Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae.

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

Interaction of the Zika virus with the cytoplasmic dynein-1.

Zika virus (ZIKV) infection is a major public health threat, making the study of its biology a matter of great importance. By analyzing the viral-host protein interactions, new drug targets may be proposed. In this work, we showed that human cytoplasmic dynein-1 (Dyn) interacts with the envelope protein (E) of ZIKV. Biochemical evidence indicates that the E protein and the dimerization domain of the heavy chain of Dyn binds directly without dynactin or any cargo adaptor. Analysis of this interactions in infected Vero cells by proximity ligation assay suggest that the E-Dyn interaction is dynamic and finely tuned along the replication cycle. Altogether, our results suggest new steps in the replication cycle of the ZIKV for virion transport and indicate a suitable molecular target to modulate infection by ZIKV.

Pseudomonas-tailed lytic phages: genome mechanical analysis and putative correlation with virion morphogenesis yield.

Aim: To unveil a putative correlation between phage genome flexibility and virion morphogenesis yield. Materials & methods: A deeper analysis of the mechanical properties of three Pseudomonas aeruginosa lytic phage genomes was undertaken, together with full genome cyclizability calculations. Results & conclusion: A putative correlation was established among phage genome flexibility, eclipse timeframe and virion particle morphogenesis yield, with a more flexible phage genome leading to a higher burst size and a more rigid phage genome leading to lower burst sizes. The results obtained are highly relevant to understand the influence of the phage genome plasticity on the virion morphogenesis yield inside the infected bacterial host cells and assumes particular relevance in the actual context of bacterial resistance to antibiotics.

A Novel Lineage of Cile-Like Viruses Discloses the Phylogenetic Continuum Across the Family Kitaviridae.

An increasing number of plant species have been recognized or considered likely reservoirs of viruses transmitted by Brevipalpus mites. A tiny fraction of these viruses, primarily those causing severe economic burden to prominent crops, have been fully characterized. In this study, based on high-throughput sequencing, transmission electron microscopy analyses of virions in plant-infected tissues, viral transmission experiments, and the morphoanatomical identification of the involved Brevipalpus mites, we describe molecular and biological features of viruses representing three new tentative species of the family Kitaviridae. The genomes of Solanum violifolium ringspot virus (SvRSV, previously partially characterized), Ligustrum chlorotic spot virus (LigCSV), and Ligustrum leprosis virus (LigLV) have five open reading frames (ORFs) > 500 nts, two distributed in RNA1 and three in RNA2. RNA1 of these three viruses display the same genomic organization found in RNA1 of typical cileviruses, while their RNA2 are shorter, possessing only orthologs of genes p61, p32, and p24. LigCSV and LigLV are more closely related to each other than to SvRSV, but the identities between their genomic RNAs were lower than 70%. In gene-by-gene comparisons, ORFs from LigCSV and LigLV had the highest sequence identity values (nt sequences: 70-76% and deduced amino acid sequences: 74-83%). The next higher identity values were with ORFs from typical cileviruses, with values below 66%. Virions of LigLV (≈ 40 nm × 55 nm) and LigCSV (≈ 54 nm × 66 nm) appear almost spherical, contrasting with the bacilliform shape of SvRSV virions (≈ 47 nm × 101 nm). Mites collected from the virus-infected plants were identified as Brevipalpus papayensis, B. tucuman, and B. obovatus. Viruliferous B. papayensis mites successfully transmitted LigCSV to Arabidopsis thaliana. SvRSV, LigCSV, and LigLV seem to represent novel sub-lineages of kitaviruses that descent on parallel evolutionary branches from a common ancestor shared with the tentative cile-like virus hibiscus yellow blotch virus and typical cileviruses. Biological and molecular data, notably, the phylogenetic reconstruction based on the RdRp proteins in which strong support for monophyly of the family Kitaviridae is observed, mark an advance in the understanding of kitavirids.

Packaging of Genomic RNA in Positive-Sense Single-Stranded RNA Viruses: A Complex Story.

The packaging of genomic RNA in positive-sense single-stranded RNA viruses is a key part of the viral infectious cycle, yet this step is not fully understood. Unlike double-stranded DNA and RNA viruses, this process is coupled with nucleocapsid assembly. The specificity of RNA packaging depends on multiple factors: (i) one or more packaging signals, (ii) RNA replication, (iii) translation, (iv) viral factories, and (v) the physical properties of the RNA. The relative contribution of each of these factors to packaging specificity is different for every virus. In vitro and in vivo data show that there are different packaging mechanisms that control selective packaging of the genomic RNA during nucleocapsid assembly. The goals of this article are to explain some of the key experiments that support the contribution of these factors to packaging selectivity and to draw a general scenario that could help us move towards a better understanding of this step of the viral infectious cycle.

