Phylogenetic analysis of Brazilian bovine viral diarrhea virus type 2 (BVDV-2) isolates: evidence for a subgenotype within BVDV-2

Phylogenetic analysis divides bovine viral diarrhea viruses (BVDV) into two different genotypes (BVDV1 and BVDV2). BVDV1 strains have been further subdivided into two to 11 subgenotypes. Phylogenetic analysis of BVDV2 isolates, however, has not been able to identify discrete subgenotypes. In this study, we identified six South American BVDV2 strains and one North American BVDV2 strain that cluster to a separate genetic group within BVDV2, thus representing a distinct subgenotype. The 5’ untranslated region (UTR) sequence homology between these six strains and other BVDV2 from North America, Europe and Asia (81.7%) is lower than the homology used to segregate BVDV1 into BVDV1a and BVDV1b (83.6%). Most nucleotide differences observed between the two subgroups of BVDV2 were concentrated in two regions, which also harbor most of the differences seen between BVDV1a and BVDV1b. To determine if this segregation was real, an additional analysis was performed comparing NS2/3 sequences. Analysis of a conserved sequence located between nucleotides 6670 and 7186 of the NS2/3 coding region also segregated these isolates to a separate group. The sequence homology between the two subgroups (86.3%) was higher than the homology in the 5’UTR (81.7%), with mean sequence homologies of 91 and 87.2% within the proposed subgroups. In contrast to the 5’UTR, alignment of the NS2/3 sequences revealed nucleotide differences distributed across the region. These results demonstrate that BVDV2 isolates cluster to two genetically distinct subgroups within BVDV2. The differences in both the 5’UTR and NS2/3 are consistent and justify this segregation. We suggest that BVDV2 may thereafter be subgenotyped into BVDV2a and BVDV2b. The existence of subgroups within the BVDV2 genotype with genetic heterogeneity similar to that seen among BVDV1 subgroups argues against BVDV2 isolates arising from BVDV1 in a recent evolutionary event. Unless the evolutionary clocks for BVDV1 and BVDV2 isolates tick along at different rates, these results indicate that BVDV2 have existed as long as BVDV1

A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate

Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis

Early molecular markers predictive of dengue hemorrhagic fever

The management of acute dengue patients during outbreaks is a challenging problem. Most of the dengue fever cases are benign, but some cases develop into a severe and possibly lethal vasculopathy, known as dengue hemorrhagic fever. Early symptoms of dengue and hemorrhagic fever are very similar. An early differential diagnosis is needed to predict which of these two clinical presentations is crucial to proper patient care and public health management. This study evaluates the predictive potential of specific mRNA expression markers of dengue hemorrhagic fever using quantitative real-time PCR assays. Six candidate "dengue hemorrhagic fever specific signature genes" were evaluated and all showed good correlation among their transcription levels at early days of infection and the later development of severe vasculopathy. The markers selected were able to indicate, at early stages of infection, the evolution of a dengue-infected patient to the severe form of the illness. Despite the fact that these results grant further validation studies, the panel of candidate prognostic markers obtained demonstrated the potential to be useful for clinical use in the form of a fast assay based in blood samples

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd

Complement factor H gene (CFH) polymorphisms C-257T, G257A and haplotypes are associated with protection against severe dengue phenotype, possible related with high CFH expression

Four genetic polymorphisms located at the promoter (C-257T) and coding regions of CFH gene (exon 2 G257A, exon 14 A2089G and exon 19 G2881T) were investigated in 121 dengue patients (DENV-3) in order to assess the relationship between allele/haplotypes variants and clinical outcomes. A statistical value was found between the CFH-257T allele (TT/TC genotypes) and reduced susceptibility to severe dengue (SD). Statistical associations indicate that individuals bearing a T allele presented significantly higher protein levels in plasma. The 257T variant is located within a NF-jB binding site, suggesting that this variant might have effect on the ability of the CFH gene to respond to signals via the NF-jB pathway. The G257A allelic variant showed significant protection against severe dengue. When CFH haplotypes effect was considered, the ancestral CG/CG promoter-exon 2 SNP genotype showed significant risk to SD either in a general comparison (ancestral all variant genotypes), as well as in individual genotypes comparison (ancestral each variant genotype), where the most prevalent effect was observed in the CG/ CG CA/TG comparison. These findings support the involvement of 257T, 257A allele variants and haplotypes on severe dengue phenotype protection, related with high basal CFH expression