Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries.

OBJECTIVES: Sample preparation for high-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences. METHODS: A total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed using different protocols. In total, 712 sequencing libraries were investigated for viral sequence contamination. RESULTS: Among sequences showing similarity to viruses, 493 were significantly associated with the use of laboratory components. Each of these viral sequences had sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components. CONCLUSIONS: We show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover, we show that detection can be problematic due to stochastic appearance and limited non-template controls. Although the exact origin of these viral sequences requires further research, our results support laboratory-component-linked viral sequence contamination of both biological and synthetic origin.

Cases of travel-acquired dengue fever in Denmark 2001-2009.

Dengue fever (DF) remains one of the most important emerging infectious diseases. Whereas DF is well recognized in endemic countries, there are indications that the disease is underdiagnosed among travellers to endemic regions. Here, we present the first descriptive survey on cases of travel-acquired DF imported to Denmark diagnosed at the national reference laboratory for dengue virus diagnostics during a 9-year period. In our study, 16 - 46 travel-acquired dengue virus infections were diagnosed per year. DF is mainly imported by adults, mostly men, returning from Southeast Asian countries. The minimum incidence of dengue virus infection among Danish travellers is estimated to be 4.9 per 100,000 travellers. Our results confirm and expand studies from other European countries, and underline the importance of surveillance based on relevant diagnostic analyses.

Construction, biological activity, and immunogenicity of synthetic envelope DNA vaccines based on a primary, CCR5-tropic, early HIV type 1 isolate (BX08) with human codons.

So far codon-optimized HIV-1 envelope genes have been investigated for the T cell line-adapted strain MN, which differs in several aspects from primary isolates. Envelopes of primary isolates may be more relevant for vaccine purposes. This article describes for the first time the engineering and characterization of four "humanized" genes encoding the secreted gp120/gp140, or the membrane-bound gp150/gp160, of a primary CCR5 tropic, clade B, clinical isolate HIV-1(BX08). The genes were built in fragments for easy cassette exchange of regions important for immunogenicity, function, and expression. The transcription and expression of the synthetic genes in mammalian cell lines were Rev independent and highly increased. Increased expression of membrane-bound gp160 induced a high cytopathic effect in U87.CD4.CCR5 cells. Gene gun and intramuscular DNA vaccination in mice induced a strong specific cytotoxic T lymphocyte response independent of the gene construct, expression level, or DNA immunization route. In contrast, the highest anti-gp120 antibody levels were induced by synthetic genes encoding the secreted glycoproteins followed by gp160/gp150. Unlike HIV-1(MN), HIV-1(BX08) V3 was not immune dominant. Despite the high antibody response only low and inconsistent neutralizing titers to the homologous HIV-1 isolate were measured. However, neutralization of SHIV89.6P could be obtained. Thus, the neutralizing epitopes on the cell line-adapted SHIV89.6P and HIV-1(MN) may be more antigenically available for the cross-neutralizing antibodies induced. In conclusion, complete "humanization" of the DNA vaccine genes failed to induce a consistent neutralizing antibody response, albeit expression and immunogenicity of the primary HIV-1 glycoproteins were greatly improved. Optimization in terms of improving neutralization may require further modifications of the DNA vaccine gene. The synthetic cassette construct described is a convenient tool developed to investigate this further.

Gene gun DNA vaccination with Rev-independent synthetic HIV-1 gp160 envelope gene using mammalian codons.

DNA immunization with HIV envelope plasmids induce only moderate levels of specific antibodies which may in part be due to limitations in expression influenced by a species-specific and biased HIV codon usage. We compared antibody levels, Th1/Th2 type and CTL responses induced by synthetic genes encoding membrane bound gp160 versus secreted gp120 using optimized codons and the efficient gene gun immunization method. The in vitro expression of syn.gp160 as gp120 + gp41 was Rev independent and much higher than a classical wt.gp160 plasmid. Mice immunized with syn.gp160 and wt.gp160 generated low and inconsistent ELISA antibody titres whereas the secreted gp120 consistently induced faster seroconversion and higher antibody titres. Due to a higher C + G content the numbers of putative CpG immune (Th1) stimulatory motifs were highest in the synthetic gp160 gene. However, both synthetic genes induced an equally strong and more pronounced Th2 response with higher IgG1/IgG2a and IFNgamma/IL-4 ratios than the wt.gp160 gene. As for induction of CTL, synthetic genes induced a somewhat earlier response but did not offer any advantage over wild type genes at a later time point. Thus, optimizing codon usage has the advantage of rendering the structural HIV genes Rev independent. For induction of antibodies the level of expression, while important, seems less critical than optimal contact with antigen presenting cells at locations reached by the secreted gp120 protein. A proposed Th1 adjuvant effect of the higher numbers of CpG motifs in the synthetic genes was not seen using gene gun immunization which may be due to the low amount of DNA used.