Structural determinants of murine leukemia virus (MLV) reverse transcriptase (RT) important for fidelity and drug resistance in vivo

Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations generating mutants that exhibit altered host tropisms, resistance to antiviral drugs, and the ability to escape the host\u27s immune system. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity and drug-resistance in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P cell line also contains an MLV-based retroviral vector (GA-1), which encodes a wild-type bacterial beta-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with X-Gal and the frequencies of inactivating mutations in lacZ were quantified. Several structural determinants of MLV RT were identified that were important for fidelity which included position V223 of the YVDD box, several residues of the dNTP-binding site, as well as residues residing in the RNase H domain of MLV RT. A number of these MLV RT mutants resulted in statistically significant decreases in fidelity (1.2 to 2.8-fold) whereas two mutants showed a statistically significant increase in fidelity (0.8-fold) relative to wild-type MLV RT. Furthermore, these amino acid residues were observed to play critical roles in catalysis and viral replication, which was exhibited by the reductions in both viral titers as well as RT activities of these mutants. In addition to identifying structural determinants important for fidelity, we also showed that the V223 position of MLV RT may not be the only structural determinant important for resistance to the antiviral nucleoside analog, 2\u27, 3\u27-dideoxy-3 \u27-thiacytidine (3TC). This is in contrast to results observed with human immunodeficiency virus type 1 (HIV-1) RT. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis as well as dug-resistance and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT

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Betydning av varierende vannføring for partikkeltransport og tilhørende forurensinger i Bølstadbekken

Partikler er viktig for spredning av næringsstoffer, spormetaller og organisk materiale i vann spesielt ved flom og kraftig overflateavrenning. Deres forekomst er knyttet til arealfordelingen samt tilstanden i nedbørsfeltet, mens spredningen til nærliggende vannforekomster påvirkes av avrenningsprosesser som følge av nedbør. I den sammenheng vil elver og bekker utgjøre viktige transportveier da de bidrar til spredning av forurensingene som kommer fra nedbørsfeltet. Tidligere studier har vist at transport av forurensinger, særlig fosfor, skjer i form av partikler. Imidlertid er det generelt en manglende forståelse av vannføringens bidrag i den totale avrenningen, noe som har ført til upresise beregninger av partikkeltransporten. Det vil derfor være et behov å undersøke vannføringens betydning for transport av partikler samt tilhørende stoffer. For å undersøke sammenhengen nærmere ble vannprøver tatt i Bølstadbekken i Ås, Akershus. Bekken befinner seg i et lavlandsområde med leire og betydelig innslag av jordbruksarealer, skog og noe bebyggelse. For å vurdere effekten av varierende vannføring, ble prøver tatt under lav vannføring (baseflow), mens for registrering av flomperioder ble prøver tatt hyppigere. Feltarbeidet ble gjennomført fra 24.08.23 til 09.11.23. Resultatene viste at selv små flomtopper bidro til økt transporten av partikler, næringsstoffer, spormetaller og organisk materiale. Dette kan i hovedsak skyldes overflateavrenning fra jordbruk og skog, gitt deres betydelige omfang i nedbørsfeltet.Particles are crucial for the dispersion of nutrients, trace metals, and organic material in water, especially during floods and heavy surface runoff. Their occurrence is linked to land distribution and land condition of the catchment area, while their distribution to nearby water bodies is affected by runoff processes as a result from precipitation. In this context, rivers and streams serve as significant transport routes, facilitating the distribution of pollutants originating from the catchment. Previous studies have shown that the transport of pollutants, particularly phosphorus, occurs in the form of particles. However, there is a general lack of understanding regarding the contribution of discharge of the overall runoff, leading to imprecise estimations of particle transport. Thus, there is a need to investigate the significance of runoff for particle transport and associated substances. To further examine this relationship, water samples were collected from Bølstadbekken in Ås, Akershus. The stream is located in a lowland area consisting of clay, and the surrounding landscape is dominated by agricultural areas, forests, and some urban areas. To assess the impact of fluctuating water-flow, samples were taken during baseflow conditions, while samples to record flood periods, were collected more frequently. The fieldwork was conducted from August 24, 2023, to November 9, 2023. The results indicated that even a small flood events contributed to increased transport of particles, nutrients, trace metals, and organic material. This can mainly be caused by surface runoff from agriculture and forestry, given their significant presence in the catchment

Intact HIV proviruses persist in children seven to nine years after initiation of antiretroviral therapy in the first year of life

In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact

Hvad vil der være tilbage til fremtiden? – virksomhedsarkiver i en digital tidsalder

Denne debatartikel ser nærmere på digital bevaring, herunder specielt på danske virksomheders praksis på dette aktuelle område. Forfatterne gør status på, hvordan virksomhederne i dag forholder sig til nogle af de mange udfordringer, som digital bevaring rummer, og der gøres opmærksom på den uensartede digitale bevaring i virksomheder. Problemerne adresseres både i et generelt perspektiv og gennem en række erfaringsbaserede scenarier. De eksisterende digitale tilbud, der i dag står til rådighed for virksomheder med et bevaringsbehov, skitseres kort. Dette leder frem til en diskussion vedrørende de fremtidige udfordringer, som både virksomheder, samfund og historikere vil stå overfor, hvis der ikke snart gøres en aktiv indsats for at gemme mere digital dokumentation for eftertiden. Slutteligt peger vi på mulige løsninger, der kan være relevante at overveje blandt historikere, arkivarer, dokumentationsspecialister og virksomheder.--- What will be left for the Future? Business Archives in the Digital Age Managing the past is a complex and difficult task. In this article we discuss digital preservation by focusing on what Danish companies do to preserve their company history. We examine how companies handle the multiple challenges that are posed by digital preservation and point to the presently uneven and lopsided preservation practices of business records. The problems associated herewith are addressed in a general perspective and through a series of experience based scenarios. Existing commercial as well as non-commercial solutions for companies interested in digital preservation are briefly outlined. This leads to an analysis of future challenges for companies and business historians alike if we do not immediately succeed in preserving more digital information. We conclude by pointing to a number of issues that calls for immediate and careful attention among historians, archivists, records managers and business leaders if we want the past to have a future

DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations

Treatment of HIV-infected individuals with antiretroviral agents selects for drug-resistant mutants, resulting in frequent treatment failures. Although the major antiretroviral resistance mutations are routinely characterized by DNA sequencing, treatment failures are still common, probably in part because undetected rare resistance mutations facilitate viral escape. Here we combined DNA bar coding and massively parallel pyrosequencing to quantify rare drug resistance mutations. Using DNA bar coding, we were able to analyze seven viral populations in parallel, overall characterizing 118 093 sequence reads of average length 103 bp. Analysis of a control HIV mixture showed that resistance mutations present as 5% of the population could be readily detected without false positive calls. In three samples of multidrug-resistant HIV populations from patients, all the drug-resistant mutations called by conventional analysis were identified, as well as four additional low abundance drug resistance mutations, some of which would be expected to influence the response to antiretroviral therapy. Methods for sensitive characterization of HIV resistance alleles have been reported, but only the pyrosequencing method allows all the positions at risk for drug resistance mutations to be interrogated deeply for many HIV populations in a single experiment

Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations

Emergence of Minor Drug-Resistant HIV-1 Variants after Triple Antiretroviral Prophylaxis for Prevention of Vertical HIV-1 Transmission

Background: WHO-guidelines for prevention of mother-to-child transmission of HIV-1 in resource-limited settings recommend complex maternal antiretroviral prophylaxis comprising antenatal zidovudine (AZT), nevirapine single-dose (NVP-SD) at labor onset and AZT/lamivudine (3TC) during labor and one week postpartum. Data on resistance development selected by this regimen is not available. We therefore analyzed the emergence of minor drug-resistant HIV-1 variants in Tanzanian women following complex prophylaxis. Method: 1395 pregnant women were tested for HIV-1 at Kyela District Hospital, Tanzania. 87/202 HIV-positive women started complex prophylaxis. Blood samples were collected before start of prophylaxis, at birth and 1–2, 4–6 and 12–16 weeks postpartum. Allele-specific real-time PCR assays specific for HIV-1 subtypes A, C and D were developed and applied on samples of mothers and their vertically infected infants to quantify key resistance mutations of AZT (K70R/T215Y/T215F), NVP (K103N/Y181C) and 3TC (M184V) at detection limits of,1%. Results: 50/87 HIV-infected women having started complex prophylaxis were eligible for the study. All women took AZT with a median duration of 53 days (IQR 39–64); all women ingested NVP-SD, 86 % took 3TC. HIV-1 resistance mutations were detected in 20/50 (40%) women, of which 70 % displayed minority species. Variants with AZT-resistance mutations were found in 11/50 (22%), NVP-resistant variants in 9/50 (18%) and 3TC-resistant variants in 4/50 women (8%). Three wome

Nevirapine- Versus Lopinavir/Ritonavir-Based Initial Therapy for HIV-1 Infection among Women in Africa: A Randomized Trial

In a randomized control trial, Shahin Lockman and colleagues compare nevirapine-based therapy with lopinavir/ritonavir-based therapy for HIV-infected women without previous exposure to antiretroviral treatment