RESUMO
Volunteer water monitoring programs generate new scientific knowledge, contribute data to decision-making processes, and increase social networks, technical knowledge, and skills of participants. Declaration of the COVID-19 pandemic threatened the ability of these programs to continue to engage volunteers to achieve such outcomes. A national water monitoring network hosted a brainstorming webinar to facilitate communication across programs to identify potential solutions to pandemic-influenced challenges. Following that webinar, a survey of United States and Canadian volunteer monitoring programs that was conducted about 3 months into the pandemic revealed that 72% of 80 responding programs planned to carry on through the 2020 field season despite most having experienced delayed starts. Other common program modifications implemented in the first months of the pandemic included adding COVID-19 safety information to program guidance, changing field team composition, monitoring timing and logistics, and adopting new communications strategies. Most programs reported loss or anticipated loss in number of data observations (74%) and volunteers (66%), while 44% reported known or anticipated losses in funding. Seventeen percent of responding programs were able to swiftly develop distance learning tools to train participants, which led to increased program capacity to reach broader audiences.
RESUMO
BACKGROUND: Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia-Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied. METHODS: We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p_gt1b). The boceprevir EC50 value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays. RESULTS: The boceprevir EC50 value for the gt6NS3p_gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p_gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir. CONCLUSIONS: A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease.
Assuntos
Farmacorresistência Viral/genética , Genótipo , Hepacivirus/genética , Hepatite C/virologia , Mutação , Prolina/análogos & derivados , Replicon , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Hepatite C/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Prolina/química , Prolina/farmacologia , Prolina/uso terapêutico , Recombinação Genética , Proteínas não Estruturais Virais/química , Replicação ViralRESUMO
Virion infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary human CD4 T lymphocytes and macrophages. Vif overcomes the HIV-inhibitory effects of cellular factor APOBEC3G, which has cytidine deaminase activity. We previously reported the isolation of a Vif-interacting ring finger protein, Triad 3, from a human leukocyte cDNA library, using the yeast two-hybrid system. The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NF-kappaB (ZIN). Sequence analysis indicates that Triad 3 protein contains all four major ring-like motifs of ZIN. We report here that ZIN binds to purified Vif in vitro and that Triad 3/ZIN interacts with HIV-1 Vif in transfected human 293T cells, as demonstrated by coimmunoprecipitation. To test the biological relevance of this interaction, we produced infectious HIV-1 NL4.3 in the presence or absence of cotransfected ZIN. HIV-1 NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were twofold less infectious in a single-cycle infectivity assay than virus produced in the absence of exogenous ZIN. It was further shown that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were impaired in viral DNA synthesis by twofold. The impairment in viral reverse transcription and the reduction in single-cycle viral infectivity were both shown to be dependent on the presence of Vif in the virus producer cells. The possible mechanisms by which ZIN interferes with the early events of HIV-1 replication are discussed.