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Indirect immunofluorescence techniques targeting the rotavirus (RV) protein VP6 are used to differentiate RV species. The ICTV recognizes RV species A to E and two tentative species, F and G. A potential new RV species, ADRV-N, has been described. Phylogenetic trees and pairwise identity frequency graphs were constructed with more than 400 available VP6 sequences and seven newly determined VP6 sequences of RVD strains. All RV species were separated into distinct phylogenetic clusters. An amino acid sequence cutoff value of 53% firmly permitted differentiation of RV species, and ADRV-N was tentatively assigned to a novel RV species H (RVH).

Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image. Using a 2.6 Å resolution single particle cryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of 100 electrons/Å2, we obtained accurate measurements of optimal exposure values over a wide range of resolutions. At low and intermediate resolutions, our measured values are considerably higher than obtained previously for crystalline specimens, indicating that both images and movies should be collected with higher exposures than are generally used. We demonstrate a method of using our optimal exposure values to filter movie frames, yielding images with improved contrast that lead to higher resolution reconstructions. This ‘high-exposure’ technique should benefit cryo-EM work on all types of samples, especially those of relatively low-molecular mass. Microscopes allow us to visualize objects that are invisible to the naked eye. One type of microscope—called the electron microscope—produces images using beams of particles known as electrons, which enables them to produce more detailed images than microscopes that use light. There are several ways to prepare samples for electron microscopy. For example, in ‘electron cryo-microscopy’—or cryo-EM for short—a sample is rapidly frozen to preserve its features before it is examined under the microscope. This technique generates images that can be analyzed by computers to produce three-dimensional models of individual viruses, proteins, and other tiny objects. Unfortunately, the samples need to be exposed to high-energy beams of electrons that will damage the sample while the images are gathered, which results in sample movement and blurry images that lack the finer details. The contrast between the sample and its background is one of the factors that determine the final quality of an image. The higher the contrast, the greater the level of structural information that can be obtained, but this requires the use of longer exposures to the electron beam. To overcome this issue, researchers found that instead of recording a single image, it is possible to record movies in which the movement of the sample under the electron beam can be tracked. After the movies are gathered, the movie frames are aligned using computer software to reduce the blurring caused by the sample moving and can then be used to make three-dimensional models. Grant and Grigorieff improved this method further by studying how quickly a large virus-like particle called ‘rotavirus double-layered particle’ is damaged under the electron beam. These experiments identified an optimum range of exposure to electrons that provides the highest image contrast at any given level of detail. These findings were used to design an exposure filter that can be applied to the movie frames, allowing Grant and Grigorieff to visualize features of the virus that had not previously been observed by cryo-EM. This method was also used to study an assembly of proteins known as the proteasome, which is responsible for destroying old proteins. Grant and Grigorieff's findings should be useful for cryo-EM studies on many kinds of samples.

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.

This crystallographic study shows the attachment of human rotavirus VP8* to histo blood group A antigen, and suggests how changes within the structure of VP8* could allow switching from sialylated to non-sialylated glycan receptor. Rotavirus surface protein targets blood-group antigen Rotaviruses are the major pathogens of infantile gastroenteritis. They attach to the surfaces of cells through interactions with specific cellular glycans. Animal rotaviruses bind to glycans with terminal sialic acid, whereas human rotavirus strains are sialidase insensitive. Venkataram Prasad and colleagues now show that certain human rotavirus strains bind to and infect cells through A-type histo-blood group antigen (HBGA), suggesting that susceptibility to specific human rotavirus strains might be influenced by different blood-group antigens, a phenomenon reported in Helicobacter pylori and norovirus infection. Crystallographic studies show how HBGA binds to the attachment protein of human norovirus (VP8), and suggest how subtle changes in the structure of VP8 might allow receptor switching. As with many other viruses, the initial cell attachment of rotaviruses, which are the major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans 1 , 2 , 3 , 4 . The distally located VP8* domain of the rotavirus spike protein VP4 (ref. 5 ) mediates such interactions. The existing paradigm is that ‘sialidase-sensitive’ animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas ‘sialidase-insensitive’ human rotavirus strains bind to glycans with internal Sia such as GM1 (ref. 3 ). Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies 1 , 3 , 6 , 7 , it is not yet known how VP8* of human rotaviruses interacts with Sia and whether their cell attachment necessarily involves sialoglycans. Here we show that VP8* of a human rotavirus strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-A-type antibodies as well as significantly enhanced in Chinese hamster ovary cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of human rotavirus. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelia and on red blood cells 8 , and are recognized as susceptibility and cell attachment factors for gastric pathogens like Helicobacter pylori 9 and noroviruses 10 . Our crystallographic studies show that the A-type HBGA binds to the human rotavirus VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific human rotavirus strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world’s population.

A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell 1 . For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4 2 , 3 . Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an ‘upright’ to a ‘reversed’ conformation. The reversed structure projects a previously buried ‘foot’ domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses. Electron cryomicroscopy and cryotomography studies reveal that rotaviruses attach to a target cell through the outer-layer protein VP4, which—following cleavage—rearranges to enable perforation of the membrane and delivery of the viral genome into the host cell.

Strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans, and to restrict type II glycan binding in the bovine counterpart. Such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses. Rotaviruses cause life-threatening gastroenteritis in children and have been shown to have variations in glycan recognition from genetic alterations. Here, the authors use X-ray crystallography to indicate that these alterations can change the specificity of the virus from cows to infant humans.

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-β1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus.

Porcine Rotavirus (PoRV) is a significant pathogen affecting swine-rearing regions globally, presenting a substantial threat to the economic development of the livestock sector. At present, no specific pharmaceuticals are available for this disease, and treatment options remain exceedingly limited. This study seeks to design a multi-epitope peptide vaccine for PoRV employing bioinformatics approaches to robustly activate T-cell and B-cell immune responses. Two antigenic proteins, VP7 and VP8*, were selected from PoRV, and potential immunogenic T-cell and B-cell epitopes were predicted using immunoinformatic tools. These epitopes were further screened according to non-toxicity, antigenicity, non-allergenicity, and immunogenicity criteria. The selected epitopes were linked with linkers to form a novel multi-epitope vaccine construct, with the PADRE sequence (AKFVAAWTLKAAA) and RS09 peptide attached at the N-terminus of the designed peptide chain to enhance the vaccine’s antigenicity. Protein-protein docking of the vaccine constructs with toll-like receptors (TLR3 and TLR4) was conducted using computational methods, with the lowest energy docking results selected as the optimal predictive model. Subsequently, molecular dynamics (MD) simulation methods were employed to assess the stability of the protein vaccine constructs and TLR3 and TLR4 receptors. The results indicated that the vaccine-TLR3 and vaccine-TLR4 docking models remained stable throughout the simulation period. Additionally, the C-IMMSIM tool was utilized to determine the immunogenic triggering capability of the vaccine protein, demonstrating that the constructed vaccine protein could induce both cell-mediated and humoral immune responses, thereby playing a role in eliciting host immune responses. In conclusion, this study successfully constructed a multi-epitope vaccine against PoRV and validated the stability and efficacy of the vaccine through computational analysis. However, as the study is purely computational, experimental evaluation is required to validate the safety and immunogenicity of the newly constructed vaccine protein.

Viroporins are small virus-encoded ion channel proteins. Most viroporins are monovalent selective cation channels, with few showing the ability to conduct divalent cations, like calcium (Ca 2+ ). Nevertheless, some viroporins are known to disrupt host cell Ca 2+ homeostasis, which is critical for virus replication and pathogenesis. Rotavirus nonstructural protein 4 (NSP4) is an endoplasmic reticulum transmembrane glycoprotein that has a viroporin domain (VPD), and NSP4 viroporin activity elevates cytosolic Ca 2+ in mammalian cells. The goal of this study was to demonstrate that the NSP4 VPD forms an ion channel and determine whether the channel can conduct Ca 2+ . Using planar lipid bilayer and liposome patch clamp electrophysiology, we show that a synthetic peptide of the NSP4 VPD has ion channel activity. The NSP4 VPD was selective for cations over anions and channel activity was observed to have both well-defined “square top” openings as well as fast current fluctuations, similar to other viroporins. Importantly, the NSP4 VPD showed similar conductance of divalent cations (Ca 2+ and Ba 2+ ) as monovalent cations (K + ), but a viroporin defective mutant lacked Ca 2+ conductivity. These data demonstrate that the NSP4 VPD is a Ca 2+ -conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca 2+ homeostasis.

Group A human rotaviruses (RVs) are a major cause of severe gastroenteritis in infants and young children. Yet, aside from the genes encoding serotype antigens (VP7; G-type and VP4; P-type), little is known about the genetic make-up of emerging and endemic human RV strains. To gain insight into the diversity and evolution of RVs circulating at a single location over a period of time, we sequenced the eleven-segmented, double-stranded RNA genomes of fifty-one G3P[8] strains collected from 1974 to 1991 at Children's Hospital National Medical Center, Washington, D. C. During this period, G1P[8] strains typically dominated, comprising on average 56% of RV infections each year in hospitalized children. A notable exception was in the 1976 and 1991 winter seasons when the incidence of G1P[8] infections decreased dramatically, a trend that correlated with a significant increase in G3P[8] infections. Our sequence analysis indicates that the 1976 season was characterized by the presence of several genetically distinct, co-circulating clades of G3P[8] viruses, which contained minor but significant differences in their encoded proteins. These 1976 lineages did not readily exchange gene segments with each other, but instead remained stable over the course of the season. In contrast, the 1991 season contained a single major clade, whose genome constellation was similar to one of the 1976 clades. The 1991 clade may have gained a fitness advantage after reassorting with as of yet unidentified RV strain(s). This study reveals for the first time that genetically distinct RV clades of the same G/P-type can co-circulate and cause disease. The findings from this study also suggest that, although gene segment exchange occurs, most reassortant strains are replaced over time by lineages with preferred genome constellations. Elucidation of the selective pressures that favor maintenance of RVs with certain sets of genes may be necessary to anticipate future vaccine needs.