Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation

Abstract INTRODUCTION: Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. METHODS: HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. RESULTS: The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. CONCLUSIONS: HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Primary dengue haemorrhagic fever in patients from northeast of Brazil is associated with high levels of interferon-β during acute phase

Dengue is an acute febrile disease caused by the mosquito-borne dengue virus (DENV) that according to clinical manifestations can be classified as asymptomatic, mild or severe dengue. Severe dengue cases have been associated with an unbalanced immune response characterised by an over secretion of inflammatory cytokines. In the present study we measured type I interferon (IFN-I) transcript and circulating levels in primary and secondary DENV infected patients. We observed that dengue fever (DF) and dengue haemorrhagic fever (DHF) patients express IFN-I differently. While DF and DHF patients express interferon-α similarly (52,71 ± 7,40 and 49,05 ± 7,70, respectively), IFN- β were associated with primary DHF patients. On the other hand, secondary DHF patients were not able to secrete large amounts of IFN- β which in turn may have influenced the high-level of viraemia. Our results suggest that, in patients from our cohort, infection by DENV serotype 3 elicits an innate response characterised by higher levels of IFN- β in the DHF patients with primary infection, which could contribute to control infection evidenced by the low-level of viraemia in these patients. The present findings may contribute to shed light in the role of innate immune response in dengue pathogenesis.

Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Potential biomarkers for the clinical prognosis of severe dengue

Currently, several assays can confirm acute dengue infection at the point-of-care. However, none of these assays can predict the severity of the disease symptoms. A prognosis test that predicts the likelihood of a dengue patient to develop a severe form of the disease could permit more efficient patient triage and treatment. We hypothesise that mRNA expression of apoptosis and innate immune response-related genes will be differentially regulated during the early stages of dengue and might predict the clinical outcome. Aiming to identify biomarkers for dengue prognosis, we extracted mRNA from the peripheral blood mononuclear cells of mild and severe dengue patients during the febrile stage of the disease to measure the expression levels of selected genes by quantitative polymerase chain reaction. The selected candidate biomarkers were previously identified by our group as differentially expressed in microarray studies. We verified that the mRNA coding for CFD, MAGED1, PSMB9, PRDX4 and FCGR3B were differentially expressed between patients who developed clinical symptoms associated with the mild type of dengue and patients who showed clinical symptoms associated with severe dengue. We suggest that this gene expression panel could putatively serve as biomarkers for the clinical prognosis of dengue haemorrhagic fever.