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Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

Influenza A viruses initiate infection by binding to glycans with terminal sialic acids present on the cell surface. Hosts of influenza A viruses variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in the majority of mammals including horses, pigs, and mice, but is absent in humans, ferrets, and birds. Intriguingly, the only known naturally occurring influenza A viruses that exclusively bind NeuGc are the extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with its NeuGc ligand and observed a high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs. We found that mutation A135E is key for binding α2,3-linked NeuGc but does not abolish NeuAc binding. Interestingly, additional mutations S128T, I130V, or a combination of T189A and K193R, converted from NeuAc to NeuGc specificity as determined by glycan microarrays. However, specific binding to NeuGc-terminal glycans on our glycan array did not always correspond with full NeuGc specificity on chicken and equine erythrocytes and tracheal epithelium sections. Phylogenetic analysis of avian and equine H7 HAs that investigated the amino acids at positions 128, 130, 135, 189, and 193 reveals a clear distinction between equine and avian residues. The highest variability in amino acids (four different residues) is observed at key position 135, of which only the equine glutamic acid leads to binding of NeuGc. The results demonstrate that avian H7 viruses, although genetically distinct from equine H7 viruses, can bind NeuGc after the introduction of two to three mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors.Author summaryInfluenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A virus, these ‘keys’ (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority binds NeuGc. NeuGc is present in species like horses, pigs, and mice, but not in humans, ferrets, and birds. Therefore, NeuGc binding could be a determinant of an Influenza A virus species barrier. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage &phi, R1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts.