Genomic and epidemiological monitoring of yellow fever virus transmission potential.

The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.

CD4+ T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes.

Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4(+) and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

Pseudo-peptides derived from isomannide: inhibitors of serine proteases.

In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown.

Enfuvirtide (T-20) resistance-related mutations in HIV type 1 subtypes B, C, and F isolates from Brazilian patients failing HAART.

The synthetic peptide T-20 (enfuvirtide, EFV) represents the first compound approved by the FDA known as entry inhibitors (EIs). The resistance mutations associated with this new class of antiretroviral drug are located in the first heptad repeat (HR1) region of gp41. Amino acid changes in codons G36D/S, I37V, V38A/M/E, Q39H/R, Q40H, N42T, and N43D can confer resistance to EFV. In this work we investigated the presence of resistance mutations that occur in patients never treated with EFV and failing HAART with protease inhibitors (PIs), nucleoside reverse transcriptase (RT) inhibitors (NRTIs), and nonnucleoside RT inhibitors (NNRTIs). This knowledge can reveal whether this salvage therapy can be effective in patients failing HAART. For this, we amplified 65 samples from plasma isolates and than sequenced a fragment of 416 nt encompassing the HR1 and HR2 regions (amino acids 33-170 of gp41). The subtype distribution among the 65 isolates was 45 (69.23%) subtype B, 9 (13.85%) subtype C, 7 (10.77%) subtype F1, and 4 (6.15%) mosaics B/F1, B/C, F1/C, and C/F1/B. We found a high prevalence (7.6%) of EFV-associated mutation G36D in this cohort of patients failing HAART therapy, five isolates from subtype B (11.11% within this group). In contrast, when 1079 sequences from drug-naive patients were analyzed, only one showed the G36D substitution. This finding indicates a strong association between the selected position G36D and HAART therapy (p < 0.0001). The isolates that possess these mutations can develop resistance to EFV more rapidly. Nevertheless, more information about the impact of these mutations in salvage therapy with EFV in patients failing HAART must still be obtained.

Drug resistance mutation profile and accumulation kinetics in human immunodeficiency virus-positive individuals infected with subtypes B and F failing highly active antiretroviral therapy are influenced by different viral codon usage patterns.

The major human immunodeficiency virus type 1 subtype circulating in Brazil is B, followed by F and C. We have genotyped 882 samples from Brazilian patients for whom highly active antiretroviral therapy failed, and we found subtype B and the unique recombinant B/F1 forms circulating. Due to codon usage variation, there is a significantly lower incidence of the substitutions L210W, Q151M, and F116Y in subtype F1 isolates than in the subtype B counterparts.

Genetic variability of HIV-1 protease from Nigeria and correlation with protease inhibitors drug resistance.

In Nigeria, the most populous country in Africa, the characterization of HIV-1 strains has been limited. In this study we evaluated the genetic diversity of the protease coding region, one of the anti-retroviral therapy target, and investigated the presence of mutations related to resistance to HIV protease inhibitors. We analyzed samples collected during 1996 and all patients were anti-retroviral drug naïves. Ten samples were evaluated by sequencing of the protease gene. The majority, 80%, were classified as subtype A and the two others were unclassified-divergent strains, something in between A and G subtypes. The gag region from these outliners were sequenced and the phylogenetic analysis classified them as subtype G. The protease amino acid consensus sequence of the Nigerian subtype A are in complete agreement with the consensus A differing from the USA subtype B consensus in 10 positions (L10V, I13V, K14R, I15V, K20I, M36I, R41K, P63L, H69K and L89M). The secondary substitutions associated with protease inhibitor resistance were observed in all Nigerian sequences at the positions L10V, M36I and L89M. The majority of sequence variation was concentrated in the interval between aminoacids 70-90 where the protease substrate binding region is located.

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity.

1. Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. 2. Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end. Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. 3. The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay. The full-length replicase is active. The aminoterminal truncated enzyme had 0.02% activity of the intact one. 4. This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase.

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli.

1. The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. 2. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10(5). The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha 32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. 3. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. 4. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses.

The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity

Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. Two constructions were developed to express either a full-lenghtt or truncated enzyme lacking the 20 aminoacids at the N-terminal en. Bacterial extr5acts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. The biological activity of these two molecules was tested using a poly(A)-dep[endent oligo(U)-primed poly(U)-polymer4ase assay. The full-lenght replicase is active. The aminoterminal truncated wnzyme had 0.02% activity o9f the intact5 one. This result indicates the importaqnce of the twenty N-terminal amino acids for the activity of FMDV RNA dependent RNMA polymerase

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli

The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10 5. The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other piconaviruses

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli.

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to rebuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid.

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid