CRISPR/Cas-9 vector system: targets UL-39 and inhibits Simplexvirus humanalpha1 (HSV-1) replication in vitro.

Simplexvirus humanalpha1 (HSV-1) affects approximately 67% of the world's population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into the plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyses revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas 9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.

Introduction of human gammaherpesvirus 8 genotypes A, B, and C into Brazil from multiple geographic regions.

Variations in the open reading frame (ORF) K1 gene sequence of human gammaherpesvirus 8 (HHV-8) has led to the identification of 6 major genotypic clades (A, B, C, D, E, and F) in specimens isolated from around the world. These clades exhibit clear clustering among individuals in different ethnic groups and from different geographic regions. The human population of Brazil varies greatly in ethnicity because of multiple immigration events from Africa, Europe, Asia, and indigenous communities. However, there is scant information about the HHV-8 genotypes currently circulating in Brazil. Here, we describe HHV-8 genotypic diversity in isolates from Brazilian HIV-infected patients living with Kaposi's sarcoma (KS) by analysis of the complete ORF-K1 region. We also identified the most likely geographic origins of these different Brazilian genotypes. We extracted HHV-8 DNA (24 positive samples) from individuals with HIV/KS from the states of São Paulo and Rio de Janeiro, amplified the ORF-K1 gene using nested PCR (about 870 base pairs), performed sequencing and phylogenetic analysis, and then calculated the mean genetic distances of Brazilian sequences from sequences in other regions of the world (523 sequences analyzed). Phylogenetic analysis showed that genotypes C, A, and B were present in 45.8 %, 29.2 % and 25 % of the isolates from Brazil, respectively. These isolates grouped into separate clades, rather than a single monophyletic cluster. Mean genetic distance analyses suggested that these genotypes were introduced into the Brazil multiple times from different geographical regions. HHV-8/A isolates appear to be from Ukraine, Russia, and the Tartar ethnic group; HHV-8/B isolates appear to be from Congo and Democratic Republic of the Congo; and HHV-8/C isolates appear to be from Australia, Algeria, England, and French Guiana. These results contribute to a better understanding of the genetic diversity and origins of HHV-8 strains circulating in Brazil, and will provide a foundation for further epidemiological and evolutionary studies of HHV-8.