The genus Phlebovirus includes the sandfly fever viruses and tick-transmitted uukuviruses. Sandfly fever group viruses have been isolated from various vertebrate species and from phlebotomines and occasionally alternative arthropods, e.g. mosquitoes, or ceratopogonids of the genus Culicoides. Uukuniemi serogroup viruses have been isolated from various vertebrate species and from ticks. Despite the public health importance of some viruses of the genus, the genomic diversity of phleboviruses that could be incriminated as causative of human or veterinary diseases remains underestimated. Here we describe the nearly complete sequences and genomic characterization of two phleboviruses belonging to the Bujaru antigenic complex: the prototype species and the Munguba virus. Furthermore, six previously unclassified phleboviruses isolated in Brazil were also sequenced and characterized: Ambe, Anhanga, Joa, Uriurana, Urucuri and Tapara viruses. The results of the phylogenetic analysis indicated that these viruses group with viruses of three antigenic complexes (Bujaru, Tapara and frijoles clades), with two unclassified phleboviruses. We also performed genomic reassortment analysis and confirmed that there were no events for the viruses described in this study, but we found a new potential reassortment in Medjerda Valley virus, which contains S and L segments of Arbia virus, and probably a unique M segment, both viruses circulate in the same geographic region, indicating these two isolates represent two distinct viruses. This study provides insights into the genetic diversity, classification and evolution of phleboviruses.

Mosquito-borne alphaviruses are widely distributed throughout the world, causing important human illnesses. Therefore, the development of methods to enable early diagnosis of infections by alphavirus is essential. We show here the development and evaluation of a quantitative real-time RT-PCR using genus-specific primers to the nsP1 viral gene of all mosquito-borne alphaviruses. The specificity and sensitivity of the assay were tested using seven alphaviruses and RNA transcribed from Venezuelan equine encephalitis virus. The detection limits of real-time RT-PCR ranged from 10 to 76 copies per ml. The melting temperature (T M ) values for amplification of the alphavirus genomes were 83.05°C and 85.28°C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with T M peaks of 84.83 to 85.28°C and encephalitic alphaviruses of 83.34°C to 84.68°C could be discriminated both diseases. Real-time RT-PCR may prove very useful for the screening and preliminary diagnosis in outbreaks and surveillance studies as well as for measuring the viral load in pathogenesis studies.

Antimicrobial peptides are critical actors of the innate immune system. Those with direct antibacterial activity often kill via cell membrane lysis, but have difficulty distinguishing between target cells and mammalian cells complicating therapeutic use. Here, we use a new technique called deep mutational surface localized antimicrobial display (dmSLAY) to rapidly elucidate single residue importance and flexibility for Protegrin-1, a potent yet toxic host-defense peptide. Subsequent biochemical analyses reveal that bacterial membrane selectivity is improved for Protegrin-1 variants which maintain membrane bound secondary structure, avoid large aromatic residues, and mutate cysteine pairs. Machine learning is used to expand our analysis to encompass over five million sequence variations and uncover full mutational profiles which promote antibacterial potency, toxicity, and specificity for bacterial or mammalian cell membranes. Our results describe an innovative, high-throughput approach for rapidly elucidating antimicrobial peptide sequence-structure-function relationships which can be used to inform future clinical antibiotic design.

Introduction:The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. Methods: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. Results: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×10 7 to 3.4×10 3 copies per ml. Conclusions: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

Parvoviruses (family Parvoviridae) are small, single-stranded DNA viruses. Many 30 parvoviral pathogens of medical, veterinary and ecological importance have been identified. In 31 this study, we used high-throughput sequencing (HTS) to investigate the diversity of parvoviruses 32 infecting wild and domestic animals in Brazil. We identified 21 parvovirus sequences (including 33 twelve nearly complete genomes and nine partial genomes) in samples derived from rodents, bats, 34 opossums, birds and cattle in Pernambuco, São Paulo, Paraná and Rio Grande do Sul states. These 35 sequences were investigated using phylogenetic and distance-based approaches, and were thereby 36 classified into eight parvovirus species (six of which have not been described previously), 37representing six distinct genera in the subfamily Parvovirinae. Our findings extend the known 38 biogeographic range of previously characterized parvovirus species, and the known host range of 39 three parvovirus genera (Dependovirus, Aveparvovirus, and Tetraparvovirus). Moreover, our 40 investigation provides a window into the ecological dynamics of parvovirus infections in 41 vertebrates, revealing that many parvovirus genera contain well-defined sub-lineages that 42 circulate widely throughout the world within particular taxonomic groups of hosts. 43