Possible recombination and gene adaptation exchanges among clinical echovirus strains: crossing the temporal and topological barriers

Six echovirus strains belonging to serotypes echovirus 6, 13, and 30 were investigated in the present work by sequencing of the whole 2C gene and about 560 nt of the 5' part of 3-dimensional genomic region. Four of the 6 echovirus strains were epidemics, whereas 2 were from sporadic cases. The whole procedure was carried out by using nucleotide distance matrices and phylogeny software. The sequences obtained strengthen the observation that recent echovirus isolates differ significantly frorn prototype strains in the downstream regions of the genome and provides further evidence that nonstructural enterovirus genes are ubiquitous and may combine freely adapting genomic sequences that are not restricted from the place of isolates' origin. For diagnostic purposes, particular emphasis is given on the utility of sequencing downstream genes and comparison of them with corresponding genomic regions front enteroviral strains that circulated all over the world. (C) 2007 Elsevier Inc. All rights reserved

Mechanistic basis for morphological damage induced by essential oil from Brazilian pepper tree, Schinus terebinthifolia, on larvae of Stegomyia aegypti, the dengue vector

BACKGROUND: Dengue has become the subject of public health programs worldwide. The lack of a vaccine and the high environmental risk of synthetic insecticides, arouse the interest in natural products against this vector. This study aimed to determine the chemical composition of the essential oil of ripe fruits and seeds of Schinus terebinthifolia Raddi; to evaluate the essential oil effect on mortality of Stegomyia aegypti (Linnaeus, 1792) larvae; and to characterize the structural damage suffered by larvae and their association with different contents of essential oil. METHODS: Ripe fruits and seeds were crunched and their essential oil was extracted through hydrodistillation, purified, and its phytochemical analysis was carried out through High Resolution Gas Chromatography, coupled with Mass Spectrometry. This essential oil was diluted in a 10-point gradient of 86.22 – 862.20 ppm, at regular intervals of 86.22 ppm. Each point received 50 larvae and the assessments of surviving were made at 24, 48 and 72 hours after inoculation. Structural damage was assessed through measurements of thickness with exoskeleton, evaluating the integrity of the head, thorax, abdominal segments, and air siphon, using ImageJ® software. Statistical data analysis was carried out through Logistic Regression and Discriminant Analysis. RESULTS: 56 substances were identified, corresponding to 81.67% of the essential oil composition. Larvae were dose-dependent susceptible to the essential oil; the concentration produced a significant effect on larval mortality. Among the major deformations found in the larvae, it was detected inhibition of chitin synthesis by the activity of the oil, thus reducing the deposition of cuticle layers. CONCLUSION: The essential oil caused death in exposed larvae after 72 hours, in a dose-dependent manner. It also changed the structure of exposed larvae, indicating a direct effect on larval exoskeleton. The results open up possibilities for the use of natural products as an alternative to control dipterans

Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece

Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties

Direct extraction and molecular characterization of enteroviruses genomes from human faecal samples

Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several enteroviruses serotypes are not easily isolated in cell cultures system used and routinely more than one passage in cell culture is performed. A total of 54 archived faecal samples were examined. The heterogeneous nature of faecal samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. PCR inhibitors are frequently encountered in stool specimens. From the three methods initially compared for extraction of viral RNA, QIAamp Viral RNA Mini Kit was retained as it yielded the highest amount of viral RNA without the interference of RTPCR inhibitors. Evaluation of 54 archived stool specimens by RT-PCR and cell culture resulted in a higher frequency of detection by RT-PCR. With the use of RT-PCR we were able to detect two additional samples otherwise considered negative for enterovirus isolation if only the cell culture standard methodology was employed. RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR in the 5'NCR (sub-grouping into distinct genetic clusters of all enteroviruses) and VP1 (reliable serotyping by sequencing) is a rapid and sensitive technique of direct poliovirus/non-polio enteroviruses recovery and molecular characterization from human faecal specimens without further passage in cell culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen. (C) 2008 Elsevier Ltd. All rights reserved

A new RT-PCR assay for the identification of the predominant recombination types in 2C and 3D genomic regions of vaccine-derived poliovirus strains

In the post-eradication era of wild polioviruses, the only remaining sources of poliovirus infection worldwide would be the vaccine-derived polioviruses (VDPVs). As the preponderance of countries certified to be polio-free has switched from OPV (oral poliovirus vaccine) to IPV (inactivated poliovirus vaccine), importation of recombinant evolved derivatives of vaccinal strains would have serious implication for public health. To test the robustness of the proposed RT-PCR screening analysis, eleven recombinant vaccine-derived polioviruses that were characterized previously by sequencing by our group, in addition to three recently identified recombinant environmental isolates were assayed. Although the most definitive characterization of VDPVs is by genomic sequencing, in this study we describe a new, inexpensive and broadly applicable RT-PCR assay for the identification of the predominant recombination types S3/Sx in 2C and S2/Sx in 3D genomic regions respectively of VDPVs, that can be readily implemented in laboratories lacking sequencing facilities as a first approach for the early detection of vaccine-derived poliovirus (VDPVs). (C) 2009 Elsevier Ltd. All rights reserved