Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.

Astroviruses are enterically transmitted viruses that cause infections in mammalian and avian species. Astroviruses are nonenveloped, icosahedral viruses comprised of a capsid protein shell and a positive-sense, single-stranded RNA genome. The capsid protein undergoes dramatic proteolytic processing both inside and outside of the host cell, resulting in a coordinated maturation process that affects cellular localization, virus structure, and infectivity. After maturation, the capsid protein controls the initial phases of virus infection, including virus attachment, endocytosis, and genome release into the host cell. The astrovirus capsid is the target of host antibodies including virus-neutralizing antibodies. The capsid protein also mediates the binding of host complement proteins and inhibits complement activation. Here, we will review our knowledge on the astrovirus capsid protein (CP), with particular attention to the recent structural, biochemical, and virological studies that have advanced our understanding of the astrovirus life cycle.

Rotavirus strains differ in their need for sialic acid (SA) for initial binding to the cell surface; however, the existence of a postattachment cell receptor, common to most, if not all, rotavirus strains, has been proposed. In the present study, antibodies to the α _v and β ₃ integrin subunits, and the α _v β ₃ ligand, vitronectin, efficiently blocked the infectivity of the SA-dependent rhesus rotavirus RRV, its SA-independent variant nar3, and the neuraminidase-resistant human rotavirus strain Wa. Vitronectin and anti-β ₃ antibodies, however, did not block the binding of virus to cells, indicating that rotaviruses interact with α _v β ₃ at a postbinding step, probably penetration. This interaction was shown to be independent of the tripeptide motif arginine-glycine-aspartic acid present in the natural ligands of this integrin. Transfection of CHO cells with α _v β ₃ genes significantly increased their permissiveness to all three rotavirus strains, and the increment of virus infectivity was reverted by incubation of these cells either with antibodies to β ₃ or with vitronectin. These findings implicate α _v β ₃ integrin as a cellular receptor common to neuraminidase-sensitive and neuraminidase-resistant rotaviruses, and support the hypothesis that this integrin could determine, at least in part, the cellular susceptibility to rotaviruses.

Adaptation to specialized diets often requires modifications at both genomic and microbiome levels. We applied a hologenomic approach to the common vampire bat (Desmodus rotundus), one of the only three obligate blood-feeding (sanguivorous) mammals, to study the evolution of its complex dietary adaptation. Specifically, we assembled its high-quality reference genome (scaffold N50 = 26.9 Mb, contig N50 = 36.6 kb) and gut metagenome, and compared them against those of insectivorous, frugivorous and carnivorous bats. Our analyses showed a particular common vampire bat genomic landscape regarding integrated viral elements, a dietary and phylogenetic influence on gut microbiome taxonomic and functional profiles, and that both genetic elements harbour key traits related to the nutritional (for example, vitamin and lipid shortage) and non-nutritional (for example, nitrogen waste and osmotic homeostasis) challenges of sanguivory. These findings highlight the value of a holistic study of both the host and its microbiota when attempting to decipher adaptations underlying radical dietary lifestyles.

In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.