The mechanism leading to reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/reinitiation process. VP2 of the feline calicivirus is encoded in the 3′-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3′-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.

Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping cistrons. The first ORF of this RNA codes for the viral major capsid protein VP1, and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a termination/reinitiation mechanism, which depends on an upstream sequence element of ∼70 nucleotides denoted “termination upstream ribosomal binding site” (TURBS). Two short sequence motifs within the TURBS were found to be essential for reinitiation. By a whole set of single site mutations and reciprocal base exchanges we demonstrate here for the first time conclusive evidence for the necessity of mRNA/18S rRNA hybridization for translation reinitiation in an eukaryotic system. Moreover, we show that motif 2 exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus forming a secondary structure that positions post-termination ribosomes in an optimal distance to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better understanding of the requirements for translation termination/reinitiation in eukaryotes.

Since November 2016, Europe witnesses another wave of incursion of highly pathogenic avian influenza (HPAI) A(H5) viruses of the Asian origin goose/Guangdong (gs/GD) lineage. Infections with H5 viruses of clade 2.3.4.4b affect wild bird and poultry populations. H5 viruses of clades 2.2, 2.3.1.2c and 2.3.4.4a were detected previously in Europe in 2006, 2010 and 2014. Clades 2.2.1.2 and 2.3.2.1.c are endemic in Egypt and Western Africa, respectively and have caused human fatalities. Evidence exists of their co-circulation in the Middle East. Subtype H5 viruses of low pathogenicity (LPAI) are endemic in migratory wild bird populations. They potentially mutate into highly pathogenic phenotypes following transmission into poultry holdings. However, to date only the gs/GD H5 lineage had an impact on human health. Rapid and specific diagnosis marks the cornerstone for control and eradication of HPAI virus incursions. We present the development and validation of five real-time RT-PCR assays (RT-qPCR) that allow sequencing-independent pathotype and clade-specific distinction of major gs/GD HPAI H5 virus clades and of Eurasian LPAI viruses currently circulating. Together with an influenza A virus-generic RT-qPCR, the assays significantly speed up time-to-diagnosis and reduce reaction times in a OneHealth approach of curbing the spread of gs/GD HPAI viruses.

A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.

Since 2014, several waves of avian influenza virus (AIV) H5N8 of clade 2.3.4.4 occurred globally on unprecedented levels. Unlike viruses in the first wave in 2014–2015 (H5N8-A), viruses in 2015–2016 (H5N8-B) exhibited unusually high pathogenicity (HP) in wild and domestic ducks.

Caliciviruses are a cause of important diseases in humans and animals. It is crucial to understand the prerequisites of efficient replication of these viruses in order to develop strategies for prevention and treatment of these diseases. It was shown before that all caliciviruses except vesiviruses have established mechanisms to achieve major capsid protein (VP1) translation from the genomic RNA. Here, we show for the first time that a member of the genus Vesivirus also has a translation initiation mechanism by which a precursor protein of the VP1 protein is expressed from the genomic RNA. This finding clearly points at a functional role of the calicivirus VP1 capsid protein in early replication, and we provide experimental data supporting this hypothesis.