cell cultures (>2,000-fold) and in MT4 cell cultures (ϳ800-fold) but weaker resistance to delavirdine (ϳ13-fold) and efavirenz (ϳ8-fold) in MT4 cell cultures. To investigate the possible mechanism for this resistance to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs), the RT coding region in pol was sequenced and compared to the consensus RT sequence of NNRTI-resistant and NNRTI-sensitive subtype A, B, and D HIV-1 isolates. D14-UG did not contain the classic amino acid substitutions conferring NNRTI resistance (e.g., Y181C, K103N, and G190A) but did have some putative sites associated with drug resistance, I135L, T139V, and V245T. Wild-type and mutated protease-RT genes from D14-UG and an NNRTI-sensitive subtype D isolate from Uganda (D13-UG) were cloned into pNL4-3 to produce recombinant viruses and to determine the effects of the mutations on susceptibility to ARV drugs, specifically, NNRTIs. The results showed that I135L and/or V245T mutations can confer high-level resistance to nevirapine and delavirdine as well as low level crossresistance to efavirenz. Finally, ex vivo fitness analyses suggested that NNRTI-resistant sites 135L and 245T in wild-type isolate D14-UG may reduce RT fitness but do not have an impact on the fitness of the primary HIV-1 isolate.Current drugs effective at inhibiting human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are classified as nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) (3,19). NRTIs interfere with enzyme activity following conversion to the triphosphate forms and incorporation into the growing DNA strand, which causes premature chain termination (4,8). In contrast, NNRTIs are noncompetitive inhibitors which bind a hydrophobic pocket adjacent to the polymerase active site, causing an allosteric change which effectively inhibits DNA polymerization (9). One of the impediments to the frequent use of NNRTIs in combination with at least two NRTIs is the rapid emergence of resistance.Studies of drug resistance have mainly focused on HIV-1 subtype B, which is dominant in developed countries (26). A paucity of data on the emergence of antiretroviral (ARV) drug resistance in individuals or populations infected with other subtypes (e.g., A, C, D, and CRF01) likely is attributable to limited access to ARV drug treatment in developing countries (1, 2, 13, 15, 30-32, 39, 47, 53). However, the rapid implementation of new ARV drug treatment programs in these regions likely will precede thorough investigations of ARV drug resistance in non-clade B HIV-1 isolates. Interestingly, nevirapine, as opposed to the more expensive p...
BackgroundIntersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection.ResultsFollowing a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.ConclusionThese findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.
Resource managers benefit from knowledge of angler support for fisheries management strategies. Factors including angler values (protection, utilitarian, and dominance), involvement (attraction, centrality, social, identity affirmation, and expression), catch-related motivations (catching some, many, and big fish, and keeping fish), satisfaction, agency trust, and demographics may relate to fisheries management preferences. Using results from a mail survey of Minnesota resident anglers, we explored how these factors were related to budget support for fish stocking relative to habitat protection/restoration. Results suggest that values, angler involvement, catch orientation, satisfaction, total and recent years fishing, age, and education influence relative support for stocking versus habitat protection/restoration. Utilitarian values, angling centrality, an orientation to catch many fish, satisfaction with the number of fish caught, number of recent years fishing, and age positively related to support for stocking over habitat management, while protection values, attraction to angling, total years fishing, and education level were negatively related to relative support for stocking.
Cryopreserved cell monolayers are a new cell culture technology intended to ensure the availability of cells in the laboratory for virus detection. Two cryopreserved cell monolayers, ELVIS for the detection of herpes simplex virus (HSV) and R-Mix for the detection of influenza virus, were evaluated. The results indicated that fresh and cryopreserved cell monolayers are comparable in sensitivity for the detection of HSV and influenza virus. The cells retain the same level of sensitivity for up to 4 months at ؊80°C.A diagnostic virology laboratory generally receives fresh cells from commercial sources once or twice a week. Receipts of commercially prepared cells can be compromised by shipping delays, mishandling of packages, and exposure to temperature stress, which may lead to suboptimal performance. Additionally, because these shipments are estimated standing orders, shortage or overage is common. Finally, the quality of cell monolayers is variable from lot to lot and difficult to control and standardize. These fundamental shortcomings of commercially prepared cells are generally accepted by clinical virology laboratories because the only functional alternative, i.e., preparing one's own cells each week, is generally impractical for technical and/or economic reasons.Recently, Diagnostic Hybrids Inc. (DHI, Athens, Ohio) developed a cryopreservation method that addresses the practical issues cited above. In this study, we compare two sensitive cell culture systems, ELVIS cells for the rapid detection of herpes simplex virus (HSV) (5, 7) and R-Mix (mixture of A549 and mink lung) cells for the detection of influenza viruses A and B (1, 2, 3, 4, 6), in both frozen and nonfrozen monolayer formats, to determine whether frozen monolayers can match the virus detection performance of fresh, commercially prepared monolayers.The cryopreserved ELVIS and R-Mix cell monolayers (ready cell frozen monolayers [RCFM]) were provided on a glass coverslip in shell vials by DHI. RCFM were shipped on dry ice and transferred quickly to storage in a Ϫ80°C chest freezer. The nonfrozen cell equivalents were commercially produced and shipped by express courier, as is routinely done. Prior to inoculation with clinical specimens, cryopreserved ELVIS RCFM and R-Mix RCFM vials were removed from the Ϫ80°C freezer and placed in an empty 24-well cluster plate. The plate was gently placed in a 35 to 37°C water bath such that the water level was just high enough to flood the plate. The vials were incubated for 4 min (Ϯ15 s) without any agitation. The thawed vials were gently removed from the water bath, and the freeze medium was immediately removed from the vials by gentle aspiration.For ELVIS RCFM, 1 ml of ELVIS replacement medium (DHI) was added to each vial and 0.2 ml of clinical specimen was inoculated into both RCFM and fresh cells. All shell vials were centrifuged at 700 ϫ g for 60 min at room temperature and incubated at 35 to 37°C for 20 to 24 h. ELVIS cells were fixed and stained for HSV detection and typing by using the ELVIS HSV ID/typing...
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