Determinación in silico de las propiedades inmunogénicas de la glicoproteína E de Zika virus / In silico determination of immunogenic properties of glycoprotein E from Zika Virus

Introducción: La infección por Zika virus (ZIKV) ha sido asociada a múltiples complicaciones y nuevas formas de transmisión. La descripción del genoma y la estructura cristalizada permiten desarrollar análisis moleculares, incluyendo las propiedades inmunológicas. Objetivos: En este trabajo, se analiza a la glicoproteína E de ZIKV, con el fin de determinar su utilidad en la creación de una vacuna proteica recombinante. Métodos: Se analizó la glicoproteína E, por medio del software DNASTAR, en base a su antigenicidad de epítopos de células B y MHC-II, estructura secundaria, hidrofilizada, flexibilidad y accesibilidad a solvente en el virión maduro e hidratado. Resultados: Se identificaron 14 sitios antigénicos para células B, de los cuales, 7 comparten su antigenicidad para MHC-II. Al tomar en cuenta los demás parámetros analizados, los sitios se reducen a 3, con longitudes de 13, 9 y 5 aminoácidos. Conclusiones: La glicoproteína E de ZIKV podría desencadenar una respuesta inmune T-dependiente, por tanto, ser útil para la creación de una vacuna proteica recombinante.

Introduction: Zika virus (ZIKV) infection have been associated with multiple complications and new ways of transmission. The description of the genome and the crystalized structure allow the performance of molecular analysis, including immunological properties. Objectives: In this paper, we analyze glycoprotein E from ZIKV to determine its utility in the development of a recombinant protein vaccine. Methods: The protein was analyzed with the software DNASTAR, through the following properties: B cells and MHC-II antigenicity, secondary structure, hydrophilicity, flexibility and solvent-accessibility in the mature and hydrated virion. Results: We identified 14 antigenic sites with B-cells antigenicity, 7 of which shared the antigenicity for MHC-II. Considering other parameters analyzed, sites were reduced to 3, with length of 13, 9 and 5 amino acids. Conclusions: Glycoprotein E, from ZIKV, could trigger a T-dependent immune response, and therefore, may be useful in the creation of a recombinant protein vaccine.

Fine structure analysis of White Spot Syndrome Virus of shrimp

White Spot Syndrome Virus (WSSV) was isolated from diseased shrimps presenting with clinical signs of WSSV infection. The seed virus was identified as WSSV by PCR, and used to inoculate to specific pathogen free (SPF) P. vannamei bloodstocks. WSSV was purified as described by Huang from infected gills from inoculated animals that were homogenized in a blender and partially purified by differential centrifugation. The final purification was carried out using density gradient in 10-40% NaBr and purified virus used for morphological analysis using transmission electron microscopy. A negative staining method using 2% PTA was used for purified virus, and electron staining with lead citrate and uranylacetate was used for ultra thin sections of infected tissues. This analysis determined that i) the spikes of intact virions could be clearly identified on the virion surface and on a partially broken envelopes, ii) the nucleocapsid structures were similar to those previously reported, even though the so called "ring" structure described previously was different and, iii) negative staining of purified WSSV fractions identified nucleosome like structures. 

Fine Structure Analysis of White Spot Syndrome Virus of Shrimp

White Spot Syndrome Virus (WSSV) was isolated from diseased shrimps presenting with clinical signs of WSSV infection. The seed virus was identified as WSSV by PCR, and used to inoculate to specific pathogen free (SPF) P. vannamei bloodstocks. WSSV was purified as described by Huang from infected gills from inoculated animals that were homogenized in a blender and partially purified by differential centrifugation. The final purification was carried out using density gradient in 10-40% NaBr and purified virus used for morphological analysis using transmission electron microscopy. A negative staining method using 2% PTA was used for purified virus, and electron staining with lead citrate and uranylacetate was used for ultra thin sections of infected tissues. This analysis determined that i) the spikes of intact virions could be clearly identified on the virion surface and on a partially broken envelopes, ii) the nucleocapsid structures were similar to those previously reported, even though the so called "ring" structure described previously was different and, iii) negative staining of purified WSSV fractions identified nucleosome like structures.(AU)