Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model.

West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal Middle-East WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 700 × in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.

Full-Genome Sequencing of Four Bluetongue Virus Serotype 11 Viruses.

Recently, a contamination incident was described in which the challenge inoculum used in a bluetongue virus serotype 8 (BTV-8) vaccination trial was contaminated with a BTV-11 virus that was closely related to the Belgian BTV-11 virus from 2008. This study reports the first complete genome sequences of four BTV-11 viruses: the BTV-11 contaminant, BTV-11 reference strain, BTV-11 vaccine strain and a recently isolated BTV-11 field strain from Martinique. Full-genome analysis showed that these viruses belong to serotype 11/nucleotype A and cluster together with other western topotype bluetongue viruses. Detailed comparisons of the genomes further indicated that the contaminant was derived from the BTV-11 reference strain, as they were distinguished by a single synonymous nucleotide substitution. The previously reported partial sequence of genome segment 2 of the Belgian BTV-11 was found to be identical to that of the BTV-11 vaccine strain, indicating that it most likely was the BTV-11 vaccine strain. These findings also suggest that the BTV-11 contaminant and the Belgian BTV-11 are not the same viruses. Finally, comparison of the reference and vaccine strain did not allow determining the amino acid substitutions that contribute to the attenuated phenotype.

False-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis.

A viral metagenomic approach using virion enrichment, random amplification and next-generation sequencing was used to investigate an undiagnosed cluster of dairy cattle presenting with high persistent fever, unresponsive to anti-microbial and anti-inflammatory treatment, diarrhoea and redness of nose and teat. Serum and whole blood samples were taken in the predicted hyperviraemic state of an animal that a few days later presented with these clinical signs. Bioinformatics analysis of the resulting data from the DNA virus identification workflow (a total of 32 757 sequences with average read length 335 bases) initially demonstrated the presence of parvovirus-like sequences in the tested blood sample. Thorough follow-up using specific real-time RT-PCR assays targeting the detected sequence fragments confirmed the presence of these sequences in the original sample as well as in a sample of an additional animal, but a contamination with an identical genetic signature in negative extraction controls was demonstrated. Further investigation using an alternative extraction method identified a contamination of the originally used Qiagen extraction columns with parvovirus-like nucleic acids or virus particles. Although we did not find any relevant virus that could be associated with the disease, these observations clearly illustrate the importance of using a proper control strategy and follow-up diagnostic tests in any viral metagenomic study.