Characterization and Whole Genome Analysis of Human Papillomavirus Type 16 E1-1374^63nt Variants

Background. The variation of the most common Human papillomavirus (HPV) type found in cervical cancer, the HPV16, has been extensively investigated in almost all viral genes. The E1 gene variation, however, has been rarely studied. The main objective of the present investigation was to analyze the variability of the E6 and E1 genes, focusing on the recently identified E1-1374^63nt variant. Methodology/Principal Findings. Variation within the E6 of 786 HPV16 positive cervical samples was analyzed using high-resolution melting, while the E1-1374^63nt duplication was assayed by PCR. Both techniques were supplemented with sequencing. The E1-1374^63nt duplication was linked with the E-G350 and the E-C109/G350 variants. In comparison to the referent HPV16, the E1-1374^63nt E-G350 variant was significantly associated with lower grade cervical lesions (p=0.029), while the E1-1374^63nt E-C109/G350 variant was equally distributed between high and low grade lesions. The E1-1374^63nt variants were phylogenetically closest to E-G350 variant lineage (A2 sub-lineage based on full genome classification). The major differences between E1-1374^63nt variants were within the LCR and the E6 region. On the other hand, changes within the E1 region were the major differences from the A2 sub-lineage, which has been historically but inconclusively associated with high grade cervical disease. Thus, the shared variations cannot explain the particular association of the E1-1374^63nt variant with lower grade cervical lesions. Conclusions/Significance. The E1 region has been thus far considered to be well conserved among all HPVs and therefore uninteresting for variability studies. However, this study shows that the variations within the E1 region could possibly affect cervical disease, since the E1-1374^63nt E-G350 variant is significantly associated with lower grade cervical lesions, in comparison to the A1 and A2 sub-lineage variants. Furthermore, it appears that the silent variation 109T>C of the E-C109/G350 variant might have a significant role in the viral life cycle and warrants further study

Metabolic characterization of serum from mice challenged with Orientia tsutsugamushi–infected mites

Scrub typhus is an acute zoonosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. To better understand the host response elicited by natural infection by chigger feeding, ICR mice were infected by Leptotrombidium chiangraiensis (Lc1) chiggers, and the metabolic profiles of their serum were examined over several time points after initiation of feeding. ICR mice were infected by either naive Lc1 chiggers (i.e. not infected by O. tsutsugamushi, NLc1) or O. tsutsugamushi–infected Lc1 chiggers (OLc1). Serum was collected from both groups of mice at 6 hours and 10 days after initiation of feeding. Metabolites were extracted from the serum and analysed by ultra performance liquid chromatography–tandem mass spectrometry. The resulting ion/chromatographic features were matched to a library of chemical standards for identification and quantification. Biochemicals that differed significantly between the experimental groups were identified using Welch's two-sample t tests; p ≤ 0.05 was considered statistically significant. A number of biochemicals linked to immune function were found to be significantly altered between mice infected by the NLc1 and OLc1 chiggers, including itaconate, kynurenine and histamine. Several metabolites linked to energy production were also found to be altered in the animals. In addition lipid and carbohydrate metabolism, bile acid and phospholipid homeostasis, and nucleotide metabolism were also found to be different in these two groups of mice. Markers of stress and food intake were also significantly altered. Global untargeted metabolomic characterization revealed significant differences in the biochemical profiles of mice infected by the NLc1 versus OLc1 chiggers. These findings provide an important platform for further investigation of the host responses associated with chigger-borne O. tsutsugamushi infections. Keywords: Chiggers, mass spectrometer, metabolites, metabolomics, mouse model, orientia tsutsugamushi, tryptopha

Design of a Bacteriophage Cocktail Active against Shigella Species and Testing of Its Therapeutic Potential in Galleria mellonella

Shigellosis is a leading global cause of diarrheal disease and travelers’ diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival