Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.

Modified vaccinia virus Ankara (MVA) is an attenuated vaccinia virus with restricted replication in human cells. The virus serves as an ideal vaccine vector suitable for safe use even in immune-compromised individuals. With its inherently large packaging capacity, expression cassettes encoding bulky genes can be inserted into deletion regions within the MVA genome. These deletion sites develop during the process of the attenuation of the virus by passage in Chicken Embryo Fibroblasts (pCEFs). Transgene stability in MVA is important to assure immunogenicity and efficacy. In the present study, we assessed the effect of substantial passage of recombinant MVA vectors on the stability of expression cassette encoding SIV Gag/Tat genes inserted at the Del-II site, as part of generating a vaccine to protect from HIV. Our data indicated that after 15 passages there was a significant loss or mutation of the inserted genes.

HCMV carries virus-encoded envelope glycoproteins important for viral attachment, entry into host cells and replication. These glycoproteins have polymorphic features resulting in distinct genotypes some of which have been associated with disease outcome. Glycoprotein complex II comprises glycoproteins M (gM) and N (gN) and is essential for viral replication. Glycoprotein N is highly polymorphic while gM is highly conserved. The existence of distinct gM genotypes has not previously been reported. PCR amplification of the entire gM gene (1298 bp) was carried out on five HCMV laboratory strains (AD169, Towne, Davis, Toledo, Merlin) and ten clinical isolates. PCR product was sequenced and nucleotide and aminoacid phylogenetic trees constructed for these samples and for an additional 287 HCMV gM sequences obtained from NCBI database. The hydrophobicity of the predicted protein sequences was compared. Three distinct genotypes were identified (gM1, gM2 and gM3) with every sequence aligning with one genotype, suggesting they are stable polymorphisms rather than random nucleotide substitutions. Aminoacid translation of the nucleotide sequences merged gM1 and gM2, but gM3 remained as a distinct and separate type. The aminoacid substitutions led to changes in the hydrophobicity of the 3 glycoprotein types particularly between gM3 and the other two types. Glycoprotein M is highly conserved, but we have identified three distinct genotypes, arising from variability in a small region of the genome. These changes altered the predicted hydrophobicity of the glycoprotein potentially altering the conformational structure of the glycoprotein and affecting its function in vivo.