Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing.

Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4-14 laboratories. Six non-target viruses were detected by three or more laboratories. The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories

Expression et caractérisation de quatre protéines du virus de la maladie de Marek homologues aux protéines majeures de tégument VP22, VP16, VP13/14 et VP11/12 du virus HSV-1

*INRA C.R. Tours, Unité de Pathologie aviaire et Parasitologie, 37380 Nouzilly (FRA) Diffusion du document : INRA C.R. Tours, Unité de Pathologie aviaire et Parasitologie, 37380 Nouzilly (FRA) Diplôme : Dr. d'Universit

Characterization of Marek's Disease Virus Serotype 1 (MDV-1) Deletion Mutants That Lack UL46 to UL49 Genes: MDV-1 UL49, Encoding VP22, Is Indispensable for Virus Growth

Experiments were conducted to investigate the roles of Marek's disease virus serotype 1 (MDV-1) major tegument proteins VP11/12, VP13/14, VP16, and VP22 in viral growth in cultured cells. Based on a bacterial artificial chromosome clone of MDV-1 (BAC20), mutant viruses were constructed in which the MDV-1 homologs of UL46, UL47, UL48, or UL49 were deleted alone and in various combinations. It could be demonstrated that the UL46, UL47, and UL48 genes are dispensable for MDV-1 growth in chicken embryonic skin and quail muscle QM7 cells, although the generated virus mutants exhibited reduced plaque sizes in all cell types investigated. In contrast, a UL49-negative MDV-1 (20Δ49) and a UL48-UL49 (20Δ48-49) doubly negative mutant were not able to produce MDV-1-specific plaques on either cell type. It was confirmed that this growth restriction is dependent on the absence of VP22 expression, because growth of these mutant viruses could be partially restored on cells that were cotransfected with a UL49 expression plasmid. In addition, we were able to demonstrate that cell-to-cell spread of MDV-1 conferred by VP22 is dependent on the expression of amino acids 37 to 187 of MDV-1 VP22, because expression plasmids containing MDV-1 UL49 mutant genes with deletions of amino acids 1 to 37 or 188 to 250 were still able to restore partial growth of the 20Δ49 and 20Δ48-49 viruses. These results demonstrate for the first time that an alphaherpesvirus UL49-homologous gene is essential for virus growth in cell culture

Marek’s disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16

International audienceGenes UL49 and UL48 of Marek's disease virus 1 (MDV-1) strain RB1B, encoding the respective homologues of herpes simplex virus type 1 (HSV-1) genes VP22 and VP16, were cloned into a baculovirus vector. Seven anti-VP22 MAbs and one anti-VP16 MAb were generated and used to identify the tegument proteins in cells infected lytically with MDV-1. The two genes are known to be transcribed as a single bicistronic transcript, and the detection of only one of the two proteins (VP22) in MSB-1 lymphoma and in chicken embryo skin cells infected with MDV-1 prompted the study of the transcription/translation of the UL49-48 sequence in an in vivo and in vitro expression system. VP16 was expressed in vitro at detectable levels, whereas it could only be detected at a lower level in a more controlled environment. It was demonstrated that VP22 is phosphorylated in insect cells and possesses the remarkable property of being imported into all cells in a monolayer. VP22 localized rapidly and efficiently to nuclei, like its HSV-1 counterpart. The DNA-binding property of VP22 is also reported and a part of the region responsible for this activity was identified between aa 16 and 37 in the N-terminal region of the protein

Evaluation of high-throughput sequencing for identifying knwon and unknown viruses in biological samples

High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and hasrapidly become a major tool for identifying viruses in biological samples, and in particular when the targetsequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of virusesin biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms.We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed ofsingle or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were addedat a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection ofquantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented,in public nucleotide sequence databases, we show that the higher output of Illumina is associated witha much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study,identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations,the number of reads generated by the Illumina platform was too small to facilitate assembly of contigswithout the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load wassufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-lengthgenomes and thus should facilitate the identification of novel viruses